Loic Ponger

Finn-Arne Weltzien

Ian Meyer

Dufour S Viii

Dufour S Ix

Herranz Jusdado

Santidrián Yebra-Pimentel

Aurora Campo

• Characterization of novel neuropeptides in eel. Application in fish breeding programs (Pechakucha)

Keywords: tachykinin-1, tachykinin-4, phylogeny, whole genome duplication, teleost, Anguilla

The aim of this PhD was to investigate the role of brain neuropeptides, such as neurokinin B, encoded by tac3 gene, in the control of reproduction of an endangered species, the European eel, Anguilla anguilla. The sexual maturation of the eel is blocked at a prepubertal stage before the oceanic migration. Due to its basal phylogenetic position among teleosts, the eel is also a relevant model for studying molecular and functional evolution of key neuropeptides. Two tac3 paralogous genes (tac3a and tac3b) were identified in the eel genome, each encoding two peptides (NKBa or b and NKB-related peptide NKB-RPa or b). Amino-acid sequence of eel NKBa is identical to human NKB, and the three others are novel peptide sequences. The four eel peptides present the characteristic C-terminal tachykinin sequence, as well as a similar alpha helix 3D structure. Tac3 genes were identified in silico in 52 species of vertebrates and a phylogeny analysis was performed on the predicted TAC3 pre-pro-peptide sequences. A synteny analysis was also done to further assess the evolutionary history of tac3 genes. Duplicated tac3 genes in eel as in other teleosts likely result from the teleost-specific whole genome duplication (3R). Among extant teleosts, TAC3b precursor sequences exhibit more divergence than TAC3a sequences, and losses of tac3b paralog would have even occurred in some teleost lineages. Comparison of vertebrate TAC3 precursor sequences also confirmed that NKB-RP peptide, encoded beside NKB by tac3 gene in actinopterygians and basal sarcopterygians, would have been lost in ancestral amniotes. Development of specific qPCR for each tac3a and tac3b paralog, allowed us to compare the tissue distribution of their transcripts. Tac3a and tac3b mRNAs showed major expression of both transcripts in the brain especially in the diencephalon. Commercially available human NKB (same sequence as eel NKBa) has been tested in vitro on primary culture of eel pituitary cells. Human NKB dose-dependently inhibited the expression of luteinizing hormone beta subunit (lhβ), while having no significant effect on the expression of other glycoprotein hormone subunits, follicle-stimulating hormone beta subunit (fshβ), thyroidstimulating hormone beta subunit (tshβ), common glycoprotein hormone alpha subunit (gpα) nor on growth hormone (gh). Human NKB/eel NKBa also dose-dependently inhibited the expression of eel GnRH receptor 2 (gnrh-r2), the other GnRH receptor types being undetectable in the cell culture. The four eel peptides were then synthesized and also tested in vitro. They all inhibited the expression of both lhβ and of gnrh-r2. This indicates a potential dual inhibitory action of the four eel peptides at the pituitary level on both LH and GnRH receptor. These studies revealed that the tachykinin 3 system may contribute to the strong inhibitory control of sexual maturation in the European eel. Further ongoing studies concern the identification in the eel of the paralogs of the other tac genes (tac1 and tac4 paralogs), and their phylogeny analysis.

 ....................................................................................................................... 1 Animals .................................................................................................................................... 2 In silico prediction .................................................................................................................... 3 Cloning and sequencing of eel tac3 cDNAs .............................................................................. 4 Phylogeny ................................................................................................................................ 5 Synteny .................................................................................................................................... 6 Tissue distribution .................................................................................................................... 7 Prediction and synthesis of eel TAC3 peptides ......................................................................... xiii8 Prediction of the three-dimensional peptide structure of eel TAC3 peptides .......................... 9 Pituitary cell culture ................................................................................................................. PCR (qPCR) ........................................................................................ 11 Statistical analysis .............................................................................................................. .......................................................................................................... 2 Article 1: Tachykinin-3 genes and peptides characterized in a basal teleost, the European eel: evolutionary perspective and pituitary role ................................................................................... ........................................................................................................ 2 Article 2: Tachykinin-1 and 4 genes and peptides in a basal teleost, Anguilla anguilla:

Identification and phylogeny analysis ............................................................................................. INTRODUCTION ..................................................................................................................... MATERIALS AND METHODS ................................................................................................. In silico prediction of tac1 and tac4 genes . ................................................................................................................................ ........................................................................................................................ ACKNOWLEDGMENTS ............................................................................................................... REFERENCES .............................................................................................................................. FIGURE LEGENDS ................................................................................................................. FIGURES : ............................................................................................................................... European eel ............................................................ 2 Evolutionary history of the tachykinins .................................................................................. 3 Reproductive role of the TAC3 in the eel ............................................................................... .......................................................................................................... 2 Tac receptors in the eel .......................................................................................................... 3 Potential receptor/ligand couples in the eel: recombinant receptors and test of peptides ... 4 Comparative evolutionary history of tac and tac receptors ................................................. Chapter I

RESULTS

Multiple tac genes and peptides in the

Introduction

I. Introduction 1 European eel

The European eel, Anguilla anguilla [START_REF] Linnaeus | Systema Naturae per Regna Tria Naturae, secundum Classes, Ordines, Genera, Species, cum Characteribus, Differentiis, Synonymis, Locis[END_REF], is a euryhaline and catadromous species with a leptocephala larva, a characteristic in common with all the species of the Elopomorpha superorder.

Taxonomy

European eel taxonomic position is Class OSTEICTI; Superorder ELOPOMORPHA; Order ANGUILLIFORME; Family ANGUILLIDAE; Gender Anguilla; Species A. Anguilla.

Fifteen eel species and three subspecies have been identified in coasts and oceans all around the world except the East coast of South America [START_REF] Minegishi | Molecular phylogeny and evolution of the freshwater eels genus Anguilla based on the whole mitochondrial genome sequences[END_REF]. The most important species in the North hemisphere are European eel, Anguilla anguilla, and American eel, Anguilla rostrata. The Japanese eel, Anguilla japonica, is the species from the Pacific Ocean. Eels are members of the elopomorphs, which are primitive teleost fishes that include silvery-coloured species such as tarpons (Elopiforma), bonefishes (Albulidae) or anguilliforms among many others. Their common characteristic is the development of leptocephala larvae as the first stage of their life cycle. The monophyletic origin of elopomorphs has been demonstrated by mitochondrial genomic analysis [START_REF] Inoue | Basal actinopterygian relationships: A mitogenomic perspective on the phylogeny of the "ancient fish[END_REF] and using multi-locus dataset composed of three nuclear and three mitochondrial genes [START_REF] Chen | Phylogeny of the Elopomorpha (Teleostei): Evidence from six nuclear and mitochondrial markers[END_REF]. The gender Anguilla, which includes the eels, belongs to the Anguilloidei suborder (Figure 1). superorder Elopomorpha whose members all possess a leptocephalus larvae [START_REF] Inoue | Basal actinopterygian relationships: A mitogenomic perspective on the phylogeny of the "ancient fish[END_REF].

The genus Anguilla is the only genus among the elopomorphs that divides its life between the sea and freshwater environments. The European eel is distributed along the North Atlantic and Mediterranean coasts and rivers (Figure 2). Adapted manually from IUCN red list [START_REF] Freyhof | Anguilla anguilla', The IUCN Red List of Threatened Species[END_REF].

Life cycle

The life cycle of European eel is catadromous, with an important growth phase in freshwater and a spawning in the sea. It includes two migratory phases associated with two major morphophysiological changes: larval metamorphosis and pre-puberty (Figure 3, for reviews: Otake 2003; [START_REF] Rousseau | Advances in eel reproductive physiology and endocrinology[END_REF].

Leptocephala larvae and larval metamorphosis

The Danish scientist, Johannes Schmidt, started a series of expeditions in the North Atlantic

Ocean for collecting leptocephala larvae, thus comparing size and abundance in each area. This data allowed deducing that the spawning areas must be located in the Sargasso sea [START_REF] Schmidt | The breeding places of the eel[END_REF].

Different expeditions during the 20th century confirmed the distribution pattern of leptocephala larvae observed by Schmidt (for review [START_REF] Miller | A century of research on the larval distributions of the Atlantic eels: A re-examination of the data[END_REF]. The leptocephala larvae have oak leave shape with a maximum size of 85mm. Leptocephala larvae below 5mm are transported passively by the currents, and after 5mm present active transport mechanisms (for review: van Ginneken and Maes 2005).

The metamorphosis from leptocephala larva to the glass eel takes place when the larvae arrive in the continental plate after a travel that takes between 8-9 months (for review: van Ginneken and , 2005). Major changes are the increase of glycosaminoglycan as energy storage, changes in internal organs structure, ossification of the vertebrae, negative phototaxis, and change of the body shape into a cylindrical one (for review: Otake 2003).

Maes

Continental growth phase

European glass eels present estuarine and fluvial upstream recruitments that are regulated by seasonality and motivated by darkness [START_REF] Tabeta | The glass eel[END_REF]. The larvae tend to enter in brackish waters, deltas and coastal lagoons mostly during new moon phase, even though they can be observed all year in the Mediterranean coast [START_REF] Arias | Estados juveniles de la ictiofauna en los caños de las salinas de la Bahía de Cádiz[END_REF]. This behaviour is possibly affected by temperature, salinity, turbidity, river flow, tidal cycle and has a seasonal component [START_REF] Tabeta | The glass eel[END_REF]. Interestingly, recent studies have detected a magnetic map in glass eels that would allow them orient depending on subtle differences in magnetic field intensity and inclination angle along the migration route, that would be also linked to tidal variations [START_REF] Cresci | Glass eels (Anguilla anguilla) have a magnetic compasslinked to the tidal cycle[END_REF][START_REF] Naisbett-Jones | A Magnetic Map Leads Juvenile European Eels to the Gulf Stream[END_REF].

Along with this upstream migration behaviour, glass eel increases in body size and changes into yellow skin colour. Therefore, the upstream freshwater phase is known as the yellow eel/stage. They swim aligned beside the riverbank at the bottom, mostly with a favourable tide. Some eels remain in coastal areas such as estuaries with variable salinity, while others ascend to the beginning of the rivers.

This yellow phase lasts a particularly variable amount of time, oscillating from 6 to 12 years in males and 10 to 20 years in females [START_REF] Beullens | Sex differentiation, changes in length, weight and eye size before and after metamorphosis of European eel (Anguilla anguilla L.) maintained in captivity[END_REF].

Silvering as a pre-puberty event

A second major morpho-physiological change known as silvering occurs in yellow eels after the appearance of a still unknown trigger [START_REF] Dufour | Reproductive endocrinology of the European eel, Anguilla anguilla[END_REF][START_REF] Rousseau | Advances in eel reproductive physiology and endocrinology[END_REF]. Morphological external changes are the increase of the eye size and silvering of the skin. Eel skin increases thickness and reduces the yellow xantophores thus decreasing the yellow colour towards a much more silver one [START_REF] Aoyama | The silver eel[END_REF]. This phase is known as the silver eel/stage. Other important changes related to morphology and migratory behaviour are a degeneration of the digestive tract accompanied of starvation and finish of growth, activation of filament chloride cells in the gills and increase of the swim bladder to support deeper depths as found in the ocean [START_REF] Aoyama | The silver eel[END_REF][START_REF] Dufour | Reproductive endocrinology of the European eel, Anguilla anguilla[END_REF][START_REF] Rousseau | Advances in eel reproductive physiology and endocrinology[END_REF][START_REF] Pelster | Swimbladder function and the spawning migration of the European eel Anguilla anguilla[END_REF]. Those changes pre-adapt the eel to face the challenges in the sea environment where spawning takes place. Particularly, osmotic adaptations previous to sea migration are regulated by changes in the expression of genes related with osmoregulation in European eel [START_REF] Kalujnaia | Transcriptomic approach to the study of osmoregulation in the European eel Anguilla anguilla[END_REF] and Japanese eel [START_REF] Lai | Transcriptomic analysis reveals specific osmoregulatory adaptive responses in gill mitochondria-rich cells and pavement cells of the Japanese eel[END_REF].

Silvering occurs after activation of the gonadotropic axis indicated by an increase of the circulating sexual steroids (Han et al., 2003a;[START_REF] Aroua | Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event[END_REF][START_REF] Durif | The silvering process of Anguilla anguilla: A new classification from the yellow resident to the silver migrating stage[END_REF][START_REF] Van Ginneken | Silvering of European eel (Anguilla anguilla L.): seasonal changes of morphological and metabolic parameters[END_REF][START_REF] Dufour | Neuroendocrinology of fish metamorphosis and puberty: Evolutionary and ecophysiological perspectives[END_REF][START_REF] Rousseau | Eel secondary metamorphosis: silvering[END_REF][START_REF] Trautner | Metamorphosis and transition between developmental stages in European eel (Anguilla anguilla, L.) involve epigenetic changes in DNA methylation patterns[END_REF]for review: Rousseau et al., 2013). Classification of sexual maturation is established from histological evaluation of oocytes. Stages I and II correspond to lipid incorporation in oocytes. Stage III corresponds to an increase in steroid production. Stage IV and V correspond to presence of nucleus or "oil droplet" in the oocytes. Since there is a marked increase in the gonadosomatic index (GSI) of 2% for females and 1% for males [START_REF] Dufour | Reproductive endocrinology of the European eel, Anguilla anguilla[END_REF], the classification of the silvering process is therefore established according to three stages of GSI: below 0.4% for a yellow eel, between 0.4% and 1.2% for an intermediate yellow-silver eel, and above 1.2% for a silver eel. This illustrates that the progression of silvering procedure is parallel to gonad maturation as a pubertal phenomenon, but sexual maturation is blocked indicating a pre-puberty [START_REF] Aroua | Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event[END_REF][START_REF] Durif | The silvering process of Anguilla anguilla: A new classification from the yellow resident to the silver migrating stage[END_REF][START_REF] Rousseau | Advances in eel reproductive physiology and endocrinology[END_REF].

The hormones inducing the changes related to eel silvering are the sexual steroids estrogens (estradiol, E2) and androgens (testosterone, T and 11-ketotestosterone, 11KT) in all studied eel species (Conger conger: [START_REF] Sbaihi | Reproductive biology of the conger eel from the south coast of Brittany, France and comparison with the European eel[END_REF]A. anguilla: Aroua et al., 2005;[START_REF] Van Ginneken | Silvering of European eel (Anguilla anguilla L.): seasonal changes of morphological and metabolic parameters[END_REF]A. japonica: Han et al., 2003c;[START_REF] Jeng | Differential expression and regulation of gonadotropins and their receptors in the Japanese eel, Anguilla japonica[END_REF][START_REF] Sudo | 11ketotestosterone Synchronously Induces Oocyte Development and Silvering-Related Changes in the Japanese Eel, Anguilla japonica[END_REF]A. rostrata: Ccottrill et al., 2001;A. diffenbachii and A. australis: Lokman et al., 1998;and A. australis: Rohr et al., 2001). Those increases have been observed as well on both gonadotropin mRNA in European and Japanese eel (Han et al., 2003b;[START_REF] Aroua | Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event[END_REF][START_REF] Jeng | Differential expression and regulation of gonadotropins and their receptors in the Japanese eel, Anguilla japonica[END_REF], with an increase of expression of the gonadotropin subunits fshβ at the beginning of the process and lhβ in the end. According to Rousseau and coworkers (2013), FSH might be related to the incorporation of lipids in the oocytes or "endogenous vitellogenesis" (stages I, II and III) while LH can be related to the second increase of sex steroid levels and late induction of Vg or "exogenous vitellogenesis" (stages IV and V). Experiments enhancing the gonadotropic axis by injection of exogenous gonadotropins (human chorionic gonadotropin, hCG or carp pituitary extract, PCe) achieved an amplification of the eye (Pankhurst 1982a ;[START_REF] Boëtius | Effects of testosterone on eye size and spermiation in silver eels, Anguilla anguilla[END_REF], increase of red muscle volume (Pankhurst 1982b), as well as a silvering and strengthening of the European eel skin [START_REF] Pankhurst | Structure and colour of the integument of the European eel Anguilla anguilla (L.)[END_REF]. Intraperitoneal injection of 17α-methyltestosterone (17MT), 11KT and T also enhanced the anatomical changes during silvering in eel species [START_REF] Olivereau | Effect of 17??-methyltestosterone on the skin and gonads of freshwater male silver eels[END_REF][START_REF] Boëtius | Effects of testosterone on eye size and spermiation in silver eels, Anguilla anguilla[END_REF][START_REF] Rohr | 11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis[END_REF][START_REF] Aroua | Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event[END_REF].

In addition, an increase of plasma cortisol levels is detected prior to migration mostly because of starvation and apparently to mobilize metabolic energy for gonadal development and active swimming during migration [START_REF] Van Ginneken | Silvering of European eel (Anguilla anguilla L.): seasonal changes of morphological and metabolic parameters[END_REF]. Huang and co-workers described these results as a synergistic action of cortisol and androgens in luteinizing hormone (LH) release [START_REF] Huang | Androgens stimulate gonadotropin-II beta-subunit in eel pituitary cells in vitro[END_REF][START_REF] Huang | Cortisol Selectively Stimulates Pituitary Gonadotropin β-Subunit in a Primitive Teleost[END_REF]for review: Rousseau et al., 2013).

Reproductive migration

Reproductive migration begins in the rivers and continues to the sea in autumn [START_REF] Tesch | The eel[END_REF]. River migrations of European eel have been tracked by acoustic transmission between freshwater to seawater transitions. Nocturnal migrations are dominant, increasing during the three moon phases with less illumination and significant preference for migration during ebb tides [START_REF] Aarestrup | Survival and progression rates of large European silver eel Anguilla anguilla in late freshwater and early marine phases[END_REF][START_REF] Barry | Freshwater and coastal migration patterns in the silver-stage eel Anguilla anguilla[END_REF].

Even though no adults have been seen in the way to the supposed spawning area [START_REF] Van Ginneken | The European eel (Anguilla anguilla, Linnaeus), its lifecycle, evolution and reproduction: A literature review[END_REF], eels are likely to abandon the continental waters and swim south using probably the Canary and North-equatorial currents with a mean direction of 250º WSW at depths of about 200m-800m (van Ginneken and Maes, 2005;[START_REF] Tesch | The eel[END_REF][START_REF] Aarestrup | Oceanic Spawning Migration of the European Eel (Anguilla anguilla)[END_REF][START_REF] Aoyama | Life History and Evolution of Migration in Catadromous Eels (Genus Anguilla )[END_REF]. A Nordic migration route for European eel was verified after observing an equal behaviour of local and translocated eels from the UK into the Baltic Sea [START_REF] Westerberg | Behaviour of stocked and naturally recruited European eels during migration[END_REF]. In the case of the American eel, satellite transmission revealed sea migratory behaviour down to the Sargasso Sea [START_REF] Béguer-Pon | Direct observations of American eels migrating across the continental shelf to the Sargasso Sea[END_REF].

The release of European silver eels in North-East of the Atlantic Ocean and nearby the Sargasso Sea confirmed daily vertical migrations in the water column nearby the spawning area [START_REF] Wysujack | The migration behaviour of European silver eels (Anguilla anguilla) released in open ocean conditions[END_REF]. These vertical migrations occurring nearby the spawning areas have been recently

observed in other eel species such as A. japonica [START_REF] Schabetsberger | Hydrographic structure of anguillid spawning areas[END_REF][START_REF] Chow | Light-sensitive vertical migration of the Japanese eel Anguilla japonica revealed by real-time tracking and its utilization for geolocation[END_REF]Chen et al., 2018;[START_REF] Higuchi | Tracking Anguilla japonica silver eels along the West Marina ridge using pop-up archival transmitting tags[END_REF], A. marmorata and A. bicolour pacifica (Chen et al., 2018). The migrations occur usually between 150-250m at night and depths to 600-800m during the day.

According to larval recruitment, European eels must arrive at the Sargasso Sea in March/April.

They must spawn in spring or summer. Adults die supposedly after reproduction since no adults have been found in the return way (for review [START_REF] Miller | A century of research on the larval distributions of the Atlantic eels: A re-examination of the data[END_REF].

Eel decline

Eel species in the northern hemisphere experienced a decrease of the glass eel recruitment after the 80's, with a big drop to 1% of the 60's-70's population size at the beginning of the 21st century (Figure 4, [START_REF] Dekker | Worldwide decline of eel ressources necessitates immediate action. Québec Declaration of Concern[END_REF]. The complexity of the life cycle and the lack of knowledge of the spawners' condition do not help in determining the cause of the population's decline. However, the growth stage and migration in the continental waters is affected by factors of anthropogenic origins, such as pollution and water quality, fisheries, habitat loss, barriers to migration and introduced parasites [START_REF] Dekker | Worldwide decline of eel ressources necessitates immediate action. Québec Declaration of Concern[END_REF][START_REF] Dekker | What caused the decline of the Lake IJsselmeer eel stock after 1960?[END_REF]). In addition, Oceanic alterations such Gulf Stream shifts can reduce survival leptocephala larvae during migration [START_REF] Feunteun | Management and restoration of European eel population (Anguilla anguilla): An impossible bargain[END_REF]. Eel life cycle can also be altered by the presence in the environment of endocrine-disrupting compounds (EDCs), pollutants derived from the plastic use and industrial activity. For example, such components (polybrominated diphenyl ethers, PBDEs and polychlorinated biphenyls, PCBs) disrupt the expression of larval metamorphosis genes tshβ, dio2 (iodothyronine deiodinase), trα (thyroid hormone receptor α) and trβ (thyroid hormone receptor β) in the European eel, that is correlated to the presence in the environment [START_REF] Couderc | Thyroid endocrine status of wild European eels (Anguilla anguilla) in the Loire (France). Relationships with organic contaminant body burdens[END_REF]. In addition, exposure to these pollutants disrupted the epigenetic programming in ovarian tissue in the European eels [START_REF] Pierron | Abnormal Ovarian DNA Methylation Programming during Gonad Maturation in Wild Contaminated Fish[END_REF]. Heavy metals present in the eel environment may also affect the eel muscle and livers (south of Spain: Usero et al., 2003;French Camargue: Oliveira Ribeiro et al., 2005) and the cause of tumours induced by genotoxicity (Sanchez-Galán et al., 2001;[START_REF] Ribeiro | Bioaccumulation and the effects of organochlorine pesticides, PAH and heavy metals in the Eel (Anguilla anguilla) at the Camargue Nature Reserve, France[END_REF].

Therefore, the neuroendocrine study of the blockade of the pre-pubertal stage of silver eel is a key factor for understanding the decline and establishing patterns for European eel conservation. rostrata). Average of juvenile eels recruited between 1960s and 1970s are the base for comparing the percentage of yearly recruited eels until 21 st Century [START_REF] Dekker | Worldwide decline of eel ressources necessitates immediate action. Québec Declaration of Concern[END_REF].

Hormonal induction of maturation

Many laboratories have searched for environmental factors such as swimming, hydrostatic pressure, photoperiod and temperature, that can induce the sexual maturation of European eel Anguilla anguilla [START_REF] Van Ginneken | Silvering of European eel (Anguilla anguilla L.): seasonal changes of morphological and metabolic parameters[END_REF][START_REF] Sébert | Effects of high hydrostatic pressure on the pituitary-gonad axis in the European eel, Anguilla anguilla (L.)[END_REF][START_REF] Palstra | Swimming physiology of European silver eels (Anguilla anguilla L.): Energetic costs and effects on sexual maturation and reproduction[END_REF]. In those studies, both swimming and hydrostatic pressure increased the gonad size and induced vitellogenesis. However, none of them reached a complete maturation thus pointing towards internal neurophysiological processes.

First studies for induction of gametogenesis in European eel took place in our laboratory in 1936.

The experiment supervised by Maurice Fontaine consisted of injecting pregnant human female urine extract in male eels thus achieving a complete spermatogenesis and sperm emission [START_REF] Fontaine | Sur la maturation complète des organes génitaux de l'anguille mâle et l'émission spontanée de ses produits sexuels[END_REF].

For inducing female oogenesis, carp pituitary extract was injected with positive results (Fontaine et al 1964). Further experiments have confirmed that LH and FSH have a particularly weak expression in pre-pubertal silver eel [START_REF] Dufour | Effects of steroid hormones on pituitary immunoreactive gonadotropin in European freshwater eel, Anguilla anguilla L[END_REF][START_REF] Aroua | Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids[END_REF]. Studies on the regulation of gonadotropin expression by our team revealed the stimulatory role of gonadotropin releasing hormone (GnRH) and the inhibitory role of dopamine. The double treatment with a GnRH agonist and an anti-dopaminergic (pimozide) could induce the synthesis and release of LH, thus producing the vitellogenesis [START_REF] Dufour | Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol[END_REF][START_REF] Vidal | Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty[END_REF].

Interest of European eel 1.5.1 Endangered species management

New measures for conservation of the European eel natural population and closing the life cycle in captivity are crucial steps to maintain the eel industry and reduce the socioeconomic impact of the eel critical situation.

The European Commission developed eel restoration plans based on escapement to the sea of at least 40% of the biomass of adult eel together with measures for decline average market prices for eels used for restocking [START_REF] Bevacqua | Assessing Management Plans for the Recovery of the European Eel : A Need for Multi-Objective Analyses[END_REF]; Commission of the European Communities, 2012).

However, no habitat restoration policies have been considered till date and monitoring of eel population does not report a population recovery [START_REF] Feunteun | Management and restoration of European eel population (Anguilla anguilla): An impossible bargain[END_REF].

Since eel life cycle has not been closed in captivity, eel exploitation is based on fattening of individuals captured in areas where stock densities were traditionally higher. The critically endangered situation of the European eel decreased the industry size and increased the market price.

Genomic interest: The base of teleost lineage

Eels are included in the super order Elopomorpha, a monophyletic group placed at the base of the teleost lineage. Eels can, therefore, reflect some ancestral characteristics. Among them, it is of special interests the teleost-specific genome duplication (TSGD) ratified by primitive duplication of their hox clusters (Henkel et al., 2012b).

It comes here to introduce whole genome duplications as of genetic importance in eel basal position. Genomic duplication events occurred two times during vertebrate radiation, mostly known as 1R and 2R [START_REF] Dehal | Two rounds of whole genome duplication in the ancestral vertebrate[END_REF][START_REF] Van De Peer | 2R or not 2R is not the question anymore[END_REF]. A third genomic duplication (3R) took place only in an ancestral teleost giving place to new physiological plasticity to the whole teleost lineage (Figure 5; [START_REF] Meyer | From 2R to 3R: Evidence for a fish-specific genome duplication (FSGD)[END_REF][START_REF] Braasch | Polyploidy in Fish and the Teleost Genome Duplication[END_REF]. The impact of this duplication on fish evolution relies upon which genes are retained in duplicate as paralogs and how the duplication modifies their evolutionary constraints [START_REF] Brunet | Gene loss and evolutionary rates following whole-genome duplication in teleost fishes[END_REF]. Even though many teleosts have lost the half of the duplicated hox genes [START_REF] Kuraku | The evolution and maintenance of Hox gene clusters in vertebrates and the teleost-specific genome duplication[END_REF], eel seems to keep many of them (Henkel et al., 2012b) thus probably increasing the fitness adapting to many different environments during its life cycle [START_REF] Pujolar | Signatures of natural selection between life cycle stages separated by metamorphosis in European eel[END_REF].

