Oliver Knapp

Elke Maier

Claudio Piselli

Roland Benz

Cezarela Hoxha

Dr Michel R Popoff
email: popoff2m@gmail.com

Central residues of the amphipathic β-hairpin loop control the properties of Clostridium perfringens epsilon-toxin channel

Keywords: Epsilon-toxin, Clostridium perfringens, pore-formation, lipid membrane, programmed cellular necrosis, ion selectivity

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Clostridium perfringens is a Gram-positive anaerobic bacterium. Its subtypes B and D are one of the etiological agent of fatal enterotoxaemia in domestic animals like sheep, goat and calves and are rarely isolated from humans [START_REF] Popoff | Epsilon toxin: a fascinating pore-forming toxin[END_REF][START_REF] Songer | Clostridial enteric diseases of domestic animals[END_REF][START_REF] Uzal | Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases[END_REF]. The major virulence factor of C. perfringens type B and D is Epsilon-toxin (ETX), a pore-forming toxin belonging to the aerolysin-like toxin family and one of the most potent clostridial toxins [START_REF] Knapp | The Aerolysin-like toxin family of cytolytic, pore-forming toxins[END_REF][START_REF] Payne | The Clostridium perfringens epsilon-toxin[END_REF]. Overgrowth of ETX-producing C. perfringens in the intestinal content of animals, notably sheep, due to change in alimentary conditions results in high levels of ETX in the intestinal content and subsequent passage through the intestinal barrier and dissemination to several organs mainly brain and kidneys. ETX can pass the blood-brain barrier and targets specific neuronal cells such as glutamatergic neurons and oligodendrocytes leading to excessive release of glutamate. Thereby, sheep enterotoxemia is characterized by acute excitatory neurological symptoms and kidney necrosis that are rapidly fatal [START_REF] Popoff | Epsilon toxin: a fascinating pore-forming toxin[END_REF][START_REF] Dorca-Arevalo | Binding of epsilon-toxin from Clostridium perfringens in the nervous system[END_REF][START_REF] Soler-Jover | Distribution of Clostridium perfringens epsilon toxin in the brains of acutely intoxicated mice and its effect upon glial cells[END_REF][START_REF] Wioland | Attack of the nervous system by Clostridium perfringens Epsilon toxin: from disease to mode of action on neural cells[END_REF][START_REF] Wioland | Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination[END_REF][START_REF] Uzal | The pathology of peracute experimental Clostridium perfringens type D enterotoxemia in sheep[END_REF][START_REF] Miyamoto | Clostridium perfringens epsilon toxin causes excessive release of glutamate in the mouse hippocampus[END_REF]. In contrast, no ETX-dependent acute neurological disease has been reported in humans. However, ETX is a demyelinating toxin [START_REF] Wioland | Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination[END_REF][START_REF] Linden | Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination[END_REF] and recent studies support that ETX might be a causative agent of multiple sclerosis [START_REF] Wagley | Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis[END_REF][START_REF] Rumah | Isolation of Clostridium perfringens type B in an individual at first clinical presentation of multiple sclerosis provides clues for environmental triggers of the disease[END_REF].

ETX is secreted as inactive protoxin monomers, which have to undergo proteolytic activation through cleavage of 13 and 29 residues at the N-and C-termini, respectively [START_REF] Minami | Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N-and C-terminal peptides[END_REF]. Proteolytic activation occurs by serine-type proteases like trypsin, α-chymotrypsin or the C. perfringens lambda protease in the gut of infected animals or in vitro [START_REF] Miyata | Cleavage of C-terminal peptide is essential for heptamerization of Clostridium perfringens ε-toxin in the synaptosomal membrane[END_REF]. Activated monomers bind to specific receptor, oligomerize into a heptameric complex which then inserts into the host cell membrane [START_REF] Miyata | Cleavage of C-terminal peptide is essential for heptamerization of Clostridium perfringens ε-toxin in the synaptosomal membrane[END_REF][START_REF] Petit | Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex[END_REF].

