Emilie Le Blévec

Jana Muroňová

Pierre Ray

Christophe Arnoult
email: christophe.arnoult@univ-grenoble-alpes.fr

Paternal epigenetics: mammalian sperm provide much more than DNA at fertilization by

Keywords: sperm, protamine, sperm compaction, epigenetic, non-coding RNA, embryonic development, environmental stress

The spermatozoon is a highly differentiated cell with unique characteristics: it is mobile, thanks to its flagellum, and is very compact. The sperm cytoplasm is extremely reduced, containing no ribosomes, and therefore does not allow translation, and its nucleus contains very closed chromatin, preventing transcription. This DNA compaction is linked to the loss of nucleosomes and the replacement of histones by protamines. Based on these characteristics, sperm was considered to simply deliver paternal DNA to the oocyte. However, some parts of the sperm DNA remain organized in a nucleosomal format, and bear epigenetic information. In addition, the nucleus and the cytoplasm contain a multitude of RNAs of different types, including non-coding RNAs (ncRNAs) which also carry epigenetic information. For a long time, these RNAs were considered residues of spermatogenesis.

After briefly describing the mechanisms of compaction of sperm DNA, we focus this review on the origin and function of the different ncRNAs. We present studies demonstrating the importance of these RNAs in embryonic development and transgenerational adaptation to stress. We also look at other epigenetic marks, such as DNA methylation or post-translational modifications of histones, and show that they are sensitive to environmental stress and transmissible to offspring. The postfertilization role of certain sperm-borne proteins is also discussed.

Introduction

Our view of what sperm cells bring to the oocyte and its contribution to human reproduction has dramatically changed since it was first understood, and remains the subject of intense research. Sperm cells, simply called sperm, were identified in the 17th and 18th century, thanks to the development of the microscope by Dutch scientist Antoni Van Leeuwenhoek (1632-1723). Van Leeuwenhoek was the first to observe these strange moving objects within semen that he called "animalcule" (1678, communication at the London Royal Society). At that time, it was hypothesized that the head of the animalcules (sperm) contained a whole tiny human curled up inside (homonculus at this time), whose development would be assured by the female, the egg was considered to act as the placenta. Nicolaas Hartsoecker (1656Hartsoecker ( -1725) ) was the first to propose an illustration of this hypothesis in 1694 [START_REF] Hartsoecker | Essay de dioptrique Paris Hertwig O (1876)[END_REF] (Figure 1). Hartsoecker in 1694 [START_REF] Hartsoecker | Essay de dioptrique Paris Hertwig O (1876)[END_REF]. We now know that this hypothesis was incorrect, but it took more than 200 years to start to decipher the contribution of sperm cells to the new living organism, and important discoveries are still being made. The fact that a spermatozoon provides half of the genetic material, and is at the origin of one of the pronuclei during fertilization was actually discovered two centuries later by several biologists between 1873 and 1883 using various animal models such as starfish [START_REF] Fol | Paternal overweight is associated with increased breast cancer risk in daughters in a mouse model[END_REF], sea urchin (Hertwig, 1876) and ascaris [START_REF] Van Beneden | Recherches sur la maturation de l'oeuf et la fécondation[END_REF]. The discovery that sperm contribute half of the heredity of the embryo was discovered simultaneously 20 years later by Theodor Boveri (1862Boveri ( -1915) ) and Walter Sutton (1877Sutton ( -1916)). In 1902In -1903, they , they proposed that chromosomes bear hereditary factors in accordance with Mendelian laws -first described 37 years previously by the monk Augustin Mendel (1822-1884) -and that sperm provide half of the chromosomes found in the zygote. Soon after this breakthrough, Jacques Loeb [START_REF] Loeb | Artificial parthenogenesis and fertilization[END_REF] rapidly recognized that the male gamete not only transmits the paternal characteristics to the developing embryo, but also induces development by activating the egg. A second major function of the male gamete, essential for fertilization, was then identified.

The characterization of the sperm-borne factor triggering egg activation was also an epic scientific journey, and culminated in the discovery of PLCzeta (PLCZ1) by teams led by Karl Swann and Antony Lay in 2002 [START_REF] Cox | Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes[END_REF], Saunders et al, 2002). A description of this sperm factor is beyond the scope of this review, and for more information about this key protein, we refer readers to a recent review [START_REF] Parrington | The eggstraordinary story of how life begins[END_REF].

For decades, the function of sperm was considered to be limited to these two roles: providing half of the hereditary material in the form of the paternal DNA, and activating the egg. In this review, we will discuss the sperm epigenome, the modifications to the genome that do not affect the DNA sequence but are capable of influencing gene expression, including non-coding RNA, DNA methylation and posttranscriptional modification of histones. We will first present the characteristics of sperm chromatin and the main elements necessary for its organization and compaction. Then, we will describe the epigenetic events occurring in the sperm, the different RNA molecules (mRNAs, regulatory non-coding RNAs) contained in sperm and their origins, and also significant sperm developmental factors including histones and proteins that are retained and transmitted. Finally, we highlight how environmental stress can alter epigenetic factors in sperm, such as DNA methylation and non-coding RNA, causing phenotypic changes in the offspring. These changes not only affect the phenotype of the F1 offspring, but also that of the following F2 generation, even though the F1 generation was not exposed to the initial environmental stress. Nevertheless, this transgenerational transmission of a new phenotype is not permanent and vanishes in most cases after the F3 generation. Readers should note that, "transgenerational transmission" is distinct from "intergenerational transmission", in that the latter only affects the F1 generation (Figure 2). (Nutritional, physical, …) induces epigenetic changes in the male reproductive organs, including testes and epididymides. These epigenetic modifications lead to an alteration of epigenetic information carried by the sperm and the progeny obtained from stressed male crossed with a nonstressed female exhibits phenotypic changes dependent of the nature of the stress. Epigenetic alterations can be maintained in the male germ cells up to F3 generation, even though the subsequent generations are not exposed to the initial (F0) environmental stress, a phenomena named "transgenerational transmission". If the epigenetic alterations of the germ cells are not transmitted to the F2 generation, then the phenotypic change is named "intergenerational transmission".

Sperm DNA compaction

Sperm cells are produced during the spermatogenesis, a very complex physiological process which can be split into three successive steps: mitosis, meiosis, and spermiogenesis and involves the expression of several thousands of genes [START_REF] Hess | Spermatogenesis and cycle of the seminiferous epithelium[END_REF]. The spermatozoon is undoubtedly the most highly differentiated cell type in mammals, with the greatest number of specific or enriched genes necessary for its biogenesis [START_REF] Uhlén | Transcriptomics resources of human tissues and organs[END_REF]. Among the many exceptional features of this cell, its DNA organization is unique, showing a high level of compaction leading to an arrest of transcription. Sperm DNA compaction involves replacing nucleosomes by donut-shaped DNA-protamine toroids, with each toroid containing 60 kbp of DNA. This rearrangement results in a highly compact and transcriptionally inactive sperm chromatin [START_REF] Steger | Transcriptional and translational regulation of gene expression in haploid spermatids[END_REF]. For a long time, the histone-to-protamine transition in mammals was considered to take place in two successive stages, with a first stage involving the replacement of histones by transition proteins, and a second stage during which transition proteins are replaced by protamines. However, discoveries in this field over the years revealed the process to be much more complex, and not as linear as it initially seemed. At the beginning of the transition, from spermatogonia to elongated spermatids, the canonical histones (H2A, H2B, H3 and H4) and the H1-linker histone undergo posttranslational modifications to facilitate their progressive replacement in nucleosomes by somatic or testicle-specific variants. Each histone variant has its own expression window that is directly related to its specific role during the process of histone-to-protamine transition. The incorporation of histone variants induces chromatin instability that favors nucleosome disassembly. Moreover, posttranslational modifications of the variants accentuate chromatin relaxation and allow DNA strand breaks that facilitate the progressive elimination of histone variants and their replacement by transition proteins and protamines [START_REF] Rathke | Chromatin dynamics during spermiogenesis[END_REF], Wang et al, 2019). Recently, it was proposed that transition proteins and protamines could be added simultaneously, rather than successively, leading to the formation of a nucleo-protamine structure [START_REF] Barral | Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells[END_REF].

1.a Histone variants

The core canonical histones (H3, H4, H2A and H2B) can be replaced by histone variants, changing the structural and functional features of the nucleosomes. Unlike canonical histones, which are incorporated during the S-phase of the cell cycle, histone variants are synthesized and incorporated throughout the cell cycle, in particular during spermiogenesis. Many histone variants have been identified in mammals for H2A, H2B and H3 canonical histones, but none for H4. The H1-linker histone, which influences the degree of compaction of the chromatin, and whose eviction leads to decondensation, also has variants [START_REF] Thoma | Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin[END_REF]. The variants are species-dependent, and human and mouse histone variants only partially overlap.

Compaction of sperm DNA is associated with intense trafficking of histone and histone variants within the nucleosome and its linker, a process that is not fully understood. Histone H1 has at least 11 variants, including the testis variants H1T, H1T2 and HILS1 (TS H1.6, TS H1.7 and TS H1.9, according to the new nomenclature) [START_REF] El Kennani | MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones[END_REF]. H1T is present from the spermatocyte I stage to the elongating spermatid stage [START_REF] Drabent | Expression of the mouse testicular histone gene H1t during spermatogenesis[END_REF]. H1T maintains a relatively open chromatin configuration, which is necessary for meiotic recombination and histone replacement [START_REF] Delucia | Histone-Induced Condensation of Rat Testis Chromatin: Testis-Specific H1t versus Somatic H1 Variants[END_REF]. H1T2 is present from the round spermatid stage to the elongated spermatid stage at the apical pole of the nucleus, under the acrosome. It is required for chromatin condensation, protamine incorporation and cell restructuring [START_REF] Martianov | Polar nuclear localization of H1T2, a histone H1 variant, required for spermatid elongation and DNA condensation during spermiogenesis[END_REF]. HILS1, present from the elongated to the condensed spermatid stages, has a higher DNA binding capacity than other histones. It could be involved in transcriptional regulation and also contribute to nuclear condensation through its interactions with transition proteins [START_REF] Yan | HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis[END_REF].