In addition to the conservation of the duplicated hox genes, eels duplicated neuropeptides and receptors are also very well preserved (Aquaporin 1: [START_REF] Tingaud-Sequeira | Structural and functional divergence of two fish aquaporin-1 water channels following teleost-specific gene duplication[END_REF]TSHβ subunit: Maugars et al., 2014;estrogen receptors: Lafont et al., 2015;leptin receptors: Morini et al., 2015;progestin receptor: Morini et al., 2017;GnRH-receptor I: Peñaranda et al., 2013; among many others).

The duplicated and preserved genes may have a particular functionality for each environment and/or phase of the eel life cycle, including puberty and sexual maturation. Many shred of evidence of differential expression of silvering related proteins and their receptors after exposure to steroids can be found in the literature. An example is the eye rhodopsin protein for the deep-sea vision that is upregulated after injection of steroids to artificially matured Japanese eels (Zhang et al. 2000).

Moving towards silvering reproductive neuropeptides our team found an increase in expression of one estrogen nuclear receptor (ESR1) in European eel pituitary thus pointing to a different proportion on the abundance of each receptor [START_REF] Lafont | Three nuclear and two membrane estrogen receptors in basal teleosts , Anguilla sp .: Identification , evolutionary history and differential expression regulation[END_REF][START_REF] Da Silva | Differential expression of gonadotropin and estrogen receptors and oocyte cytology during follicular maturation associated with egg viability in European eel (Anguilla anguilla)[END_REF]. In the same line, Peñaranda and co-workers found upregulated expression of GnRH receptor 1a in female eel and GnRH receptor 2 for both males and females during experimental maturation [START_REF] Peñaranda | Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development[END_REF]. Further general expression studies in male eel during artificial maturation have detected an upregulation of neuropeptides and G protein-coupled receptors in midbrain and forebrain suggesting a re-organization of the signalling pathways after exposure to chorionic gonadotropin. Among them, they found both up and downregulation of two different uncharacterized pro-dynorphin precursor genes that can be interpreted as dominance in the expression of a different paralog gene before and after the silvering process [START_REF] Churcher | Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation[END_REF]. Those pieces of evidence suggest an increase of paralog neuropeptides expressed after maturation compared to those expressed in a premature stage.

The Brain Pituitary Gonad axis

The hypothalamic/brain -pituitary -gonad axis (BPG) refers to the neuroendocrine axis involved in the control of reproduction in vertebrates (Figure 6). The Gonadotropin-releasing Hormone (GnRH) is the major hypothalamic peptide during puberty and sexual maturation [START_REF] Yamamoto | Preoptic gonadotropin-releasing hormone (GnRH) neurons innervate the pituitary in teleosts[END_REF][START_REF] White | Ontogeny of gonadotropin-releasing hormone (GnRH) gene expression reveals a distinct origin for GnRH-containing neurons in the midbrain[END_REF][START_REF] González-Martínez | Immunohistochemical localization of three different prepro-GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides[END_REF][START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF][START_REF] Zohar | Neuroendocrinology of reproduction in teleost fish[END_REF].

In mammals, the secreted GnRH is conducted to the pituitary by the brain-pituitary portal system, a vascular system. However, in teleost fishes GnRH neurons innervate directly the pars distalis of the pituitary [START_REF] Yamamoto | Preoptic gonadotropin-releasing hormone (GnRH) neurons innervate the pituitary in teleosts[END_REF]for review: Zohar et al., 2010). GnRH interacts with the pituitary GnRH-receptors and induces the synthesis and secretion of the pituitary gonadotropins LH and FSH, which in turn stimulate the production of the sexual steroids, estrogens and androgens In the gonads, the gonadotropins interact with specific receptors in different cell types thus producing the sex steroids testosterone (T) and estrogen (E2) (steroidogenesis) and inducing gametogenesis (for review: [START_REF] Zohar | Neuroendocrinology of reproduction in teleost fish[END_REF]. In teleosts fishes, the progestagen steroid 17K,20L-Dihydroxy-4-pregnen-3-one (17K,20L-DP) induces oocyte maturation and spermiation [START_REF] Nagahama | Endocrine regulation of gametogenesis in fish[END_REF].

In oviparous female animals such as birds or fishes, this BPG axis is connected to the liver (Figure 6), where chorionic proteins for the formation of the yolk, such as vitellogenin, are synthetized [START_REF] Mommsen | 5 Vitellogenesis and oocyte assembly[END_REF]. In teleosts, the sexual steroid E2 induce the production of vitellogenin (Vg) that takes place in the liver [START_REF] Arukwe | Eggshell and egg yolk proteins in fish: Hepatic proteins for the next generation: Oogenetic, population, and evolutionary implications of endocrine disruption[END_REF].

GnRH system

GnRH peptides

The Gonadotropin-releasing hormone (GnRH) is a decapeptide with the characteristic sequence of QxWSxGxWxPG, where x is a variable aminoacid [START_REF] Sherwood | The GnRH family of peptides[END_REF][START_REF] Lethimonier | Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish[END_REF][START_REF] Millar | GnRHs and GnRH receptors[END_REF][START_REF] Chen | GnRH and GnRH receptors: Distribution, function and evolution[END_REF][START_REF] Roch | Evolution of GnRH: Diving deeper[END_REF]Roch et al., 2014a).

First, GnRH was called luteinizing hormone-releasing hormone (LHRH) and was isolated from the porcine [START_REF] Schally | Isolation and properties of the FSH and LH-releasing hormone[END_REF] and ovine [START_REF] Amoss | Purification, amino acid composition and N-terminus of the hypothalamic luteinizing hormone releasing factor (LRF) of ovine origin[END_REF] hypothalamus. Amoss and co-workers (1971) reported that in vitro treatment with ovine LHRH performed on rat pituitaries induced the release of LH and FSH.

More than 20 different GnRHs have been characterized till date. The different GnRHs have been named according to the species where they were found the first time [START_REF] Sherwood | The GnRH family of peptides[END_REF][START_REF] Dubois | Evolutionary development of three gonadotropin-releasing hormone (GnRH) systems in vertebrates[END_REF][START_REF] González-Martínez | Immunohistochemical localization of three different prepro-GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides[END_REF][START_REF] Lethimonier | Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish[END_REF][START_REF] Millar | GnRHs and GnRH receptors[END_REF], the mammalian GnRH being the only exception to this rule.

GnRH is encoded by a gene with 3 introns and 4 exons [START_REF] Okubo | Molecular cloning and tissuespecific expression of a gonadotropin-releasing hormone receptor in the Japanese eel[END_REF][START_REF] Chen | GnRH and GnRH receptors: Distribution, function and evolution[END_REF][START_REF] Roch | Evolution of GnRH: Diving deeper[END_REF][START_REF] Roch | GnRH receptors and peptides: Skating backward[END_REF][START_REF] Tostivint | General and Comparative Endocrinology Evolution of the gonadotropin-releasing hormone ( GnRH ) gene family in relation to vertebrate tetraploidizations[END_REF]X. Zhou et al., 2012). GnRH-genes encode the pre-pro-GnRH that contains a signal peptide of about 23 amino acids, the GnRH decapeptide, a cleavage site (Gly-Lys-Arg) and its associated peptide of 40-60 amino acids (GAP) (Figure 7 A).

Phylogeny studies have demonstrated the existence of 4 GnRH families generated during the 1R and 2R in non-teleost vertebrates. From these, three GnRH families are preserved in teleost fishes [START_REF] Lethimonier | Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish[END_REF] with duplications following the 3R teleost-specific genome duplication. [START_REF] Fernald | Gonadotropin-releasing hormone genes: phylogeny, structure, and functions[END_REF] grouped different GnRH variants by their tissue distribution thus naming them GnRH I, II and III in agreement with phylogenetic studies (Figure 7, B and C). A fourth GnRH (GnRH-IV) have been deduced from whole genome duplication events and would be lost in the vertebrate lineage:

• GnRH-I (or GnRH): hypothalamic variant secreted by the POA GnRH-neurons, [START_REF] Okubo | Structural and functional evolution of gonadotropin-releasing hormone in vertebrates[END_REF]. GnRH-neurons in POA innervate the teleost pituitary [START_REF] Yamamoto | Preoptic gonadotropin-releasing hormone (GnRH) neurons innervate the pituitary in teleosts[END_REF]for review: Zohar et al., 2010). This GnRH is species-specific and in teleost corresponds mostly to seabream, medaka or salmon GnRH (Figure 7C, [START_REF] Soga | Testosterone differentially regulates expression of GnRH messenger RNAs in the terminal nerve, preoptic and midbrain of male tilapia[END_REF][START_REF] Yamamoto | Preoptic gonadotropin-releasing hormone (GnRH) neurons innervate the pituitary in teleosts[END_REF][START_REF] Lethimonier | Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish[END_REF][START_REF] Mohamed | Developmental expression of the G protein-couple receptor 54 and three GnRH mRNAs in the teleost fish cobia[END_REF]. In the eel, it releases gonadotropins during sexual maturation thus inducing gonads development after stimulation with steroids [START_REF] Montero | Differential REgulation of the Two Forms of Gonadotropin-Releasing Hormone (mGnRH and cGnRH-II) by Sex Steroids in the European Female Silver Eel (Anguilla anguilla)[END_REF].

• GnRH-II: mesencephalic variant, a regulator of sexual behaviour, food intake and energy balance [START_REF] Kauffman | A critical role for the evolutionarily conserved gonadotropin-releasing hormone II: Mediation of energy status and female sexual behavior[END_REF][START_REF] Temple | An evolutionarily conserved form of gonadotropin-releasing hormone coordinates energy and reproductive behavior[END_REF], also named chicken GnRH-II in teleosts [START_REF] Mohamed | Developmental expression of the G protein-couple receptor 54 and three GnRH mRNAs in the teleost fish cobia[END_REF].

• GnRH-III: the telencephalic variant, identified only in teleosts and lost in the tetrapod lineage [START_REF] Tostivint | General and Comparative Endocrinology Evolution of the gonadotropin-releasing hormone ( GnRH ) gene family in relation to vertebrate tetraploidizations[END_REF]. It is not found in the eel till date [START_REF] Okubo | Structural and functional evolution of gonadotropin-releasing hormone in vertebrates[END_REF].

• GnRH-IV: was generated after the 2R in early vertebrates according to syntenic analysis of the paralogon region [START_REF] Decatur | General and Comparative Endocrinology Insight from the lamprey genome : Glimpsing early vertebrate development via neuroendocrine-associated genes and shared synteny of gonadotropinreleasing hormone ( GnRH )[END_REF][START_REF] Roch | GnRH receptors and peptides: Skating backward[END_REF], it was lost almost immediately after the second duplication and no alive vertebrate has it [START_REF] Gaillard | Characterization of Gonadotropin-Releasing Hormone (GnRH) Genes From Cartilaginous Fish: Evolutionary Perspectives[END_REF]. GnRH and GAP. B: Conservation of the three GnRH lineages along vertebrate radiation, being GnRH1 and GnRH2 the ones present in the eel. C: Phylogenetic tree explaining the specific names that the peptide GnRH1 receives in major vertebrate lineages [START_REF] Okubo | Structural and functional evolution of gonadotropin-releasing hormone in vertebrates[END_REF].

In summary, GnRH-I is expressed in POA, GnRH-II is expressed in the midbrain and GnRH-III is found in the olfactory bulbs. The conserved distribution of GnRH along vertebrates indicates that GnRH appeared very early in the evolution and has a very important function. Studies from Roch and co-workers (2014) concluded that GnRH superfamily has a monophyletic origin with a common ancestor for vertebrate and invertebrate GnRH, corazonins and adipokinetic hormones.

Each vertebrate species expresses 2 or 3 different variants of GnRH with different receptors thus illustrating the pleiotropic actions of GnRH [START_REF] Lethimonier | Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish[END_REF].

GnRH receptors

The biological activity of GnRH is mediated by a G-protein coupled receptor (GPCR) called GnRHreceptor (GnRH-R). The GPCR receptors are a family of receptors derived from the rhodopsin structure.

Teleost GnRH-R has been isolated in many species thus finding four or five GnRH-R isoforms [START_REF] Gopurappilly | Functional significance of GnRH and kisspeptin, and their cognate receptors in teleost reproduction[END_REF]. Those isoforms have been classified into four GnRH-R major lineages or types: mammalian type I and II and non-mammalian type I and II. Non-mammalian type I is duplicated in teleosts. A non-mammalian type III has been found in teleosts and is grouped together with mammalian type II. These four types of GnRH further subdivide into six classes:

• Non-mammalian type I: teleost GnRH-Rn1 (class 1) and GnRH-Rn1b (class 2)

• Non-mammalian type II: teleost GnRH-Rn2 (class 3)

• Non-mammalian type III/mammalian type II: teleost GnRH-Rn3 (class 4) and GnRH-Rn3b/ mammalian GnRH-Rm2 (class 5)

• Mammalian type I: mammalian GnRH-Rm1 (class 6)

Dopaminergic system

Dopamine (DA) is an organic chemical of the catecholamine and phenylalanine families synthesized from the aminoacid L-DOPA or L-3,4-dihydroxyphenylalanine, which is synthesized from the aminoacid tyrosine. The aminoacid is processed in the dopaminergic neurons by tyrosinehydroxylase and transformed into L-dihydroxy-phenylalanine (L-DOPA). Finally, the enzyme DOPAdecarboxylase transforms it into dopamine [START_REF] Wahbe | Endogenous dopamine synthesis and dopa-decarboxylase activity in rat renal cortex[END_REF][START_REF] Gjedde | Dopa decarboxylase activity of the living human brain[END_REF].

Dopamine is a multifunctional neurotransmitter acting mostly on the pituitary and in mammals particularly on prolactin secretion [START_REF] Enjalberts | Angiotensis II and Dopamine Modulate Both cAMP and Inositol Phosphate Productions in Anterior Pituitary Cells[END_REF][START_REF] Elsholtz | Inhibitory Control of Prolactin and Pit-1 Gene Promoters by Dopamine[END_REF][START_REF] Bole-Feysot | Prolactin (PRL) and its receptor:Actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice[END_REF]. However, in other sarcopterygians such as amphibians [START_REF] Sotowska-Brochocka | Dopaminergic inhibition of gonadotropic release in hibernating frogs, Rana temporaria[END_REF][START_REF] Trudeau | Hormonal induction of spawning in 4 species of frogs by coinjection with a gonadotropin-releasing hormone agonist and a dopamine antagonist[END_REF] and actinopterygians such sturgeons [START_REF] Pavlick | Dopaminergic influence on gonadotropin secretion in white sturgeon (Acipenser transmontanus)[END_REF], dopamine is characterized as a strong inhibitor of the BPG axis. In teleosts, DA inhibits basal and GnRH-stimulated LH release in a wide range of teleosts including cyprinids [START_REF] Lin | Effects of [d-Arg6, Trp7, Leu8, Pro9 NEt]-luteinizing hormonereleasing hormone (sGnRH-A) and [d-Ala6, Pro9NEt]-luteinizing hormone-releasing hormone (LHRH-A), in combination with pimozide or domperidone, on Gonadotropin release and ovulation in the Chine[END_REF][START_REF] Fontaine | Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes[END_REF], silurids [START_REF] De Leeuw | The dopminergic inhibition of the gonadotropin-releasing hormone-induced gonadotropin release: an in vitro study with fragments and cell suspensions from pituitaries of the African catfish, Clarias gariepinus (Burchell)[END_REF], salmonids [START_REF] Saligaut | Brain aminergic systems in salmonids and other teleosts in relation to steroid feedback and gonadotropin release[END_REF], cichlids [START_REF] Yaron | Regulation of Fish Gonadotropins[END_REF][START_REF] Bryant | Dopaminergic inhibition of gonadotropin-releasing hormone neurons in the cichlid fish Astatotilapia burtoni[END_REF] and also in the European eel [START_REF] Dufour | Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol[END_REF][START_REF] Vidal | Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty[END_REF]Levavi-Sivan 2004b;for reviews: Dufour et al., 2005;[START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF][START_REF] Zohar | Neuroendocrinology of reproduction in teleost fish[END_REF][START_REF] Fontaine | Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes[END_REF].

The inhibitory activity of DA in teleosts is generally mediated by dopamine receptors type 2, or D2-R, placed in the pituitary [START_REF] Chang | Differential actions of dopamine receptor subtypes on gonadotropin and growth hormone release in vitro in goldfish[END_REF][START_REF] Yu | Adrenergic and dopaminergic regulation of gonadotropin-releasing hormone release from goldfish preoptic-anterior hypothalamus and pituitary in vitro[END_REF][START_REF] Vacher | Modulation of pituitary dopamine D1 or D2 receptors and secretion of follicle stimulating hormone and luteinizing hormone during the annual reproductive cycle of female rainbow trout[END_REF][START_REF] Fontaine | Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes[END_REF]. However, some teleosts use the dopamine receptor type 1, or D1-R [START_REF] Roche | D1, but not D2, dopamine receptor regulates steroid levels during the final stages of pikeperch gametogenesis[END_REF], thus illustrating the metabolic plasticity of teleosts BPG.

Pituitary gonadotropins

The gonadotropins I (GTH-I; follicle stimulating hormone or FSH) and II (GTH-II; luteinizing hormone or LH) are glycoproteic hormones together with chorionic gonadotropin (hCG). Those peptides are characterized by post-translational glycosylations. Gonadotropic cells in the pituitary produce FSH and LH, while hCG comes from the placenta. All these peptides have two subunits α and β; sharing the same α-subunit, the glycoprotein-α (GP α). The β-subunit peptides are specific for each hormone and are encoded by different genes: fshβ and lhβ. GPα is encoded in the gpα gene and this subunit is common for gonadotropins and for TSH β-subunit. The biological activity of gonadotropins requires the association of both subunits [START_REF] Vaitutaitis | Gonadotropins and Their Subunits : Basic and Clinical Studies[END_REF][START_REF] Hagen | Identification of human luteinizing hormone, follicle-stimulating hormone, luteinizing hormone beta-subunit and gonadotrophin alphasubunit in foetal and adult pituitary glands[END_REF], and the β-subunit offers the specific function to each hormone [START_REF] Themmen | Mutations of gonadotropins and gonadotropin receptors: Elucidating the physiology and pathophysiology of pituitary-gonadal function[END_REF].

Sex steroids

Gonadal/sex steroids (estrogens, androgens and progesterone) are synthesized mostly by the gonads and with less importance by adrenal glands, brain and other tissues. Steroids can act as regulators of neuronal plasticity and brain development, control of feedback loops and neuroprotection against malign agents [START_REF] Mcewen | Estrogen actions throughout the brain[END_REF]. Those sex steroids act through androgen, progesterone and estrogen receptors. The sex steroids regulate the gonadotropic axis with negative and positive feedback loops. In mammal juveniles, steroids induce a negative feedback loop that inhibits GnRH release [START_REF] Ramirez | Inhibitory Effect of Testosterone on Luteinizing Hormone Secretion in Immature and Adult Rats[END_REF][START_REF] Goodman | Pulsatile Secretion of Luteinizing Hormone: Differential Suppression by Ovarian Steroids*[END_REF] for review: [START_REF] Sisk | The neural basis of puberty and adolescence[END_REF]. This negative feedback decreases with puberty and GnRH increases its release thus up regulating the luteinizing hormone and therefore inducing the gonads maturation (for review: [START_REF] Sisk | The neural basis of puberty and adolescence[END_REF]. Estrogen is particularly important since it acts as a switcher thus inverting negative LH and GnRH regulation feedback loops into positive ones [START_REF] Moenter | The Estradiol-Induced Surge of Gonadotropin-Releasing Hormone in the Ewe*[END_REF]) via estrogen receptor [START_REF] Scully | Role of Estrogen Receptor-a in the Anterior Pituitary Gland[END_REF]. However, this negative feedback may be different in fishes, being more a modulator feedback [START_REF] Weltzien | The brain-pituitary-gonad axis in male teleosts, with special emphasis on flatfish (Pleuronectiformes)[END_REF].

Main regulators of BPG-axis 2.5.1 Gonadal peptides

The gonadal peptides, inhibins, activins and follistatins, are hormones that also regulate the LH and FSH release in the pituitary and have a paracrine action on the gonads in mammals [START_REF] Ling | Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin[END_REF][START_REF] Meunier | Gonadal and extragonadal expression of inhibin alpha, beta A, and beta B subunits in various tissues predicts diverse functions[END_REF][START_REF] Gospodarowicz | Pituitary follicular cells secrete both vascular endothelial growth factor and follistatin[END_REF][START_REF] Nakamura | Activinbinding protein from rat ovary is follistatin[END_REF][START_REF] Bilezikjian | Autocrine / paracrine regulation of pituitary function by activin , inhibin and follistatin[END_REF]for review: Namwanje and Brown, 2016) and teleosts (zebrafish: [START_REF] Wu | Activin, Inhibin, and Follistatin in Zebrafish Ovary: Expression and Role in Oocyte Maturation1[END_REF]eel: Aroua et al., 2012;for review: Petrino et al., 2007). Inhibins and activins A, B and AB are dimeric proteins from the transforming growth factor superfamily. Activins are dimers formed by two activin-β subunits, βA and βB. The combination of pairs of these two subunits results in activin A (βA+βA), activin B (βB+βB) and activin AB (βA+βB). Inhibins are also dimers with one inhibin-β subunit and one inhibinα subunit. There is one inhibin-α subunit and there are two inhibin-β subunits, βA and βB. The inhibin A is formed by inhibin-α and inhibin-βA subunits and the inhibin B is formed by inhibin-α and inhibin-βB. While the beta subunit in inhibin is more similar to the beta subunit in activin, the alpha subunit is more divergent [START_REF] Burger | Inhibin: definition and nomenclature, including related substances[END_REF][START_REF] Robertson | Inhibin / activin and ovarian cancer[END_REF]. These protein complexes are produced in many tissues but especially in the ovary and pituitary. Activins stimulate directly FSH secretion but no effect on LH has been found [START_REF] Ling | Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin[END_REF][START_REF] Vale | Purification and characterization of an FSH releasing protein from porcien ovarian follicular fluid[END_REF]. Interestingly, inhibins have the contrary effect, downregulating FSH synthesis and inhibiting the secretion [START_REF] Van Zonneveld | Do cycle disturbances explain the age-related decline of female fertility? Cycle characteristics of women aged over 40 years compared with a reference population of young women[END_REF].

Follistatins are glycoproteins with a single chain that binds to activin [START_REF] Nakamura | Activinbinding protein from rat ovary is follistatin[END_REF][START_REF] Robertson | Follistatin/activin-binding protein[END_REF][START_REF] Suginos | Molecular Heterogeneity of Follistatin , an Activin-binding Protein[END_REF] and to inhibin [START_REF] Shimonaka | Follistatin binds to both activin and inhibin through the common beta-subunit[END_REF]. In mammals, follistatins present an alternative splicing that generates one protein of 315 aa (follistatin 315) and 288 aa (follistatin 288). They are synthesized in the pituitary and the ovary among other tissues, where they act as paracrine factors [START_REF] Gospodarowicz | Pituitary follicular cells secrete both vascular endothelial growth factor and follistatin[END_REF]. Follistatins 315 and 288 have the capacity to inhibit, in vitro and in vivo, FSH release through blocking the activity of activins in rat [START_REF] Nakamura | Activinbinding protein from rat ovary is follistatin[END_REF].

Activin A (βA+βA), inhibin A (α+βA) and follistatin have been found in the zebrafish ovary (Wu et al., 2000). In these studies, activin and inhibin induced final oocyte maturation in a dose dependent manner. Follistatin blocked the effect of activin/inhibin and also the final oocyte maturation.

Therefore, activins/inhibins have been proposed as paracrine regulators of ovarian function in fishes.

Additionally, inhibin A is upregulated by FSH, which in turn is inhibited by inhibin A thus suggesting a negative feedback loop between pituitary and teleost ovary [START_REF] Poon | Characterization of inhibin α subunit (inha) in the zebrafish: Evidence for a potential feedback loop between the pituitary and ovary[END_REF].

RF-amides

This large family contains peptides with a characteristic arginine (R) followed by a phenylalanine (F) that is amidated in the C-terminal end. The first molecule of this family has been characterized by Price and Greenberg as a cardiac excitatory in the mollusc Macrocallista nimbosa [START_REF] Price | Structure of a molluscan cardioexcitatory neuropeptide[END_REF]. Many peptides with the RF-amide motif have been characterized in other metazoans [START_REF] Walker | A review of FMRFamide-and RFamide-like peptides in metazoa[END_REF][START_REF] Osugi | Evolutionary origin of GnIH and NPFF in chordates: Insights from novel amphioxus RFamide peptides[END_REF][START_REF] Osugi | RFamide peptides in agnathans and basal chordates[END_REF][START_REF] Pasquier | Molecular evolution of GPCRs: Kisspeptin/kisspeptin receptors[END_REF][START_REF] Tsutsui | Review: Structure, function and evolution of GnIH[END_REF].

Thanks to phylogenetic studies, RFamides are classified in 5 groups (GnIH, kisspeptin, NPFF, 26RFa and PrRP [START_REF] Fukusumi | Recent advances in mammalian RFamide peptides: The discovery and functional analyses of PrRP, RFRPs and QRFP[END_REF][START_REF] Tsutsui | Gonadotropin-inhibitory hormone (GnIH) and its control of central and peripheral reproductive function[END_REF]), two of which have been involved in the control of reproduction.

GONADOTROPIN INHIBITORY HORMONE (GnIH)

: is a dodecapeptide identified in birds [START_REF] Tsutsui | A novel avian hypothalamic peptide inhibiting gonadotropin release[END_REF]. This peptide is also present in amphibians (fGRP and fGRP-RP-2, [START_REF] Tsutsui | A new key neurohormone controlling reproduction, gonadotropininhibitory hormone (GnIH): Biosynthesis, mode of action and functional significance[END_REF], mammals (RFRP) and invertebrates (for reviews: [START_REF] Kriegsfeld | Identification and characterization of a gonadotropininhibitory system in the brains of mammals[END_REF][START_REF] Pelster | Swimbladder function and the spawning migration of the European eel Anguilla anguilla[END_REF], Tsutsui et al., 2006, 2007[START_REF] Page | Neurokinin B and pre-eclampsia: A decade of discovery[END_REF], 2013, 2018;[START_REF] Iwasa | Developmental changes in the mammalian gonadotropin-inhibitory hormone (GnIH) ortholog RFamide-related peptide (RFRP) and its cognate receptor GPR147 in the rat hypothalamus[END_REF]. In birds, this peptide is expressed mainly in hypothalamic neurons and inhibits the synthesis and release of gonadotropins by acting directly in the pituitary [START_REF] Tsutsui | The general and comparative biology of gonadotropin-inhibitory hormone (GnIH)[END_REF]. In vertebrates, environmental signals such as photoperiod regulate GnIH and produce the seasonal breeding behaviour [START_REF] Kriegsfeld | Seasonal control of gonadotropin-inhibitory hormone (GnIH) in birds and mammals[END_REF]. In amphibians, GnIH is suggested to be more a GH-releasing factor [START_REF] Ukena | Novel neuropeptides related to frog growth hormonereleasing peptide: Isolation, sequence, and functional analysis[END_REF].