However, ETX binds only to a restricted number of cell lines that include MDCK, human renal leiomyoblastoma G-402, and mouse renal mpkCCD [START_REF] Shortt | An assessment of the in vitro toxicology of Clostridium perfringens type D epsilon-toxin in human and animal cells[END_REF]. ETX recognizes the myelin and lymphocyte protein (MAL) as a specific receptor [START_REF] Blanch | The Cytotoxicity of Epsilon Toxin from Clostridium perfringens on Lymphocytes Is Mediated by MAL Protein Expression[END_REF][START_REF] Rumah | The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens epsilon-Toxin[END_REF], and the lipid environment of the membrane receptor plays also a role in ETX interaction with cell membranes [START_REF] Gil | Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide[END_REF]. ETX pores in target cell membranes lead to a rapid change in membrane permeability and ultimately cell death [START_REF] Miyata | Clostridium perfringens ε-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby Canine Kidney Cells and rat synaptosomes[END_REF][START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF][START_REF] Shimamoto | Changes in ganglioside content affect the binding of Clostridium perfringens epsilon-toxin to detergent-resistant membranes of Madin-Darby canine kidney cells[END_REF]. The channel-forming domain of ETX was revealed through the comparison of the primary structure of ETX with that of the channel-forming stretches of a variety of binding components of A-B-types of toxins such as Anthrax protective antigen (PA), C2II of C2-toxin and Ib of Iota-toxin and through the introduction of point mutations in the putative channel-forming domain [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF] and (Fig. 1). These findings are in agreement with the structure of ETX monomers and the recently resolved structure of the ETX pore assembled on membrane [START_REF] Cole | Clostridium perfringens ε-toxin shows structural similarity to the pore-forming toxin aerolysin[END_REF][START_REF] Savva | The pore structure of Clostridium perfringens epsilon toxin[END_REF]. ETX monomer shares a similar structure with that of aerolysin, which is the prototype of this toxin family, except that ETX lacks the N-terminal domain 1 of aerolysin [START_REF] Cole | Structural studies on epsilon toxin from Clostridium perfringens[END_REF][START_REF] Parker | Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states[END_REF]. Moreover, ETX and aerolysin assemble in a similar heptameric structure on target cell membranes [START_REF] Savva | The pore structure of Clostridium perfringens epsilon toxin[END_REF][START_REF] Degiacomi | Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism[END_REF][START_REF] Iacovache | Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process[END_REF]. ETX and the other aerolysin family toxins are potent cytotoxins which are active at nM range. They recognize distinct receptors and thus target different cell types. Aerolysin and C. septicum alpha toxin which belong to the aerolysin toxin family, bind to different sets of GPI-anchored proteins and target a broad range of cell types. However, ETX which recognizes a more specific receptor (MAL), is only cytotoxic for a restricted number of cell types [START_REF] Gordon | Clostridium septicum alpha-toxin uses glycosylphosphatidylinositol-anchored protein receptors[END_REF][START_REF] Hong | Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha-toxin[END_REF][START_REF] Kennedy | Programmed cellular necrosis mediated by the pore-forming alpha-toxin from Clostridium septicum[END_REF][START_REF] Knapp | Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis[END_REF][START_REF] Abrami | The glycan core of GPI-anchored proteins modulates aerolysin binding but is not sufficient: the polypeptide moiety is required for the toxin-receptor interaction[END_REF][START_REF] Gurcel | Aerolysin and related Aeromonas toxins[END_REF][START_REF] Nelson | Channel formation by the glycosylphosphatidylinositol-anchored protein binding toxin aerolysin is not promoted by lipid rafts[END_REF]. ETX has a much more potent lethal activity (50% lethal dose LD50 70 ng/kg of mice by intraperitoneal route) [START_REF] Minami | Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N-and C-terminal peptides[END_REF] than aerolysin and C. septicum alpha toxin (LD50 7 µg/kg and 10-14 µg/kg of mice by intravenous route, respectively) [START_REF] Amimoto | Protective effect of Clostridium septicum alpha-toxoid vaccine against challenge with spores in guinea pigs[END_REF][START_REF] Bernheimer | Partial characterization of aerolysin, a lytic exotoxin from Aeromonas hydrophila[END_REF][START_REF] Ballard | Purification and characterization of the lethal toxin (alpha-toxin) of Clostridium septicum[END_REF], likely due to the fact that ETX interacts with critical cells in the host such as kidney cells, oligodendrocytes and some neuronal cells [START_REF] Soler-Jover | Distribution of Clostridium perfringens epsilon toxin in the brains of acutely intoxicated mice and its effect upon glial cells[END_REF][START_REF] Wioland | Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination[END_REF][START_REF] Fernandez Miyakawa | Clostridium perfringens epsilon toxin is cytotoxic for human renal tubular epithelial cells[END_REF][START_REF] Lonchamp | Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release[END_REF][START_REF] Morris | Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes[END_REF][START_REF] Soler-Jover | Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo[END_REF].