Several variants of the histone H2A (tH2A, H2A.L.1-3, H2A.B.1-3, H2AP) and the histone H2B (tH2B) have been specifically identified in mouse testis, but their specific roles have not been fully described. The histone variants tH2A and tH2B (TS H2A.1 and TS H2B.1, according to the new nomenclature) are present from the spermatogonia stage and gradually disappear during condensation of spermatid nuclei [START_REF] El Kennani | MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones[END_REF][START_REF] Montellier | Chromatin-to-nucleoprotamine transition is controlled by the histone H2B variant TH2B[END_REF][START_REF] Trostle-Weige | Isolation and characterization of TH2A, a germ cell-specific variant of histone 2A in rat testis[END_REF][START_REF] Van Roijen | Immunoexpression of testis-specific histone 2B in human spermatozoa and testis tissue[END_REF]. They induce chromatin instability and could guide the placement of transition proteins and protamines. These variants also seem to play a role in epigenetic reprogramming [START_REF] Padavattan | Structural and functional analyses of nucleosome complexes with mouse histone variants TH2a and TH2b, involved in reprogramming[END_REF]. The class of histone variants H2A.L and H2B.L are present from the round spermatid stage to the elongated spermatid stage, and H2A.L.1, H2A.L.2 and H2B.L.1 are still present in the mature sperm [START_REF] Govin | Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis[END_REF]. H2A.L.2 allows the chromatin to open, and could play a crucial role in the incorporation of transition proteins and protamines [START_REF] Barral | Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells[END_REF]. H2A.B.3 is present from the spermatocyte I stage to the round spermatid stage [START_REF] Soboleva | A unique H2A histone variant occupies the transcriptional start site of active genes[END_REF]. H2A.B.3 could also promote the chromatin reorganization necessary for histone-toprotamine transition, by modulating the incorporation and removal of H2A.L.2 and TP1 from chromatin [START_REF] Anuar | Gene editing of the multi-copy H2A.B gene and its importance for fertility[END_REF]. It could also play a role in pre-mRNA splicing [START_REF] Soboleva | A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B[END_REF].

Histone H3 (H3.1 and H3.2) has a testicle-specific histone variant (H3t), and a testicle-enriched variant (H3.3) in mice. H3t (TS H3.4, according to the new nomenclature) is present from the spermatogonia stage to the early spermatid stage [START_REF] El Kennani | MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones[END_REF][START_REF] Trostle-Weige | Isolation and characterization of TH3, a germ cell-specific variant of histone 3 in rat testis[END_REF].

H3t could contribute to destabilizing chromatin, and play another as yet unknown role in later stages of spermiogenesis that cannot be compensated for by other members of the H3 family [START_REF] Tachiwana | Structural basis of instability of the nucleosome containing a testisspecific histone variant, human H3T[END_REF][START_REF] Ueda | Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis[END_REF]. H3.3 is a ubiquitous protein encoded by two genes, H3f3a and H3f3b.

These genes have different nucleotide sequences, but produce the same protein. H3f3a transcripts are present in small amounts from the spermatogonia stage to the elongated spermatid stage, whereas H3f3b transcripts are only detected during the spermatocyte I stage [START_REF] Bramlage | Differential expression of the murine histone genes H3.3A and H3.3[END_REF].

H3.3 allows destabilization of the chromatin, promotes transcription and plays an essential role in the histone-to-protamine transition by modulating the incorporation of TP1 and P1 in chromatin [START_REF] Ahmad | The Histone Variant H3.3 Marks Active Chromatin by Replication-Independent Nucleosome Assembly[END_REF][START_REF] Thakar | H2A.Z and H3.3 Histone Variants Affect Nucleosome Structure: Biochemical and Biophysical Studies[END_REF][START_REF] Yuen | Histone H3.3 regulates dynamic chromatin states during spermatogenesis[END_REF]. Moreover, H3.3 is involved in Meiotic Sex Chromosome Inactivation (MSCI) at the pachytene stage. Sex chromosomes are located at the periphery of the nucleus in the sexual body and are inactivated during meiosis. Induction of MSCI is associated with both removal of conventional H3 and accumulation of H3.3 and γH2AX in the sexual body, suggesting that these histone variants are needed for sex chromosome condensation and silencing [START_REF] Mahadevaiah | Recombinational DNA double-strand breaks in mice precede synapsis[END_REF][START_REF] Van Der Heijden | Chromosome-wide nucleosome replacement and H3.3 incorporation during mammalian meiotic sex chromosome inactivation[END_REF]. The human-specific H3.5 variant is present from the spermatogonia to the spermatocyte I stages [START_REF] Urahama | Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis[END_REF]. H3.5 could also be involved in chromatin destabilization. Moreover, another, as yet poorly characterized, role related to DNA synthesis has been described [START_REF] Shiraishi | Roles of histone H3.5 in human spermatogenesis and spermatogenic disorders[END_REF][START_REF] Urahama | Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis[END_REF].

Overall, this short description of the numerous histone variants involved in sperm DNA compaction highlights the complexity of the process.

1.b Transition proteins

Transition proteins are basic nuclear proteins (more basic than histones and less basic than protamines) that are synthesized during the advanced stages of spermiogenesis and disappear before the final stage of spermiogenesis [START_REF] Alfonso | Immunohistochemical Localization of Spermatid Nuclear Transition Protein 2 in the Testes of Rats and Mice[END_REF]. There are four types of transition proteins: TP1, TP2, TP3 and TP4 (or TNP1, TNP2, TNP3 and TNP4). The first two are present in all species, and are consequently the best characterized. TP1 is a 54-amino-acid protein that is highly conserved in mammals. It is rich in serine, arginine, histidine and lysine residues [START_REF] Kremling | Nucleotide sequences and expression of cDNA clones for boar and bull transition protein 1 and its evolutionary conservation in mammals[END_REF]. TP2 is a larger protein, with 117 to 138 amino acids depending on the species, it is rich in the same basic amino acids as TP1, and also contains some cysteine residues [START_REF] Kleene | Characterization of a cDNA clone encoding a basic protein, TP2, involved in chromatin condensation during spermiogenesis in the mouse[END_REF][START_REF] Schlüter | The gene for human transition protein 2: Nucleotide sequence, assignment to the protamine gene cluster, and evidence for its low expression[END_REF]. Its N-terminal part contains two zinc finger domains allowing it to bind to unmethylated CpG sites on DNA, whereas its C-terminal part is very basic [START_REF] Baskaran | Mammalian spermatid specific protein, TP2, is a zinc metalloprotein with two finger motifs[END_REF][START_REF] Cole | Nuclear transition protein 2 (TP2) of mammalian spermatids has a very basic carboxyl terminal domain[END_REF][START_REF] Meetei | Identification of Two Novel Zinc Finger Modules and Nuclear Localization Signal in Rat Spermatidal Protein TP2 by Site-directed Mutagenesis[END_REF]. Animals lacking TP1 or TP2 produce normal sperm but have reduced fertility, demonstrating that there is some functional redundancy between TP1 and TP2. The absence of both TP1 and TP2 causes male sterility, associated with protamine defects and major DNA breaks. The cells produced are incompatible with fertilization, even by intracytoplasmic sperm injection (ICSI) [START_REF] Shirley | Abnormalities and Reduced Reproductive Potential of Sperm from Tnp1-and Tnp2-Null Double Mutant Mice[END_REF][START_REF] Yu | Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice[END_REF][START_REF] Zhao | Nucleoprotein Transitions During Spermiogenesis in Mice with Transition Nuclear Protein Tnp1 and Tnp2 Mutations[END_REF][START_REF] Zhao | Targeted Disruption of the Transition Protein 2 Gene Affects Sperm Chromatin Structure and Reduces Fertility in Mice[END_REF]. Although the two proteins can partially compensate for each other, they play unique roles in the histone-toprotamine transition. During the histone-to-protamine transition, TP1 could be involved in DNA destabilization and thus promote histone removal; it could also contribute to DNA strand break repair [START_REF] Caron | Stimulation of DNA repair by the spermatidal TP1 protein[END_REF][START_REF] Singh | Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies[END_REF]. TP2 could be necessary for stabilizing and initiating DNA condensation [START_REF] Baskaran | Interaction of spermatid-specific protein TP2 with nucleic acids, in vitro. A comparative study with TP1[END_REF]. Finally, transition proteins also carry a significant number of post-translational modifications (acetylation, phosphorylation and methylation) on their serine, lysine, and arginine residues. These modifications are inserted at specific times during nuclear condensation in spermatids and are crucial for the histone-to-protamine transition [START_REF] Gupta | Mapping of Post-translational Modifications of Transition Proteins, TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2[END_REF].

1.c Protamines

Protamines were first isolated and discovered in salmon sperm at the end of the 19th century by Friedrich Miescher [START_REF] Miescher | Das Protamin, eine neue organische Base aus den Samenfäden des Rheinlachses[END_REF]. There are three types of protamines: protamine 1 (P1), protamine 2 (P2) and protamine 3 (P3), but only P1 and P2 are involved in sperm DNA compaction.

P1 is expressed in all vertebrates, whereas P2 is expressed only in certain mammalian species (Primates, Rodents, Lagomorphs and equine and bovine species) [START_REF] Balhorn | The protamine family of sperm nuclear proteins[END_REF][START_REF] Corzett | Protamine 1: Protamine 2 stoichiometry in the sperm of eutherian mammals*[END_REF], Hamilton et al, 2019b). Protamines, proteins of low molecular weight, are highly basic (more basic than histones and TPs), and containing extensive proportions of arginine (47-70% of the amino acids) and cysteine (8-16% of the amino acids) residues in placental mammals [START_REF] Balhorn | The protamine family of sperm nuclear proteins[END_REF][START_REF] Calvin | Comparative analysis of the nuclear basic proteins in rat, human, guinea pig, mouse and rabbit spermatozoa[END_REF][START_REF] Mckay | The amino acid sequence of human sperm protamine P1[END_REF][START_REF] Mckay | Human sperm protamines[END_REF][START_REF] Queralt | Evolution of protamine P1 genes in mammals[END_REF]. In placental mammals, P1 consists of around 50 amino acids organized in three characteristic domains. A central arginine-rich DNA binding domain, flanked on either side by short segments containing phosphorylation sites composed of serine and threonine residues, and cysteine residues necessary for the formation of inter-protamine disulfide bridges [START_REF] Balhorn | The protamine family of sperm nuclear proteins[END_REF][START_REF] Mckay | The amino acid sequence of human sperm protamine P1[END_REF][START_REF] Queralt | Evolution of protamine P1 genes in mammals[END_REF]. P2 contains 54 to 63 amino acids depending on the species [START_REF] Mckay | Human sperm protamines[END_REF][START_REF] Yelick | Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not[END_REF]. Unlike P1, a precursor to P2 is synthesized with about 100 amino acids. The mature form of P2 is obtained by cleavage, resulting in the removal of about 40% of its N-terminus [START_REF] Balhorn | The protamine family of sperm nuclear proteins[END_REF][START_REF] Carré-Eusèbe | Processing of the precursor of protamine P2 in mouse. Peptide mapping and N-terminal sequence analysis of intermediates[END_REF][START_REF] Yelick | Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not[END_REF]. In humans, two mature forms of P2 have been described, P2a with 57 amino acids and P2b with 54 amino acids [START_REF] Mckay | Human sperm protamines[END_REF]. Both bull and boar possess the PRM2 gene, and P2 mRNA transcripts have been detected, but no P2 protein has yet been identified in mature sperm from these animals [START_REF] Ganguly | Differential expression of protamine 1 and 2 genes in mature spermatozoa of normal and motility impaired semen producing crossbred Frieswal (HF×Sahiwal) bulls[END_REF][START_REF] Maier | The lack of protamine 2 (P2) in boar and bull spermatozoa is due to mutations within the P2 gene[END_REF][START_REF] Mazrimas | A corrected primary sequence for bull protamine[END_REF][START_REF] Tobita | Isolation and characterization of nuclear basic protein (protamine) from boar spermatozoa[END_REF]. They are synthesized during the last stages of spermatogenesis to replace histones at the DNA, and highly contribute to its compaction [START_REF] Rathke | Chromatin dynamics during spermiogenesis[END_REF].