The role in teleost is ambiguous. Studies in tilapia Oreochromis niloticus showed that GnIH peptides stimulate both FSH and LH and inhibits GH (Biran et al., 2014b) and lh and fsh mRNA levels in goldfish Carassius auratus [START_REF] Moussavi | Seasonal effects of GniH on basal and GnRH-induced goldfish somatotrope functions[END_REF]. Controversially, LH is inhibited in serum of the same goldfish [START_REF] Qi | Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus[END_REF], common carp Cyprinus carpio [START_REF] Peng | GnIH plays a negative role in regulating GtH expression in the common carp, Cyprinus carpio L[END_REF] in cavefish Astyanax altiparanae [START_REF] Branco | Effects of GnRH and the dual regulatory actions of GnIH in the pituitary explants and brain slices of Astyanax altiparanae males[END_REF] and in orange spotted grouper Epinephelus coioides [START_REF] Wang | Molecular identification of GnIH/GnIHR signal and its reproductive function in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides)[END_REF], among many others. Interestingly, in vitro studies with pituitary cells from different stages of goldfish show that secretion of LH and expression of lh mRNA is dissociated after administration of GnIH and depends on the status of recrudescence. In this way, while GnIH enhances the secretion of LH in pre-spawning fish, it decreases the lh expression in early recrudescence, increases it at mid recrudescence to decrease it again at pre-spawning stage [START_REF] Moussavi | Seasonal effect of GnIH on gonadotrope functions in the pituitary of goldfish[END_REF].

In addition, a regulation of hypothalamic GnRH by GnIH during the sexual maturation has been described for goldfish [START_REF] Qi | Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus[END_REF] and orange spotted grouper [START_REF] Wang | Molecular identification of GnIH/GnIHR signal and its reproductive function in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides)[END_REF]. Therefore, GnIH can be a regulator of the BPG axis during sexual maturation in teleosts.

KISSPEPTIN (Kiss):

The system of kisspeptin peptides and receptors (Kiss/KissR) was identified initially in cancer processes. A Kiss peptide, (Kiss1) has been identified as a suppressor of metastasis in a melanoma cell line and the kiss mRNA have been overexpressed in tumour cells with suppressed metastasis, therefore called "metastin" [START_REF] Lee | KiSS-1, a Novel Human Malignant Melanoma Metastasis-suppressor Gene[END_REF]. The gene for the receptor kissR was isolated later and cloned in rat brain. It was first identified as an orphan receptor with 40% homology to Galanin (GAL) receptor that could not be activated by GAL [START_REF] Lee | Discovery of a receptor related to the galanin receptors[END_REF]. Finally, in 2001, the kisspeptin peptides were associated with the Gpr54 receptor and later called KissR [START_REF] Kotani | The Metastasis Suppressor Gene KiSS-1 Encodes Kisspeptins, the Natural Ligands of the Orphan G Protein-coupled Receptor GPR54[END_REF][START_REF] Muir | AXOR12, a Novel Human G Protein-coupled Receptor, Activated by the Peptide KiSS-1[END_REF][START_REF] Ohtaki | Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor[END_REF].

Four kiss genes originated in the ancestral vertebrate after the two rounds of whole genome duplication events. Two kiss genes, kiss1, encoding Kiss1 peptide, and kiss2, encoding Kiss2 peptide, were retained in the ancestor actinopterygian after gene loss and pseudogenization events. Later, those genes duplicated with the teleost-specific whole genome duplication, but duplicates were lost immediately after, being two genes present in the eel (for review: [START_REF] Pasquier | Molecular evolution of GPCRs: Kisspeptin/kisspeptin receptors[END_REF]. Genome duplication and gene loss events along vertebrate radiation produced a very different conservation pattern of the kissR genes. While the frog X. tropicalis conserved three receptors, in the bird clade they disappeared [START_REF] Pasquier | Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family[END_REF]. The system Kiss/KissR is characterized along all the vertebrates species with an expression that differs in location and temporal patterns, but it is generally related to the reproduction function [START_REF] Pasquier | Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family[END_REF], for review: Pasquier et al., 2014).

The reproductive function of the Kiss peptides is the pulsatile release of GnRH in the blood stream in mammals [START_REF] Clarke | Control of GnRH secretion: One step back[END_REF][START_REF] Ramaswamy | Evidence from the agonadal juvenile male rhesus monkey (macaca mulatta) for the view that the action of neurokinin b to trigger gonadotropin-releasing hormone release is upstream from the kisspeptin receptor[END_REF][START_REF] Navarro | New insights into the control of pulsatile GnRH release: The role of Kiss1/neurokinin B neurons[END_REF][START_REF] Hanchate | Kisspeptin-GPR54 Signaling in Mouse NO-Synthesizing Neurons Participates in the Hypothalamic Control of Ovulation[END_REF].

This pulse is regulated from a particular neuron in the arcuate nucleus that expresses Kiss, Neurokinin B (NKB or TAC3) and dynorphin (Dyn), and it is called KNDy neuron [START_REF] Navarro | New insights into the control of pulsatile GnRH release: The role of Kiss1/neurokinin B neurons[END_REF][START_REF] Merkley | KNDy (kisspeptin/neurokinin B/dynorphin) neurons are activated during both pulsatile and surge secretion of LH in the ewe[END_REF][START_REF] Mittelman-Smith | Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight[END_REF][START_REF] Goodman | Kisspeptin, neurokinin B, and dynorphin act in the arcuate nucleus to control activity of the GnRH pulse generator in ewes[END_REF][START_REF] Ruka | Regulation of arcuate neurons coexpressing kisspeptin, neurokinin b, and dynorphin by modulators of neurokinin 3 and kopioid receptors in adult male mice[END_REF][START_REF] Skorupskaite | The kisspeptin-GnRH pathway in human reproductive health and disease[END_REF].

BPG axis in the eel

The prepubertal silver eel is blocked in the sexual maturation process by many different inhibitory mechanisms thus reducing the number of GnRH receptors (GnRH-Rs) and pituitary gonadotropins released [START_REF] Dufour | Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol[END_REF][START_REF] Vidal | Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty[END_REF]Sébert et al., 2008b;[START_REF] Peñaranda | Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development[END_REF][START_REF] Jolly | Functional Characterisation of Eel Dopamine D 2 Receptors and Involvement in the Direct Inhibition of Pituitary Gonadotrophins Neuroendocrinology[END_REF][START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF].

Two different GnRH forms have been characterized in European eel (Dufour et al., 1993b;[START_REF] Montero | Immunocytochemical localization of mammalian GnRH (gonadotropin-releasing hormone) and chicken GnRH-II in the brain of the European silver eel (Anguilla anguilla L.)[END_REF] and Japanese eel [START_REF] Okubo | Expression of two gonadotropin-releasing hormone (GnRH) precursor genes in various tissues of the Japanese eel and evolution of GnRH[END_REF] and three splicing variants have been found [START_REF] Okubo | Three mRNA species for mammalian-type gonadotropin-releasing hormone in the brain of the eel Anguilla japonica[END_REF]. One of the GnRH is similar to the mammalian (mGnRH or GnRH-I) while the other is similar to the chicken one (cGnRH or GnRH-II). GnRH-III would have been lost in the eel clade independently from the other teleost lineages [START_REF] Okubo | Structural and functional evolution of gonadotropin-releasing hormone in vertebrates[END_REF]. Studies determined the brain distribution of those peptides using radioimmunoassay (RIA) [START_REF] Montero | Immunocytochemical localization of mammalian GnRH (gonadotropin-releasing hormone) and chicken GnRH-II in the brain of the European silver eel (Anguilla anguilla L.)[END_REF]. Eel GnRHs present a differential localization in the brain, with mGnRH (GnRH-I) most extended in the brain and in axonal endings in the pituitary gland (Dufour et al., 1993b). GnRH-I (mammaltype) and GnRH-II (chicken GnRH) have been characterized for Japanese eel and both genes are expressed in different areas of the brain of the Japanese eel [START_REF] Okubo | Expression of two gonadotropin-releasing hormone (GnRH) precursor genes in various tissues of the Japanese eel and evolution of GnRH[END_REF]Okubo et al. 2000, Figure 7). After artificial maturation of female eels, GnRH-I protein levels were upregulated in the hypothalamus and pituitary while GnRH-II was downregulated (Dufour et al., 1993b).

Three GnRH-receptors have been recently characterized in European eel Anguilla anguilla by Peñaranda and co-workers in 2013, namely GnRH-RIa, GnRH-RIb, GnRH-RII. GnRH-RIa is found in the gonads and kidney, while it is not present in liver, fin, heart and gill. GnRH-RIb is present in all tissues except male liver, fin and kidney, being more expressed in females than in males for all the tissues.

The highest difference between females and males is observed in the olfactory bulbs, di-and mesencephalon, and cerebellum. GnRH-RII shows also differential expression between males and females with no expression detected in female pectoral fin or male cerebellum. The highest transcript concentration is found in olfactory bulbs, telencephalon, di-and mesencephalon, and medulla oblongata in both sexes. This receptor is not present in liver, anterior and posterior kidney, heart, and gill [START_REF] Peñaranda | Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development[END_REF]. Peñaranda studies have demonstrated sexually differential upregulation of GnRH receptors during artificial maturation GnRH-RIa being upregulated in female and GnRH-RII upregulated in both sexes in the midbrain, with a peak of expression in the pituitary during early vitellogenesis. In the pituitary, the receptor II is upregulated in the final period of the sexual maturation in both males and females, in parallel to an upregulation of lhβ gonadotropin subunit [START_REF] Peñaranda | Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development[END_REF].

Many experiments demonstrated that it is necessary a long-term treatment with estradiol (E2)

and the combination of GnRH and DA antagonist to induce experimental maturation and therefore that steroid feedback acts also on the DA hypophysiotropic system [START_REF] Dufour | Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol[END_REF][START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF][START_REF] Vidal | Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty[END_REF][START_REF] Weltzien | Androgen-dependent stimulation of brain dopaminergic systems in the female European eel (Anguilla anguilla)[END_REF][START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the european eel: Molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF][START_REF] Jolly | Functional Characterisation of Eel Dopamine D 2 Receptors and Involvement in the Direct Inhibition of Pituitary Gonadotrophins Neuroendocrinology[END_REF] Dopamine neurons are distributed in the eel telencephalon, but only the ones in the anteroventralis Nucleus Preopticus (NPOav) innervate the pituitary pars distalis (Sébert et al., 2008b; for review: [START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF]. As described previously for other teleosts, DA inhibits the gonadotropin production in the eel [START_REF] Vidal | Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty[END_REF]. DA receptors (D1A, D1B, D1C, D2A and D2B) are identified in the European eel, but only the D2 is present in the pituitary, being the D2B ten times more expressed than the D2A [START_REF] Kapsimali | Distribution of the mRNA encoding the four dopamine D1 receptor subtypes in the brain of the european eel (Anguilla anguilla): Comparative approach to the function of D1 receptors in vertebrates[END_REF][START_REF] Vidal | Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty[END_REF][START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the european eel: Molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF]for review: Dufour et al., 2005;[START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF]. In addition, in situ hybridization on pituitary cells revealed that D2B receptor is located in LH-producing cells and D2A is collocated with FSH, and DA highly inhibited basal and testosterone-stimulated lh expression and less strongly basal and activin-stimulated fsh expression by primary culture of eel pituitary cells [START_REF] Jolly | Functional Characterisation of Eel Dopamine D 2 Receptors and Involvement in the Direct Inhibition of Pituitary Gonadotrophins Neuroendocrinology[END_REF].

In opposition to other teleosts, the kisspeptin system is inhibitory for gonadotropins in the eel.

Two Kiss (Kiss1 and Kiss2) and three Kiss-receptors (KissR1, KissR2 and KissR3) have been characterized in European eel [START_REF] Pasquier | First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla[END_REF][START_REF] Pasquier | Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family[END_REF][START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF]. In vitro experiments with silver eel pituitary cell culture demonstrated that the expression of gnrh-rII gene and lhβ are downregulated by heterologous and eel kisspeptins [START_REF] Pasquier | First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla[END_REF][START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF]. The three kissR genes are differentially expressed in the BPG axis and differentially regulated in experimentally matured eels, as compared to prepubertal controls [START_REF] Pasquier | Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family[END_REF]. Kissr-1 mRNA was highly expressed in brain, pituitary and in both ovary and testis. Lower expression was detected in muscle, retina and fat tissue. Kissr-2 mRNA was expressed in the brain and pituitary, with the highest concentration of mRNA in the telencephalic and di-/mes-encephalic areas. Kissr-3 mRNA was highly expressed in the di-/mes-encephalic area and lowly expressed in the other areas of the brain as well as in the gonads, ovary and testis, and in the muscle. According to these studies, the differences in tissue distribution and regulation indicate a subfunctionalization of those receptors in the eel as an evolutionary constraint for the conservation of multiple KissR paralogs [START_REF] Pasquier | Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family[END_REF].

Many stimulators of LH have been found (androgens: [START_REF] Huang | Androgens stimulate gonadotropin-II beta-subunit in eel pituitary cells in vitro[END_REF]; Insulin-like growth factor: [START_REF] Huang | Insulin-like growth factor-I stimulates gonadotrophin production from eel pituitary cells: A possible metabolic signal for induction of puberty[END_REF]cortisol: Huang et al., 1999). Additionally, activins A and B upregulate fshβ subunit expression and inhibit the expression of lhβ with no effect in other pituitary hormones [START_REF] Aroua | Pituitary gonadotropins FSH and LH are oppositely regulated by the activin/follistatin system in a basal teleost, the eel[END_REF].

Levels of GnRH-I in the brain and gonadotropins in the pituitary increase under endogenous steroids, while GnRH-II decreases (Dufour et al., 1993b). Experimentation with eel suggests that steroids may have a negative feedback regulation on GnRH-I controlled by administration of androgens, and a positive feedback effect on GnRH-II and GTH secretion dependent on administration of estradiol (Montero and Dufour, 1996). The positive feedback of steroids on the GnRH-I-gonadotropin axis in the eel can amplify the pubertal stimulation of gonadotropic function in these species (Montero and Dufour, 1996). Intracellular nuclear receptor 1 for estrogens (ESR1) are upregulated in vitro by estradiol in eel hepatocyte primary cultures [START_REF] Lafont | Three nuclear and two membrane estrogen receptors in basal teleosts , Anguilla sp .: Identification , evolutionary history and differential expression regulation[END_REF]. EST are then also related with eel sexual maturation, being the ESR1 consistently upregulated in vivo in female eel during induced sexual maturation [START_REF] Lafont | Three nuclear and two membrane estrogen receptors in basal teleosts , Anguilla sp .: Identification , evolutionary history and differential expression regulation[END_REF].

Tachykinin peptides

Tachykinins (TK or TAC) are members of a large family of peptides present in all the animal kingdom from cnidarian (for review: Hu et al., 2014b) to a wide distribution in bilaterian (for review:

Vanden [START_REF] Vanden Broeck | Tachykinin-like peptides and their receptors. A review[END_REF]Severini et al., 2002;[START_REF] Pennefather | Tachykinins and tachykinin receptors: a growing family[END_REF][START_REF] Liu | Tachykinin peptides and receptors: Putting amphibians into perspective[END_REF][START_REF] Van Loy | Tachykinin-related peptides and their receptors in invertebrates: A current view[END_REF][START_REF] Satake | Insight into molecular and functional diversity of tachykinins and their receptors[END_REF]Hu et al., 2014b). Tachykinins are brain and gut peptides, but are also found in endostyle and gonad of sea squirt [START_REF] Satake | Insight into molecular and functional diversity of tachykinins and their receptors[END_REF]. In these tissues, they act as neurotransmitters of endocrine and local autocrine/paracrine regulations (for reviews: Severini et al., 2002;[START_REF] Satake | Insight into molecular and functional diversity of tachykinins and their receptors[END_REF]. In addition, they are observed in the skin of amphibians, as well as the salivary gland of mosquito and octopus, where they serve as exocrine secretion (for review : Severini et al., 2002;[START_REF] Liu | Tachykinin peptides and receptors: Putting amphibians into perspective[END_REF][START_REF] Satake | Insight into molecular and functional diversity of tachykinins and their receptors[END_REF]Hu et al., 2014b). Their sequence is normally 10 to 14 aa long (Severini et al., 2002) but exceptionally they can reach up to 45 aa by differential cleavage events as human endokinins (EK) A and B [START_REF] Page | Characterization of the endokinins : Human tachykinins with cardiovascular activity[END_REF][START_REF] Page | Hemokinins and endokinins[END_REF] or be 7 aa long as in the oyster tachykinin related peptides [START_REF] Dubos | Characterization of a tachykinin signalling system in the bivalve mollusc Crassostrea gigas[END_REF]. Their C-terminal end is a characteristic motif of 5 aa with a constant glycine in its third position: xxGxx and an α-amidation (for review: [START_REF] Nakanishi | Mammalian Tachykinin Receptors[END_REF][START_REF] Almeida | Tachykinins and tachykinin receptors: structure and activity relationships[END_REF][START_REF] Page | Hemokinins and endokinins[END_REF][START_REF] Satake | Insight into molecular and functional diversity of tachykinins and their receptors[END_REF]Hu et al., 2014b). This C-terminal determines the activation of the receptor (for review [START_REF] Nakanishi | Mammalian Tachykinin Receptors[END_REF][START_REF] Maggi | The mammalian tachykinin receptors[END_REF][START_REF] Almeida | Tachykinins and tachykinin receptors: structure and activity relationships[END_REF][START_REF] Page | New challenges in the study of the mammalian tachykinins[END_REF].

The typical tac gene contains the information for the pre-pro-tachykinin peptide that includes the tachykinin peptides. The pre-pro-tachykinin uses to be large with a signal peptide at 16-30 residues at the N-terminal and can encode a variable number of tachykinin peptides. It is translated in the endoplasmatic reticulum and the signal peptide directs the pre-pro-peptide towards the Golgi apparatus to be secreted. The signal peptide is cleaved off by proteolysis. The spacer parts are split off by endopeptidases at specific sites of single or two basic residues in both extremes, as reported generally for neuropeptides [START_REF] Harris | Processing of pro-hormone precursor proteins[END_REF]. The inactive peptides are further amidated in the Cterminal glycine site for activation [START_REF] Harris | Processing of pro-hormone precursor proteins[END_REF][START_REF] Regoli | Receptors Antagonists for Substance P and Related Peptides *[END_REF]. After, the activated neuropeptide is packed in the secretory granules and transferred from the Golgi apparatus to the nerve terminal through the axon [START_REF] Krause | Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A[END_REF][START_REF] Kurtz | Identification, localization and receptor characterization of novel mammalian substance-P peptides[END_REF][START_REF] Page | Characterization of the endokinins : Human tachykinins with cardiovascular activity[END_REF]for review: Pennefather et al., 2004).

Radiata

A tachykinin-like was found by histochemistry in the Hydra attenuata using antibodies against the human tachykinin known as substance P (SP). The fluorescence was mostly found in the nerve cells of the ectoderm [START_REF] Taban | Localization of substance P-like immunoreactivity in Hydra[END_REF] and was verified as substance P-like by chromatography [START_REF] Grimmelikhuijzen | Substance P-like immunoreactivity in the nervous system of hydra[END_REF]. The SP-like staining was found in the ectoderm of basal disk and tentacles and not found in the foot [START_REF] Taban | Localization of substance P-like immunoreactivity in Hydra[END_REF][START_REF] Grimmelikhuijzen | Substance P-like immunoreactivity in the nervous system of hydra[END_REF][START_REF] Pierobon | Substance p and hydra: An immunohistochemical and physiological study[END_REF]. These studies detected the tachykinin peptides in nerve cells bodies (sensory neurons and ganglion cells) and in nerve fibres. Positive staining was found also in nests of differentiating small interstitial cells and in nematoblasts at various stages of differentiation [START_REF] Pierobon | Substance p and hydra: An immunohistochemical and physiological study[END_REF]. Others found staining also in interstitial and gastrodermal cells [START_REF] Taban | Localization of substance P-like immunoreactivity in Hydra[END_REF].

The exposure to SP induced the contraction of the tentacles [START_REF] Pierobon | Substance p and hydra: An immunohistochemical and physiological study[END_REF]. More recently, studies in sea anemone Nematostella vectensis demonstrated the presence of two tachykinin-kind peptides, namely Nv-TAC-I (YQVIFEGVR) and Nv-TAC-II (VGRTLQVGRR) [START_REF] Anctil | Chemical transmission in the sea anemone Nematostella vectensis: a genomic perspective[END_REF]. While Nv-TAC-I presents a tachykinin-kind C-terminal motif, Nv-TAC-II is more related to collagen peptidic sequence (see collagen A1, Uniprot P02452) (for review: Hu et al., 2014b). 3.2 Bilateria

Protostome

Two tachykinin-like peptide types, invertebrate tachykinins (Inv-TKs) and tachykinin-related peptides (TKRPs), have been identified in insects [START_REF] Siviter | Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A[END_REF][START_REF] Predel | Tachykinin-related peptide precursors in two cockroach species: Molecular cloning and peptide expression in brain neurons and intestine[END_REF], molluscs [START_REF] Kanda | Isolation and characterization of novel tachykinins from the posterior salivary gland of the common octopus Octopus vulgaris[END_REF][START_REF] Kanda | A novel tachykininrelated peptide receptor of Octopus vulgaris -Evolutionary aspects of invertebrate tachykinin and tachykinin-related peptide[END_REF], echiuroid worms [START_REF] Kawada | Characterization of a novel cDNA sequence encoding invertebrate tachykinin-related peptides isolated from the echiuroid worm, Urechis unicinctus[END_REF] and coelenterates [START_REF] Taban | Localization of substance P-like immunoreactivity in Hydra[END_REF][START_REF] Anctil | Chemical transmission in the sea anemone Nematostella vectensis: a genomic perspective[END_REF].

Inv-TKs

The first TK-like, eledoisin, received its name from the species it was isolated, the octopus Eledone moschata, in 1961 [START_REF] Erspamer | Structure and pharmacological actions of eledoisin, the Active endecapeptide of the posterior salivary glands of eledone[END_REF][START_REF] Anastasi | Occurrence and Some Properties of Eledoisin in Extracts of Posterior Salivary Glands of Eledone[END_REF]. The gene of another Inv-TK was characterized in the mosquito, Aedes aegypti, and named sialokinin I [START_REF] Beerntsen | Characterization of the Sialokinin I gene encoding salivary vasodilator of the yellow fever mosquito, Aedes aegypti[END_REF]. Both Inv-TK were isolated from salivary glands and restricted to this tissue (Table 1).

These peptides are stored in the salivary glands for exocrine secretion as toxin-like compounds (for review: [START_REF] Satake | Insight into Tachykinin-Related Peptides, Their Receptors, and Invertebrate Tachykinins: A review[END_REF][START_REF] Satake | Insight into molecular and functional diversity of tachykinins and their receptors[END_REF][START_REF] Satake | Overview of the primary structure, tissue-distribution, and functions of tachykinins and their receptors[END_REF].

TKRPs

Tachykinin and tachykinin-related peptides have been found in protostomes by radioimmunoassay and immunohistochemistry: locust Lousta migratoria [START_REF] Benedeczky | Light and electron microscopic localization of substance P-like immunoreactivity in the cerebral ganglion of locust with a monoclonal antibody[END_REF], cockroach Periplaneta americana [START_REF] Verhaert | Substance P-like immunoreactivity in the central nervous system of the blattarian insect Periplaneta americana L. revealed by a monoclonal antibody[END_REF], lobster Panulirus interruptus [START_REF] Mancillas | Immunocytochemical localization of enkephalin and substance P in retina and eyestalk neurones of lobster[END_REF], crab Uca pucillator [START_REF] Fingerman | Localization of substance P-like, leucine-enkephalin-like, methionine-enkephalin-like, and FMRF amide-like immunoreactivity in the eyestalk of the fidder crab, Uca pugilator[END_REF], among many others.

Later, sequencing techniques revealed that these mature peptides exist as tandem repeats in their precursors (Schoofs et al., 1990a;Schoofs et al., 1990b) and the number of repeats/copies goes from five in the fruitfly, Drosophila melanogaster [START_REF] Siviter | Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A[END_REF] up to 13/14 in the cockroach species Leucophaea maderae and Periplaneta americana [START_REF] Predel | Tachykinin-related peptide precursors in two cockroach species: Molecular cloning and peptide expression in brain neurons and intestine[END_REF].

Common C-terminal motive characteristics were discovered with the wide distribution of tachykinin sequences along the protostome species (Table 2): in ecdysozoa (Van Loy et al., 2010b):

the fruit fly Drosophila melanogaster [START_REF] Siviter | Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A[END_REF][START_REF] Winther | Tachykinin-related peptides modulate odor perception and locomotor activity in Drosophila[END_REF], and also in lophotrochozoans: worm Urechis unitincus [START_REF] Ikeda | Two novel tachykinin-related neuropeptides in the echiuroid worm, Urechis unicintus[END_REF][START_REF] Kawada | A novel tachykinin-related peptide receptor: Sequence, genomic organization, and functional analysis[END_REF], octopus Octopus vulgaris [START_REF] Kanda | A novel tachykininrelated peptide receptor of Octopus vulgaris -Evolutionary aspects of invertebrate tachykinin and tachykinin-related peptide[END_REF], worm Paranemertes sp. [START_REF] Chung | Preliminary report of a neurokinin-like receptor gene sequence for the Nemertean Paranemertes sp[END_REF], and more recently in the oyster Crassostea gigas (Dubos et al., 2018a), among many others.

Protostomes TKRPs are distributed in brain and the innervations arrive till guts and oviducts (for review: [START_REF] Satake | Overview of the primary structure, tissue-distribution, and functions of tachykinins and their receptors[END_REF]. The tachykinins are also present in the cockroach Leucophaea maderae (Lem-TAC). LemTAC-reactive fibres are located nearby proctolin-immunoreactive fibres from the terminal ganglion to the hind gut [START_REF] Nässel | Tachykinin-related peptides in invertebrates: a review[END_REF][START_REF] Nässel | Neuronal co-localization of different isoforms of tachykinin-related peptides (LemTRPs) in the cockroach brain[END_REF]. The corpora cardiaca in the locust is connected with the LomTAC-fibers from the lateral neurosecretory cells and related to the release of the adipokinetic hormone [START_REF] Nässel | Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca[END_REF]. In the fruitfly, the distribution in mid-gut endocrine cells and neuronal tissues was revealed by in situ hybridization [START_REF] Siviter | Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A[END_REF].