Heptamers of aerolysin family toxins form small pores (about 1 nm diameter) in the plasma membrane of target cells which are the initial step of cell death by necrosis [START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF][START_REF] Iacovache | Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process[END_REF][START_REF] Chakraborty | Aerolysin of Aeromonas sobria: evidence for formation of ion-permeable channels and comparison with alpha-toxin of Staphylococcus aureus[END_REF][START_REF] Sellman | Generation of a membrane-bound, oligomerized pre-pore complex is necessary for pore formation by Clostridium septicum alpha toxin[END_REF][START_REF] Navarro | Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins[END_REF]. They use a common mechanism of pore formation consisting of binding of toxin monomers to cell surface receptor, heptamerization and formation of a prepore, conformational change of the trans-membrane domain and insertion of β-amphipathic hairpins assembled in β-barrel into the lipid membrane [START_REF] Podobnik | Molecular mechanism of pore formation by aerolysin-like proteins[END_REF].

ETX rapidly decreases intracellular K+, increases Cl-as well as Na+, followed by a later entry of calcium [START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF]. This results in massive ATP depletion, stimulation of the AMP-activated protein kinase (AMPK), mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of the apoptosis-inducing factor (AIF) [START_REF] Chassin | Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells[END_REF]. However, the specific signaling pathway from pore formation to cell necrosis is not fully understood.

We previously determined that the ETX pore forming domain segment is localized from His151 to Ala181 [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. We confirmed that Lys162 and Pro166 are located in the loop between the two β-strands forming the transmembrane β-hairpin of the channel. Indeed, when Lys162 and Pro166 were replaced by histidine, the channel activity was blocked with anti-His6-tag antibodies. In addition, Ser156 and Thr175 were localized in the hydrophilic side of the channel lumen since when they were replaced by Cys these residues were accessible to methanethiolsulfonate [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. In this study, we further investigated the role of the central residues of each β-strand of the amphipathic β-hairpin and facing the channel lumen by replacement with charged residues. Our experiments allow a better understanding of the mechanism of ETX interaction with target cells and of fundamental function of pore forming toxins in general.

Materials and Methods

Generation of Mutants and Isolation of ETX Proteins

ETX mutants were generated by the "Megaprime Mutagenesis" method and cloned into the expression plasmid pGEX-T2 (Amersham Pharmacia). A detailed description of the method was recently given by Knapp et al., 2009 [25]. Primers used for the generation of the mutants are shown in Table 1. DNA sequence of the mutants was checked by DNA sequencing and recombinant plasmids were transformed into Escherichia coli strain BL21. Recombinant strains were grown in LB medium supplemented with ampicillin (100 µg/mL) at 37°C until OD 0.8 was reached. Protein production was induced with isopropyl-1-thio-β-D-galacto-pyranoside (0.5 mM IPTG) while cells were grown at 32.5°C for an additional 18-20 h. Bacteria were collected by centrifugation, suspended in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, and lyzed by sonication. It is noteworthy that GST-ETX and GST-ETX mutants did not need to be activated because the peptides removed by the activation process from the N-and C-termini of ETX were already missing in the recombinant GST-ETX molecules.

Purification occurred with the help of a glutathione Sepharose column (Amersham Bioscience) according to the manufacturer recommendations. The obtained glutathione-S-transferase (GST) fusion proteins were dialyzed against phosphate buffered saline/glycerol (1:1) and analyzed by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE). Total protein content was assayed with the Bradford reagent. The purity of the proteins was analyzed by SDS-PAGE as previously described (Sup Fig. S1) [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF].