1.d Organization of sperm chromatin

The presence of a large number of positively-charged amino acids in protamines, and in particular the arginine clusters, constitute zones for DNA tethering, thanks to the electrostatic bonds created by their opposing charges. These proteins therefore form very stable complexes with DNA [START_REF] Balhorn | The protamine family of sperm nuclear proteins[END_REF][START_REF] Brewer | Dynamics of Protamine 1 Binding to Single DNA Molecules[END_REF][START_REF] Derouchey | Role of Amino Acid Insertions on Intermolecular Forces between Arginine Peptide Condensed DNA Helices IMPLICATIONS FOR PROTAMINE-DNA PACKAGING IN SPERM[END_REF]. Protamine binding will thus neutralize the charge on the DNA molecules and allow them to pack more closely. The structural organization of the protamine-DNA interaction is still unclear, but it seems that protamines wrap around the doublestranded DNA by positioning themselves in one of the grooves of the double helix [START_REF] Balhorn | The protamine family of sperm nuclear proteins[END_REF][START_REF] Fita | X-ray diffraction study of DNA complexes with arginine peptides and their relation to nucleoprotamine structure[END_REF][START_REF] Hud | Evidence of Novel Secondary Structure in DNA-Bound Protamine Is Revealed by Raman Spectroscopy[END_REF]. P1 can bind about 11 bp of DNA, whereas the larger P2 can bind up to 15 bp, at a rate of one protamine per turn of the helix [START_REF] Bench | DNA and total protamine masses in individual sperm from fertile mammalian subjects[END_REF]. Protamine binding in the DNA grooves induces curvature, resulting in the formation of a toroidal loop-like structure or "donut loop", with each toroidal loop connecting the DNA to the nuclear matrix [START_REF] Allen | AFM analysis of DNA-protamine complexes bound to mica[END_REF], Allen et al, 1993[START_REF] Balhorn | The protamine family of sperm nuclear proteins[END_REF][START_REF] Björndahl | Human sperm chromatin stabilization: a proposed model including zinc bridges[END_REF][START_REF] Hud | Identification of the Elemental Packing Unit of DNA in Mammalian Sperm Cells by Atomic Force Microscopy[END_REF][START_REF] Ward | Function of sperm chromatin structural elements in fertilization and development[END_REF]. Each toroidal loop can pack up to 60 kb of DNA, and the sperm nucleus can contain up to 50,000 toroidal loops by the end of spermiogenesis [START_REF] Hud | Identification of the Elemental Packing Unit of DNA in Mammalian Sperm Cells by Atomic Force Microscopy[END_REF].

Protamines also have cysteine residues, the thiol (-SH) groups of which are oxidized to disulfide (-SS-) by nuclear glutathione peroxidase 4 (nGPx4) to form a three-dimensional network of disulfide bridges [START_REF] Puglisi | The nuclear form of glutathione peroxidase 4 is associated with sperm nuclear matrix and is required for proper paternal chromatin decondensation at fertilization[END_REF]. The intra-protamine disulfide bridges stabilize the folding of different protamine domains and are essential for DNA condensation. The inter-protamine disulfide bridges, in complement, serve to increase the structural stability of the nucleoprotamines and to maintain the strained conformation of the structure, which further enhances the level of DNA compaction [START_REF] Hutchison | Role of Disulfide Bonds on DNA Packaging Forces in Bull Sperm Chromatin[END_REF]. The chromatin stability is proportional to the number of disulfide bridges formed, and this stabilization process is progressive, beginning in the testis and continuing as sperm transit through the epididymis [START_REF] Balhorn | Formation of intraprotamine disulfides in vitro[END_REF][START_REF] Gosálvez | Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species[END_REF][START_REF] Saowaros | The formation of disulfide bonds in human protamines during sperm maturation[END_REF][START_REF] Yossefi | Chromatin condensation in hamster sperm: A flow cytometric investigation[END_REF].

In P2, zinc finger domains composed of cysteine and histidine residues and stabilized by zinc salt bridges contribute to the formation of P2-DNA complexes [START_REF] Bench | Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2[END_REF][START_REF] Bianchi | P2 protamines from human sperm are zinc -finger proteins with one Cys2His2 motif[END_REF]. In addition to its role in stabilizing the nucleoprotamine structure as effectively as disulfide bridges, zinc also prevents superstabilization by reducing the availability of free thiol groups to form disulfide bridges [START_REF] Björndahl | Human sperm chromatin stabilization: a proposed model including zinc bridges[END_REF]. Zinc salt bridges are also easier to disrupt than disulfide bridges, and could allow faster decondensation of the chromatin upon oocyte fertilization [START_REF] Björndahl | Human sperm chromatin stabilization: a proposed model including zinc bridges[END_REF]. This setup probably explains why species expressing only P1 have the most extensively compacted chromatin, and why species expressing more P2 are more sensitive to DNA damage [START_REF] Gosálvez | Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species[END_REF].

In addition to their structural and binding residues, protamines have serine and threonine residues which are phosphorylated immediately after protamine synthesis by SRPK1 (serine/arginine-rich protein-specific kinase-1) for P1 and Camk4 (Calcium/calmodulin-dependent protein kinase type IV) for P2 [START_REF] Papoutsopoulou | SR protein-specific kinase 1 is highly expressed in testis and phosphorylates protamine 1[END_REF][START_REF] Wu | Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4[END_REF]. These phosphorylation events are necessary to initially establish correct binding between the protamines and the DNA. Phosphorylation reduces the affinity of the protamines for DNA (by increasing the negative charge of protamines), to give the protamines time to position themselves optimally with respect to the DNA, thus reducing mismatching. Subsequently, the progressive dephosphorylation of protamines gradually increases the affinity of protamines for DNA and induces chromatin condensation [START_REF] Green | Synthesis and processing of mammalian protamines and transition proteins[END_REF]. Optimal positioning of protamines is essential for the appropriate formation of inter-protamine disulfide bridges [START_REF] Marushige | Phosphorylation of sperm histone during spermiogenesis in mammals[END_REF]. Other post-translational modifications of protamines have been identified and could contribute to the protamine-DNA binding or protamine removal process after fertilization, suggesting the existence of a protamine code equivalent to the histone code [START_REF] Brunner | Epigenetic marking of sperm by post-translational modification of histones and protamines[END_REF].

During spermatogenesis, not all histones are replaced by protamines, and variable fractions of histones remain associated with DNA in the mature sperm (1-8% for mouse [START_REF] Balhorn | Mouse sperm chromatin proteins: quantitative isolation and partial characterization[END_REF][START_REF] Jung | Chromatin States in Mouse Sperm Correlate with Embryonic and Adult Regulatory Landscapes[END_REF], 13% for bull [START_REF] Samans | Uniformity of Nucleosome Preservation Pattern in Mammalian Sperm and Its Connection to Repetitive DNA Elements[END_REF], 3-15% for human [START_REF] Bench | DNA and total protamine masses in individual sperm from fertile mammalian subjects[END_REF][START_REF] Samans | Uniformity of Nucleosome Preservation Pattern in Mammalian Sperm and Its Connection to Repetitive DNA Elements[END_REF][START_REF] Tanphaichitr | Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man[END_REF], and 25% in some marsupial species [START_REF] Soon | Isolation of histones and related chromatin structures from spermatozoa nuclei of a dasyurid marsupial, Sminthopsis crassicaudata[END_REF]). These residual histones were first considered a defect in the replacement of histones by protamines, but it is now clear that persistent histones (canonical or variant) in mature sperm are not randomly distributed and have a role to play. Histone-enriched regions appear to be distributed either in large 10-100-kb regions corresponding to the solenoids present between toroidal loops, or in short domains dispersed throughout the genome, corresponding to stretches of DNA anchoring the toroidal loops to the nuclear matrix [START_REF] Arpanahi | Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences[END_REF][START_REF] Hammoud | Distinctive Chromatin in Human Sperm Packages Genes for Embryo Development[END_REF][START_REF] Ward | Function of sperm chromatin structural elements in fertilization and development[END_REF]. The precise location of persistent histones in the genome remains a subject of debate. Some studies found that nucleosomes are enriched at the level of hypomethylated CpG sites corresponding to promoters and genes that are important for embryogenesis (transcription factors, signaling factors, Homeobox gene clusters, globin genes, imprinted gene clusters, genes encoding microRNAs, etc.) [START_REF] Arpanahi | Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences[END_REF][START_REF] Erkek | Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa[END_REF][START_REF] Gardiner-Garden | Histone-and Protamine-DNA Association: Conservation of Different Patterns within the β-Globin Domain in Human Sperm[END_REF][START_REF] Hammoud | Distinctive Chromatin in Human Sperm Packages Genes for Embryo Development[END_REF][START_REF] Royo | Alternative Computational Analysis Shows No Evidence for Nucleosome Enrichment at Repetitive Sequences in Mammalian Spermatozoa[END_REF][START_REF] Yoshida | Mapping of histone-binding sites in histone replacement-completed spermatozoa[END_REF]. Nevertheless, other authors have reported that the nucleosomes persist at intergenic regions, regions rich in repeated sequences (telomeres, subtelomeres, centromeres, pericentromeres, LINE1 and SINE) and rRNA and SRP RNA (RNA component of Signal Recognition Particle) gene clusters [START_REF] Benjamin | High-Resolution Mapping of Chromatin Packaging in Mouse Embryonic Stem Cells and Sperm[END_REF][START_REF] Hoghoughi | RNA-Guided Genomic Localization of H2A.L.2 Histone Variant[END_REF][START_REF] Meyer-Ficca | Alteration of poly(ADP-ribose) metabolism affects murine sperm nuclear architecture by impairing pericentric heterochromatin condensation[END_REF][START_REF] Samans | Uniformity of Nucleosome Preservation Pattern in Mammalian Sperm and Its Connection to Repetitive DNA Elements[END_REF][START_REF] Sillaste | A novel hypothesis for histoneto-protamine transition in Bos taurus spermatozoa[END_REF][START_REF] Zalenskaya | Chromatin Structure of Telomere Domain in Human Sperm[END_REF]. These divergent results could be due to the fact that different protocols were used to isolate histones, but could ultimately be complementary: even if the majority of persistent histones are localized in gene deserts, some of the histones are also deposited in specific regions that are important for embryonic development [START_REF] Yamaguchi | Reevaluating the Localization of Sperm-Retained Histones Revealed the Modification-Dependent Accumulation in Specific Genome Regions[END_REF]. Importantly, histones retained in sperm carry epigenetic marks that are transferred to the embryo, and they could be involved in embryonic development [START_REF] Bui | Essential role of paternal chromatin in the regulation of transcriptional activity during mouse preimplantation development[END_REF][START_REF] Ihara | Paternal Poly (ADP-ribose) Metabolism Modulates Retention of Inheritable Sperm Histones and Early Embryonic Gene Expression[END_REF][START_REF] Van Der Heijden | Sperm-derived histones contribute to zygotic chromatin in humans[END_REF].