In vitro studies demonstrated that tachykinins have a role in the protostome gut activity regulation and skeletal muscle contraction (Schoofs et al., 1990a;Schoofs et al., 1990b;[START_REF] Ikeda | Two novel tachykinin-related neuropeptides in the echiuroid worm, Urechis unicintus[END_REF][START_REF] Palamiuc | A tachykinin-like neuroendocrine signalling axis couples central serotonin action and nutrient sensing with peripheral lipid metabolism[END_REF]. Recent studies in the oyster Crassostrea gigas [START_REF] Dubos | Characterization of a tachykinin signalling system in the bivalve mollusc Crassostrea gigas[END_REF] found a decrease of tachykinin receptor along the sexual maturation in the gonads, which indicates a role in sexual maturation inhibition. These functions are analogous to vertebrate tachykinins (for review : Hu et al., 2014b). Interestingly, tachykinin expression increased in starved oysters, thus pointing to a possible role in feeding behaviour too [START_REF] Dubos | Characterization of a tachykinin signalling system in the bivalve mollusc Crassostrea gigas[END_REF].

Tachykinin related peptides (pTACRP)

Locust

Lom-TAC I APSLGFHGVR Lom-TAC II APMRGFQSVR Lom-TAC III ALKGFFGTR Lom-TAC IV APQAGFYGVR Lom-TAC V GPSGFYGVR Lom-TAC VI APLSGFYGVR Lom-TAC VII APVGFYGTR Fruitfly D-TAC I APTSSFIGMR D-TAC II APLAFVGLR D-TAC III FIPINNRLSDVLQSLEEERLRDSLLQDFFDRVAGRDGSAV D-TAC IV APTGFTGMR D-TAC V APVNSFVGMR D-TAC VI AALSDSYDLRGKQQRFADFNSKFVAVR D-TAC VII APNGFLGMR Echiuroid worm Uru-TAC I LRQSQFVGAR Uru-TAC II AAGMGFFGAR Uru-TAC III AAPSGFFGAR Uru-TAC IV SFSEELDEEFDMPKKPRAAYSGFFGAR Uru-TAC V APSMGFFGAR Uru-TAC VI APHMRFYGSR Uru-TAC VII APKMGFFGAR Oyster Cragi-TAC I FGFAPMR Cragi-TAC II ARFFGLR Cragi-TAC III FRFTALR

Deuterostome

In deuterostomes, tachykinin peptides have been found in echinoderms, urochordates and vertebrates. A general motif for deuterostome-non-echinoderm tachykinins at C-Terminal is FxGLM, where x is a variable amino acid, aromatic or aliphatic (for review: [START_REF] Pennefather | Tachykinins and tachykinin receptors: a growing family[END_REF]. Some exceptions are found, for example the human endokinins (EK) C and D that present a substitution of the final M by L [START_REF] Naono | Leucine at the carboxyl-terminal of endokinins C and D contributes to elicitation of the antagonistic effect on substance P in rat pain processing[END_REF][START_REF] Nishimori | New tachykinin peptides and nociception[END_REF].

Echinodermata

Tachykinins have been found in all studied echinoderms except sea urchins, indicating that asterozoa have conserved this signalling system but echinozoa has lost it [START_REF] Semmens | Transcriptomic identification of Starfish Neuropeptide precursors yields new insights into neuropeptide evolution[END_REF][START_REF] Zandawala | Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms[END_REF] 3). The absence of central nervous system and the pentaradial symmetry of the echinoderms indicate that tachykinin function is conserved in simple nervous systems and is not related to bilaterian symmetry [START_REF] Semmens | Transcriptomic identification of Starfish Neuropeptide precursors yields new insights into neuropeptide evolution[END_REF]. In deuterostomes echinoderms, the tachykinin C-terminal motif is GxF where x is a leucine or an isoleucine (GLF or GIF) [START_REF] Semmens | Transcriptomic identification of Starfish Neuropeptide precursors yields new insights into neuropeptide evolution[END_REF][START_REF] Zandawala | Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms[END_REF]. Further studies in tissue distribution of tachykinin in echinoderms will help to understand their physiological roles.

Tunicata

In an Urochordate, the ascidian Ciona intestinalis, a tachykinin precursor was isolated from the neural complex and encoded two peptides, Ci-TACRP and Ci-TAC (Table 4), which share the vertebrate FXGLM motif in C-terminal [START_REF] Satake | Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: Evolutionary origin of the vertebrate tachykinin family[END_REF]. The sequences of the two peptides are located in the same exon, indicating that both peptides are transcribed at the same moment and no alternative splicing would induce any differential transcription of both encoded peptides.

Ci-TAC precursor expression was reported to be predominant in the brain, gut and endostyle [START_REF] Satake | Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: Evolutionary origin of the vertebrate tachykinin family[END_REF]Kawada et al., 2011), and in the ovarian [START_REF] Aoyama | A novel biological role of tachykinins as an up-regulator of oocyte growth: Identification of an evolutionary origin of tachykininergic functions in the ovary of the ascidian, Ciona intestinalis[END_REF][START_REF] Kawada | Transcriptomes of the premature and mature ovaries of an ascidian, Ciona intestinalis[END_REF] suggesting that the tachykinins may function as brain/gut peptides and endocrine/paracrine hormones as well as be involved in oocyte maturation. These functions might have been established

in the ancestral chordate (for review: [START_REF] Satake | Insight into molecular and functional diversity of tachykinins and their receptors[END_REF].

Ascidian

Ciona intestinalis

Ci-TACRP QRDLYEKNKRHVRHFYGLM Ci-TAC SIGDQPSIFNERASFTGLM

Vertebrata

The evolutive storyline for tachykinin in chordates is that an ancestral tac gene and a tacreceptor gene in proto-chordates promoted four paralogs each after the two whole genome duplication rounds (1R/2R) in early vertebrates (Figure 5; [START_REF] Donoghue | Genome duplication, extinction and vertebrate evolution[END_REF][START_REF] Dehal | Two rounds of whole genome duplication in the ancestral vertebrate[END_REF]. This event proceeds from the possible loss of one of the four paralogs, the tac2 gene (For review: Hu et al., 2014). Further divergence caused after genome duplication events [START_REF] Jaillon | Changing by doubling", the impact of Whole Genome Duplications in the evolution of eukaryotes[END_REF] opened diverse evolutionary pathways for tachykinins. The teleost specific third genome duplication produced a tetraploidization (Figure 5; [START_REF] Meyer | From 2R to 3R: Evidence for a fish-specific genome duplication (FSGD)[END_REF][START_REF] Braasch | Polyploidy in Fish and the Teleost Genome Duplication[END_REF] Postlethwait, 2012) that could be followed by rediploidization and gene loss in some cases or physiology subfunctionalization in some others.

Vertebrate tac genes consist in five to seven exons that encode a pre-pro-tachykinin peptide.

Two peptides are cleaved from the pre-pro-tachykinin (Figure 8, for review: Hu et al., 2014b;[START_REF] Nässel | Tachykinin-related peptides in invertebrates: a review[END_REF]. The tachykinin peptide placed close to the N-terminal end will be here called TAC-related peptide or TACRP and the second peptide closer to the C-terminal extreme will be called TAC. The transcription of the pre-pro-peptide may encode one or both TAC peptides by alternative splicing events (Human, Table 7, [START_REF] Page | Differential mRNA splicing and precursor processing of neurokinin B in neuroendocrine tissues[END_REF]. The splicing of the gene can determine the isoform of the pre-pro-peptide, which in turn can include different lengths for the N-terminal cleavage sequence.

Hence, the length of the two encoded peptides can vary in such a manner that more than two different peptides can be translated and cleaved from the same gene.

Four peptides can be translated from the gene tac1:

• Substance P (SP) or TAC1RP

• Neurokinin A (NKA) or TAC1

• Neuropeptide K (NPK) or TAC1-NPK

• Neuropeptide gamma (NPγ) or TAC1-NPγ

In the gene tac3 there are two peptides encoded:

• TAC3 related peptide TAC3RP

• Neurokinin B (NKB) or TAC3.

In the gene tac4 there are up to five possibilities for a peptide depending on the splicing. Three peptides can be translated from the TAC4RP and two from the TAC4 site:

• Hemokinin (HK) or TAC4RP-HK

• Endokinin A (EKA) or TAC4RP-EKA

• Endokinin B (EKB) or TAC4RP-EKB

• Endokinin C (EKC) or TAC4RP-EKC

• Endokinin D (EKD) or TAC4RP-EKD
The meaning of this alternative splicing is unknown, but it is likely a vertebrate characteristic since the tac gene in C. intestinalis is transcribed with both peptides included in the same exon [START_REF] Satake | Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: Evolutionary origin of the vertebrate tachykinin family[END_REF].

The conservation or loss of the tachykinin genes and peptides in each clade is described here from lungfishes to teleosts for illustrating the phylogenetic relations during the vertebrate radiation after genome duplication events and opening pharmacological possibilities for each peptide.

Agnathan

A tachykinin immunoreactive to NKA antibodies was found in the brain of the sea lamprey Petromyzon marinus [START_REF] Waugh | Novel tachykinins from the brain of the sea lamprey, Petromyzon marinos, and the skate, Raja rhina[END_REF] with N-terminal similar to SP (RKPHPKEWGLM). Later, the identical sequence was purified from gastrointestinal extract in the river lamprey Lampetra fluvialitis (David Waugh et al., 1995). A second tachykinin of 9 aa (HFDEFVGLM) more similar to NKA was found in these same extract. Further immunohistochemical analysis in the sea lamprey brain determined that tachykinins are widely distributed and there are at least two different kind of peptides [START_REF] Auclair | Immunohistochemical distribution of tachykinins in the CNS of the lamprey Petromyzon marinus[END_REF]. Studies in the locomotor network of the isolated spinal chord determined that endogenous tachykinins contribute to the activity of this network [START_REF] Pérez | Endogenous tachykinin release contributes to the locomotor activity in lamprey[END_REF] by reducing the calcium and potassium entry in commissural interneurons and in motor neurons [START_REF] Pérez | Modulation of calcium currents and membrane properties by substance P in the lamprey spinal cord[END_REF][START_REF] Pérez | Substance P depolarizes lamprey spinal cord neurons by inhibiting background potassium channels[END_REF]. The number of tachykinin genes in the lamprey is still unknown and its research will contribute to construct the genetic history of the lamprey genome.

Chondrichthyan

Tachykinin peptides have been found in elasmobranchs. A substance P immunoreactive peptide (KPRPGQFFGLM) was isolated from a brain extract from the dogfish Scylorhinus canicula [START_REF] Waugh | from the brain of the dogfish[END_REF]. Later, Waugh and co-workers found a NKA-like peptide in skate Raja rhina (HKLGSFVGLM) (1994) and NPγ in shark Sphyrna lewini (ASGPTQAGIVGRKRQKGEMFVGLM) (1995).

Sarcopterygian

Sarcopterygians conserved the tachykinin genes and peptides. A substance P-like peptide (KPRPDEFYGLM) was found in the intestine of the lungfish Neoceratodus forsteri [START_REF] Liu | Purification, characterization, and biological activity of a substance P-related peptide from the gut of the Australian lungfish, Neoceratodus forsteri[END_REF].

This peptide has a contractile action on gut circular muscle. The sequence found in lungfish is identical to a tachykinin peptide found in the intestine of cane toad Bufo marinus [START_REF] Conlon | Bufokinin: A substance P-related peptide from the gut of the toad, Bufo marinus with high binding affinity but low selectivity for mammalian tachykinin receptors[END_REF], hence pointing to a common ancestor.

Amphibia

A review has been dedicated to tachykinins in amphibians [START_REF] Liu | Tachykinin peptides and receptors: Putting amphibians into perspective[END_REF]. These peptides have been identified in various anoures and one urodele. In amphibians, tachykinins have been isolated, like in other vertebrates from the intestine or the brain, but mostly from the skin (syncytial cells of the granular glands) (for review: Severini et al., 2002). Many amphibian tachykinins have no known mammalian counterparts; they can be divided into aromatic and aliphatic groups (for review:

Skin amphibian tachykinins

Skin tachykinins, while conserving the C-terminal tachykinin motif, exhibit unique sequences in their N-terminus and show pharmacological profiles distinct from SP, NKA and NKB (for reviews: Severini et al., 2002;[START_REF] Liu | Tachykinin peptides and receptors: Putting amphibians into perspective[END_REF]. They are thought to serve as exogenous factors and to have emerged from the amphibian SP, NKA and NKB genes exclusively during the evolution of amphibians (for review: [START_REF] Satake | Insight into molecular and functional diversity of tachykinins and their receptors[END_REF]. Some examples are given below. In 1977, a new peptide was isolated from the skin of the amphibian, the African frog Kassina senegalensis, and named kassinin (Anastasi et al., 1977). This dodecapeptide (DVPKSDQFVGLM) possesses the characteristic C-terminal FVGLM but differs in the aa composition in its N-terminus (Anastasi et al., 1977).

Hylambatin (DPPDPDRFYGMM), isolated from the skin of the South-African frog Hylambates maculatus, is a tachykinin having a GMM at C-terminal end (Yasuhara et al., 1981). Peptides, isolated from the skin of the Costa Rican frog Agalychnis callidryas (GPPDPDRFYPGM and EPDPDRFYPGM),

have either PGM at their C-terminus [START_REF] Mignogna | Tachykinins and Other Biologically Active Peptides From the Skin of the Costa Rican Phyllomedusid Frog Agalychnis callidryas[END_REF].

Brain/gut amphibian tachykinins

A SP-related peptide, termed bufokinin (KPRPDEFYGLM), was isolated from the gut of the toad Bufo marinus [START_REF] Conlon | Bufokinin: A substance P-related peptide from the gut of the toad, Bufo marinus with high binding affinity but low selectivity for mammalian tachykinin receptors[END_REF] and it is identical in African clawed frog Xenopus laevis [START_REF] Johansson | The primary structures and myotropic activities of two tachykinins isolated from the African clawed frog, Xenopus laevis[END_REF]. In Xenopus laevis, a second peptide was isolated from intestine (TLTTGKDFVGLM)

whose N-terminal region shows no structural similarity to previously known tachykinins [START_REF] Johansson | The primary structures and myotropic activities of two tachykinins isolated from the African clawed frog, Xenopus laevis[END_REF]. In the European green frog Rana ridibunda brain, a SP-related peptide, named ranakinin (KPNPERFYGLM), as well as a NKB-related peptide (DMHDFFVGLM), were isolated [START_REF] O O'harte | Ranakinin: A Novel NK1 Tachykinin Receptor Agonist Isolated with Neurokinin B from the Brain of the Frog Rana ridibunda[END_REF]. In Rana ridibunda, a NKA-related peptide was also isolated from the intestine (HKLDSFIGLM) (Wang et al., 1992b). In Rana catesbeiana brain and intestine, four novel tachykinins, named ranatachykinin A (KPSPDRFYGLM), B (YKSDSFYGLM), C (HNPASFIGLM) and D (KPNPERFYAPM)

were isolated (Kozawa et al.,1991). In the urodele, Amphiuma tridactylum, SP-related peptide showed many substitutions compared to mammalian SP (DNPSVGQFYGLM) and NKA-related peptide (HKDAFIGLM) showed two substitutions (David Waugh et al., 1995).

Tissue distribution

As we have seen, amphibian tachykinins were isolated from brain, gut and skin and their distribution in these organs have been well documented (for reviews: Severini et al., 2002;[START_REF] Liu | Tachykinin peptides and receptors: Putting amphibians into perspective[END_REF]. In the gastrointestinal tract, tachykinin-immunoreactive fibres have been observed in both the extrinsic (bufokinin and SP in the toad Bufo marinus: [START_REF] Osborne | A pharmacological and immunohistochemical study of the splanchnic innervation of ileal longitudinal muscle in the toad Bufo marinus[END_REF][START_REF] Murphy | Ceil & Tissue An immunohistochemical study of the innervation of the large intestine of the toad ( Bufo marinus )[END_REF][START_REF] Liu | Bufokinin: immunoreactivity, receptor localization and actions in toad intestine and mesenteric circulation[END_REF] and intrinsic neurons (SP/NKA in Xenopus laevis: [START_REF] Johansson | The primary structures and myotropic activities of two tachykinins isolated from the African clawed frog, Xenopus laevis[END_REF]. In amphibians, tachykinin-immunoreactivity have been reported in endocrine cells (Hyla arborea, Ambystoma mexicanum, Cynops hongkongensis and Atelopus oxyrhynchus: [START_REF] Buchan | An immunocytochemical study of regulatory peptides in the amphibian gastrointestinal tract[END_REF] salamanders Necturus: [START_REF] Holmgren | Neuropeptide functions in the fish gut[END_REF]; Bufo marinus: [START_REF] Liu | Bufokinin: immunoreactivity, receptor localization and actions in toad intestine and mesenteric circulation[END_REF]Xenopus laevis: Johansson et al., 2002). In the endocrine cells, amphibian tachykinin have contractile effects on smooth muscle.

Amphibian tachykinins have also been detected in cardiovascular system, adrenal gland, bladder and lung (for review: [START_REF] Liu | Tachykinin peptides and receptors: Putting amphibians into perspective[END_REF].

Further research in the amphibian genomes will provide information about the total duplicated tachykinin genes and peptides that occur in this clade and their possible functions.

Sauropsida

Tachykinin 1

In sauropsids, tachykinin peptides encoded by tac1 gene (SP, NKA) have been characterized

(Table 5). In the chicken, antisera raised against N-terminal and C-terminal regions of SP and against C-terminal region of NKA were used to isolate tachykinin-like immunoreactive peptides from extracts of small intestine [START_REF] Conlon | Arg3]substance P and neurokinin A from chicken small intestine[END_REF]. In 1992, Wang and collaborators isolated, using the same antisera, SP and NKA from brain of alligator Alligator mississipiensis (Wang et al., 1992a). In the Burmese python, Python molurus, tachykinin-like immunoreactive peptides isolated from intestine were also three: SP, NKA and NPγ [START_REF] Conlon | Tachykinins (Substance P, Neurokinin A and Neuropeptide γ) and neurotensin from the intestine of the burmese python, python molurus[END_REF], from which NKA was identical to human/chicken/alligator NKA. Finally, SP and NPγ were isolated from the intestine of a tortoise, Gopherus agassizii [START_REF] Wang | Tachykinins ( Substance P and Neuropeptide γ ) from the Brains of the Pallid Sturgeon , Scaphirhynchus albus and the Paddlefish[END_REF].

TAC1 (NKA) HKTDSFVGLM

In the intestine of the alligator, SP-immunoreactivity was confined to the peripheral innervation with fibres in the lamina propria and muscle layers, and cell bodies in the submucosa and myenteric plexuses [START_REF] Buchan | Regulatory peptides in the gastrointestinal tract of Alligator mississipiensis[END_REF]. In the turtle Chrysemys scripta, SP-like immunoreactivity was located in brain, spinal cord, dorsal root ganglion, and retina [START_REF] Korte | Substance P-like immunoreactivity in cerebellar mossy fibers and terminals in the red-eared turtle, Chrysemys scripta elegans[END_REF].

SP immunoreactive nerve fibres innervate blood vessels in the chicken skin as well as being localized in both epidermis and dermis [START_REF] Sann | Substance P and calcitonin gene-related peptide in the chicken skin: distribution and cardiovascular effects[END_REF]. SP/NKA-like immunoreactive neurons were observed in crocodile (C. porosus) salt gland and saltwater acclimation resulted in a reduction of this immunoreactivity [START_REF] Cramp | The effects of saltwater acclimation on neurotransmitters in the lingual salt glands of the estuarine crocodile, Crocodylus porosus[END_REF].

Tachykinin 3

Page and collaborators identified TAC3 pre-pro-peptide precursors (containing NKB peptide sequence) in two reptiles (alligator Alligator mississipiensis and anole lizard Anolis carolinensis), as well as in one bird (chicken Gallus gallus) by bioinformatics database analysis [START_REF] Page | Differential mRNA splicing and precursor processing of neurokinin B in neuroendocrine tissues[END_REF]. The sequence of the chicken is coincident with the alligator (DMHDFVGLM) and with human, while the lizard has an insertion of a phenylalanine (DMHDFFVGLM).

Mammalia

The three tachykinin genes tac1, tac3 and tac4 are conserved in all the mammalian clade and the tac3 gene is common to the studied mammals (Table 6). The peptides encoded in the tac1 and tac4

gene are divergent for some studied mammal species (Article 2, Supplementary figures 1 and 2; for review: [START_REF] Page | Hemokinins and endokinins[END_REF]).

Tachykinin 1

In 1931, a substance, found in an extract of horse brain and intestine and first called « preparation/powder P », induced in vitro atropine-resistant contraction of the rabbit isolated intestine and lowered the arterial blood pressure of atropinized rabbit [START_REF] Euler | An unidentified depressor substance in certain tissue extracts[END_REF].

Forty years will be needed to determine its chemical structure [START_REF] Chang | Amino-acid Sequence of Substqnce P[END_REF].

Meanwhile, a new factor partially purified from bovine and rat hypothalamic tissue and named sialogen was shown to stimulate salivary secretion in rat [START_REF] Leeman | Stimulation of Salivary Secretion by a Factor Extracted from Hypothalamic Tissue1[END_REF]. In 1968,

Lembeck and Starke reported that substance P was also able to stimulate salivary secretion in rats and suggested that sialogen and substance P may be the same peptide [START_REF] Lembeck | Substanz P und Speichelsekretion[END_REF], confirmed later by [START_REF] Chang | Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P[END_REF]. Its primary structure was further determined as an amidated undecapeptide [START_REF] Chang | Amino-acid Sequence of Substqnce P[END_REF] and its chemical synthesis obtained [START_REF] Tregear | Synthesis of Substance P[END_REF] in the same volume of Nature in 1971 (Table 6).

In 1983, two studies led to the identification of a novel peptide with a sequence homologous to that of amphibian kassinin and SP [START_REF] Kimura | Novel neuropeptides, neurokinin α and β, isolated from porcine spinal cord[END_REF][START_REF] Nawa | Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor[END_REF]. The first one isolated this peptide from an extract of porcine spinal cord and named it neurokinin α [START_REF] Kimura | Novel neuropeptides, neurokinin α and β, isolated from porcine spinal cord[END_REF]. In 1984, a novel mammalian tachykinin designated neuromedin L was isolated from porcine spinal cord by using bioassay for a tachykinin-like effect on contractility of smooth muscle preparation from guinea pig ileum [START_REF] Minamino | Neuromedin L: a novel mammalian tachykinin identified in porcine spinal cord[END_REF]. Neuromedin L, substance K and neurokinin α correspond to the same peptide that will be named neurokinin A (NKA, Table 6).

Two biologically active N-terminally extended forms of NKA were later identified (Table 6) and named neuropeptide K (NPK, [START_REF] Tatemoto | Neuropeptide K: Isolation, structure and biological activities of a novel brain tachykinin[END_REF] and neuropeptide γ (NPγ, [START_REF] Kage | Neuropeptide-gamma: a peptide isolated from rabbit intestine that is derived from gamma-preprotachykinin[END_REF].

The tac1 gene consists of seven exons and there are different alternative splicing that produce different pre-pro-tachykinins. The β PPT-A is the pre-pro-tachykinin peptide encoded in the seven exons, while α PPT-A lacks exon 6, γ PPT-A exon 4 and δ PPT-A exons 4 and 6 (Table 7, for reviews: and 6; NPK in exons 3, 4, 5 and 6 (bovine: [START_REF] Nawa | Tissue-specific generatrion of two preprotachykinin mRNAs from one gene by alternative RNA splicing[END_REF]human: Harmar et al., 1986;[START_REF] Harmar | Identificaiton and cDNA sequence of delta-preprotachykinin, a fourth splicing variant of the rat substance P precursor[END_REF]rat: Carter and Krause, 1990;For reviews: Nakanishi 1986;[START_REF] Pennefather | Tachykinins and tachykinin receptors: a growing family[END_REF]. The first study to compare the tissue distribution of α-tac1 and β-tac1 isophorm mRNAs was performed in bovine by Nawa and collaborators in 1984. While α-tac1 mRNAs are mainly observed in the nervous system, β-tac1 mRNAs are found in both nervous system and peripheral tissues [START_REF] Nawa | Tissue-specific generatrion of two preprotachykinin mRNAs from one gene by alternative RNA splicing[END_REF]. Highest expression of tac1 mRNA was reported in brain, heart, spleen, salivary, thyroid and adrenal glands, while a minor expression was observed in cerebellum, kidney, prostate, skeletal muscle, testis, trachea, uterus and bone marrow [START_REF] Pinto | mRNA expression of tachykinins and tachykinin receptors in different human tissues[END_REF]. Tac1 mRNA was undectable in thymus, fetal and adult liver, lung and placenta [START_REF] Pinto | mRNA expression of tachykinins and tachykinin receptors in different human tissues[END_REF].

Both SP and NKA were found in rat primary sensory neurons [START_REF] Dalsgaard | Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P[END_REF] and splice variants γ-tac1 including TAC1RP and TAC1, and δ-tac1 encoding only TAC1RP have also been identified in rat [START_REF] Sivam | Neonatal and Adult 6-Hydroxydopamine-Induced Lesions Differentially Alter Tachykinin and Enkephalin Gene Expression[END_REF][START_REF] Carter | Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma[END_REF]).

In the pituitary, high levels of tac1 mRNA was reported in rat [START_REF] Jonassen | Developmental and Hormonal Regulation of the Sex Difference in Preprotachykinin Gene Expression in Rat Anterior Pituitariesa[END_REF]. SP and NKA but no NPK in rat anterior pituitary [START_REF] Brown | Gonadal Steroid Regulation of Substance P (SP) and SP-Encoding Messenger Ribonucleic Acids in the Rat Anterior Pituitary and Hypothalamus*[END_REF].

Tachykinin 3

In 1983, two Japanese laboratories described the isolation from porcine spinal cord and primary structure of a novel tachykinin peptide [START_REF] Kangawa | Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord[END_REF][START_REF] Kimura | Novel neuropeptides, neurokinin α and β, isolated from porcine spinal cord[END_REF]. This peptide possessed structural homology to tachykinin (especially to amphibian kassinin) and had the ability to induce contraction of the guinea pig ileum as SP. Kimura and collaborators named it neurokinin β, while Kangawa chose neuromedin K. The structure and expression of the gene for the bovine neuromedin K precursor (preprotachykinin B or PPT-B) were reported 3 years later [START_REF] Kotani | Immunoglobulin Autoantibodies G Subclasses of Anti-Thyroid in Human Autoimmune Thyroid Peroxidase Diseases *[END_REF]. cDNA encoding tac3 was cloned and sequenced in rat [START_REF] Bonner | A cDNA encoding the precursor of the rat neuropeptide, neurokinin B[END_REF], mouse (Kako et al 1993) and human [START_REF] Page | Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia[END_REF].

The tachykinin peptide derived from the tac3 gene, called alternatively TAC3, NKB or neuromedin K is a decapeptide, with the common tachykinin sequence FXGLM, where X here is a valine. Alternative splice events have been described for this gene (Table 7, [START_REF] Page | Differential mRNA splicing and precursor processing of neurokinin B in neuroendocrine tissues[END_REF]. During some time, the gene tac3 was called tac2 in rodents [START_REF] Duarte | Incongruent pattern of neurokinin B expression in rat and mouse brains[END_REF], but it is now referred as a synonym in the bioinformatics databases such as NCBI or ENSEMBL and tac3 is the common form used.

The tac3 gene consists of seven exons and NKB is encoded in exon 5 (bovine: [START_REF] Kotani | Immunoglobulin Autoantibodies G Subclasses of Anti-Thyroid in Human Autoimmune Thyroid Peroxidase Diseases *[END_REF] For review: [START_REF] Pennefather | Tachykinins and tachykinin receptors: a growing family[END_REF].