Mass spectrometry of GST-ETX T175E, GST-ETX S156E-Q158E and GST-ETX S156E-Q158E-S173E-T175E

To verify that the 55 kDa bands in the SDS-PAGE of Sup. Figure 1 are indeed derivatives of recombinant ETX, the 55 kDa bands from a similar SDS-PAGE of GST-ETX, GST-ETX 175E, the double mutant GST-ETX S156E-Q158E and the quadruple mutant GST-ETX S156E-Q158E-S173E-T175E were excised from the gel and were subjected to trypsin treatment followed by mass spectrometry. Nineteen different peptides were identified as partial peptides of the ETX part of the protein with the accession number WP_006248023.1 of GST-ETX T175E as it is shown in Sup. Figure 2 for the sequence of ETX/recombinant ETX. The partial peptides covered the sequence of recombinant ETX in this case by 49%. For the other three ETX mutants the coverage was 40%. The results of the mass spectrometry verified that we were indeed here working with GST-ETX mutants of C. perfringens.

Lipid Bilayer Experiments

Black lipid bilayer membranes were formed of a 1% solution of diphytanoyl phosphatidylcholine (DiPh-PC) (Avanti Polar Lipids, Alabaster, AL) in n-decane, as described previously [START_REF] Benz | Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli[END_REF]. GST-ETX and its mutants (1 to 5 nM) were added to the aqueous phase bathing membranes in the black state.

The electrical measurements were performed using Ag/AgCl electrodes (with salt bridges) connected in series to a voltage source and a home-made current-to-voltage converter. The amplified signal was recorded on a strip chart recorder (Rikadenki, Freiburg, Germany). Zero-current membrane potential measurements were performed by establishing fivefold KCl gradients across membranes containing 100 to 1000 channels as described earlier [START_REF] Benz | Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli[END_REF].

Cell Culture

Madin-Darby canine kidney (MCDK) cells, were grown in Dulbecco`s modified Eagel + GlutaMax TM -I (+4.5 g/l Glucose, -Pyruvate) (GIBCO ® )-Medium (DMEM) containing 100 units/ml penicillin G sodium and 100 µg streptomycin sulfate (Penicillin-Streptomycin, GIBCO ® ). Cell culture medium was supplemented with 10 % fetal bovine serum (GIBCO ® ) and cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Cell viability assay

The cytotoxic effect of GST-ETX and GST-ETX mutants were analyzed on MDCK cells using the MTT test as previously described [START_REF] Soler-Jover | Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo[END_REF][START_REF] Petit | Clostridium perfringens Epsilon Toxin rapidly decreases membrane barrier permeability of polarized MDCK Cells[END_REF]. MTT (Sigma Aldrich). MDCK cells were grown until confluence and then treated for five hours with different toxin concentrations (starting concentration of 25 µg/ml). Control cells were incubated only with DMEM. Thereafter, 50 µl of 0.5 mg/ml MTT-solution was added and the cells were incubated for 1 h at 37°C. Cells were lyzed and transformation of MTT to blue Formazan was stopped by adding 100µl of lysis buffer (20% SDS, 50% dimethylformamide, pH 4.6) [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF][START_REF] Petit | Clostridium perfringens Epsilon Toxin rapidly decreases membrane barrier permeability of polarized MDCK Cells[END_REF]. Formazan concentration was measured after 18 h at 540 nm in a 96-microtiter plate.

The experiments were performed three times for each protein, and means and standard failure were calculated from the results.

Propidium iodide influx studies

Confluent cells grown on 96-well plates were incubated with or without GST-ETX and GST-ETX mutants and with propidium iodide (PI, 5 µg ml -1 ). At the indicated times, the plates were read with a spectrofluorimeter (Fluoroskan II) (excitation 380 nm and emission 620 nm). The results were expressed as the percentage of fluorescence obtained compared to non-intoxicated cells incubated with 0.5% Triton X100 at 37°C.