In summary, sperm DNA compaction is highly complex and involves a multitude of steps and molecules. Interestingly, the transition from nucleosome to nucleoprotamine does not result in total erasure of epigenetic markers, as evidenced by the presence of histones and histone variants in mature sperm. Histones are only one facet of epigenetic control over transcription/translation, and the question of the significance of other markers in paternal inheritance remains to be evaluated.

Paramutations in sperm and the discovery of the importance of

RNA in paternal heritage

Paramutations were first described in plants as an epigenetic modification of one allele induced by crosstalk between the two alleles of the same gene, giving rise to a new phenotype for the modified allele [START_REF] Chandler | Paramutation: from maize to mice[END_REF]. Importantly, despite its name, paramutation causes changes in the expression of the affected allele without any DNA mutation. Nevertheless, the modified locus can induce a similar change in homologous sequences, and consequently this change of allelic expression is transmitted to future generations and is thus heritable. In fact, paramutation allows the transmission of heritable characteristics through epigenetic mechanisms. Paramutation was reported in mice for the Kit gene [START_REF] Rassoulzadegan | RNA-mediated nonmendelian inheritance of an epigenetic change in the mouse[END_REF]. The Kit gene, coding for a tyrosine kinase receptor, is essential for the maintenance of primordial germ cells (PGCs) in both sexes in the embryo, but it is also expressed in adult testis where it is required for DNA synthesis in differentiating spermatogonia [START_REF] Rossi | Role of c-kit in mammalian spermatogenesis[END_REF]. In addition, Kit is expressed in melanocytes and is necessary for melanin expression. Kit-null animals (insertion of a lacZ cassette within the gene, Kit tm ) are not viable, in contrast to heterozygous animals Kit +/tm , which have a white tail tip and white fingers [START_REF] Rassoulzadegan | RNA-mediated nonmendelian inheritance of an epigenetic change in the mouse[END_REF] . Interestingly, the authors reported that WT animals (Kit +/+ ) obtained from crosses between two heterozygote animals (Kit +/tm ) or a heterozygote animal and a WT animal also had a white tail tip and white fingers, despite their full WT genotype (Figure 3). This observation suggests that the Kit + allele from the heterozygous animal has been modified (called Kit*). The whitespotted phenotype was transmitted to the F2 generation, even though no Kit tm allele was present, but vanished in subsequent generations. Levels of the polyadenylated messenger RNA for Kit were generally decreased in animals with white-spotted fur and a full WT genotype (paramutated animals), but spliced fragments of the full Kit transcript with multiple sizes were strongly expressed in the testis. More surprisingly, extensive deregulation of the temporal window of Kit mRNA expression was observed, with strong expression in postmeiotic cells in heterozygous males compared to WT animals, where Kit mRNA was barely detectable. Moreover, Kit mRNA is not eliminated in the residual bodies during the sperm cytoplasmic reduction, and remains present in mature epididymal sperm, suggesting a role for Kit mRNA fragments in the transmission of the white-spotted phenotype. This role for fragmented Kit mRNA in the paramutation was demonstrated by injecting sperm mRNA extracts from heterozygous males into WT oocytes. The offspring expressed the white-spotted phenotype, in contrast to offspring from WT males. The role of fragmented Kit mRNA was also supported by the fact that an injection of 2 microRNA species (miR-221 and miR-222) targeting Kit mRNA mimicked the effect of sperm RNA injection. Importantly, animals generated from WT sperm and miRNA-injected WT oocytes could transmit the white-spotted phenotype to the F2 generation.

These results allowed the authors to propose that the modified phenotype is induced by the transfer of cleaved Kit RNA molecules to embryos. These findings demonstrated that microRNA injected into the zygote can induce paramutation by altering gene expression, and thus confirmed that sperm miRNA can alter the phenotype of the progeny. This idea was supported by Grandjean and coll. [START_REF] Grandjean | The miR-124-Sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth[END_REF], who showed that injection of miR-124 induced a large body size as a result of modification of Sox9 RNA, and Wagner and coll. [START_REF] Wagner | RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse[END_REF], who demonstrated that microinjection of miR-1 led to deregulation of the transcript Cdk9, a transcription factor, and cardiac hypertrophy in mice. In both cases, the phenotypes were transmitted to subsequent generations. Heterozygous mice carry a wild-type (+) and a modified/inactivated Kit allele (tm), and bear a whitespotted phenotype with white fingers and white tale tips, while wild-type homozygotes have solid color. The heterozygote males produce abnormal Kit RNA from the Kit tm allele, which are packaged in the sperm and delivered to the egg upon fertilization. Some resulting progeny with two wild-type alleles develop the heterozygote white-spotted phenotype (Kit*). The aberrant RNAs are transmitted to the progeny and thought to regulate expression of the wild-type allele in the zygote, thus propagating the paramutation to the progeny. The establishment and transgenerational inheritance of the paramutation also requires the expression of an RNA methyltransferase Dnmt2 [START_REF] Kiani | RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2[END_REF], but its role remains unclear. The paramutation might gradually disappear over successive generations due to the reduction in aberrant Kit RNAs.

3. Sperm contains numerous messenger RNA (mRNA) and noncoding RNA (ncRNA)

The coding region of DNA represents only 2.5% of the genome. Nevertheless, most of the genome (~85%) can be transcribed as non-coding RNA (ncRNA). There are two types of ncRNAs, housekeeping RNA such as transferRNA (tRNA) and ribosomalRNA, and regulatory ncRNAs, which have many functions, including post-transcriptional regulation of mRNA. For a detailed review of the numerous classes of regulatory ncRNA and more detail on their structure and functions, we refer readers to [START_REF] Hammond | An overview of microRNAs[END_REF][START_REF] Hombach | Non-coding RNAs: Classification, Biology and Functioning[END_REF]. Here, we will focus only on short ncRNAs, including Piwiinteracting RNA (piRNA), microRNA (miRNA), small interfering RNAs (endo-siRNAs), and transferRNAderived fragments (tRFs).

Short ncRNAs can be split into two classes: those coded at specific loci in the genome, and those resulting from endonucleolytic cleavage of other types of RNA. The first class corresponds to piRNA, miRNA, and endo-siRNA. piRNA and miRNA genes, located throughout the genome, are non-coding genes which exclusively generate ncRNA. Some miRNAs are located within introns or untranslated regions (UTR) of protein coding genes. Endo-siRNAs have only been mapped to genomic areas that produce transcripts capable of forming double-stranded RNA (dsRNA) structures, such as retrotransposons or bidirectional transcription sites. tRFs, in contrast, are generated through the cleavage of transfer RNA by endonucleases such as Angiogenin, DICER, and probably some uncharacterized proteins.

During the cytoplasmic sperm compaction occurring at the end of spermiogenesis, most of the cytoplasm, including ribosomes, the endoplasmic reticulum, and the Golgi apparatus, are discarded in the residual body. Moreover, as explained above, the sperm DNA becomes highly compacted as nucleosomes are replaced by protamines, eventually resulting in closed chromatin. As a result of these changes, transcription and translation are considered to be arrested in sperm. Nevertheless, RNA was first detected in sperm over 30 years ago [START_REF] Pessot | Presence of RNA in the sperm nucleus[END_REF]. The more recent development of high-throughput sequencing technologies has revealed the vast diversity of RNA present, with more than 10,000 distinct transcripts identified [START_REF] El Fekih | Sperm RNA preparation for transcriptomic analysis: Review of the techniques and personal experience[END_REF][START_REF] Selvaraju | Occurrence and functional significance of the transcriptome in bovine (Bos taurus) spermatozoa[END_REF][START_REF] Sendler | Stability, delivery and functions of human sperm RNAs at fertilization[END_REF]. Various types of RNA were identified, including messenger RNAs and several classes of ncRNAs, including short and long ncRNAs [START_REF] Dadoune | Spermatozoal RNAs: what about their functions?[END_REF]. Moreover, RNA-seq technology identified both full and truncated RNAs, which is an interesting result when considering Kit-induced paramutation results. A multispecies sperm-borne RNA database to allow comparative annotation of all sperm RNA has been established [START_REF] Schuster | SpermBase: A Database for Sperm-Borne RNA Contents[END_REF]. When sperm RNAs were discovered, they were at first considered to be remnants of spermatogenesis. However, the studies showing that microinjections of RNAs into the zygote alter the heredity of the progeny over several generations [START_REF] Grandjean | The miR-124-Sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth[END_REF][START_REF] Wagner | RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse[END_REF] 3.a MicroRNA (miRNA) and small interfering RNAs (endo-siRNAs) MicroRNA (miRNA) and small endogenous interfering RNAs (endo-siRNAs) are small ncRNAs characterized by transcript sizes smaller than 200 nt. Single-stranded miRNAs are generally the most abundant class of small ncRNAs, mediating gene silencing at the post-transcriptional level. Doublestranded siRNAs are key players in the RNA interference (RNAi) pathway, an alternative pathway for post-transcriptional gene silencing. Considerable light into the molecular processes governing small RNA biogenesis pathways has been shed by several teams: for miRNA, the canonical pathway involves two endoribonucleases, DROSHA in the nucleus and DICER in the cytoplasm, which sequentially generate the active, ~22-nucleotide mature miRNA from the hairpin shaped primary miRNA [START_REF] Hammond | An overview of microRNAs[END_REF] (Figure 4). siRNA are produced from double-stranded RNA by DICER, without input from DROSHA. Both ncRNA are loaded onto the RNA-induced silencing complex (RISC), which cleaves the target complementary mRNA to induce RNA decay and gene silencing (Figure 3).