A wide tissue distribution of tac3 was reported in the bovine, as soon as 1986, with a high expression in the hypothalamus and intestine [START_REF] Kotani | Immunoglobulin Autoantibodies G Subclasses of Anti-Thyroid in Human Autoimmune Thyroid Peroxidase Diseases *[END_REF]. In situ hybridization study in the rat brain showed that tac3 was widely distributed in a pattern distinct from that of SP (tac1) [START_REF] Bonner | A cDNA encoding the precursor of the rat neuropeptide, neurokinin B[END_REF]. The large brain distribution, including KNDy neurons [START_REF] Lehman | Minireview: Kisspeptin/neurokinin B/dynorphin (KNDy) cells of the arcuate nucleus: A central node in the control of gonadotropin-releasing hormone secretion[END_REF], of tac3 has been reviewed many times (Severini et al., 2002;[START_REF] Rance | Neurokinin B and the hypothalamic regulation of reproduction[END_REF]. Using radioimmunoassay and immunocytochemistry, Tateishi and collaborators investigated the distribution and localisation of NKB-like immunoreactivity (NKB-LI) in rat peripheral tissues [START_REF] Tateishi | Distribution and localization of neurokinin A-like immunoreactivity and neurokinin B-like immunoreactivity in rat peripheral tissue[END_REF]. According to these studies, all tissues examined contained NKB-LI, except thyroid; they found high levels of NKB-LI in oesophagus, head of pancreas, adrenal and kidney. Tac3 expression was reported in human placenta [START_REF] Page | Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia[END_REF]. Tac3 gene expression in human was also found to be strong in brain, placenta, testis and kidney, while lower in prostate, bone marrow, salivary gland, skeletal muscle, thymus and adrenal gland, and undetectable in cerebellum, liver and spleen [START_REF] Pinto | mRNA expression of tachykinins and tachykinin receptors in different human tissues[END_REF]. Other reproductive organs (uterus, oviduct) than placenta, prostate and gonads have been shown to express NKB (for review: Hu et al., 2014b). In intestine, while works described the localization of NKB at this level, no study has yet provided evidence for the existence of tac3 gene (for review : Severini et al., 2002;[START_REF] Lecci | Tachykinins and tachykinin receptors in the gut, with special reference to NK2 receptors in human[END_REF].

Tachykinin 4

In 2000, a third tachykinin gene, tac4, was identified in mammals, while investigating genes differentially expressed during the transition from pro-B to pre-B stages of hematopoietic cells in mouse bone marrow (Zhang et al., 2000). Tac4 mRNA was restricted to hematopoietic cells and the 11-aa peptide encoded by tac4 was thus termed hemokinin (HK-1, table 6) (Zhang et al., 2000). The tac4 gene was then isolated from rat brain and shown to encode a predicted HK-1, identical to mouse HK-1 [START_REF] Kurtz | Identification, localization and receptor characterization of novel mammalian substance-P peptides[END_REF]. In human, two groups isolated and characterized tachykinin 4 gene, also named tac4 from hypothalamus and thymus [START_REF] Kurtz | Identification, localization and receptor characterization of novel mammalian substance-P peptides[END_REF][START_REF] Page | Characterization of the endokinins : Human tachykinins with cardiovascular activity[END_REF]. [START_REF] Kurtz | Identification, localization and receptor characterization of novel mammalian substance-P peptides[END_REF] found that tac4 gene encoded a different 11-aa peptide termed human HK-1 and a truncated form HK-1(4-11), with FFGLM (instead of FYGLM) at their C-terminal [START_REF] Kurtz | Identification, localization and receptor characterization of novel mammalian substance-P peptides[END_REF]. In contrast, [START_REF] Page | Characterization of the endokinins : Human tachykinins with cardiovascular activity[END_REF] identified that the tac4 gene transcript was spliced into four different variants that encode four different peptides named endokinins (EK) (Table 6, for reviews: [START_REF] Patacchini | Newly discovered tachykinins raise new questions about their peripheral roles and the tachykinin nomenclature[END_REF][START_REF] Page | Gene regulation of neurokinin B and its receptor NK3 in late pregnancy and pre-eclampsia[END_REF]. Similarly, in rabbit, cloning of tac4 cDNA from pooled lung, spleen and thymus, revealed a structure comparable to that of human, with alternative splicing, giving two transcripts, α and β-tac4 [START_REF] Page | Hemokinins and endokinins[END_REF]). Both transcripts encoded a decapeptide identical to the C-terminal of EKA and EKB and termed EK-1 (Table 6). A second peptide, a dodecapeptide with a divergent C-terminal and named EK-2, was characterized only in the α-tac4 isomorph with a different C-terminal (QRGLL), which were characterised as EKC and EKD [START_REF] Page | Hemokinins and endokinins[END_REF]).

The tac4 gene consists of five exons and HK-1 is encoded in exon 2 (human and rat: [START_REF] Kurtz | Identification, localization and receptor characterization of novel mammalian substance-P peptides[END_REF]human: Page et al., 2003;For review: Pennefather et al., 2004).

In mouse, tac4 mRNAs were detected, by RT-PCR, at the highest levels in bone marrow (hematopoietic cells), at a weaker level in thymus, but were absent in brain and spleen (Zhang et al., 2000). In another study, however, moderate to strong expression were reported in the brain, spleen, stomach, skin and lactating breast, while weak tac4 signal was detected in heart, kidney, thymus, small intestine, muscle and lung [START_REF] Kurtz | Identification, localization and receptor characterization of novel mammalian substance-P peptides[END_REF]. Using qPCR, [START_REF] Duffy | Centrally administered hemokinin-1 (HK-1), a neurokinin NK1 receptor agonist, produces substance Plike behavioral effects in mice and gerbils[END_REF] showed that tac4 mRNA was detected in a wide range of peripheral tissues (heart, kidney, liver, lung, stomach, spleen, adrenal, gonads and uterus) and also in numerous brain regions [START_REF] Duffy | Centrally administered hemokinin-1 (HK-1), a neurokinin NK1 receptor agonist, produces substance Plike behavioral effects in mice and gerbils[END_REF]. Tac4 mRNA was also found in the uterus, granulosa cells and blastocyst-stage embryos in the mouse [START_REF] Pintado | A Role for Tachykinins in Female Mouse and Rat Reproductive Function1[END_REF].

In human, tac4 was weakly expressed in kidney, liver, lung, stomach, testis, placenta, prostate and fetal liver, while a stronger expression was observed in the heart, skeletal muscle, skin and thyroid, as shown by RT-PCR [START_REF] Kurtz | Identification, localization and receptor characterization of novel mammalian substance-P peptides[END_REF]. Using specific primers to overlap each unique exon junction, Page and collaborators showed that while α and β had very restricted expression patterns, γ and δ were ubiquitously expressed in a range of human tissues [START_REF] Page | Characterization of the endokinins : Human tachykinins with cardiovascular activity[END_REF]. Α-tac4 was found only in the adrenal gland, fetal liver, and very weakly in the spleen, while β-tac4 was observed in the heart, liver, bone marrow, prostate, adrenal gland and testis [START_REF] Page | Characterization of the endokinins : Human tachykinins with cardiovascular activity[END_REF].

Even if expressed in the brain, tac4 mRNA is found at low levels in this tissue compared to other tachykinin genes, suggesting major peripheral roles of human HK-1 and EKs. [START_REF] Ogawa | Cloning and expression of tachykinins and their association with kisspeptins in the brains of zebrafish[END_REF]Zhou et al., 2012;[START_REF] López-Bellido | Substance P mRNA expression during zebrafish development: influence of mu opioid receptor and cocaine[END_REF] and grass carp Ctenopharyngodon idella [START_REF] Hu | TAC1 gene products regulate pituitary hormone secretion and gene expression in prepubertal grass carp pituitary cells[END_REF]. The 3R paralogon of tac1 would be lost in teleost fish lineage (Zhou et al., 2012). [START_REF] Moons | Comparative distribution of substance P (SP) and cholecystokinin (CCK) binding sites and immunoreactivity in the brain of the sea bass (Dicentrarchus labrax)[END_REF]. In goldfish, carassin immunoreactivity was localized in a single nucleus, the nucleus pretectalis posterioris, with fibres in the preoptic area [START_REF] Rao | Brain and Pituitary Immunocytochemistry Of Carassin In the Goldfish, Carassius Auratus -a New Neurohormone Peptide[END_REF].

In goldfish, tac1 mRNA was reported in most brain regions (olfactory bulbs, telencephalon and preoptic region, hypothalamus, optic tectum-thalamus and cerebellum-medulla), as well in peripheral tissues such as intestine, testis and pituitary [START_REF] Lin | Goldfish γ-Preprotachykinin mRNA Encodes the Neuropeptides Substance P, Carassin, and Neurokinin A1[END_REF]. In medaka Oryzias latipes, tac1 mRNA was localized by in situ hybridization in different parts of the brain:

telencephalon, olfactory bulbs and hypothalamus [START_REF] Suehiro | General and Comparative Endocrinology Mass spectrometric map of neuropeptide expression and analysis of the c -prepro-tachykinin gene expression in the medaka ( Oryzias latipes ) brain[END_REF]. Tac1 mRNA was observed in most regions of the zebrafish brain (olfactory bulb, telencephalon, preoptic region, hypothalamus, mesencephalon and rhombencephalon) [START_REF] Ogawa | Cloning and expression of tachykinins and their association with kisspeptins in the brains of zebrafish[END_REF], while in grass carp, high levels were detected in cerebellum, medulla oblongata and spinal cord, lower levels in telencephalon, optic tectum and hypothalamus, but no signal in the olfactory bulb [START_REF] Hu | TAC1 gene products regulate pituitary hormone secretion and gene expression in prepubertal grass carp pituitary cells[END_REF]. Transcript signals for tac1 in peripheral tissues were found in the gills, heart, intestine, liver, muscle and gonad, but not in the kidney and blood of grass carp [START_REF] Hu | TAC1 gene products regulate pituitary hormone secretion and gene expression in prepubertal grass carp pituitary cells[END_REF].

In the pituitary, carassin-immunoreactive granules were observed in some cells of proximal pars distalis and adjacent sections reacted with growth hormone and gonadotropin antibodies [START_REF] Rao | Brain and Pituitary Immunocytochemistry Of Carassin In the Goldfish, Carassius Auratus -a New Neurohormone Peptide[END_REF]. High levels of tac1 mRNA were reported in grass carp [START_REF] Hu | TAC1 gene products regulate pituitary hormone secretion and gene expression in prepubertal grass carp pituitary cells[END_REF] and goldfish (Lin and Peter 1997) pituitary.

Tachykinin 3

In teleost fishes, the tac3 gene has been characterized in zebrafish Danio rerio [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF]Zhou et al., 2012;[START_REF] Ogawa | Anatomy of the kisspeptin systems in teleosts[END_REF], Nile tilapia Oreochromis niloticus (Biran et al., 2014a;[START_REF] Jin | Neurokinin B-related Peptide Suppresses the Expression of GnRH I , Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus[END_REF], grass carp Ctenopharyngodon idella (Hu et al., 2014a), goldfish Carassius auratus (Qi et al., 2015a), striped bass Morone saxatilis [START_REF] Zmora | Neurokinin B regulates reproduction via inhibition of kisspeptin in a teleost, the striped bass[END_REF], and orange-spotted grouper Epinephelus coioides (Chen et al., 2018). The teleost specific whole genome duplication has led to the duplication of the gene tac3, leading to two genes, tac3a and tac3b. However, one duplicated gene seems to be lost in the case of tilapia (Biran et al., 2014a;[START_REF] Jin | Neurokinin B-related Peptide Suppresses the Expression of GnRH I , Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus[END_REF], grass carp (Hu et al., 2014a), striped bass [START_REF] Zmora | Neurokinin B regulates reproduction via inhibition of kisspeptin in a teleost, the striped bass[END_REF] and grouper (Chen et al., 2018). A related peptide to TAC3 has been found in the tac3 gene and named neurokinin F (NKF) (zebrafish: [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF]tilapia: Biran et al., 2014;striped bass: Zmora et al., 2017) or tachykinin 3 related peptide (NKBRP or TAC3RP) (carp: Hu et al., 2014a;grouper: Chen et al., 2018). Thus the two tac3 genes, tac3a and tac3b encode four peptides (Table 8), namely TAC3RPa, TAC3a, TAC3RPb and TAC3b (or NKBa-13, NKBa-10, NKBb-13 and NKBb-11 according to their aa length in zebrafish: Zhou et al., 2012).

Low similarity among fish TAC3 pre-pro-peptide precursors was observed, but TAC3 peptides were well conserved across fish species (zebrafish: [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF]Zhou et al., 2012;tilapia: Biran et al., 2014;carp: Hu et al., 2014a;goldfish: Qi et al., 2015;striped bass: Zmora et al., 2017;grouper: Chen et al., 2018). All the encoded peptides possessed the tachykinin signature motif (FXGLM)

flanked by potential dibasic cleavage sites and an adjacent glycine at the C-terminus for amidation. In this signature motif, X represents valine (V) in all fish except cichlid NKB (tilapia and other cichlids: Biran et al., 2014) and cyprinid NKBRP/NKB-13 (goldfish: [START_REF] Qi | Goldfish neurokinin B: Cloning, tissue distribution, and potential role in regulating reproduction[END_REF] for which X represents isoleucine (I). Exception was NKB encoded by tac3b gene in zebrafish (Zhou et al., 2012;[START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF] and goldfish [START_REF] Qi | Goldfish neurokinin B: Cloning, tissue distribution, and potential role in regulating reproduction[END_REF], which presented a modification of that motif with FVGLL at the Cterminus.

A wide brain expression of tac3 was also observed in teleosts (zebrafish: [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF][START_REF] Ogawa | Cloning and expression of tachykinins and their association with kisspeptins in the brains of zebrafish[END_REF], Zhou et al., 2012;goldfish: Qi et al., 2015;grass carp: Hu et al., 2014a;striped bass: Zmora et al., 2017;orange-spotted grouper: Chen et al., 2018). In the pituitary, a weak expression of tac3a but no detectable expression of tac3b was observed in the goldfish [START_REF] Qi | Goldfish neurokinin B: Cloning, tissue distribution, and potential role in regulating reproduction[END_REF], while both genes were expressed in zebrafish pituitary [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF][START_REF] Ogawa | Cloning and expression of tachykinins and their association with kisspeptins in the brains of zebrafish[END_REF].

In teleost species, where only tac3a has been identified, a weak expression was found in the pituitary (orange-spotted grouper: Chen et al., 2018;grass carp: Hu et al., 2014a). In the ovary, weak expression of tac3a (and none for tac3b) was found in goldfish [START_REF] Qi | Goldfish neurokinin B: Cloning, tissue distribution, and potential role in regulating reproduction[END_REF] and orange-spotted grouper (Chen et al., 2018). In zebrafish, contradictory results were obtained with sexually mature fish: while tac3b gene was found to be expressed in the ovary according to some authors [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF]Zhou et al., 2012), it was tac3a gene in another study [START_REF] Ogawa | Cloning and expression of tachykinins and their association with kisspeptins in the brains of zebrafish[END_REF]. Intestine expressed tac3a (and not tac3b) in zebrafish (Jakob [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF] and goldfish (Qi et al., 2014), and the single tac3 gene (tac3a) in grass carp (Hu et al., 2014a) and orange-spotted grouper (Chen et al., 2018).

Tachykinin 4

By genomic data mining, Zhou and collaborators (2012) were the first, and up to now, the only group to reveal the existence of tac4 genes in non-mammals: tac4a in stickleback, tac4b in rainbow smelt and northern pike, as well as tac4a and tac4b in zebrafish and medaka (Zhou et al., 2012)

Physiological role in reproduction

In vertebrates, tachykinin peptides bind to receptors belonging to the first class rhodopsin-like Gprotein coupled receptors (GPCR). There are three tachykinin receptors in human, namely NK1/TACR1, NK2/TACR2 and NK3/TACR3 (for review [START_REF] Maggi | The mammalian tachykinin receptors[END_REF][START_REF] Pennefather | Tachykinins and tachykinin receptors: a growing family[END_REF][START_REF] Steinhoff | Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease[END_REF].

Considering the large tissue distribution of tachykinins (and their receptors), one may expect involvement of these peptides in many various physiological functions. Severini and collaborators, in 2002, reviewed not less than six different systems in which regulation tachykinins were involved: cardiovascular system, gastrointestinal tract, airways system, urogenital tract, immune system and central nervous system (Severini et al., 2002). They also reported a role of these peptides in pain, neurogenic inflammation and lachrymal secretion (Severini et al., 2002). In this Introduction, we will focus on the involvement of tachykinins in the control of reproductive function in vertebrates.

At the central level

On GnRH

Tac1 peptides

Using a double-chamber perfusion system, Ohtsuka and collaborators (1987) were able to show that SP stimulated the in vitro release of LH-releasing hormone by rat mediobasal hypothalamus.

A lot of in vivo studies investigated the effect of tac1-encoded peptides (SP and NKA) on LH secretion in mammals via central pathway, using intravenous or intracerebroventricular (icv) injections (for review: [START_REF] Debeljuk | Modulation of the hypothalamo-pituitary-gonadal axis and the pineal gland by neurokinin A, neuropeptide K and neuropeptide γ[END_REF][START_REF] Fergani | Expanding the role of tachykinins in the neuroendocrine control of reproduction[END_REF]. While most of these studies reported a stimulatory effect of SP on LH release, absence of effect was also observed.

Concerning NKA, stimulation, absence of effect or even inhibition was noted. This discrepancy was related to the presence or absence of sex steroids (intact or castrated animals) or to speciesdifference. NPK was also shown to have an effect on LH as icv injection of the peptide produced a suppression of LH release in ovariectomized rats; in contrast, injection of NKA was ineffective and of NPγ less effective compared to NPK [START_REF] Sahu | Effects of Tachykinins on luteinizing hormone release in Female Rats : Potent inhibitory action of neuropeptide K[END_REF].

In 2003, Pintado and collaborators treated neonatally female mice or rats with capsaicin (that induces depletion of SP) and observed a reduced fertility. In addition, they injected tachykinin TACR3 receptor antagonist SR 142801 to female rats and showed a reduction in the litter size [START_REF] Pintado | A Role for Tachykinins in Female Mouse and Rat Reproductive Function1[END_REF]. More recently, it was reported that female tac1 -/-mice displayed delayed puberty (Simavli et al., 2015).

Tac3 peptide (NKB)

Mutations in the tac3 gene or in the tacr3 lead to hypogonadotropic hypogonadism [START_REF] Guran | Hypogonadotropic Hypogonadism due to a Novel Missense Mutation in the First Extracellular Loop of the Neurokinin B Receptor[END_REF][START_REF] Topaloglu | TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction[END_REF][START_REF] Fukami | Hypothalamic Dysfunction in a Female with Isolated Hypogonadotropic Hypogonadism and Compound Heterozygous TACR3 Mutations and Clinical Manifestation in Her Heterozygous Mother[END_REF][START_REF] Gianetti | TAC3/TACR3 mutations reveal preferential activation of gonadotropin-releasing hormone release by neurokinin B in neonatal life followed by reversal in adulthood[END_REF][START_REF] Young | TAC3 and TACR3 defects cause hypothalamic congenital hypogonadotropic hypogonadism in humans[END_REF][START_REF] Francou | Normosmic congenital hypogonadotropic hypogonadism due to TAC3/TACR3 mutations: Characterization of neuroendocrine phenotypes and novel mutations[END_REF], that could reverse in adulthood [START_REF] Gianetti | TAC3/TACR3 mutations reveal preferential activation of gonadotropin-releasing hormone release by neurokinin B in neonatal life followed by reversal in adulthood[END_REF]. Likewise, tac3 or tacr3 null mice showed abnormal estrous cyclicity even though still fertile [START_REF] Yang | Uncovering novel reproductive defects in neurokinin B receptor null mice: Closing the gap between mice and men[END_REF][START_REF] True | Neurokinin B is critical for normal timing of sexual maturation but dispensable for adult reproductive function in female mice[END_REF].

The first analysis of TACR3 expression showed its presence in GnRH neurons indicating a possible direct effect of TAC3 on GnRH [START_REF] Krajewski | Morphologic evidence that neurokinin B modulates gonadotropin-releasing hormone secretion via neurokinin 3 receptors in the rat median eminence[END_REF]. Later, further analysis in sheep have demonstrated the existence of many neurons and immunoreactive fibres to TACR3 in POA and in other hypothalamic regions as the arcuate nucleus (ARC) [START_REF] Amstalden | Neurokinin 3 receptor immunoreactivity in the septal region, preoptic area and hypothalamus of the female sheep: Colocalisation in neurokinin B cells of the arcuate nucleus but not in gonadotrophin-releasing hormone neurones[END_REF]. Moreover, in this same area, TAC3 is co-expressed with kisspeptin (Kiss) and dynorphin (Dyn) in the so-called KNDy neurons (for reviews: [START_REF] Lehman | Minireview: Kisspeptin/neurokinin B/dynorphin (KNDy) cells of the arcuate nucleus: A central node in the control of gonadotropin-releasing hormone secretion[END_REF]. KNDy neurons project to GnRH neurons and regulate their activity [START_REF] Navarro | New insights into the control of pulsatile GnRH release: The role of Kiss1/neurokinin B neurons[END_REF][START_REF] Grachev | The role of neurokinin B signalling in reproductive neuroendocrinology[END_REF][START_REF] Fergani | Expanding the role of tachykinins in the neuroendocrine control of reproduction[END_REF] and ablation of those neurons in female rats induces hypogonadotropic hypogonadism [START_REF] Mittelman-Smith | Ablation of KNDy neurons results in hypogonadotropic hypogonadism and amplifies the steroid-induced lh surge in female rats[END_REF].

In vivo studies in different mammals have shown the stimulatory effect of TAC3 on GnRH/LH secretion (rat: [START_REF] Navarro | Interactions between kisspeptin and neurokinin B in the control of GnRH secretion in the female rat[END_REF]mice: Navarro et al., 2011;[START_REF] Pelster | Swimbladder function and the spawning migration of the European eel Anguilla anguilla[END_REF]sheep: Billings et al., 2010;monkey: Ramaswamy et al., 2010;human: (Skorupskaite et al., 2017a;2017b).

In contrast to mammals, tachykinins and kisspeptins are expressed in different neurons in teleosts (zebrafish: [START_REF] Ogawa | Cloning and expression of tachykinins and their association with kisspeptins in the brains of zebrafish[END_REF]striped bass, Morone saxatilis: Zmora et al., 2017). However, TAC3 peptides down-regulated kiss2 gene expression (striped bass: [START_REF] Zmora | Neurokinin B regulates reproduction via inhibition of kisspeptin in a teleost, the striped bass[END_REF], suggesting that tachykinin peptides may act indirectly on GnRH via the kisspeptin system, as in mammals. The expression pattern of TAC3 and TAC3RP and the paralog receptors differs consistently from the one of the mammals. TAC3 and TAC3RP are expressed in the nuclear lateralis tuberis (NLT), which is the teleost homologous structure to the ARC [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF]. However, Kiss2 expression has not been found in this area in zebrafish [START_REF] Servili | Organization of two independent kisspeptin systems derived from evolutionary-ancient kiss genes in the brain of zebrafish[END_REF]. When administered in vivo, NKB peptides could increase gonadotropin expression and/or release (zebrafish: [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF]tilapia: Biran et al., 2014;goldfish: Qi et al., 2015;NKB in grouper: Chen et al., 2018). In contrast, some studies reported no effect on gonadotropin expression (tilapia: [START_REF] Jin | Neurokinin B-related Peptide Suppresses the Expression of GnRH I , Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus[END_REF]NKB-RP in grouper: Chen et al., 2018).

On DA

Future studies should aim at investigating the potential regulation of dopaminergic neurons by products of tachykinin genes, especially in teleosts, amphibians and seasonal mammals for which puberty and/or adult sexual maturation is controlled by dopamine, in addition to GnRH. Indeed, many studies in mammals reported interactions between tachykinin peptides and DA at the hypothalamic level. For example, it was suggested that tachykinins could exert an inhibitory influence on PRL release by stimulating DA release at the hypothalamic level (for review: [START_REF] Debeljuk | Tachykinins and the control of prolactin secretion[END_REF]; thus it could also the case for gonadotropin release and expression.

At the pituitary level

A direct action of tachykinins at the pituitary level is possible as nkr/tacr expression has been detected in pituitary cells/gonadotropes in mammals (rat: Larsen et al., 1992;sheep: Dupre et al., 2010) and teleosts (zebrafish: [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF]tilapia: Biran et al., 2014a;carp: Hu et al., 2017).

Moreover, SP/NKA/NKB fibres have been reported surrounding hypophyseal blood capillary vessels in the median eminence in mammals (monkeys: [START_REF] Kalil | The Distribution of Substance P and Kisspeptin in the Mediobasal Hypothalamus of the Male Rhesus Monkey and a Comparison of Intravenous Administration of These Peptides to Release GnRH as Reflected by LH Secretion[END_REF].

Tac1 peptides

The first reported study on the in vitro effect of tachykinin on gonadotropins dates back to 1974. [START_REF] Fisher | Synthesis and some biological activities of substance P[END_REF] observed that SP induced release of LH and FSH from whole rat pituitaries cultured in vitro. This preliminary data using a few pituitaries and high dose of SP was followed by a contradictory one, that reported no effect of SP on LH and FSH release by hemipituitaries of OVX rats [START_REF] Vijayan | In vivo and in vitro effects of substance P and neurotensin on gonadotropin and prolactin release[END_REF]. Using a double-chamber (hypothalamus-pituitary) perfusion system, Ohtsuka and collaborators (1987) were able to show that SP stimulated the in vitro release of LH by rat pituitary; this effect was significant only in the presence of hypothalamus, as well as estrogen, and abolished when SP antagonist was also added [START_REF] Ohtsuka | Substance P stimulates gonadotropin-releasing hormone release from rat hypothalamus in vitro with involvement of oestrogen[END_REF]. Kalra and collaborators communicate unpublished data in intact male rats showing that NPK and NPγ significantly stimulated LH release in vitro from hemipituitaries [START_REF] Kalra | Diverse effects of tachykinins on luteinizing hormone release in male rats: mechanism of action[END_REF] for review: [START_REF] Kalra | Neuropeptide K (NPK) suppresses copulatory behavior in male rats[END_REF]. In cultured porcine gonadotropes, SP was reported to stimulate LH release without affecting intracellular LH

Tac3 peptide (NKB)

To the best of our knowledge, only one in vitro study has investigated the potential direct effect of NKB on gonadotropin, by using a gonadotroph cell line [START_REF] Mijiddorj | Role of Neurokinin B and Dynorphin A in pituitary gonadotroph and somatolactotroph cell lines[END_REF]. The authors reported no effect of NKB on lhβ and fshβ mRNA expression, even if NKB receptor was detected in this cell line.