Results

Channel formation of ETX mutants in artificial lipid bilayer membranes

ETX and aerolysin share a similar structure of their transmembrane domain. The β-strands of the β-amphipathic hairpin are rich in the polar residues serine and threonine which likely facilitate membrane insertion. However, in contrast to aerolysin which contains glutamic residues in the amphipathic β-strands, ETX has no charged residues in the corresponding structure. A conserved glutamic residue lies in the C-terminus of the loop connecting the two amphipathic β-strands in ETX and aerolysin (Fig. 1). Thus, we investigated the role of charged residues in the amphipathic β-strands of ETX on its channel properties. Mutations were introduced in central residues of each β-strand forming the amphipathic β-hairpin and facing the channel lumen that were supposed to be critical for the channel activity (Fig. 1 and2). ETX wild type and mutants were produced as recombinant proteins with GST-tag (Fig. S1). Experiments with lipid bilayer membranes represent an excellent tool to directly study channel formation by ETX because this toxin forms small ion-permeable channel in artificial membranes without the need of any receptor [START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF][START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. Channel formation occurred in lipid bilayer membranes made from DiPh-PC/n-decane by both wt ETX and recombinant GST-ETX with identical channel properties [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF], as confirmed in this study (Table 2, Sup Fig. S3 andS4). In addition, no significant difference in cytotoxic activity was observed between wt ETX and GST-ETX as previously reported [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. Therefore, we used the different ETX-mutants directly as GST fusion proteins in defined concentrations (1 to 5 nM) to study their channel-forming properties in lipid bilayer membranes. All recombinant GST-ETX mutants were able to increase the conductance of lipid bilayer membranes except the ETX-mutant with the β-hairpin removed. This mutant with β-hairpin deletion did not show any channel-forming activity (data not shown). The GST-ETX-mutant increased membrane conductance by several orders of magnitude during approximately 30 minutes over the conductance of membranes without channels (about 50 to 100 GΩ, corresponding to a specific conductance of about 0.1 µS/cm 2 ). We observed some deviation in channel-forming activity as the overview given in Table 2 demonstrates. In particular, the ETX double-mutants GST-ETX S156E-Q158E and GST-ETX S156E-T175E and the GST-ETX triple-mutant GST-ETX S156E-S173E-T175E exhibited some lower activity when compared to the other mutants (Table 2).

However, since also these mutants showed the formation of regular channels (see below) the reason for this lower activity is not clear, in particular as mutants containing the same two mutations and an additional third one had a higher channel-forming ability than the mutants with only two point mutations did. • Conductance and zero-current membrane potentials for wild type ETX were taken from [START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF] and those for wild type GST-ETX and the mutant GST-ETX S156E from [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF].

b The ground level of specific conductance of unmodified DiPh-PC membranes (i.e. in the absence of GST-ETX and its mutants) was about 0.1 µS/cm 2 . The increased conductance ++ corresponded to a specific conductance in a range of 10 µS/cm 2 and +++ in a range of 100 µS/cm 2 for the addition of about 1 nM GST-ETX and its mutants to one side of the lipid bilayers. Addition of the ETX-mutants at lower concentration (around 1 nM) allowed the observation of conductance steps, similar to those shown in Fig. 3 for all mutants. The conductance steps for all the mutants were quite homogeneous as the histograms shown in Fig. 4 for the single mutant ETX GST-ETX T175E and the quadruple mutant GST-ETX S156E-Q158E-S173E-T175E. These two histograms were chosen as examples for the histograms obtained for the conductance fluctuations of all the other mutants (Sup Fig. S5). Most histograms were narrow as that of GST-ETX T175E shows.

We observed only for the quadruple mutant GST-ETX S156E-Q158E-S173E-T175E a relatively broad distribution (Fig. 4). The single channel conductance (± SD) of all GST-ETX mutants studied here in are given in Table 2 for two different KCl concentrations. The results demonstrate that regular channels with relatively small deviations were observed for both KCl concentrations. In 1 M KCl solution the mutant GST-ETX T175E had approximately 510 pS in 1 M KCl a similar single channel conductance as wt ETX. All the other mutants showed a much smaller conductance around 350 to 400 pS in 1 M KCl (Table 2 and Sup Fig. S5). Similar results were also obtained at 0.1 M KCl, where the single channel conductance was about 40 pS approximately one tenth of the conductance at 1 M KCl, which seems to be reasonable, because the conductance of non-selective channels such as wt ETX, GST-ETX, and the GST-ETX mutants should show an almost linear dependence on the aqueous salt concentration. Only in the case of the mutant GST-ETX S173E-T175E, the single-channel conductance was about one-half of that expected, but further mutations of this mutant had a similar conductance in 0.1 M KCl as all the other GST-ETX mutants (Table 2). The decreased conductance of this mutant at low KCl concentration remains to be determined, it could not result from fewer oligomers than heptamers forming the pore.