As they target the 3′ untranslated regions of mRNAs, miRNA are not very specific, and a single miRNA is predicted to induce the cleavage of hundreds of mRNAs. In contrast, siRNAs are more specific.

More than 2000 distinct miRNAs have been characterized so far, targeting around 1/3 of all mRNAs. [START_REF] Chu | Small RNAs: regulators and guardians of the genome[END_REF][START_REF] Lam | siRNA Versus miRNA as Therapeutics for Gene Silencing[END_REF], [START_REF] Chu | Small RNAs: regulators and guardians of the genome[END_REF] and Clancy, S (2008)). AGO: Argonaute; endo-siRNA: endogenous small interfering RNA; miRNA: micro-RNA; pre-miRNA: precursor miRNAs; ORF: open reading frame; RISC: RNA-induced silencing complex RNA interference pathways, and particularly those involving miRNAs, are emerging as important regulators of spermatogenesis and epididymal maturation. Complete and ubiquitous deletions of DICER or DROSHA are lethal, but their deletion in germ cells only causes male and female infertility, demonstrating the importance of ncRNAs during gametogenesis. Around 600 distinct miRNAs have been described in the mouse testis [START_REF] Schuster | SpermBase: A Database for Sperm-Borne RNA Contents[END_REF], and both miRNAs and siRNAs are abundantly expressed during spermatogenesis [START_REF] Song | Male germ cells express abundant endogenous siRNAs[END_REF]. The nature and copy number of miRNAs changes during sperm maturation [START_REF] Nixon | The microRNA signature of mouse spermatozoa is substantially modified during epididymal maturation[END_REF]; their origins and importance will be discussed below.

3.b Transfer RNA-derived fragments (tRFs)

Transfer RNAs, key elements of the transcriptional / translational machinery, have a characteristic shape, with several domains, organized from 3' to 5': the TψC arm, variable loop, anticodon loop and the D arm. Endonucleases can generate two types of transfer RNA-derived fragments (tRFs). In response to stress, Angiogenin cuts the anticodon loop and produces halved fragments. Other cleavage events, orchestrated by DICER, Angiogenin, and other uncharacterized enzymes near the Dor TψC-arms produce small RNA known as 5' tRFs and 3' tRFs, respectively [START_REF] Keam | tRNA-Derived Fragments (tRFs): Emerging New Roles for an Ancient RNA in the Regulation of Gene Expression[END_REF] (Figure 5). Numerous functions have been attributed to tRFs. The first to be identified was a function similar to miRNA (see above), silencing gene expression by binding to the 3' UTR of mRNAs. Other functions were since identified, such as direct regulation of translation by tRFs binding to ribosomes.

Finally, tRFs can also form complexes with RNA-binding proteins such as Ybx1 to regulate RNA degradation and stability. Mature sperm from the cauda epididymis contain numerous ncRNA, and 80% of them are tRFs, mostly 5' tRFs [START_REF] Peng | A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm[END_REF][START_REF] Sharma | Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals[END_REF]. Processing of mature tRNA can give rise to small RNA fragments. tRNA-derived fragments are shorter than tRNA halves and are produced by the action of Angiogenin and Dicer. In mammalian cells under stress, a cleavage in the anticodon loop produces tRNA halves by the action of the nuclease Angiogenin (ANG). tRNA fragments identified in mouse cauda sperm are predominantly enriched in fragments derived from the 5' ends of tRNAs (in red), as shown by [START_REF] Sharma | Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals[END_REF] 3.c Piwi-interacting RNA (piRNA)

Numerous transposable elements, likely derived from viruses, are found in mammalian genomes.

Their movement can disrupt gene expression when transposed into exons, introns, or into 5' or 3' -UTRs. It is thus crucial to inhibit their dissemination to preserve the integrity of the genome [START_REF] Parhad | Rapid evolution and conserved function of the piRNA pathway[END_REF]. Dissemination is prevented in germ cells by the expression of 22-27-nt small-ncRNA, known as piRNA, the primary function of which is transposon silencing. piRNA, located in clusters, are expressed from the pre-pachytene stage, and their production is DICER-independent (Figure 6). The piRNA silencing pathway involves several proteins from the PIWI clade of Argonaute proteins, such as MILI, MIWI2, and MIWI. Mutations that disrupt this pathway cause transposon overexpression, leading to sterility. Interestingly, a second wave of piRNA synthesis occurs over the course of the pachytene stage, during which several hundred piRNAs with unique sequences are expressed from piRNA clusters containing few transposons and repeat sequences. The piRNAs expressed during this second wave control the expression of genes coding for proteins involved in meiosis [START_REF] Goh | piRNAdirected cleavage of meiotic transcripts regulates spermatogenesis[END_REF], and massively eliminate the corresponding mRNAs at the elongated spermatid stage [START_REF] Gou | Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis[END_REF].

Figure 6: A simplified mechanism of piRNA biosynthesis and function. OR The mechanism of piRNA biosynthesis and function.

Mammalian piRNAs derive from distinct transposon sequences known as piRNA clusters. They can be transcribed in the sense or antisense direction, and give rise to single-stranded long non-coding RNA transcripts. The precursors are transported into the cytoplasm by an unknown mechanism and processed in a Dicer-independent manner. Sense and antisense transcripts are in turn loaded onto PIWI clade proteins and trimmed to the length of mature piRNA, their 5' and 3' ends are modified, and they are used in an amplification loop to generate other complementary mature sense and antisense piRNAs. Mature piRNAs are mainly involved in transposon silencing by repressing their transcription or by cleaving their mRNAs. Transposon transcriptional silencing is thought to occur when a piRNA-guided PIWI protein recognizes a nascent transposon transcript in the nucleus and recruits a DNA and histone methylation machinery to the transposon locus, which silences transcription. In the cytoplasm, piRNA induces post-transcriptional silencing of target transposon transcripts in an RNAi-like manner. In mouse spermatocytes, pachytene piRNAs are depleted of transposon sequences, and these piRNAs are thought to regulate gene expression during meiosis and late spermiogenesis, but the exact mechanism is unknown. (Adapted from [START_REF] Meister | Argonaute proteins: functional insights and emerging roles[END_REF][START_REF] Ozata | PIWI-interacting RNAs: small RNAs with big functions[END_REF]).

Origin of sperm ncRNA

Some of these RNAs appear to be deeply embedded in the sperm nucleus, since harshly purified nuclei devoid of acrosomes, plasma membrane, perinuclear theca, and nuclear envelope still contain ncRNA and mRNA [START_REF] Yan | Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei[END_REF]. Due to their deep localization, they are probably produced during spermatogenesis and not discarded during sperm cytoplasm compaction. Interestingly, when these purified sperm heads were injected into artificially activated oocytes, 2-cell embryos developed, 17% of which produced live pups when re-implanted in foster mothers. Furthermore, the purification of epididymal sperm heads and flagella indicate that ncRNAs are present in both subcellular compartments, but that the composition of these ncRNAs differs between the two compartments.

Whereas the heads contain mostly tRFs, the flagella are comparatively enriched in piRNA and miRNAs [START_REF] Sharma | Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm[END_REF].

Sperm released into the lumen of the seminiferous tubules transit through the epididymis and are then stored in the cauda epididymis until ejaculation. The epididymis is separated into several functional areas. This very long epithelial tubule is composed of several cell types, including secretory cells. It is well known that during epididymal transit the composition of sperm changes dramatically, and that these changes are necessary for sperm to become competent. For instance, the lipid composition of the plasma membrane is extensively changed as a result of phospholipase A2 secretion, in addition group III sPLA2 [START_REF] Sato | Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice[END_REF] and proteins important for sperm/egg binding are inserted into the plasma membrane of the sperm as it travels [START_REF] Caballero | CD9-Positive Microvesicles Mediate the Transfer of Molecules to Bovine Spermatozoa during Epididymal Maturation[END_REF][START_REF] Da Ros | From the epididymis to the egg: participation of CRISP proteins in mammalian fertilization[END_REF]. Epididymal proteins are transferred to the sperm by the fusion of small membrane vesicles known as epididymosomes. Interestingly, Belleannée and coll. [START_REF] Belleannee | Epididymosomes convey different repertoires of microRNAs throughout the bovine epididymis[END_REF] showed that these vesicles can transport miRNA, and hypothesized that via this route miRNA synthesized and secreted in one epididymal region could modulate the function of epididymal cells in another region.

Since these epididymosomes can fuse with sperm, they could also provide new ncRNAs to sperm. To verify this hypothesis, several types of experiments were performed.

First, the composition of ncRNA in sperm was measured using RNA sequencing on immature sperm (testicular sperm) and maturing sperm located in different regions of the epididymis. These analyses showed that the ncRNA composition of sperm changes dramatically, with loss and acquisition of ncRNA, mostly in the distal part of the epididymis [START_REF] Nixon | The microRNA signature of mouse spermatozoa is substantially modified during epididymal maturation[END_REF]. A study tracking the composition of the ncRNA in the testis from pachytene spermatocytes to immature sperm cells, and in the epididymis from caput sperm to cauda sperm indicated that the predominant ncRNA in testis are piRNA, and that transit through the epididymis is associated with a loss of almost all piRNAs and a remarkable increase in tRFs [START_REF] Sharma | Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm[END_REF]. The results of this study also confirmed a notable qualitative and quantitative increase in miRNAs during transit through the caput epididymis (Figure 7A).

Second, to demonstrate that these changes were due to uptake of epididymosomes, the concentration of ncRNA was measured in immature sperm before and after co-incubation with caput epididymosomes. Reilly and coll. [START_REF] Reilly | Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome[END_REF] showed that this co-incubation actually increased the RNA copy numbers for miR-191, miR-375, miR-467a, miR-467d, miR-467e demonstrating that the sperm RNA payload is strongly influenced by secretions in the epididymis. Similarly, another comparison of ncRNA content between testicular sperm and testicular sperm incubated with caput epididymosomes indicated that incubation with caput epididymosomes changes the composition of ncRNA, with notable increases in miRNA, tRFs and a decrease in piRNA [START_REF] Sharma | Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals[END_REF][START_REF] Sharma | Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm[END_REF].