In teleosts, most of in vivo and in vitro (primary cell culture of pituitary cells) data showed increase of gonadotropin release and expression after treatment with TAC3 peptides (in vivo, zebrafish: [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF]in vivo and in vitro, tilapia: Biran et al., 2014;in vivo, goldfish: Qi et al., 2015;in vitro, striped bass: Zmora et al., 2017;in vivo, orange-spotted grouper: Chen et al., 2018).

However, some studies reported either no effect (in vitro, grass carp: Hu et al., 2014a;in vivo and in vitro, tilapia: Jin et al., 2016;in vitro, orange-spotted grouper: Chen et al., 2018) or inhibitory effect (in vitro, tilapia: [START_REF] Jin | Neurokinin B-related Peptide Suppresses the Expression of GnRH I , Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus[END_REF] of TAC3 peptides on gonadotropins. The effect of the TAC3 peptides varies from species to species, between sexes and during development. For example, zebrafish TAC3a and TAC3RPa induce only LH release when injected to mature females [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF].

Similarly, when injected to goldfish female in mid-vitellogenesis and male in late-spermatogenesis, all three TAC3 peptides (TAC3a, TAC3RPa and TAC3RPb) increase expression of lhβ but also fshβ in the pituitary and GnRH in the brain (Qi et al., 2015b). In tilapia, when applied in vitro to mature female pituitary cells, the two TAC3 (TAC3 and TAC3RP) induce higher increase of FSH and LH release than TAC3RP. Intra peritoneal injections of TAC3 to mature females also increases both FSH and LH plasma levels, while TAC3RP induces only LH release (Biran et al., 2014a). However, if the TAC peptides are applied in vitro to juvenile mixed sexes pituitary cells the effect is inhibitory towards expression of fshβ and lhβ subunits [START_REF] Jin | Neurokinin B-related Peptide Suppresses the Expression of GnRH I , Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus[END_REF]. In the same study, TAC3RP injected to mature male inhibits the expression of the next gonadotropic genes: gnrh-rI, kiss2 and the same tac3 gene. TACR3 have been found in LH pituitary cells in tilapia (Biran et al., 2014a) and is likely responsible for direct regulation of lhβ expression by endogenous tilapia TAC3 peptides.

At the peripheral level 3.3.3.1. Uterus

Genes encoding tachykinin peptides (tac1, tac3 and tac4) and receptors (tacr1, tacr2 and tacr3) were all expressed in the uterus of mouse [START_REF] Cintado | Increase in neurokinin B expression and in tachykinin NK(3) receptor-mediated response and expression in the rat uterus with age[END_REF][START_REF] Pintado | A Role for Tachykinins in Female Mouse and Rat Reproductive Function1[END_REF][START_REF] Patak | Functional and Molecular Characterization of Tachykinins and Tachykinin Receptors in the Mouse Uterus1[END_REF], rat (tac3: [START_REF] Pinto | Expression of preprotachykinin-B, the gene that encodes neurokinin B, in the rat uterus[END_REF] and human [START_REF] Patak | Tachykinins and tachykinin receptors in human uterus[END_REF][START_REF] Pinto | mRNA expression of tachykinins and tachykinin receptors in different human tissues[END_REF], and their expression changed during the estrous cycle and during pregnancy (mouse: [START_REF] Patak | Functional and Molecular Characterization of Tachykinins and Tachykinin Receptors in the Mouse Uterus1[END_REF]rat: Hamlin et al., 2000;[START_REF] Candenas | Changes in the expression of tachykinin receptors in the rat uterus during the course of pregnancy[END_REF].

SP, NKA and NKB, when applied to isolated myometrium from non-pregnant women, produced contractions, while TACR2 receptor-selective antagonist abolished the uterotonic effect of NKA agonist [START_REF] Patak | Tachykinins and tachykinin receptors in human uterus[END_REF]. Tachykinins (SP, NKA and NKB) produce a direct contractile effect on uterine smooth muscle in mouse [START_REF] Patak | Functional characterization of tachykinin NK 1 receptors in the mouse uterus[END_REF][START_REF] Pennefather | Tachykinins and tachykinin receptors: a growing family[END_REF], rat: [START_REF] Fisher | Potencies of agonists acting at tachykinin receptors in the oestrogen-primed rat uterus: effects of peptidase inhibitors[END_REF][START_REF] Candenas | Changes in the expression of tachykinin receptors in the rat uterus during the course of pregnancy[END_REF][START_REF] Cintado | Increase in neurokinin B expression and in tachykinin NK(3) receptor-mediated response and expression in the rat uterus with age[END_REF], and human (Patak, 2000b;[START_REF] Patak | Tachykinins and tachykinin receptors in human uterus[END_REF]. Human HK-1 is also a uterine stimulant in the human [START_REF] Pennefather | Regulation of the Stimulant Actions of Neurokinin A and Human Hemokinin-1 on the Human Uterus: A Comparison with Histamine1[END_REF]. TACR2

receptor is mediating tachykinin actions on the myometrium of human (Patak and Story, 2000a;[START_REF] Patak | Tachykinins and tachykinin receptors in human uterus[END_REF][START_REF] Pennefather | Regulation of the Stimulant Actions of Neurokinin A and Human Hemokinin-1 on the Human Uterus: A Comparison with Histamine1[END_REF], rat [START_REF] Moodley | NK2 receptors mediate tachykinin-induced contractions of rat uterus during the oestrous cycle[END_REF].

Placenta

In the placenta of pre-eclampsia women, elevated circulating NKB, as well as increased tac3 expression, are reported as compared to placenta of normal pregnant women [START_REF] Page | Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia[END_REF][START_REF] Page | Gene regulation of neurokinin B and its receptor NK3 in late pregnancy and pre-eclampsia[END_REF][START_REF] Page | Neurokinin B and pre-eclampsia: A decade of discovery[END_REF].

Gonads and gametes

Expression of tac1 and tac3 was detected in the mammalian ovary, oocytes and granulosa cells [START_REF] Pintado | A Role for Tachykinins in Female Mouse and Rat Reproductive Function1[END_REF][START_REF] Debeljuk | Tachykinins and the control of prolactin secretion[END_REF]. Isolated cumulus granulosa cells expressed tac1, tac3 and tac4 [START_REF] Pintado | A Role for Tachykinins in Female Mouse and Rat Reproductive Function1[END_REF]. Tachykinins control ovary steroid secretion and may have an ancient role in stimulating oocyte growth as in Ciona intestinalis Ci-TACRP enhanced oocyte growth from the vitellogenic stage to the post-vitellogenic stage [START_REF] Aoyama | A novel biological role of tachykinins as an up-regulator of oocyte growth: Identification of an evolutionary origin of tachykininergic functions in the ovary of the ascidian, Ciona intestinalis[END_REF].

In the male reproductive system (for review: [START_REF] Debeljuk | Tachykinins and their possible modulatory role on testicular function: A review[END_REF], tac1, tac3 and tac4 genes are expressed in the human sperm. Tachykinins enhance the sperm motility by TACR1 and TACR2 dependent mechanisms [START_REF] Pinto | Autocrine regulation of human sperm motility by tachykinins[END_REF]. Tachykinins also potentiate contractility dependent mechanisms [START_REF] Pinto | Autocrine regulation of human sperm motility by tachykinins[END_REF] of the vas deferens and of seminal vesicles mediated by TAC1RP (SP) [START_REF] Candenas | Tachykinins and tachykinin receptors: effects in the genitourinary tract[END_REF]. TAC1RP (SP) and TAC1 (NKA) are present in prostate of Guinea pig and rat in low levels, abundant in dog and absent in humans [START_REF] Candenas | Tachykinins and tachykinin receptors: effects in the genitourinary tract[END_REF], but tac1, tac3 and tac4

have been detected in human prostate.

PhD objectives

The aim of this PhD is to investigate the presence and the role of brain neuropeptides; such as neurokinin B encoded by tac3 gene, in the control of reproduction in an endangered species, the European eel, Anguilla anguilla.

The eel presents a unique life cycle with a pre-pubertal blockade of sexual maturation as long as the reproductive oceanic migration does not take place. Raising basic knowledge on eel reproductive neuroendocrinology contributes to decipher the regulatory mechanisms of its complex life cycle.

Furthermore, due to its basal phylogenetic position among teleosts, as a representative of Elopomorphs, the eel is also a key-species for addressing molecular and functional evolution of endocrine systems.

The objectives of this PhD are:

1. To identify in silico tac3 gene(s) in Anguilla species and predict the sequence and 3D structure of encoded peptides.

2. To analyse the tissue distribution of the tac3 paralog transcripts in the European eel.

3. To synthesize eel predicted TAC3 peptides and test their biological activity on the expression of pituitary hormones by primary culture of eel pituitary cells.

4. To retrace the evolutionary scenario of tac3 genes in vertebrate and specially teleosts, by phylogeny and synteny analyses, with a special focus on the impact of whole genome duplication events.

5. To extend in silico identification and evolutionary scenario to the other tac genes (tac1 and tac4)

JTT substitution matrix. The TAC3 pre-pro-peptide sequence from the elasmobranch elephant shark (Callorhinchus milii) was chosen as outgroup. The resulting phylogenetic tree was displayed using Figtree v1.4.3. Nodes were collapsed for bootstrapping values below 50% using Mesquite v2.1.

Phylogeny analyses were also performed on some neighbouring genes of tac3 genomic region : b4galnt1b and c1galt1b.

Same procedure applied to phylogenetic analysis of TAC1 and TAC4 pre-pro-peptide sequences, where salmonid species were included.

Synteny

Tac3 genomic region of a non-teleost actinopterygian, spotted gar, (Lepisosteus oculatus) was chosen as reference, using Genomicus PhyloView v91.01

(http://www.genomicus.biologie.ens.fr/genomicus-91.01/cgi-bin/search.pl). The genomic regions of eel tac3a and tac3b were manually analysed with CLC DNA Workbench 6 software (Qiagen Bioinformatics) on de novo assembled European eel draft genome (Henkel et al., 2012b;[START_REF] Jansen | Rapid de novo assembly of the European eel genome from nanopore sequencing reads[END_REF]. The draft genomes of the Japanese (Henkel et al., 2012a) and American [START_REF] Pavey | Draft genome of the American Eel (Anguilla rostrata)[END_REF] eels were also used.

Tissue distribution

Tissues from ten female silver eels were collected to study the expression of eel tac3 genes. They were stored in RNA later at 4°C (24h), then -20ºC, until extraction. The sampled tissues were brain, pituitary, gill, eye, intestine, liver, spleen, and ovary. The brain was dissected in olfactory bulbs, telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. Tissue RNA extraction was performed as described in the Cloning section. 500 ng of total RNA were reversetranscribed using SuperScript III First Strand cDNA Synthesis Kit. Samples were then stored at -20ºC until qPCR.

Prediction and synthesis of eel TAC3 peptides

SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) was used for prediction of signal peptide (Petersen et al., 2011), and Neuropred (http://stagbeetle.animal.uiuc.edu/cgi-bin/neuropred.py) for cleavage and amidation sites (Southey et al., 2006).

Predicted eel TAC3 peptides [NKBa (10 aa), NKBRPa (13 aa), NKBb (10 aa) and NKBRPb (13aa)] were synthesized (0.1-mmol scale) by the solid-phase methodology on a Rink amide 4methylbenzhydrylamine resin (Biochem, Meudon, France) using a 433A peptide synthesizer (Applied Biosystems, Courtaboeuf, France) and the standard procedure, as previously described (30). The synthetic peptides were purified by reversed-phase (RP) HPLC on a 2.2 cm × 25 cm Vydac 218TP1022

C 18 column (Alltech, Templemars, France), using a linear gradient (20-40% over 60 min) of acetonitrile/TFA (99.9:0.1, v/v) in water, at a flow rate of 10 ml/min. Analytical RP-HPLC, performed on a 0.46 cm × 25 cm Vydac 218TP54 C 18 column, showed that the purity of the peptide was greater than 99%. The molecular mass of the peptide was verified by mass spectrometry on a MALDI-TOF Voyager DE-PRO instrument (Applied Biosystems).

Prediction of the three-dimensional peptide structure of eel TAC3 peptides

Secondary protein structures of eel TAC3 peptides were modelled using the I-TASSER server, an automated protein-modelling server (Yang et al., 2014). Only models with the C-score between 2 and -5 were considered. The visualization of the predicted three-dimensional structures was performed using the RasWin Molecular Graphics software v. 2.7.5.2 (http://www.rasmol.org/). 9 Pituitary cell culture

Dispersion and culture

Dispersion and primary culture of pituitary cells, using 30-40 female eel pituitaries per cell culture experiment, were performed as described in (Montero et al., 1996) and as recently used for the test of eel kisspeptins [START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF]. Cultures were performed in serum-free culture medium (M199 with Earle's salt, sodium bicarbonate, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml fungizone (Gibco, Illkirch, France) at 18 ºC under 3% CO2 and saturated humidity.

Treatments

Human NKB (Sigma) and eel TAC3 peptides stock solutions (10 -4 M) were prepared in NaOH 0.1M and stored at -20ºC. Stock solutions were diluted in the culture medium just before the addition to the culture wells. The experiment started at day 0 (24h after cell plating) and five wells (62,500 cells/well) for control and each treatment were used as replicates. Culture medium was changed and peptide treatment solution was added again on days 4 and 7. Culture was stopped at day 10. The effects of the treatment were tested on independent experiments performed on cells from different batches of fish and figures display the results of representative experiments.

RNA extraction and cDNA synthesis

Total RNA was directly extracted as previously described [START_REF] Pasquier | First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla[END_REF][START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF]. Briefly, cells were washed with sterile PBS (Gibco, Illkirch, France) and lysed with Cell-to-cDNA ™ II Cell Lysis II Buffer (Ambion; 80µl/well). The lysates were digested with RNase-free DNase I (Roche). Eight microliters of RNA solution of each sample were then reversed transcribed with a SuperScript III First Strand cDNA Synthesis Kit (Invitrogen) and stored at -80ºC. The cDNA samples obtained were stored at -20ºC until qPCR.

10 Quantitative real-time PCR (qPCR)

Primers

Primers for eel tac3a and tac3b were designed based on sequences of European eel TAC3 propeptides using Primer 3 (Table S1) and purchased at Eurofins. Amplicon sizes were 140 bp for tac3a and 190 bp for tac3b. Forward and reverse primers of each couple were located in different exons to prevent amplification of genomic DNA. To assess the specificity of the qPCR primers, each couple was tested for its inability to amplify the transcript of the other tac3 gene.

The housekeeping gene was -actin as previously reported [START_REF] Aroua | Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids[END_REF][START_REF] Pasquier | First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla[END_REF]. Primers for eel lhβ, fshβ, β subunit of thyroid-stimulating hormone (tshβ), common α subunit of glycoprotein (gpα), type 2 GnRH receptor (gnrh-r2) and growth hormone (gh) have already been described (Table S1). European eel possesses three GnRH receptors (GnRH-R1a, GnRH-R1b and GnRH-R2) [START_REF] Peñaranda | Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development[END_REF], but gnrh-r1a and gnrh-r1b expression was below the limit of detection in cultures of pituitary cells [START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF], and thus were not assayed in this study.

SYBR green assay

Quantitative PCR assays were performed using the LightCycler® System (Roche) with SYBR Green I sequence-unspecific detection as previously described [START_REF] Pasquier | First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla[END_REF][START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF]. Briefly, the qPCRs were prepared with 2µl of RNase-free water (Ambion, Texas, USA), 2µl of SYBR Green master mix (Roche), 1µl of each forward and reverse primer (500nM final concentration) and 4µl of diluted cDNA template. The protocol was: an initial step of polymerase activation for 10 min at 95ºC; then 41 cycles ( -actin, gh, gp , lhβ, fshβ and tshβ) or 45 cycles (tac3a and tac3b) of 10 s at 95ºC for denaturing, 5 s at 60ºC for annealing, 10 s at 72ºC for primer extension and a single final extension step of 5 min at 72ºC. For gnrh-r2, the protocol was an initial step of polymerase activation for 10 min at 95ºC; 42 cycles of 10 s at 95ºC, 7 s at 61ºC, 4 s at 72ºC and a single final extension step of 5 min at 72ºC. Each program ended with a melting curve analysis by slowly increasing the temperature (0.01°C/s) from 68 to 95°C with a continuous registration of changes in fluorescent emission intensity. Serial dilutions of cDNA pool of brain (tissue distribution) or pituitary cells (cell culture)

were used as a standard curve. One chosen dilution was also included in each run as a calibrator.

Normalization of data was performed using total RNA levels (tissue distribution) and -actin mRNA level (cell culture experiments).

Statistical analysis

Data are presented as the mean ± SEM. Mean values were compared by Student's t-test or oneway ANOVA followed by Tukey's multiple comparison test, using Instat (GraphPad Software Inc., San Diego, CA, USA). Differences between groups with P<0.05 were considered statistically significant.

Normality was tested with Wilk.-Shaphiro test using R.

Introduction to article 1

The first gene to be characterized in the eel genome was the tachykinin 3 because of its reproductive role in mammals and teleosts. Without regard to the presence of the tac3 gene and diverse TAC3 peptides in other teleost, its presence in the eel was unknown. Therefore, a first in silico search for the tachykinin 3 gene in the eel genome was the beginning of this research.

In a practical point of view, the possible role of the tachykinin peptides in the sexual maturation of the eel was still unexplored. Although the effect of the tachykinin peptides in the regulation of gonadotropin release was proven already for mammals and teleosts, there was risk of no reproductory role in the eel.

Thus, the objective of this first study was to reveal the existence of tachykinin genes and peptides in the eel that could take part in the regulation of the sexual maturation. Thanks to the eel genome and bioinformatic tools, the tac3 gene system was characterised in three eel species. These genes encoded TAC3 peptides that were synthesized and tested in vitro in pituitary cells with the objective of discovering any possible role on gonadotropin release. Our qPCR analysis revealed the dual inhibition of lhβ and gnrh-rII genes in the pituitary cells of silver pre-pubertal eel.

To that end, this chapter is focused on the first characterisation of the tachykinin genes and peptides in the European eel and their implication in the sexual maturation of the pre-pubertal eel.

2 Article 1: Tachykinin-3 genes and peptides characterized in a basal teleost, the European eel: evolutionary perspective and pituitary role

IV Article 2 1 Introduction to article 2

The bioinformatic analysis of the eel species and other teleosts for the search of the tachykinin 3

genes revealed an impact of the teleost whole genome duplication or 3R in the teleost tachykinins. In this scenario, we decided to widen the perspective of the tachykinin genes and peptides in teleost.

Our in silico analysis discovered that the tachykinin genes tac1 and tac4 in the eel species, Anguilla anguilla, have been affected by the whole teleost duplication. Together with the tac3 gene, the European eel has conserved up to 6 tachykinin genes. Each of these genes encodes two peptides with preserved cleavage and amidation sites, hence likely active peptides. Consequently, the eel has potentially 12 tachykinin peptides with hidden pharmacologic potential.

These genes have also been duplicated and preserved in most of the studied teleosts, likely after the teleost whole genome duplication. We could report only independent losses and most of the clades preserve also the two encoded peptides in each gene.

To better understanding the impact of genome duplications in the tachykinin system, we analysed the duplications occurred after the salmonids whole genome duplication event, or 4R. Our results indicate that the tachykinin peptides in salmonids were also preserved in duplicate after their specific genome duplication and a total of 24 tachykini peptides were found in most of the salmonid species studied.

In conclusion, the impact of the teleost whole genome duplication in the tachykinin system extended the possibilities for tachykinin peptides.

2 Article 2: Tachykinin-1 and 4 genes and peptides in a basal teleost,

Anguilla anguilla: Identification and phylogeny analysis

INTRODUCTION

Tachykinins (TK or TAC) are members of a large family of peptides, mainly produced by brain and gut in mammals. Their C-terminal end was characterized by a motif of five residues (FxGLM) and an α-amidation. The most known tachykinin peptides are neurokinin A (NKA), substance P (SP) and neurokinin B (NKB). While the tac1 gene encodes NKA and SP, NKB is coded by the tac3 gene (also named tac2 in rodents) (for reviews: Severini et al., 2002;[START_REF] Pennefather | Tachykinins and tachykinin receptors: a growing family[END_REF]Hu et al., 2014b). In 2000, a third gene, tac4 gene was identified in mammals and shown to encode other tachykinins: hemokinin-1 (HK-1) and endokinins (EKs) (for reviews: [START_REF] Patacchini | Newly discovered tachykinins raise new questions about their peripheral roles and the tachykinin nomenclature[END_REF][START_REF] Page | Differential mRNA splicing and precursor processing of neurokinin B in neuroendocrine tissues[END_REF]. Tac 1, 3 and 4 paralogous genes are likely resulting from the whole genome duplication events that occurred in ancestral vertebrates (1R and 2R, for "first and second rounds on whole genome duplication"), while the fourth paralog would have been lost early after the 2R.

We recently characterized in the European eel two tac3 paralogous genes (tac3a and tac3b)

resulting from the additional, teleost-specific, whole genome duplication (TSWGD or 3R for "third round of whole genome duplication"). Each paralog encodes two peptides, thus leading to four TAC3 peptides in the eel: TAC3a (or NKBa) with a sequence identical to that of human NKB, TAC3b (or NKBb), TAC3-RPa (or NKB-RPa) and TAC3-RPb (or NKB-RPb) [START_REF] Campo | Tachykinin-3 genes and peptides characterized in a basal teleost, the European eel: evolutionary perspective and pituitary role[END_REF]. Few studies addressed the other tachykinins in teleosts. Zhou et al (2012) reported the presence of two tac4 genes (tac4a and b) in zebrafish and medaka, but the presence of a single tac1 gene in these species.

The present study aimed at identifying European eel tac1 and tac4 genes by in silico data mining, and at predicting the sequences of the respective encoded peptides. We also searched for tac1

and tac4 genes in various other species and performed phylogeny analyses of tac1 and tac4 with a special focus on teleosts.

Phylogeny analysis

Chondrichthyan, sarcopterygian, and actinopterygian TAC1 or TAC4 pre-pro-peptide aminoacid sequences were aligned using CLUSTAL included in SeaView v. 4.6. Alignments were manually adjusted for optimization of key regions such as cleavage sites and signal peptide.

Phylogeny analyses of the TAC1 or TAC4 alignments were achieved using a maximum likelihood method, RAxML black-box (https://www.phylo.org/, with 1,000 bootstrap replicates and JTT substitution matrix. The TAC1 or TAC4 pre-propeptide sequences from the elasmobranch elephant shark (Callorhinchus milii) were chosen as outgroup, respectively. The resulting phylogenetic trees were displayed using Figtree v1.4.3. Nodes were collapsed for bootstrapping values below 30% using Mesquite v2.1.

RESULTS

Identification of European eel tac1 and tac4 genes and encoded peptides

Two tac1 genes and two tac4 genes were identified in the European eel genome. Predicted CDS and corresponding pre-pro-peptide sequences are shown in Figures 1 and2.

Each TAC1 and TAC4 pre-pro-peptide sequence encoded two putative peptides, TAC and TAC-Related Peptide (TAC-RP) (Figures 1 and2). These peptides were delimited by cleavage sequences at both ends. The C-terminal end of each peptide sequence showed a glycine before the cleavage site, which indicates an amidation site.

Predicted sequences of European eel peptides TAC1a, TAC1-RPa, TAC1b, TAC1-RPb, TAC4a, TAC4-RPa, TAC4b, TAC4-RPb are given in Table 1, as well as the corresponding human tachykinin peptides, TAC1 (also called Neurokinin A, NKA), TAC1-RP (also called Substance, SP), TAC4 (also called Endokinin, EKC/D), TAC4-RP (also called Hemokinin, HK, and Endokinin EKA/B). The eight eel predicted peptides are identical in the three eel species studied, European eel, Anguilla anguilla, American eel, Anguilla rostrata, Japanese eel, Anguilla japonica, and represent novel peptide sequences (Table 1; Figure S1 andS2).

Phylogeny analysis of TAC1

Based on an alignment of 90 TAC1 pre-propeptide amino-acid sequences (Figure S1), and assuming the elephant shark Callorhinchus milii sequence as outgroup, a phylogenetic tree was generated using the Maximum Likelihood method (Figure 3).

As shown in Figure 3, the TAC1 pre-propeptide sequences of osteichtyans clustered into two well-supported clades: sarcopterygians and actinopterygians. The coelacanth TAC1 pre-propeptide sequence branched at the base of the sarcopterygian clade, in agreement with its phylogenetic position.

Similarly, the TAC1 pre-propeptide sequence of the spotted gar Lepisosteus oculatus, a holostean species, branched at the base of the actinopterygian TAC1 clade.

In most teleosts, including zebrafish and medaka, two tac1 genes (a and b) were found. One clade grouped all teleosts TAC1a pre-propetide sequences, including the TAC1 previously reported in zebrafish Danio rerio and some other teleosts (Zhou et al., 2012) RP). The sequence of eel TAC1 peptides, TAC1a and b, TAC1-RPa and b, differed from human NKA and SP are not common to any other studied species. Therefore, they represent novel sequences.

To our knowledge, this is the first demonstration of two tac1 genes in teleosts, since the previous study by Zhou et al. (2012) reported the presence of a single tac1 gene in the studied teleosts, namely zebrafish Danio rerio, medaka Oryzias latipes, Atlantic salmon Salmo salar and rainbow smelt Osmerus mordax.

Based on our finding of two tac1 genes in a basal teleost, the eel, we further searched and retrieved duplicated tac1 genes in many other teleost species (Figure 3 and Figure S1) including the above mentioned, differently from the previous report (Zhou et al., 2012). However, we identified only a single tac1 gene (tac1a) in the available genomes of some other teleosts, fugu and stickleback.

Origin and fate of duplicated tac1a and b paralogs in teleosts

In order to investigate the origin and relationships of the teleost tac1a and b paralogs, we performed a phylogenetic analysis of TAC1 pre-propeptide sequences. A single tac1 gene was identified in chondrichtian as well as in sarcopterygian species such as coelacanth or human. We also retrieved a single tac1 gene in the non-teleost actinopterygian, a holostean species, the spotted gar. In contrast, we found that two tac1 genes are present in the eel as well as in most other teleost species investigated. This suggests that the duplicated tac1 paralogs in teleosts may result from the teleostspecific whole genome duplication (TSWGD or 3R for "Third Whole genome duplication").

Our phylogeny analysis supported this hypothesis. The spotted gar TAC1 pre-propeptide sequence branched at the base of all TAC1a and TAC1b teleost sequences, indicating a common actinopterygian ancestral tac1 gene for teleost duplicated tac1a and tac1b genes. Furthermore, teleost sequences split into two clades, one clade encompassing all TAC1a pre-propeptide sequences and the other one all TAC1b pre-propeptide sequences, with the exception of osteoglossomorph TAC1b that were basal to all teleost sequences. Preliminary synteny analysis on some available teleost genomes (zebrafish, medaka) (data not shown) indicated that tac1a and tac1b genomic regions correspond to duplicated paralogons, supporting that the duplicated tac1a and tac1b genes are issued from the 3R

and not from a local specific gene duplication. Synteny analysis of osteoglossomorph tac3a and tac3b genomic regions and comparison with the other teleosts would be required to confirm the classification of the paralogs in this group.