Zero-current measurements and ion selectivity of the ETX mutants

The other important information that also could be obtained from electrophysiological experiments with lipid bilayer membranes in the presence of the different GST-ETX mutants was the ion selectivity. Wildtype ETX is slightly anion selective for anions and cations with the same aqueous mobility such as potassium ions and chloride [START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF][START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. Exchange of strategically positioned amino acids (i.e. in or near the pore-forming domain) through negatively charged ones should change the selectivity. To obtain information about the selectivity of the different GST-ETX mutant channels we performed zero-current membrane potential measurements in the presence of 5-fold KCl gradients.

After reconstitution of a considerable number of mutant channels into the lipid bilayers at 0.1 M KCl we generated a five-fold KCl gradient across the membranes by addition of concentrated KCl solution to the cis-side under stirring. The zero-current membrane potential was measured about 5 minutes after generation of the KCl gradient. The results of these measurements are also given in Table 1.

Only the single ETX mutant GST-ETX S175E and the double mutant GST-ETX S156E-T175E were anion-selective similar to wildtype ETX. All the other mutants formed cation-selective channels with increasing selectivity for potassium ions for increasing number of mutations. The ETX mutant GST-ETX S156E-Q158E-S173E-T175E, where four neutral amino acids were replaced by glutamic acids showed the highest cation selectivity of all ETX mutants.

Pore-forming activity on MDCK cells and cytotoxicity

GST-ETX wt and mutant activity on MDCK cells was checked by monitoring the cell viability using the MTT test (Fig. 5A and5B) and the entry of propidium iodide (PI) as previously investigated [START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF][START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. GST-ETX wt showed a concentration-dependent cytotoxicity with an EC50 around 10 -8 M (Fig. 5) and induced PI entry overtime starting 60-90 min after incubation with 10 -8 M toxin concentration (Fig. 5C and5D). The mutant GST-ETX S156E showed the same level of activity in PI entry and only a low decrease in cytotoxicity compared to ETX-GST wt. The double mutant GST-ETX S173E-T175E exhibited a slightly lower activity in PI entry and cytotoxicity compared to the wt. The double mutants GST-ETX S156E-Q158E and GST-ETX S156E-T175E as well as the triple and quadruple mutants were drastically impaired in the cytotoxicity and PI entry (Fig. 5).

Surprisingly, the single mutant GST-ETX T175E did not show any activity in PI entry and cytotoxicity even at high concentration (10 -6 M). The reason of the loss of activity on cells of GST-T175E was unclear. Four experiments with freshly prepared GST-ETX T175E in order to avoid protein degradation gave the same negative results on cells. The same GST-ETX T175E preparation showing a unique band in SDS-PAGE, which formed pores in lipid bilayers, was not active in MDCK cells indicating a possible degradation in the presence of cells such as by membrane-bound protease. GST-ETX T175E incubated with MDCK cell membranes and analyzed by SDS-PAGE and western blot with anti-ETX antibodies showed partial monomer degradation and no or weak formation of oligomers (Fig. 6). This indicated that GST-ETX 175E retained the ability to bind to MDCK cell membranes, but apparently oligomerized poorly if at all and formed no functional channels in cell membranes.

Discussion

ETX is a pore-forming toxin that displays three structural domains showing similarities to the pore-forming toxin aerolysin [START_REF] Cole | Clostridium perfringens ε-toxin shows structural similarity to the pore-forming toxin aerolysin[END_REF]. Domain II contains an antiparallel amphipathic loop with alternate hydrophilic-hydrophobic residues that is predicted to insert into the cell membrane [START_REF] Cole | Clostridium perfringens ε-toxin shows structural similarity to the pore-forming toxin aerolysin[END_REF]. We have previously shown that this region encompassing amino acids 151 to 181 is critical for ETX channel formation and subsequent cytotoxicity [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. Here, we further investigated the properties of the ETX pore-forming domain.