Epididymosomes not only provide new epididymal ncRNA to sperm but also increase the copy number of ncRNA in sperm. These observations reinforce the overall potential epigenetic influence of sperm after fertilization. The composition of sperm ncRNA and their evolution from the testis to the cauda epididymis is shown in figure 7A.

Functions of sperm-borne ncRNA

5.a Role of sperm-borne ncRNA in embryonic development

Although the presence of several types of ncRNA, including tRFs, miRNA, siRNA, and piRNA was clearly demonstrated in mature cauda epididymis sperm, the function of the sperm ncRNA upon fertilization and early embryo development may not be so clear-cut because (i) the quantity of sperm-borne ncRNA delivered at fertilization into the oocyte remains very low compared to the amount already present within the oocyte, (ii) the most abundant sperm miRNA are also present in the oocyte before fertilization [START_REF] Amanai | A restricted role for sperm-borne microRNAs in mammalian fertilization[END_REF], (iii) oocyte maturation and preimplantation embryonic development are not affected by complete depletion of maternal miRNA, as demonstrated by Dgcr8 oocyte-deficient females, (of note, this observation suggests that the miRNAdependent gene silencing pathway is not activated at these stages [START_REF] Suh | MicroRNA function is globally suppressed in mouse oocytes and early embryos[END_REF]) and (iv) In several species including mouse and Human, injection of round spermatids and testicular sperm, with a very different miRNA contents than fully mature sperm, permits to obtain live healthy offspring [START_REF] Tanaka | Fourteen babies born after round spermatid injection into human oocytes[END_REF] Altogether, these results raise questions about any role of sperm-borne ncRNA, and in particular miRNA function in the development of mammalian zygotes and embryos. A. The composition of sperm ncRNA changes dramatically as cells travel from the testis to the cauda epididymis. In the testis, the most abundant type of ncRNA is piRNA, which plays a crucial role in transposon control. After spermiation, sperm transit through the caput epididymis, where tRFs and miRNAs are gained. piRNAs are no longer present, but how they are eliminated remains unknown. In the cauda epididymis, the miRNA composition is also strongly modified. B. In the absence of miRNAs and tRFs, embryo development is abnormal. The miRNA are also key nucleic acids for transgenerational adaptation in response to paternal environmental stress. Adapted in part from [START_REF] Sharma | Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals[END_REF][START_REF] Sharma | Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm[END_REF] To address these questions, several approaches were used to investigate the role of sperm ncRNAs in fertilization and embryo development:

Although tissue-specific inactivation of DICER or DROSHA in male germ cells leads to infertility, some non-motile and abnormal sperm are produced that can be used for ICSI. Interestingly, embryos generated with sperm lacking DICER or DROSHA and WT oocytes developed poorly in comparison to those generated with WT sperm and oocytes, but their development was improved when KO sperm were co-injected with small amounts of ncRNA from WT sperm [START_REF] Yuan | Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development[END_REF]. The absence of DICER affected around 47% of sperm miRNAs and 8% of sperm siRNAs, and the absence of DROSHA had an impact on around 52% of sperm miRNAs (DROSHA is not involved in siRNA biogenesis),

suggesting that some ncRNAs produced during spermatogenesis play a crucial role in embryonic development. Interestingly, DICER and DROSHA-deficient sperm did not perform equally. Embryos generated with DICER-deficient sperm developed poorly at all stages (from 2-cell to blastocyst), whereas those generated with DROSHA-deficient sperm developed almost normally up to the 4-cell stage, from which point they showed marked developmental defects. These results suggest that sperm-borne siRNA plays a role in the early stages of embryonic development, whereas sperm-borne miRNA are more important at later stages, beyond the 4-cell stage. These results also highlight a long-term effect of these sperm-borne ncRNAs, after embryonic gene activation, which occurs at the 2-cell stage in mice. It is interesting to note that the improvement was greater when total WT RNA was injected than with small RNA only, suggesting that sperm not only deliver regulatory RNA molecules but also mRNA.

To assess the importance of the different types of ncRNA in embryo development, and in particular ncRNAs provided by epididymosomes, embryos were generated by ICSI with testicular, caput and cauda sperm, and embryo development was studied. mRNA from embryos were sequenced at various stages (4-cell, 8-cell, morula, and blastocyst). Overexpression of transcripts coding for RNAbinding proteins (Hnrnpab, Hnrnpu, Pcbp1, Eif3b, Yrdc, Ythdf1, Srsf2, and Ybx1) and chromatinassociated factors (Smarcd2, Smarca5, Smarcc1, Trim28, and Ezh2) was observed only in embryos generated with caput sperm and not those generated with cauda sperm [START_REF] Conine | Small RNAs Gained during Epididymal Transit of Sperm Are Essential for Embryonic Development in Mice[END_REF]. These changes affected embryo development: 50% of embryos were absent at day 7.5 of gestation, suggesting that their post-implantation development was abnormal, and finally no pups were obtained at term. This result suggests that the ncRNA gained as sperm pass through the caput transiently make sperm unable to sustain embryo development and that the ncRNA payload is drastically modified in the cauda epididymis. Interestingly, co-incubation of caput sperm with epididymosomes purified from the cauda epididymis rescued the level of expression of transcripts for RNA-binding and chromatin-associated factors in embryos, allowing the embryos to develop to term [START_REF] Conine | Small RNAs Gained during Epididymal Transit of Sperm Are Essential for Embryonic Development in Mice[END_REF]. Remarkably, only the injection of miRNAs, but not tRFs, purified from cauda sperm restored normal embryo development following fertilization with caput sperm. This observation suggests that miRNAs gained during epididymal transit are involved in embryo development, and that the tRFs acquired play a distinct role. Several sperm-borne miRNAs were identified as crucial for early embryo development, such as miR-34c, which is involved in DNA synthesis and inhibition of Bcl-2 expression in the zygote. Inhibition of miR-34c leads to failure of the first mitosis [START_REF] Liu | Sperm-borne microRNA-34c is required for the first cleavage division in mouse[END_REF].

5.b Roles of ncRNAs in the environmental stress-response

Environmental injuries suffered by the parents and their impact on phenotypes in the next generation were first discovered by a study monitoring the health of adults whose mothers lived through the Dutch famine during the Second World War. The results showed that the adults exposed to famine in utero during the first trimester of pregnancy had a higher risk of obesity and those exposed in the third trimester had a lower risk [START_REF] Ravelli | Obesity in young men after famine exposure in utero and early infancy[END_REF]. Most early studies on transgenerational effects of parental stress focused on the influence of metabolic stress over the course of pregnancy. The effects of preconceptional stress on female gametes were more difficult to demonstrate since any preconceptional stress that affects oogenesis may also have an impact on the subsequent pregnancy. Nevertheless, a recent study showed that corticotropin releasing factor type 1 (CRF1) mRNA, a key component in the stress response, is increased in eggs harvested from stressed female rats, and that offspring obtained after mating stressed females with control males exhibited higher levels of CRF1 in the brain and behavioral defects [START_REF] Zaidan | Prereproductive stress to female rats alters corticotropin releasing factor type 1 expression in ova and behavior and brain corticotropin releasing factor type 1 expression in offspring[END_REF]. Thus, female germ cells appear to be able to transmit stress-induced traits to their progeny. Similar experiments aiming to demonstrate transgenerational transmission of environmental stress by sperm were easier to carry out since stress suffered by the male impacts neither full embryo development nor the raising of pups, which are mother-dependent.

Obesity is a significant health issue, and several epidemiologists have compared the increase in the rate of obesity in Western countries to an epidemic-like phenomenon, suggesting possible transmission of this condition. The first observations that a father's diet can affect his daughters' health, leading to obesity and diabetes, were reported quite recently [START_REF] Ng | Chronic high-fat diet in fathers programs beta-cell dysfunction in female rat offspring[END_REF]. This paternal diet-induced phenotype is associated with reprogramming of metabolic gene expression in liver due to alteration of epigenetic marks [START_REF] Carone | Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals[END_REF]. These first reports were followed by numerous studies investigating the transgenerational impact of paternal diet on the progeny's susceptibility to metabolic disorders, and seeking to identify the molecular basis for its transgenerational transmission. The transmission of the phenotype was observed up to the third generation. A high-fat diet (HFD) leads to extensive modifications to the ncRNA content in both testis and sperm cells [START_REF] Fullston | Paternal obesity initiates metabolic disturbances in two generations of mice with incomplete penetrance to the F2 generation and alters the transcriptional profile of testis and sperm microRNA content[END_REF] and miRNAs, piRNAs, and tRFs were found to be differentially expressed in sperm from animals consuming the HFD, piRNA being the most likely (de Castro [START_REF] De Castro Barbosa | High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring[END_REF].

Interestingly, Let-7c, one miRNA that is upregulated in sperm from males fed with HFD , was also upregulated in adipose tissues of their F1 offspring. This miRNA is known to regulate glucose homeostasis and insulin sensitivity, and has been linked to a predisposition for type-2 diabetes. The importance of ncRNA in paternal transmission of obesity and diabetes was also supported by an experiment where ncRNA were injected into zygotes: the injection of spermatic ncRNA from a male fed with the HFD into a control zygote led to metabolic disorders in the progeny, whereas injection of spermatic ncRNA from a male fed with the control diet had no effect [START_REF] Grandjean | RNA-mediated paternal heredity of diet-induced obesity and metabolic disorders[END_REF].

Interestingly, the injection of miR19b into a control zygote mimicked the phenotype observed after injection of ncRNA from the HFD male. It is worth noting that the various reports on miRNAs involved in the paternal transmission of metabolic disorders fail to agree. Even more surprisingly, the change in miRNA content induced by HFD is not observed in sperm from male progeny, despite the fact that they can transmit the metabolic phenotype to their own offspring, the grand-children of HFD males [START_REF] Fullston | Sperm microRNA Content Is Altered in a Mouse Model of Male Obesity, but the Same Suite of microRNAs Are Not Altered in Offspring's Sperm[END_REF]. This unexpected result strongly suggests that components other than miRNA are also important for transgenerational paternal effects. This hypothesis was recently confirmed by Sharma and coll. [START_REF] Sharma | Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals[END_REF], who showed that sperm from males consuming a low-protein diet contains higher levels of tRF-Gly-GCC, that regulates the endogenous retroelement MERVL.

Importantly, the long terminal repeat (LTR) in MERVL regulates a subset of genes expressed at high levels in preimplantation embryos. The impact of the low-protein diet on gene expression in preimplantation embryos was reproduced by microinjection of a synthetic tRF-Gly-GCC oligo [START_REF] Sharma | Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals[END_REF]. A HFD also deregulates several thousand long ncRNAs in sperm, and it was thus suggested that these long ncRNA could be involved in paternal heredity (An et al, 2017). Taken together, these results suggest that stress activates the production of numerous ncRNA types in the testis and/or epididymis, and that it consequently affects gene expression in post-fertilization embryos at multiple levels.