Duplicated tac1a and tac1b have been conserved throughout teleost radiation. Phylogeny analysis showed long branches for TAC1b pre-propeptide sequences in clupeocephala, suggesting larger sequence divergence of tac1b, as compared to tac1a. Furthermore, tac1b paralog would have been lost in some species, such as the stickleback and fugu, in the genomes of which we could identify only a single tac1 gene. We classified their single gene as tac1a, according to the phylogeny analysis.

This suggests independent and recurrent losses of tac1b paralog in some teleosts subgroups, gasterosteiforms and tetraodontiforms. Synteny analysis would allow us to further assess the loss of tac1b in these species.

Concerning the additional tac1 genes identified in salmonids (up to four tac1 genes), phylogeny analysis supports that they come from the additional whole genome duplication specific to the salmonid lineage (4R).

Two tac4 genes and four TAC4 peptides in the eel

We identified two tac4 genes in the European eel, as well as in the American eel and Japanese eel. Each tac4 gene encodes two predicted peptides. This organization of the TAC4 precursor coding for two peptides TAC and TAC-RP is a conserved feature as seen for human TAC4 pre-propeptide encompassing Endokinin (EK) (TAC4) and Hemokinin (HK) (TAC4-RP). The sequence of eel TAC4 peptides, TAC4a and b, TAC4-RPa and b are not common to any other studied TAC4 peptide and represent novel sequences.

The finding of two tac4 genes in the eel is in agreement with the previous study by Zhou et al. (2012) who reported the presence of duplicated tac4a and b genes in zebrafish and medaka. We identified two tac4 genes in most teleost species investigated, with the exception of osteoglossomorphs in which we retrieved only a single tac4 gene (Figure 4 and Figure S2).

Origin and fate of duplicated tac4a and b paralogs in teleosts

In order to trace the origin and relationships of the teleost tac4a and b paralogs, we performed a phylogeny analysis of TAC4 pre-propeptide sequences. A single tac4 gene was identified in chondrichthyan as well as in sarcopterygian species such as coelacanth or human. We also found a single tac4 gene in the non-teleost actinopterygian, the spotted gar. In contrast, we found two tac4 genes in the eel as well as in most other teleost species investigated. This suggests that duplicated tac4a and b paralogs may result from teleost 3R.

As described above for TAC1, phylogeny analysis of TAC4 pre-propeptide sequences showed that the spotted gar TAC4 branched at the base of all TAC4a and TAC4b teleost sequences, indicating a common actinopterygian ancestral tac4 gene for teleost duplicated tac4a and tac4b genes.

Furthermore, teleost sequences split into two clades, one clade encompassing all TAC4a prepropeptide sequences and the other one all TAC4b pre-propeptide sequences. A previous synteny analysis of zebrafish tac4a and b genomic regions supports the 3R origin of teleost tac4a and b (Zhou et al., 2012).

Duplicated tac4a and tac4b have been conserved throughout teleost radiation, with the possible exception of osteoglossomorphs, in which we identified only a single tac4 gene. Phylogeny analysis showed a basal position of this single tac4 in the tac4b clade. Synteny analysis would allow us to further assess the loss of tac4a paralog in osteoglossomorphs. In contrast, as for tac1, we found up to four tac4 genes in salmonids, likely resulting from 4R.

Full complement of tachykinin genes and peptides in the eel

In conclusion, we identified in a basal teleost, the European eel, two tac1 genes encoding four TAC1 peptides, and two tac4 genes encoding four TAC4 peptides. Our previous study also demonstrated the presence in the eel of two tac3 genes, encoding four TAC3 peptides, and resulting from teleost 3R as shown by phylogeny and synteny analyses [START_REF] Campo | Tachykinin-3 genes and peptides characterized in a basal teleost, the European eel: evolutionary perspective and pituitary role[END_REF]. Altogether these results indicate that the eel has conserved all 3R-duplicated tac paralogs, leading to six tac genes and twelve peptides, as compared to three genes and five peptides in human. This provides novel peptide sequences for analyses of tachykinin structure activity relationships. As previously reported for TAC3

peptides [START_REF] Campo | Tachykinin-3 genes and peptides characterized in a basal teleost, the European eel: evolutionary perspective and pituitary role[END_REF], future studies will aim at synthesizing eel TAC1 and TAC4 predicted peptides, and testing their biological activity in the eel.

As for tachykinin paralogs, we previously reported the remarkable conservation of duplicated paralogs issued from 1R/2R or 3R in the eel as compared to other teleost model species: for instance hox genes (Henkel et al., 2012b); kisspeptins and receptors [START_REF] Pasquier | Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family[END_REF], leptin and receptors [START_REF] Morini | Duplicated leptin receptors in two species of eel bring new insights into the evolution of the leptin system in vertebrates[END_REF] and steroid receptors [START_REF] Lafont | Three nuclear and two membrane estrogen receptors in basal teleosts, Anguilla sp.: Identification, evolutionary history and differential expression regulation[END_REF]. The present results on tachykinins comfort the key position of the eel for tracing the evolutionary history of endocrine systems in teleosts. We are currently extending our investigation to the tachykinin receptors and our preliminary results indicate the presence in the eel of all 3R-duplicated paralogs of tachykinin receptors, leading to six receptors as compared to three in human The teleost TAC4a and TAC4b sequences are coloured in orange and green, respectively.

FIGURES :

Figure 1 :

Chapter V

General discussion

V General Discussion 1 Multiple tac genes and peptides in the European eel

Our studies have found six tachykinin genes in the European eel, two tac1 genes, two tac3 genes and two tac4 genes. These genes are duplicated when compared with the three genes found in the spotted gar and in human. Our synteny studies revealed that the whole paralogon genomic region including the tac3 gene has been duplicated in teleosts when compared to spotted gar. This duplication was most likely generated in the whole genome teleost duplication or 3R event reported previously [START_REF] Meyer | From 2R to 3R: Evidence for a fish-specific genome duplication (FSGD)[END_REF][START_REF] Volff | Genome evolution and biodiversity in teleost fish[END_REF][START_REF] Braasch | Polyploidy in Fish and the Teleost Genome Duplication[END_REF]. Further synteny analysis developed on the tac1 and tac4 paralogons will assess the impact of the 3R on the duplication of these two genes.

Each of the tachykinin genes in the eel encoded two peptides, what makes a total of 12 tachykinin peptides. Four of those peptides belong to the tac1 genes, four are encoded in the tac3 genes and four are present in the tac4 genes. However, not all human tachykinin genes encode two peptides. The human gene tac3 encodes for a single tachykinin peptide, the neurokinin B (TAC3). The aminoacid sequence of this single TAC3 peptide is equal to all other studied sarcopterygian except the frog. It is also identical to the TAC3 peptide from the actinopterygian spotted gar and to the TAC3a peptide found in eel. Therefore, we can assume that the eel TAC3a peptide encoded in the tac3a gene is generally present in the sarcopterygian and ancestral actinopterygian species.

Interestingly, the other eel TAC3 peptides are different and unique in the Anguilla genus. The Cterminal ends conserve a full tachykinin formula, which indicates their capacity to activate the tachykinin receptors. On the other hand, the aminoacid sequences of the eel TAC3 peptides are divergent for the N-terminal end and have different lengths. While TAC3a and TAC3b are decapeptides, both TAC3RP peptides are made of 14 aminoacids. In addition, these related peptides present a tyrosine (Y) in the N-terminal end. This feature is conserved in most of the TAC3RP peptides from the studied teleost species, the spotted gar, the coelacanth and in the elasmobranchs elephant shark (Callorhinchus milii), the small-spotted cat shark (Scyliorhinus canicula) and the little skate (Leucoraja erinacea). This conserved pattern in the N-terminal end indicates a possible role in the function of the tachykinins. In the human TAC1RP or substance-P, specific aminoacids indicating proteolytic sites are found in the N-terminal end [START_REF] Regoli | Receptors Antagonists for Substance P and Related Peptides *[END_REF]). These sites are different from those of the TAC1 peptide. Both peptides are encoded in the same gene and can be co-expressed.

The differences in the aminoacid composition would induce different peptide degradation methods, which in turn produce different lifetime between the two peptides (for review: Severini et al., 2002).

This phenomenon has been described previously as a method for modulating the response of each tachykinin peptide when co-released with other tachykinins, as indicated by Maggi previously (2000).

Therefore, the conserved tyrosine in the N-terminal of TAC3RP peptides and not found in the TAC3 peptides can also be a modulatory system for the tachykinin response. Post-transcriptional modifications in the N-terminal end must be also studied for a possible differential response between peptides.

None of the peptides encoded in the eel tac1 and tac4 genes are equal to the human peptides.

TAC1RPb is a common peptide in the actinopterygian spotted gar and the basal teleosts, the eels and the arowanas. Those peptides are also in common with the TAC1RPa that is encoded in the tac1a gene of northern pike and the studied salmonids species. The eel TAC1b is also in common with the TAC1a from rainbow smelt, Osmerus mordax. These results indicate a convergence between genes tac1b from eel and tac1a from osmeriformes, esociformes and salmonids. This convergence needs to be verified by further synteny analysis of the tac1 genomic region.

While the peptides TAC4RPa and TAC4b are unique to the eel species, TAC4a and TAC4RPb are shared with other species. TAC4RPb is common to the eels and the basal teleosts bony tongue elephantfish (Paramormyrops kingsleyae) and pirarucu (Arapaima gigas). Therefore, eel TAC1RPb, and TAC4RPb have a conserved sequence.

Interestingly, TAC4a is conserved between most of the studied teleost species including the eel (Article 2, table1). Another teleost tachykinin, TAC3a peptide found in Atlantic herring with sequence EMHDIFVGLM-NH2 is also conserved by many of the clupeocephala species used in our analysis, but not in the eels or the arowanas (Article 1, Supplemental Figure 1). These findings indicate that the tachykinin peptides in teleosts have possibly developed a particular functioning for TAC3a and TAC4a peptides that has been conserved strictly among the wide teleost radiation. In contrast, for some species such as northern pike and zebrafish, the TAC3b peptide shows a substitution of the final previously for other tachykinin peptides (Severini et al., 2002). We can assume that the peptides TAC1a and TAC4RPa are divergent tachykinin-like peptides, exclusive to the Anguilla genus. These sequences must be verified by cloning.

The mid regions of the TAC1 and the TAC4 peptides have similar aminoacids marked with the presence of one or two glutamines (Q), a non-polar aminoacid (Article 2, Table 1). In contrast, TAC3 peptides are characteristic for including one or two aspartic acids (D) in this same mid regions (Article 1, Table 1). According to our modelling results, this aminoacid is also involved in the alpha-helix structure of the eel peptides and is placed also in a highly hydrophobic area of the peptide.

Differently from glutamine, the aspartic acid has a negative charge that may affect the charge of the whole TAC3 peptide. This charge may be a particular indicator for receptor recognition and affinity, which may explain the preference of the mammalian TACR3 receptor for TAC3 peptides when compared to the other tachykinin peptides [START_REF] Buck | The tachykinins: a family of peptides with a brood of "receptors[END_REF].

The new tachykinin peptides found in the eel open the possibility for different combinations of peptide-receptor. Differential selectivity of all tachykinin receptors has been reported for the human TACRP1 (substance P), TAC1 (Neurokinin A) and TAC3 (Neurokinin B) and TAC4RP (Hemokinin)

peptides [START_REF] Buck | The tachykinins: a family of peptides with a brood of "receptors[END_REF][START_REF] Morteau | Hemokinin 1 is a full agonist at the substance P receptor[END_REF]for review: Regoli et al., 1987;[START_REF] Quirion | Multiple neurokinin receptors: Recent developments[END_REF][START_REF] Maggi | The mammalian tachykinin receptors[END_REF][START_REF] Pennefather | Tachykinins and tachykinin receptors: a growing family[END_REF][START_REF] Borbély | Role of hemokinin-1 in health and disease[END_REF]. As suggested previously for kisspeptin and its receptors [START_REF] Pasquier | Molecular evolution of GPCRs: Kisspeptin/kisspeptin receptors[END_REF], the conservation of the receptor activation site in the C-terminal end of the peptide, may facilitate certain flexibility of peptide/receptor association in the tachykinin systems along the vertebrate radiation. Therefore, the peptides found in the eel and in the studied teleost species can enlarge the already wide pharmacological possibilities of the tachykinins.

Evolutionary history of the tachykinins

We found that the conservation of all the duplicated genes in the eel applies also to the Japanese and the American eel. Previous analysis of the hox cluster demonstrated already a wide conservation of the genes in the European eel genome (Henkel et al., 2012a), that was confirmed also for the Japanese eel (Henkel et al., 2012b). Other genes have been conserved in duplicate in the eel after the 3R: dopamine D2 receptors [START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the european eel: Molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF], GnRH-receptor type I [START_REF] Peñaranda | Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development[END_REF], β-subunit of thyroid-stimulating-hormone and its receptor [START_REF] Maugars | Multiple thyrotropin b-subunit and thyrotropin receptor-related genes arose during vertebrate evolution[END_REF], leptin [START_REF] Morini | Duplicated leptin receptors in two species of eel bring new insights into the evolution of the leptin system in vertebrates[END_REF], progestin receptor (Morini et al., 2017), intracellular nuclear receptor-2 and G-coupled membrane receptors for estrogen [START_REF] Lafont | Three nuclear and two membrane estrogen receptors in basal teleosts, Anguilla sp.: Identification, evolutionary history and differential expression regulation[END_REF]Morini et al., 2017). Therefore, there are evidences that the eel is a particular teleost with a considerable retention of the teleost duplicated genes and in particular the tachykinins.

Not all the studied teleosts have conserved the tachykinin genes. The basal teleosts from the Osteoglossomorpha clade have all lost one of the duplicates of the tac4 gene. Nevertheless, the tac4 gene has been preserved in duplicate in the genome of other key species, namely Atlantic herring (Clupeiforme), zebrafish (Cypriniformes), stickleback (Gasterosteiformes), medaka (Beloniformes), tilapia (Perciformes) and fugu (Tetraodontiformes). Additionally, the tac3b gene is not present in the Atlantic herring and the medaka, and the tac1b gene can't be found in the genome of stickleback nor in fugu. We can therefore assume that the tachykinin genes went through independent loses along the teleost radiation (Figure 9). However, our results indicate that many teleosts have conserved the duplicated tachykinin peptides after the 3R: northern pike, zebrafish and tilapia, among others. During our research we discovered a tac1b gene in zebrafish and a tac3b gene in tilapia that were not reported in previous studies (zebrafish : Zhou et al., 2012;tilapia: Biran et al., 2014). Hence, the multiple-gene loss for genome reshaping after the 3R [START_REF] Inoue | Rapid genome reshaping by multiple-gene loss after whole-genome duplication in teleost fish suggested by mathematical modeling[END_REF] didn't affect the tachykinin genes in teleosts.

According to the phylogeny studies, the pre-pro-tachykinin peptide that is encoded in the tac3b gene is divergent for the clade of neoteleosts, where tilapia belongs. Species within this group have highly divergent TAC3b peptides: no signs of the tachykinin motive, the cleavage sequence and/or the amidation site (fugu, stickleback, the mangrove killifish Kryptolebias marmoratus, the zebrambuna Maylandia zebra and the pundamilia P. nyererei). These species would have lost the possibility to cleave the TAC3b peptide or to amidate it in a sort of non-functionalization event possibly due to lack of selective constraint as reported for teleost duplicated genes [START_REF] Glasauer | Whole-genome duplication in teleost fishes and its evolutionary consequences[END_REF] . However, the TAC3RPb includes a complete tachykinin peptide with cleavage sequence and amidation signal, which may be translated and could prevent the disappearance of the tac3b gene.

Other species show a different C-terminal with substitution of the final methionine by a leucine (zebrafish, goldfish Carassius auratus, northern pike and cavefish Astyanax mexicanus among others). The phylogenetic tree indicates that this paralog tac3b is more divergent than the other paralogon. Therefore, tac3b follows a more accelerated evolutionary rate than the paralog tac3a, in agreement with previous studies on paralogs after the teleost whole genome duplication [START_REF] Brunet | Gene loss and evolutionary rates following whole-genome duplication in teleost fishes[END_REF].

Similarly, tac1 gene is paralog to tac4 from the second round of genome duplication events (2R) that underwent the vertebrate lineage. The human TAC4 peptide has a substitution of methionine by leucine in the tachykinin C-terminal end [START_REF] Naono | Leucine at the carboxyl-terminal of endokinins C and D contributes to elicitation of the antagonistic effect on substance P in rat pain processing[END_REF]. In this case, the peptide TAC4 developed a new function by activating the same receptor than the TAC1RP peptide. While TAC4RP induces hyperalgesia, TAC4 counteracts its activity in an analgesic action [START_REF] Yang | Effects of Endokinin A/B and Endokinin C/D on the modulation of pain in mice[END_REF]for review: Borbély and Helyes, 2017). The promiscuity of the tachykinin receptor and its different responses could ease the neofunctionalization of the tachykinin peptides. Further studies on the eel tachykinin receptors may be of interest for understanding the function of each tachykinin peptide in the eel.

The tachykinin genes have been pretty conserved in sarcopterygians and especially in mammals.

Losses are mostly found in the tac4 gene, for the frog, snakes and birds, but not in turtles. In addition, the tac3 gene is preserved in the chicken, the great tit Parus major, the quail Coturnix japonica and the blue-crowned manakin Lepidothrix coronata. However, it is lost on other species as the duck Annas platyrhynchos. This indicates that the tachykinin genes in sarcopterygians might undergo independent losses. In addition, the tac1 gene is the most widely present in the sarcopterygian lineage.

Reproductive role of the TACin the eel

Effect on pituitary gonadotropins

Our experiments in vitro resulted in a dual inhibition of the mRNA expression of gonadotropin βsubunit, lhβ, and the receptor II of GnRH, gnrh-rII. The dual regulation of lhβ and gnrh-rII in the eel pituitary has been reported also for kisspeptin [START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF]. These results reveal a direct regulation by the TAC3 peptides on pituitary gonadotropins of the pre-pubertal silver eel. The peptide product of the genes lhβ and gnrh-rII work together during the sexual maturation regulating the inhibition process in the silver eel in two different manners: by decreasing the gonadotropin level and by reducing the sensitivity to GnRH. Further in situ analysis of the lhβ and gnrh-rII genes expression will demonstrate co-expression of these genes in the same cells or in different cell-types.

The in vitro effect of TAC3 peptides in the pituitary gonadotropins has been described for other teleosts species (tilapia: Biran et al., 2014;[START_REF] Jin | Neurokinin B-related Peptide Suppresses the Expression of GnRH I , Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus[END_REF]striped bass: Zmora et al., 2017;orange spotted grouper: Chen et al., 2018). Studies in grass carp revealed the effect of TAC3 peptides on somatolactin-α expression mediated by TACR3, prolactin expression mediated by TACR2 and also the translation and the release of the prolactin (Hu et al., 2014a). The peptides encoded in the tac1 gene induced the release of somatolactin-α and prolactin, and interestingly, also the release of LH. The TAC1RP peptide induced the release of LH after 3 hours of activity and the attenuation of lhβ expression after 24h exposition to this peptide [START_REF] Hu | TAC1 gene products regulate pituitary hormone secretion and gene expression in prepubertal grass carp pituitary cells[END_REF]. This activity on LH was mediated by the receptor TACR1. Our preliminary tests on the pre-pubertal silver eel pituitary cells determined that the TAC3 peptides do not have any effect on prolactin in cultured pituitary cells. Further research with the discovered eel peptides encoded in the tac1 and tac4 genes will assess other possible direct effect of the tachykinin peptides on pituitary expression.

The TAC3 peptides inhibited the expression of lhβ but didn't affect the expression of fshβ. The expression of fshβ in the pituitary cell culture revealed a pattern of decrease in all the experimental groups and the control after 4 days in culture (Figure 10). This pattern can indicate an in vivo stimulation of fshβ in the pre-pubertal silver eel. Because the fshβ expression maybe was very low during the cell culture, the possible inhibitory effect of the TAC3 peptides couldn't be assessed. Next experiments may be needed to look for the response of fshβ in presence of a stimulator such as activin to counteract the natural inhibitory phenomena. In order to assess the possible direct effect on gonadotropin cells, studies on the expression and activation of tachykinin receptors are necessary. In situ hybridization of the TACR3 indicated that it is co-expressed with lhβ in the pituitary of tilapia (Biran et al., 2014a) thus indicating the direct effect on gonadotropin. In addition, in situ hybridisation in carp pituitary cells revealed that tachykinin receptors may be specialized in the direct regulation of secretion for gonadotropes (TACR1) lactotropes (TACR2) and somatotropes (TACR3) [START_REF] Hu | TAC1 gene products regulate pituitary hormone secretion and gene expression in prepubertal grass carp pituitary cells[END_REF].

Our preliminary analysis by PCR of both eel tacr3 receptor genes indicate that they are both expressed in the brain, but only one paralog, the tacr3a, is likely expressed in the pituitary (Figure 11). We can then raise the hypothesis that tacr3a paralogon and not tacr3b is implicated in the inhibitory effect of TAC3/TAC3RP peptides in lh and gnrh-rII on the pituitary. Further analysis by in situ hybridisation in the eel will be of interest for revealing the possible co-expression of the tacr3 genes with the lhβ subunit.

Effect on the brain

The brain distribution of the eel tac3 genes indicates that there is a wide expression of the two paralogons in the diencephal (Article 1, Figure 4). Kisspeptin peptides, in particular Kiss2 have also shown to be widely expressed in this area [START_REF] Pasquier | Eel Kisspeptins: Identification, functional activity, and inhibition on both pituitary LH and GnRH receptor expression[END_REF], where the three GnRH-receptors of the eel are also expressed [START_REF] Peñaranda | Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development[END_REF]. In mammals, the diencephalon includes the arcuate nucleus (for review: Hu et al., 2014b) where the KNDy neurons are found [START_REF] Mittelman-Smith | Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight[END_REF]. The possible action of tachykinin and kisspeptin peptides on GnRH in teleosts would be hence analogue to the one of the mammal KNDy neurons. Kiss and TAC3 neurons are expressed in distinct neurons in the stripped bass but TAC3 peptides downregulated the expression of Kiss2 thus reducing the content of GnRH1 in the pituitary [START_REF] Zmora | Neurokinin B regulates reproduction via inhibition of kisspeptin in a teleost, the striped bass[END_REF].

The preliminary finding of the expression of tacr3, both paralogs, in the brain (Figure 11) opens the possibility for a local action on expression or release of GnRH, involving or not Kiss2, in a direct or indirect manner. Further analysis of tachykinins and their receptors in the diencephal area will be of interest for understanding the tachykinergic role in the diencephal and the possible regulation of GnRH release to the pituitary.

whole tachykinin system, including tac1 and tac4 peptides and all three receptors is expressed in the human granulosa cells and in the cumulus cells for regulation of the ovary function. In addition, the tachykinin expression and effect is modulated by kisspeptin [START_REF] García-Ortega | Expression of Tachykinins and Tachykinin Receptors and Interaction with Kisspeptin in Human Granulosa and Cumulus Cells1[END_REF].

Our preliminary analysis in the eel suggests that both paralogs tac3a and tac3b are more expressed in mature ovaries than in ovaries of the pre-pubertal stage. Therefore, eel tachykinins may have a potential role in gonads maturation. Since our preliminary PCR study did not detect any tacr3 in the ovary (figure 11), the function of the TAC3 peptides may be exerted via another receptor. The characterization of the tachykinin system in the ovary of the eel will open a new aspect of the eel sexual maturation process.

VI Perspectives

The tachykinin peptides characterized in the European eel during this thesis is the beginning of a long experimental path till full understanding of the tachykinin system in the eel. The next projects will allow a wider comprehension of this system in the eel, the vertebrates radiation and the sexual maturation process.

Tacand 4 in the eel

For completing the characterization of the new tachykinin peptides discovered in the eel it will be necessary to investigate the localization of expression by qPCR (tissue distribution).

The synthesis of the four predicted TAC1 peptides and the four predicted TAC4 peptides of the eel, will allow to test their biological effects on eel pituitary cell culture to investigate the possible tachykinin regulations on gonadotropins or other pituitary hormones and receptors.

Tac receptors in the eel

The in silico characterization of the tachykinin receptors in spotted gar and in the eel was initiated during this thesis. As in human, three tachykinin receptors were identified in the gar.

Consistently, six tachykinin receptors were found in the genome of the three eel species, likely generated in the 3R. Further synteny analysis must be performed to assess the impact of the whole genome duplication event in the whole tachykinin system. In addition, sequences must be verified by cloning.

Phylogeny studies must be performed for comparative research of the tachykinin system in vertebrates and in teleost radiation.

Later, preparation of qPCR assay for tissue distribution of those 6 receptors will characterize the potential target tissues of the tachykinin system in the eel.

xii 3 .

 3 2.2.3.4.2 Holostean .......................................................................................................... 3.2.2.3.4.3 Teleostean ......................................................................................................... 3.2.2.3.4.3.1 Tachykinin 1 ............................................................................................... 3.2.2.3.4.3.2 Tachykinin 3............................................................................................... 3.2.2.3.4.3.3 Tachykinin 4 ............................................................................................... 3.3 Physiological role in reproduction .................................................................................... 3.3.1 At the central level .................................................................................................................. 3.3.1.1 On GnRH ........................................................................................................................... 3.3.1.1.1 Tac1 peptides ............................................................................................................ 3.3.1.1.2 Tac3 peptide (NKB) ................................................................................................... 3.3.1.2 On DA ............................................................................................................................... 3.3.2. At the pituitary level ............................................................................................................... 3.3.2.1 Tac1 peptides ................................................................................................................... 3.3.2.2 Tac3 peptide (NKB) ........................................................................................................... 3.3.3. At the peripheral level ............................................................................................................ 3.3.3.1. Uterus .............................................................................................................................. 3.3.3.2. Placenta ........................................................................................................................... 3.3.3.3. Gonads and gametes ....................................................................................................... 4 PhD objectives .

  .. II Materials and Methods ........................................................................................................

9. 1

 1 Dispersion and culture ...................................................................................................... 9.2 Treatments ....................................................................................................................... 9.3 RNA extraction and cDNA synthesis ................................................................................. 10 Quantitative real-time

10. 1

 1 Primers ............................................................................................................................ 10.2 SYBR green assay ............................................................................................................

  ... III Article 1 ............................................................................................................................... 1 Introduction to article 1 .

  ... IV Article 2 ............................................................................................................................ 1 Introduction to article 2 .

  .......................................................................... Prediction of eel TAC1 and TAC4 peptides ........................................................................... Phylogeny analysis ................................................................................................................

  .. Identification of European eel tac1 and tac4 genes and encoded peptides ......................... Phylogeny analysis of TAC1 .................................................................................................. Phylogeny analysis of TAC4 .................................................................................................. xiv DISCUSSION .

  .. Two tac1 genes and four TAC1 peptides in the eel .............................................................. Origin and fate of duplicated tac1a and b paralogs in teleosts ............................................ Two tac4 genes and four TAC4 peptides in the eel .............................................................. Origin and fate of duplicated tac4a and b paralogs in teleosts ............................................ Full complement of tachykinin genes and peptides in the eel .............................................

  FIGURES: ..................................................................................................................................

  .

3. 1

 1 Effect on pituitary gonadotropins ................................................................................... 3.2 Effect on the brain .......................................................................................................... 3.3 Effect on the gonads ....................................................................................................... VI Perspectives ...................................................................................................................... 1 Tac1 and 4 in the eel ....

  ............................................................................................................................... xv xvi

Figure 1 :

 1 Figure 1: Phylogenetic position of the anguilliformes. Mitochondrial genomic evidence for the monophyly of the

Figure 2 :

 2 Figure 2: Distribution pattern of Anguilla anguilla. Extant eels presence area (orange) and introduced eels area (purple).

Figure 3 :

 3 Figure 3: The life cycle of the European eel (with permission from Henckel)..

Figure 4 :

 4 Figure 4: Trends in glass eel recruitment. European eel (A. anguilla), Japanese eel (A. japonica) and American eel (A.

Figure 5 :

 5 Figure 5: Genome duplication events in the deuterostome radiation. 1R : First whole genome duplication event; 2R : Second whole genome duplication event ; 3R : Teleost whole genome duplication event.

Figure 6 :

 6 Figure6: Brain Pituitary Gonad Liver axis in teleosts[START_REF] Rousseau | Advances in eel reproductive physiology and endocrinology[END_REF]. GnRH: Gonadotropin releasing hormone; DA: dopamine; FSH: follicle stimulating hormone; LH: luteinizing hormone; Vg: Vitellogenin[START_REF] Rousseau | Advances in eel reproductive physiology and endocrinology[END_REF]

Figure 7 :

 7 Figure 7: Structural and phylogenetic studies on GnRH. A: structure of the GnRH pre-pro-peptide and encoded peptides:

Figure 8 :

 8 Figure 8: Processing of a tachykinin peptide. Cell processing of a vertebrate tachykinin gene; translation and peptide activation. BS: basic site for cleavage; TACRP: tachykinin-related peptide; TAC: tachykinin peptide; αA: α-amidation site.

  [START_REF] Liu | Tachykinin peptides and receptors: Putting amphibians into perspective[END_REF]. The common feature of amphibian aromatic tachykinins is a Tyr residue (Y) at position 4 from the C-terminus, while amphibian aliphatic tachykinins contain either Val (V) or Ile (I) residue at position 4 from the C-terminus (for review:[START_REF] Liu | Tachykinin peptides and receptors: Putting amphibians into perspective[END_REF]. Most of amphibian tachykinins possess the consensus C-terminal FxGLM, where x is an aromatic (F or Y) or hydrophobic (V or I) residue, but exceptions occur as we are going to see.

  . TAC1a sequences from basal teleosts, Elopomorphs (eels) and Osteoglossomorph, branched at the base of the teleost TAC1a clade, in agreement with their basal phylogenetic position. Another well-supported clade encompassed eel TAC1b sequence and clupeocephala TAC1b sequences. Long branches suggested important TAC1b pre-propetide sequence divergence in clupeocephala. Osteoglossomorph TAC1b pre-propeptide sequences branched at the base of all teleost TAC1a and b pre-propeptide sequences. Tac1b genecould not be retrieved in the genome of some teleost species, such as stickleback Gasterosteus aculeatus and fugu Takifugu rubripes. In contrast, up to four tac1 genes were identified in salmonids, with two tac1a and two tac1b paralogs in several Onchorynchus species and in Salvelinus alpinus.

Figure 1 :

 1 Figure 1: Nucleotide and deduced amino-acid sequences of tac1a and tac1b. Nucleotides (top) are

Figure 2 :

 2 Figure 2: Nucleotide and deduced amino-acid sequences of tac4a and tac4b. Nucleotides (top) are

Figure 3 :

 3 Figure 3: This phylogenetic tree was based on 90 deduced amino-acid sequences of tac1 (see Fig

Figure 4 :

 4 Figure 4: This phylogenetic tree was based on 90 deduced amino-acid sequences of tac4 (see Fig

  methionine (M) by a similar aminoacid, the leucine (L). Other teleost species TAC3b have a nontachykinin C-terminal end (LADLL-NH 2 in the perch Perca fluvialitis; LAALL-NH 2 in sea bass Dicentrarchus labrax; LAELL-NH 2 in the barramundi Lates calcarifer). The differences in the sequence maybe associated with the different pattern of receptor recognition and activation (Jakob Biran et al., 2012).In the eel species, the peptides TAC1a and TAC4RPa present different tachykinin motives. TAC1a has a single substitution of the glutamine (G) by serine (S), being the C-terminal formula FVSLM-NH 2 . TAC4RPa presents a substitution of G by alanine (A) in the tachykinin motive FHALM-NK 2 . The second position of this C-terminal end has an aromatic aminoacid, the histidine (H), as reported

Figure 9 :

 9 Figure 9: Conservation of tachykinin peptides in sarcopterygian and actinopterygians.

Figure 10 :

 10 Figure 10: Fshβ expression in the cultured pituitary cells. Control group.

Figure 11 :

 11 Figure 11: PCR analysis of the tacr3a and tacr3b in the brain, pituitary and ovary of two European eel individuals, one of them silver (Silv.) and the other artificially matured (Mat.).

Table 1 :

 1 Invertebrate tachykinins.

	Invertebrate tachykinins (Inv-TAC)
	Eledone
	Ele-TAC I	EPSKDAFIGLM
	Ele-TAC II	KPPSSSEFIGLM
	Octopus
	Oct-TAC I	KPPSSSEFIGLM
	Oct-TAC II	KPPSSSEFVGLM
	Mosquito
	Sialokinin 1	NTGDKFYGLM
	Sialokinin 2	DTGDKFYGLM

Table 2 :

 2 Protostome tachykinin related peptides.

  . Echinoderm tachykinins show only a single tac gene (Starfish Asterias rubens: Semmens et al., 2016; ophiura Ophiura victoriae: Zandawala et al., 2017) that have several TAC peptides encoded, being up to five in the ophiura. Echinoderm TAC peptides have modified the C-terminal end into an xxGxF motif (Table

Table 3 :

 3 Echinoderm tachykinins.

	Echinoderms
	Ophiura victoriae
	Ophiura TAC I	KNNVFSAGLF
	Ophiura TAC II	NGWSQGQQSGLF
	Ophiura TAC III	QRWNQNQQPGLF
	Ophiura TAC IV	SSGTNNMGRVRTKSSGQHVFRSGGLF
	Ophiura TAC I (R)	RKNNVFSAGLF
	Starfish
	Starfish TAC I	QLWANQQSGLF
	Starfish TAC II	GGGVPHVFQSGGIF

Table 4 :

 4 Ascidian tachykinins.

Table 5 :

 5 Tachykinin 1 peptides in sauropsids

	Tac1 derived peptides
	Chicken
	TAC1RP (SP)	RPRPQQFFGLM
	TAC1 (NKA)	HKTDSFVGLM
	Alligator
	TAC1RP (SP)	RPRPQQFFGLM
	TAC1 (NPγ) DAGYGQISHKRHKTDSFVGLM
	TAC1 (NKA)	HKTDSFVGLM
	Python
	TAC1RP (SP)	RPRPQQFYGLM
	TAC1 (NPγ) DAGYSPLSHKRHKTDSFVGLM
	TAC1 (NKA)	HKTDSFVGLM
	Tortoise
	TAC1RP (SP)	RPRPQQFYGLM
	TAC1 (NPγ) DAGYSPLSHKRHKTDSFVGLM

Table 6 :

 6 Mammalian tachykinins.

Table 7 :

 7 Human alternative splice events in tachykinin genes.

	Gene name Chromosome location Isoform Accession number NCBI Encoded peptides
	tac1	Ch. 7	α	NM_013996	SP
			β	NM_003182	SP and NKA (NPK)
			γ	NM_013997	SP and NKA (NPγ)
			δ	NM_013998	SP
	tac3	Ch. 12	α	AF537115	NKB
			β	AF537118	NKB
			γ	AF537121	Unknown
	tac4	Ch. 17	α	NM_170685	EK-A and EK-C
			β	-	EK-B and EK-D
			γ	-	EK-B
			δ	-	EK-B

Table 8 :

 8 Examples of teleost tachykinins.

	Tac1 derived peptides
	Zebrafish
	TAC1RP (SP)	KPRPHQFIGLM
	TAC1 (NKA)	IYLHKINSFVGLM
	Tac3 derived peptides
	Zebrafish
	TAC3RPa (NKFa)	YNDIDYDSFVGLM
	TAC3a (NKBa)	EMHDIFVGLM
	TAC3RPb (NKFb)	YDDIDYDSFVGLM
	TAC3b (NKBb)	PNMNDIFVGLL
	Tilapia
	TAC3RP (NKF)	YNDLDYDSFVGLM
	TAC3 (NKB)	EMDDIFIGLM
	Goldfish
	TAC3RPa (NKFa)	YNDIDYDSFVGLM
	TAC3a (NKBa)	EMHDIFVGLM
	TAC3RPb (NKFb)	YNDIDYDSFIGLM
	TAC3b (NKBb)	PNMNDIFVGLL
	Medaka
	TAC3RP (NKF)	YTDLDYDSFVGLM
	TAC3 (NKB)	DMDDIFVGLM
	Tac4 derived peptides
	Zebrafish
	TAC4RPa (HK)	SKSQHFHGLM
	TAC4a (EK)	NKGEIFVGLM
	TAC4RPb (HK)	SKSHQFYGLM
	TAC4b (EK)	HKGDMFVGLM
	First studies investigating the tissue distribution of SP and NKA in teleosts used

immunohistochemistry with antibodies directed against mammalian tachykinins. SP-like immunoreactivity showed extensive localization throughout the brain (goldfish Carassius auratus:

[START_REF] Sharma | Distribution of substance Plike immunoreactivity in the goldfish brain[END_REF] rainbow trout Oncorhynchus mykiss: Vecino et al., 1989; Apteronotus leptorhynchus: Weld and Maler, 1992

; and sea bass Dicentrarchus labrax:

Table 1 .

 1 . These studies open new research avenues for comparative evolutionary histories of tachykinin and tachykinin receptors across vertebrate and teleost radiation. AP, Pelster B, Spaink HP, et al. Rapid de novo assembly of the European eel genome from nanopore Sequences of European eel (Anguilla anguilla) predicted TAC1 and TAC 4 peptides and comparison with human peptides.

	sequencing reads. Scientific Reports (2017) 7:1-13. doi:10.1038/s41598-017-07650-
	Lafont A-G, Rousseau K., Tomkiewicz K., Dufour S. (2016) Three nuclear and two membrane
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	expression regulation. General and Comparative Endocrinology, 235: 177-191. PMID: 26654744
	Morini M., Pasquier J., Dirks R., van den Thillart G., Tomkiewicz J., Rousseau K., Dufour S.,
	Lafont A.G. (2015). Duplicated leptin receptors in a vertebrate, the eel, bring new insights into the
	evolutionary	history	of	the	leptin	system,	PLoS	ONE	10(5):	e0126008
	doi:10.1371/journal.pone.0126008.
	Page NM, Morrish DW, Weston-Bell NJ. Differential mRNA splicing and precursor processing of
	neurokinin	B	in	neuroendocrine	tissues.	Peptides	(2009)	30:1508-1513.
	doi:10.1016/j.peptides.2009.04.023
	Pasquier J., Lafont A-G, Tostivint H., Vaudry H., Rousseau K., Dufour S. (2012). Comparative
	evolutionary histories of kisspeptins and kisspeptin receptors reveal both parallel and divergent
	features. Frontiers in Endocrinology. 3:173. doi: 10.3389/fendo.2012.00173. eCollection 2012
	Pasquier J, Cabau C, Nguyen T, Jouanno E, Severac D, Braasch I, Journot L, Pontarotti P, Klopp
	C, Postlethwait JH, et al. Gene evolution and gene expression after whole genome duplication in fish:
	the PhyloFish database. BMC Genomics (2016) 17:368. doi:10.1186/s12864-016-2709-z
	Patacchini R, Lecci A, Holzer P, Maggi CA. Newly discovered tachykinins raise new questions
	about their peripheral roles and the tachykinin nomenclature. Trends in Pharmacological Sciences
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Acknowledgements

I am using this opportunity to express my deepest gratitude and special thanks to the members of the Jury, Prof. Sylvain Milla and Prof. Luz Pérez who in spite of being extraordinarily busy with her/his duties, took time out to correct my thesis manuscript. I would like to thank also to Prof. Hervé Tostivint, Dr, Bruno Quérat and Dr. Ron Dirks that honoured me accepting the evaluation of my work the day of my thesis defence.

It is a pleasure to thank all those who made this thesis possible. First and foremost I want to express my grade to my thesis advisors, Dr. Sylvie Dufour, Dr. Karine Rousseau and Dr. Nédia Kamech, for allowing me to join their team, for their expertise, patience and kindness. The enthusiasm and discipline they have for their research was contagious and motivational for me, thank you for your support and your humanity, for getting me up always till the very end.

I would like to acknowledge to the IMPRESS ITN project and the European Commission for the funding provided during this PhD thesis.

ACKNOWLEDGMENTS

This work was supported by a grant from the European Community, Innovative Training Network, ITN IMPRESS MSCA-ITN-2014-ETN N° 642893.

! A ellas va dedicada esta tesis." Aurora vi Avant-propos This PhD was done in the frame of International Training Network (ITN) of the Marie Sklodowska-Curie Actions entitled IMPRESS, « Improved production strategies for endangered freshwater species », funded by the EU Research and Innovation Programme Horizon 2020 for the period 2015-2018. ESR leader • Representing 14 ESRs part of the IMPRESS European training Network of the Marie Sklodowska-Curie Actions funded by the EU Research and Innovation Programme Horizon 2020 for the period 2015-2018.

(1 year term, October 2015-October 2016)

content [START_REF] Hidalgo-Díaz | A modulatory role for substance P on the regulation of luteinizing hormone secretion by cultured porcine gonadotrophs[END_REF]. It also potentiated GnRH-stimulated LH release and reversed GnRH-induced decrease of gonadotrop LH stores, these effects not being blocked by the use of GnRH-receptor antagonist. A few years later, the same group demonstrated that SP direct effect on pig gonadotropes was extracellular Ca 2+ -dependent and did not concern effect on lhβ transcript levels [START_REF] Hidalgo-Díaz | Role of Ca2+ in the secretory and biosynthetic response of porcine gonadotropes to substance P and gonadotropin-releasing hormone[END_REF].

In the urodele, crested newt Triturus carnifex, an indirect measurement of the effect of SP on gonadotropins was performed using a perfusion system combining pituitary and testis/ovary [START_REF] Gobbetti | Substance P Downregulates Basal and Gonadotropin-Releasing Hormone-Induced Gonadotropin In Vitro Secretion by Pituitary Gland of Crested Newt, Triturus carnifex[END_REF]. Considering the lack of antisera against gonadotropins in this species, the authors measured gonadotropins indirectly through their effects on the secretion of testicular androgens and ovarian progesterone from gonads superfused with the preincubated pituitaries. They concluded that SP downregulated gonadotropin release in both sexes, as pituitaries of both sexes preincubated with SP did not change basal steroid secretion, while pituitaries incubated with medium alone increased their steroid secretion.

In teleost, to date, only one study investigated the direct effects of peptides encoded by tac1 gene on pituitary hormone expression and release [START_REF] Hu | TAC1 gene products regulate pituitary hormone secretion and gene expression in prepubertal grass carp pituitary cells[END_REF]. Using primary culture of prepubertal carp pituitary cells, Hu and collaborators showed that carp SP and NKA could elevate prolactin (PRL) and somatolactin-α (SL α) mRNAs and secretion, without any effect on POMC, fshβ, tshβ, gp-α, gh and slβ expression. For lhβ, SP nut not NKA could induce a dose-dependent inhibition of mRNA levels (after 24h of treatment) while both induced a dose-dependent stimulation of LH release (after 3h). This induction of lhβ release was blocked by the use of TACR1 antagonist, but not TACR2 or TACR3 antagonists, while the inhibition of lhβ mRNA by SP was totally abolished in the presence of the same TACR1 antagonist. Moreover, SP was able to partially suppress GnRH-induction of lhβ mRNA while co-treatment with TACR1 antagonist enhanced this induction by GnRH.

Chapter II

Material and Methods

II Materials and Methods 1 Animals

European female eels (Anguilla anguilla) were at the pre-pubertal "silver" stage, corresponding to the end of the continental stage of the eel life cycle, previous to migration to the ocean for reproduction. They were purchased from Gebr. Dil import-export BV (Akersloot, The Netherlands) and transferred to MNHN, France. Animals were anesthetized by cold and then killed by decapitation under the supervision of authorized person (KR; N°R-75UPMC-F1-08) according to the protocol approved by Cuvier Ethic Committee France (N°68-027).

In silico prediction

Tac3 sequences from vertebrate species were retrieved from the Ensembl release 91 (www.ensemble.org) and NCBI (https://www.ncbi.nlm.nih.gov/) databases. Additional blasts were performed using TBLASTN algorithm of the CLC DNA Workbench 6 software (CLC Qiagen, Aarhus, Denmark) in the teleost genomes and multi-organ transcriptomes downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/), Ensembl and Phylofish (http://phylofish.sigenae.org/index.html, [START_REF] Pasquier | Gene evolution and gene expression after whole genome duplication in fish: the PhyloFish database[END_REF]. The sequences of zebrafish tac3a (Gene ID: 100320280) and tac3b (Gene ID: 569642) (J. [START_REF] Biran | Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction[END_REF] were translated with the EXPASY online tool (https://web.expasy.org/translate/) and used as queries. The obtained predicted sequences of the tachykinin pre-pro-peptide were added to the query list for a new multiblast in the next species, up to 52 species in total.

For the European eel, blast analyses were performed on both available draft genomes (Illumina: (Henkel et al., 2012a) and nanopore: [START_REF] Jansen | Rapid de novo assembly of the European eel genome from nanopore sequencing reads[END_REF] and on multi-organ transcriptome (ZFscreens B.V (http://www.zfgenomics.com/sub/eel))). In addition, the draft genomes of the Japanese (Anguilla japonica) and American (Anguilla rostrata) eels (Henkel et al., 2012a;[START_REF] Pavey | Draft genome of the American Eel (Anguilla rostrata)[END_REF] were used for prediction of the tac3 genes using the European eel transcripts as a query.

Cloning and sequencing of eel taccDNAs

The anterior part of the brain including olfactory bulbs, telencephalon and di-/mes-encephalon was dissected and stored in RNA later (Ambion Inc., Austin, TX, USA) at 4°C (24h), then -20°C until extraction. Total RNA was extracted using mechanical homogenization in Trizol Reagent (Invitrogen SARL, Cergy-Pontoise, France), according to manufacturer's instructions. Samples were homogenized by TissueLyser II (QIAGEN, Hilden, Germany) and further treated with deoxyribonuclease I (Roche).

RNA quantifications have been performed using a nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA). One microgram of total RNA was reverse-transcribed using SuperScript III First Strand cDNA Synthesis Kit (Invitrogen) and stored at -20ºC.

Nested PCRs were performed on anterior brain cDNA using two couples of primers (see Article 1, Table S1). The primers were designed in the 5' and

Phylogeny

18 sarcopterygian, 1 chondrichthyan and 59 actinopterygian TAC3 pre-pro-peptide amino-acid sequences were aligned using MUSCLE and CLUSTAL included in SeaView v. 4.6.1 (Gouy et al., 2010).

Alignment was manually adjusted for optimization of key regions such as cleavage sites and signal peptide.

Phylogeny analysis of the TAC3 alignment was achieved using a maximum likelihood method, RAxML black-box (https://www.phylo.org/, [START_REF] Stamatakis | RAxML version 8: A tool for phylogenetic analysis and postanalysis of large phylogenies[END_REF], with 1,000 bootstrap replicates and

Chapter III Article 1

Chapter IV Article 2 For the European eel, blast analyses were performed on both available draft genomes (Illumina, Henkel et al., 2012a andnanopore, Janson et al., 2017) and on multi-organ transcriptome (ZF-screens B.V http://www.zfgenomics.com/sub/eel). In addition, the draft genomes of the Japanese (Anguilla japonica) (Henkel at al., 2012b) and American (Anguilla rostrata) eels [START_REF] Pavey | Draft genome of the American Eel (Anguilla rostrata)[END_REF] were used.

MATERIALS AND METHODS

In silico prediction of tac1 and tac4 genes

Prediction of eel TAC1 and TAC4 peptides

SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) was used for prediction of signal peptide (Petersen et al., 2011), and Neuropred (http://stagbeetle.animal.uiuc.edu/cgi-bin/neuropred.py)

for cleavage and amidation sites (Southey et al., 2006).

Phylogeny analysis of TAC4

Based on an alignment of 64 TAC4 pre-propeptide amino-acid sequences (Figure S2), and assuming the elephant shark Callorhinchus milii sequence as outgroup, a phylogenetic tree was generated using the Maximum Likelihood method (Figure 4).

The osteichtyans TAC4 pre-propeptide sequences clustered into two well-supported clades:

sarcopterygians and actinopterygians. The coelacanth Latimeria chalumnae TAC4 pre-propeptide sequence branched at the base of the sarcopterygian clade, in agreement with its phylogenetic position.

Long branches suggested important TAC4 pre-propeptide sequence divergence in amniotes.

The holostean (spotted gar) TAC4 pre-propeptide sequence branched at the base of the actinopterygian TAC4 clade, in agreement with its phylogenetic position. Two tac4 genes were found in various teleost species, including in zebrafish Danio rerio and medaka Oryzias latipes as previously reported (Zhou et al., 2012). Teleost sequences split into two clades, TAC4a and TAC4b. Only a single tac4 gene was retrieved in osteoglossomorph genomes, branching at the base of the teleost TAC4b clade. Up to four tac4 genes were identified in salmonids, with two tac4a and two tac4b paralogs in several Onchorynchus species.

DISCUSSION

Two tac1 genes and four TAC1 peptides in the eel

Our results showed that the European eel, a basal teleost representative of Elopomorphs, as well as other eel species, the American eel and the Japanese eel, possess two tac1 paralogous genes, each of them encoding two peptides, TAC1a or b and TAC-Related-Peptide (TAC-RPa or b). This organization of the precursor coding for two peptides TAC and TAC-RP is a conserved feature for tachykinins, as exemplified by human TAC1 pre-propeptide including NKA (TAC1) and SP (TAC1-Supplemental figure 1 : Alignment of 90 sequences of tac1 gene collected and deduced from genome. Alignment was performed using CLUSTAL algorithm and manual reordering.

Supplemental figure 2 : Alignment of 64 sequences of tac4 gene collected and deduced from genome. Alignment was performed using CLUSTAL algorithm and manual reordering.

Effect on the gonads

In silver pre-pubertal eel, qPCR analysis on tac3 gene distribution in the ovary shows the weak presence of the expression of tac3a gene and no presence of the tac3b. Nevertheless, the PCR analysis for a preliminary tissue distribution on artificially matured female eel identified both paralogons in the ovary (Figure 12). The role of tachykinin peptides in the ovary have been determined in the ascidian C. intestinalis [START_REF] Aoyama | A novel biological role of tachykinins as an up-regulator of oocyte growth: Identification of an evolutionary origin of tachykininergic functions in the ovary of the ascidian, Ciona intestinalis[END_REF]. The ascidian has a single tachykinin gene encoding two peptides and a single tachykinin receptor. The TACR is present in the ovary and the TAC peptides are necessary for progression from vitellogenic to post-vitellogenic stage by increasing the expression of proteases and increasing also their enzymatic activities. Studies in the ovary function of tac3 and the tacr3 genes revealed a role of the human TAC3 peptide in the acceleration of follicle development, upregulation of the aromatase genes and an increase of the estradiol production in vivo and in vitro on zebrafish [START_REF] Qi | Neurokinin B exerts direct effects on the ovary to stimulate estradiol production[END_REF]. Human TAC3 peptide applied in vitro to human granulosa cell line upregulated also the expression of aromatase in the same studies. In mouse, the steroids produced in the ovaries regulate the presence of the tachykinin peptides in the uterus [START_REF] Pinto | Ovarian steroids regulate tachykinin and tachykinin receptor gene expression in the mouse uterus[END_REF]; the role in the uterus may be related with contractility [START_REF] Patak | Tachykinins and tachykinin receptors in human uterus[END_REF]. Recent studies determined that the

Chapter VI Perspectives 3 Potential receptor/ligand couples in the eel: recombinant receptors and test of peptides

In order to determine receptor preferences for ligand, experiences the six recombinant eel tachykinin receptors will be expressed in cell lines (CHO) cells must be carried.

Comparative evolutionary history of tac and tac receptors

A comparative evolutionary history of the tachykinin and receptors will reveal the conservation and loss of tachykinins and receptors in vertebrate key species.

This analysis can also reveal the possible couples of peptide and receptor that are bound in a coevolution constraint.

To test the ability of the twelve eel TAC peptides to bind to the receptors and activate second messenger pathways.

Bibliography

Characterization of tachykinin system and role in reproduction in the European eel

Résumé

L'objectif de cette thèse est d'étudier le rôle de neuropeptides cérébraux, telle que la Neurokinin B codée par le gène tac3, dans le contrôle de la reproduction d'une espèce en danger, l'anguille Européenne, Anguilla anguilla. La maturation sexuelle de l'anguille est bloquée à un stade prépubertaire avant la migration océanique. Etant donnée sa position phylogénétique basale parmi les téléostéens, l'anguille est un modèle pertinent pour étudier l'évolution moléculaire et fonctionnelle de neuropeptides d'intérêt. Deux gènes paralogues tachykinine 3 (tac3) ont été identifiés dans le génome de l'anguille, chacun codant pour deux peptides. Ces gènes paralogues résultent de la duplication complète du génome spécifique aux téléostéens, comme le montrent les analyses phylogénétiques et synténiques. Les analyses de qPCR montrent que les deux gènes sont exprimés dans le cerveau. Les quatre peptides d'anguille ont été synthétisés et testés sur des cultures primaires de cellules hypophysaires d'anguille. Les quatre peptides inhibent l'expression de l'hormone lutéinisante et d'un récepteur à la gonadolibérine, révélant un double rôle inhibiteur dans le contrôle de la reproduction.

Mots clés : Tachykinine, Anguilla, téléostéens, évolution, reproduction

Abstract

The aim of this PhD is to investigate the role of brain neuropeptides, such as neurokinin B, encoded by tac3 gene, in the control of reproduction of an endangered species, the European eel, Anguilla anguilla. The sexual maturation of the eel is blocked at a prepubertal stage before the oceanic migration. Due to its basal phylogenetic position among teleosts, the eel is also a relevant model for studying molecular and functional evolution of key neuropeptides. Two tachykinin 3 (tac3) paralogous genes were identified in the eel genome, each encoding two peptides. These paralogs result from the teleost-specific whole genome duplication, as shown by phylogeny and synteny analyses. Both genes are expressed in the brain as shown by qPCR. The four eel peptides were synthesized and tested on primary cultures of eel pituitary cells. The four peptides inhibited the expression of luteinizing hormone and gonadotropin-releasing hormone receptor, revealing a dual inhibitory role in the control of reproduction. Key-words: Tachykinin, Anguilla, teleost, evolution, reproduction