It is worth noting the plasticity of ncRNA production. The transgenerational metabolic phenotype can easily be reversed by exercise. Progeny produced from males fed a HFD exhibit metabolic disorders than can be restored when obese fathers engage in low-to moderate-intensity exercise for 2 months [START_REF] Mcpherson | An Exercise-Only Intervention in Obese Fathers Restores Glucose and Insulin Regulation in Conjunction with the Rescue of Pancreatic Islet Cell Morphology and MicroRNA Expression in Male Offspring[END_REF][START_REF] Mcpherson | Preconception diet or exercise intervention in obese fathers normalizes sperm microRNA profile and metabolic syndrome in female offspring[END_REF]. This phenotype reversion following exercise is supported by the fact that physical training modifies sperm ncRNA, and in particular a subset of piRNAs in humans [START_REF] Ingerslev | Endurance training remodels sperm-borne small RNA expression and methylation at neurological gene hotspots[END_REF] as well as a subset of miRNA (miR-19b, miR-455 and miR-133a) and two species of tRF derived from tRNA-Gly and tRNA-Pro in mice [START_REF] Short | Exercise alters mouse sperm small noncoding RNAs and induces a transgenerational modification of male offspring conditioned fear and anxiety[END_REF]. As with diet, there is no consensus on the type and identity of the ncRNA induced in sperm by physical training.

Transgenerational stress transmission by sperm is not restricted to metabolic stress, and several reports suggest that physical stress (loud noises, predator odor, physical constraints, etc.) or hormonal stress (cortisone injection) experienced by the father alters behavioral responses to stress in the progeny, partly due to deregulation of the hypothalamic-pituitary-adrenal axis [START_REF] Rodgers | Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation[END_REF][START_REF] Short | Elevated paternal glucocorticoid exposure alters the small noncoding RNA profile in sperm and modifies anxiety and depressive phenotypes in the offspring[END_REF]. Importantly, the stress experienced by the father modifies a specific subset of spermatic miRNAs: nine miRNA (miR-193-5p, miR-204, miR-29c, miR-30a, miR-30c, miR-32, miR-375, miR-532-3p, and miR-698) were determined to be over-expressed by (Rodgers et al., 2013), and three (miR-98, miR-144 and miR-190b) were reported by [START_REF] Short | Elevated paternal glucocorticoid exposure alters the small noncoding RNA profile in sperm and modifies anxiety and depressive phenotypes in the offspring[END_REF]. Another observation of the transgenerational impact of stress applied early during life reported effects on 73 miRNAs, including miR-375-3p and -5p, miR-200b-3p, miR-672-5p, and miR-466-5p, which were upregulated [START_REF] Gapp | Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice[END_REF]). Yet another report indicated that more than 100 miRNAs were modified in response to hormonal stress [START_REF] Chan | Reproductive tract extracellular vesicles are sufficient to transmit intergenerational stress and program neurodevelopment[END_REF]. It is important to highlight that there was no overlap in deregulated miRNA expression in sperm between these reports, although all linked the effects to the hypothalamic-pituitary-adrenal axis. Furthermore, injection of the nine miRNA reported by [START_REF] Rodgers | Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation[END_REF] into a control zygote fully reproduced the disruption of the hypothalamic-pituitary-adrenal axis observed with sperm from stressed fathers, confirming the importance of these miRNAs [START_REF] Rodgers | Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress[END_REF]. Interestingly, injection of just one miRNA partly reproduced the stress phenotype, suggesting that the combination of several miRNAs is important for transgenerational stress adaptation. The injection of the nine miRNAs modified the expression of 75 mRNAs present in oocytes, including genes playing roles in chromatin remodeling. This epigenetic control induced long-term modifications of gene expression in the target tissue, with 298 genes found to have altered expression in the paraventricular nucleus of the hypothalamus [START_REF] Rodgers | Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress[END_REF]. Interestingly, changes to miRNA levels were observed up to 12 weeks after a short initial stress, suggesting that stress has a long-term effect on the transcriptomic program of epididymal cells [START_REF] Chan | Reproductive tract extracellular vesicles are sufficient to transmit intergenerational stress and program neurodevelopment[END_REF]. Thus, physical stress experienced by the father induces altered stress responsivity in offspring, but physical exercise produces a compensatory anxiolytic effect in the progeny [START_REF] Short | Exercise alters mouse sperm small noncoding RNAs and induces a transgenerational modification of male offspring conditioned fear and anxiety[END_REF].

Finally, stress-induced ncRNA modifications of the father's sperm modify the cancer susceptibility of the progeny, in particular for breast cancer following metabolic stress [START_REF] Da Cruz | Paternal malnutrition programs breast cancer risk and tumor metabolism in offspring[END_REF], Fontelles et al, 2016), and medulloblastoma following irradiation [START_REF] Paris | Transgenerational inheritance of enhanced susceptibility to radiation-induced medulloblastoma in newborn Ptch1(+)/(-) mice after paternal irradiation[END_REF].

In conclusion, the composition of sperm ncRNA is very sensitive to environmental stress, and these changes can have long-term effects on the expression of genes in various tissues, allowing rapid adaptability of the progeny without the need for DNA mutations, which are rare and random and therefore require a large population and many generations to achieve efficient adaptability.

The functions of the different ncRNAs are summarized in figure 7B.

6. Importance of sperm DNA methylation in the environmental stress-response DNA methylation, which consists in the methylation of cytosine residues in CpG dinucleotides to form 5-methylcytosine, is a well-characterized mechanism driving gene expression. DNA methylation of a gene promotor is known to inhibit transcription of the corresponding gene. Consequently, DNA methylation is an important regulator of spermatogenesis, and its deregulation is associated with multiple forms of male infertility [START_REF] Gunes | The role of epigenetics in idiopathic male infertility[END_REF]. DNA methylation of male germ cells is mostly acquired in utero, and is completed by the end of the pachytene spermatocyte stage [START_REF] Oakes | Developmental acquisition of genomewide DNA methylation occurs prior to meiosis in male germ cells[END_REF].

There is now some evidence that the sperm methylation profile influences the phenotype of the progeny. First, several researchers modified the level of folate in the paternal diet [START_REF] Lambrot | Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes[END_REF][START_REF] Ly | Intergenerational impact of paternal lifetime exposures to both folic acid deficiency and supplementation on reproductive outcomes and imprinted gene methylation[END_REF]. Folate is a precursor of co-enzymes necessary for several synthetic pathways, including those producing purine bases or thymidine monophosphate. These co-enzymes are also methyl donors and are involved in DNA methylation among other things. When the paternal diet lacks folate, a significant change in sperm DNA methylation is observed, with 57 genomic regions showing altered methylation profiles [START_REF] Lambrot | Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes[END_REF]. Notably, these different methylated sites are close to the TSS of genes involved in development. These changes were associated with developmental abnormalities in the progeny, supporting the importance of sperm DNA methylation in the phenotype of the progeny. Second, reproduction in dairy cattle mainly relies on artificial insemination, and the best bull sires can have several thousand offspring. It is thus possible to study any relationship between sperm methylation and complex phenotypes in the offspring. In the study by [START_REF] Liu | Analyses of inter-individual variations of sperm DNA methylation and their potential implications in cattle[END_REF], the levels of methylation of variably methylated regions (VMRs) in sperm from 19 sires correlated with complex traits in their daughters including reproduction, milk production, and health. A high level of correlation was observed between methylation levels of VMR and reproductive traits, clearly demonstrating the intergenerational transmission of traits through DNA methylation [START_REF] Liu | Analyses of inter-individual variations of sperm DNA methylation and their potential implications in cattle[END_REF].

We showed above that ncRNA are involved in the paternal epigenetic legacy, and in particular in the transgenerational transmission of environmental insults. Is there any evidence showing that methylation of sperm DNA also contributes to the paternal legacy? First, the possible role of sperm DNA methylation in the transgenerational transmission of obesity was observed in several studies.

Obesity in rats induces a change in the methylation profile of epididymal sperm, and 18 regions were identified as differentially methylated between control rats and rats fed a HFD. Interestingly, the same regions were also differentially methylated in sperm from F1 offspring, but not in control progeny. Some regions were found to be hyper-or hypo-methylated (de Castro [START_REF] De Castro Barbosa | High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring[END_REF].

Similarly, physical training modified the methylation profile of sperm in humans, and around 300 differentially-methylated regions were identified following a 6-week exercise regimen [START_REF] Ingerslev | Endurance training remodels sperm-borne small RNA expression and methylation at neurological gene hotspots[END_REF]. Second, paternal stress can also modify the behavior of offspring in rodents. For instance, males separated from their mothers at an early age showed anxiety in adulthood. This separation anxiety induced a change in the methylation of genes, including MeCP2, cannabinoid receptor 1 (CB1), and corticotropin releasing factor receptor 2 (CRFR2) in sperm. The offspring and grandoffspring of these males, reared normally, also showed anxious behavior and similar alterations of the methylation of these genes in neural tissues [START_REF] Franklin | Epigenetic transmission of the impact of early stress across generations[END_REF].

The transgenerational transmission of methylation profiles and their contribution to the phenotype of progeny remain a mystery when considering the two waves of general erasing of methylation marks. The first wave occurs just after fertilization and preserves only imprinted marks [START_REF] Abdalla | Active demethylation of paternal genome in mammalian zygotes[END_REF][START_REF] Jenkins | Dynamic alterations in the paternal epigenetic landscape following fertilization[END_REF] ), and the second wave of DNA demethylation occurs before meiosis in PGCs in the developing embryo, corresponding, respectively, to embryonic days E11.5 and E12.5 in mice [START_REF] Yamaguchi | Tet1 controls meiosis by regulating meiotic gene expression[END_REF]; for review, see [START_REF] Cantone | Epigenetic programming and reprogramming during development[END_REF]. It is worth highlighting that in most studies relating to transgenerational transmission of environmental stress through paternal germ cells, simultaneous changes were observed in both methylation profiles and ncRNA content in sperm. It is therefore difficult to weigh up the respective contributions of DNA methylation and ncRNA in the paternal legacy. Surprisingly, the changes in ncRNA observed in the father are generally not observed in the subsequent generation even though the phenotype is transmitted to the grandoffspring [START_REF] Gapp | Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice[END_REF]. It is therefore possible that the paternal stress-induced ncRNA signal is converted into DNA methylation or histone post-translational modifications. Are ncRNA and DNA methylation two sides of the same coin? If DNA methylation is involved in such a process, it suggests that specific methylation marks made in response to environmental events are protected from the first wave of demethylation occurring within the zygote, in a similar way to imprinted marks, or that the transmitted ncRNA could contribute to apposing these marks after the demethylation wave. In contrast to most of the genome, which is compacted with protamines, imprinted genes remain associated with histones in nucleosomes. The presence of nucleosomes at these loci allows them to escape from the general erasing of marks. The localization of stress-dependent methylation marks in regions of compacted DNA in sperm with nucleosomes, and their escape from the first demethylation wave in zygotes remain to be formally demonstrated. Moreover, it is expected that species such as humans containing a larger proportion of sperm chromatin packaged in nucleosomes are more sensitive to environmental stress than species with a more limited proportion of their DNA compacted in nucleosomes, such as murine or bovine species. This hypothesis deserves further exploration.

Histones and other proteins as sperm-borne factors

As indicated in the first paragraph above, nucleosomes retained in sperm are important for paternal genome activation, and subsequent embryo development. Histones play key roles in epigenetics, and their post-translational modifications (PTM) such as methylation, acetylation, crotonylation, …, allow transcription to be adapted by changing the relaxation state of DNA. The histones retained in sperm carry several types of PTM (La [START_REF] Spina | Heterogeneous distribution of histone methylation in mature human sperm[END_REF][START_REF] Steilmann | Presence of histone H3 acetylated at lysine 9 in male germ cells and its distribution pattern in the genome of human spermatozoa[END_REF], and these histone marks have been described as repressive or active [START_REF] Brykczynska | Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa[END_REF].

As expected from the localization of the histones retained in the promotors of genes important for development (see above), changing the histone marks in sperm has a negative impact on embryo development. For instance, reducing H3K4 and H3K9 methylation in sperm by overexpression of histone H3 lysine 4 demethylase (KDM1A) increased embryo resorption during pregnancy and the rate of morphological defects in live offspring [START_REF] Perez-Cerezales | Elimination of methylation marks at lysines 4 and 9 of histone 3 (H3K4 and H3K9) of spermatozoa alters offspring phenotype[END_REF][START_REF] Siklenka | Disruption of histone methylation in developing sperm impairs offspring health transgenerationally[END_REF].

More surprisingly, developmental and morphological defects were trans-generationally transmitted from the F1 to the F2 generation, even though germ cells in F1 males lacked KDM1A overexpression [START_REF] Siklenka | Disruption of histone methylation in developing sperm impairs offspring health transgenerationally[END_REF]. This finding suggests that epigenetic marks at the histone level also support transgenerational adaptation to stress. Modification of histone marks was also observed with Vinclozolin, an agricultural fungicide used as a model to study transgenerational inheritance of chemical stress. Vinclozolin is an endocrine disruptor that decreases sperm count and viability, causing decreased male fertility [START_REF] Anway | Epigenetic transgenerational actions of endocrine disruptors and male fertility[END_REF]. In this model, the F1 generation is treated at embryonic stages, and thus the F2 is produced from treated sperm. Studying transgenerational inheritance in this model revealed extra areas of histone H3 retention in sperm from the F3 generation of Vinclozolin-exposed lineages compared to a control lineage (Ben [START_REF] Maamar | Epigenetic Transgenerational Inheritance of Altered Sperm Histone Retention Sites[END_REF].

Unexpectedly, the number of histone retention sites was normal in sperm from the F2 generation but increased in sperm from the F3 generation and beyond [START_REF] Skinner | Alterations in sperm DNA methylation, non-coding RNA and histone retention associate with DDT-induced epigenetic transgenerational inheritance of disease[END_REF], suggesting that histone modifications may be induced by other epigenetic factors. In addition to histone modifications, Vinclozolin treatment induces DNA methylation and alters the ncRNA payload [START_REF] Skinner | Alterations in sperm DNA methylation, non-coding RNA and histone retention associate with DDT-induced epigenetic transgenerational inheritance of disease[END_REF], and the various contributions and interactions of these epigenetic modifications to the infertility phenotype remain to be clarified.

Finally, apart from the sperm factor PLCzeta, which triggers oocyte activation and the absence of which leads to human infertility [START_REF] Escoffier | Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP[END_REF][START_REF] Parrington | The eggstraordinary story of how life begins[END_REF], other sperm-borne proteins have been shown to play a role in embryo development. For instance, glutathione-Stransferase omega 2 (GSTO2), localized in the perinuclear theca of the sperm head, in conjunction with GSTO from the oocyte speeds up sperm DNA decondensation. Selective inhibition of GSTO2 in sperm before ICSI delays sperm DNA decondensation, leading to abnormal cleavage timings, and ultimately to a decreased rate of blastocyst formation (Hamilton et al, 2019a).

Perspectives and discussion

The discovery of the role of ncRNA in embryo development and adaptation to environmental stress has potentially significant medical consequences and technical applications.

8.a Medical implications

Several reports indicated that analysis of the ncRNA composition of semen may be predictive of the fertilization competence of sperm [START_REF] Cui | Spermatozoa micro ribonucleic acid-34c level is correlated with intracytoplasmic sperm injection outcomes[END_REF], Munoz et al, 2015[START_REF] Salas-Huetos | Spermatozoa from patients with seminal alterations exhibit a differential micro-ribonucleic acid profile[END_REF].

ncRNA profiling of infertile males may therefore be of interest to identify possible epigenetic causes for couples presenting with embryonic development failures after several cycles of ovarian stimulation. Nevertheless, all sperm-borne miRNAs necessary for optimal embryo development in humans remain to be characterized. Because of extensive variability in ncRNA between species, the short list identified so far in mice may not be relevant for human fertility. This identification step is required before any attempt to rescue or optimize embryonic development using synthetic ncRNA can be made.

We have also shown above that environmental insults can modify the ncRNA load in sperm and have dramatic effects on the physiopathology of the progeny, including the emergence of metabolic disorders and stress responses. A transient change in lifestyle, with a balanced diet, physical exercise and stress management approaches will likely modify the sperm ncRNA payload and could be recommended to couples consulting for infertility, to optimize the chances of fertilization, embryo development, and the health of any child born.

Finally, it would be interesting, for ICSI with testicular sperm to co-inject the most important and specific epididymal ncRNAs to improve embryo development. Again, this necessitates to characterize them in the human species because of species variability.

8.b Technical applications

The most promising application of the discovery of the role of ncRNA in embryo development is in embryo cloning from somatic cells. It is well known that embryos obtained through somatic cell nuclear transfer (SCNT) show abnormal development, with disrupted kinetics of the first cleavage and defects in histone methylation, leading to low live birth rates and overweight offspring.

Interestingly, co-injection of purified sperm ncRNAs along with a somatic cell nucleus decreases histone H3 methylation (H3K9me3), and increases K40 acetylation of tubulin, both of which are associated with increased SCNT yields [START_REF] Qu | Sperm-borne small RNAs regulate alpha-tubulin acetylation and epigenetic modification of early bovine somatic cell nuclear transfer embryos[END_REF]. Among the classes of miRNAs involved in embryo development, miR-449a seems to be relevant since, when SCNT is performed with coinjection of miR-449a, the cleavage rate increases, epigenetic reprogramming of the embryo is improved, and the apoptotic index of blastocysts decreases [START_REF] Wang | Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine[END_REF]. Sperm ncRNA is also a marker of the quality of sperm and could be used in animal reproduction to select bulls with a high fertility index as, for example, the level of expression of miR-216b in sperm is inversely correlated with the fertility index for bulls [START_REF] Alves | Sperm-borne miR-216b modulates cell proliferation during early embryo development via K-RAS[END_REF].

Conclusion

The data presented in this review show that the spermatozoon not only provides the paternal DNA component, but also a large number of epigenetic regulators, which influence embryonic development and allow rapid adaptation of the offspring to environmental stress. Much remains to be discovered, such as the identity and function of all the ncRNAs involved in embryonic development and stress adaptation. The targets of these ncRNAs and the signaling pathways

activated have yet to be characterized, and also need to be investigated.

Figure 1 .

 1 Figure 1. Illustration of the homunculus 49 hypothesis proposed by Antoni Van 50 Leeuwenhoek in 1678 and illustrated by 51Hartsoecker in 1694[START_REF] Hartsoecker | Essay de dioptrique Paris Hertwig O (1876)[END_REF].52"Courtesy of The Linda Hall Library of Science, 53Engineering & Technology" 54

  Figure 1. Illustration of the homunculus 49 hypothesis proposed by Antoni Van 50 Leeuwenhoek in 1678 and illustrated by 51Hartsoecker in 1694[START_REF] Hartsoecker | Essay de dioptrique Paris Hertwig O (1876)[END_REF].52"Courtesy of The Linda Hall Library of Science, 53Engineering & Technology" 54 55 56

Figure

  Figure 2. Stress (Nutritional, physical, …) induces epigenetic changes in the male reproductive organs,

Figure 3 :

 3 Figure 3: Model for transgenerational inheritance of paramutated Kit.

  stimulated a reevaluation of the role of the various endogenous sperm RNAs in embryo development and epigenetic inheritance, as explained below.

Figure 4 :

 4 Figure 4: The mechanism of miRNA and endo-siRNA biosynthesis and function. miRNA and endo-siRNA are involved in post-transcriptional gene silencing through translational inhibition and mRNA decay, or mRNA cleavage by Argonaute and subsequent degradation. (Adapted from[START_REF] Chu | Small RNAs: regulators and guardians of the genome[END_REF][START_REF] Lam | siRNA Versus miRNA as Therapeutics for Gene Silencing[END_REF],[START_REF] Chu | Small RNAs: regulators and guardians of the genome[END_REF] andClancy, S (2008)). AGO: Argonaute; endo-siRNA: endogenous small interfering RNA; miRNA: micro-RNA; pre-miRNA: precursor miRNAs; ORF: open reading frame; RISC: RNA-induced silencing complex

Figure 5 :

 5 Figure 5: tRNA fragments derived from mature tRNAs.Processing of mature tRNA can give rise to small RNA fragments. tRNA-derived fragments are shorter than tRNA halves and are produced by the action of Angiogenin and Dicer. In mammalian cells under stress, a cleavage in the anticodon loop produces tRNA halves by the action of the nuclease Angiogenin (ANG). tRNA fragments identified in mouse cauda sperm are predominantly enriched in fragments derived from the 5' ends of tRNAs (in red), as shown by[START_REF] Sharma | Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals[END_REF]

Figure 7 .

 7 Figure 7. Origin and functions of sperm ncRNA.