ETX is an anion selective pore-forming toxin as are aerolysin and C. septicum alpha-toxin from the aerolysin toxin family, but ETX retains a much more potent lethal activity than the other heptameric pore-forming toxins [START_REF] Knapp | The Aerolysin-like toxin family of cytolytic, pore-forming toxins[END_REF][START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF]. We found that the residues located in the central part of each β-strand forming the amphipathic β-hairpin and facing the channel lumen are critical for the channel properties. Indeed, replacement of GST-ETX S156 and GST-ETX T175 by a Cys induced a slightly higher cation selectivity, but did not greatly impair the cytotoxicity [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. Addition of methanethiosulfonate reagents resulted in increased cation selectivity and decreased conductance supporting the localization of GST-ETX S156 and GST-ETX T175 in the interior of the hydrophilic channel face and their role in the control of the channel characteristics [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. To better understand the function of these residues, we introduced charged residues at these positions. Replacement of GST-ETX S156 and GST-ETX T175 by the negatively charged glutamic acid, respectively, did not significantly alter the ion selectivity of the mutant T175E and induced a higher cation selective mutant GST-ETX S156E with a lower conductance for GST-ETX S156E. However, GST-ETX S156E showed the same level of activity in PI entry into MDCK cells and only a weak decrease in cytotoxicity compared to the wt, whereas the T175E mutant was totally inactive on cells. The discrepancy between the decreased cytotoxicity of GST-ETX S156E and its conserved activity on PI permeability on cells compared to GST-ETX wt indicates that the general ETX channel permeability as monitored by PI entry is not the only parameter involved in cytotoxicity. There are also other parameters involved in ETX toxicity. Binding to a surface receptor and oligomerization are also prerequisites for channel formation as the key process. If one of these processes is disturbed, intoxication of target cells is impaired. However, even the formation of a small number of pores may be sufficient to start inflammatory disease processes such as respiratory bursts and the release of inflammatory mediators in target cells together with apoptotic processes. In this respect, it is noteworthy that receptors are not needed for the interaction between lipid bilayers and toxins [START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF][START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF][START_REF] Knapp | Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis[END_REF], whereas specific receptor is required on cell membrane. Indeed, only a few cell lines containing specific receptor are sensitive to ETX [START_REF] Petit | Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex[END_REF][START_REF] Blanch | The Cytotoxicity of Epsilon Toxin from Clostridium perfringens on Lymphocytes Is Mediated by MAL Protein Expression[END_REF][START_REF] Rumah | The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens epsilon-Toxin[END_REF].

Permeability to specific ions such as loss of K + and influx of Ca ++ likely contributes more directly to cytotoxicity. Indeed, K + efflux is a major signal induced by pore-forming toxin that triggers cell necrosis and inflammasome activity [START_REF] Broz | Inflammasomes: mechanism of assembly, regulation and signalling[END_REF][START_REF] Conos | Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner[END_REF][START_REF] Gonzalez-Juarbe | Pore-forming toxin-mediated ion dysregulation leads to death receptor-independent necroptosis of lung epithelial cells during bacterial pneumonia[END_REF][START_REF] Gutierrez | MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1beta Independently of Gasdermin-D[END_REF][START_REF] Kloft | Pore-forming toxins activate MAPK p38 by causing loss of cellular potassium[END_REF]. ETX was found to very rapidly and dramatically decrease intracellular K + in MDCK cells that is likely the initial signal leading to cell death [START_REF] Chassin | Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells[END_REF][START_REF] Petit | Clostridium perfringens Epsilon Toxin rapidly decreases membrane barrier permeability of polarized MDCK Cells[END_REF]. All together, these results show that the amino acids located on the central part of the β-strands forming the ETX pore and facing the channel lumen control the size and the ion selectivity of the channel with the 151-161 β-strand residues playing a major role. The modulation of the characteristics of the ETX channel impact the subsequent cytotoxic activity. However, there was no direct correlation between the decrease in conductance levels of change in ion selectivity and the intensity of the cytotoxic activity. This is in agreement with previous findings that ETX channels are general diffusion pores allowing the passage of both anion and cations [START_REF] Knapp | Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX)[END_REF]. Pore formation on cell membrane is a required initial step in ETX cytotoxicity. However, the precise mechanism and signaling pathway between pore formation and subsequent cell death by necrosis are not completely understood [START_REF] Chassin | Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells[END_REF].The initial ETX interaction events include leakage of intracellular K + and ATP, as well as cell entry of Na + and Ca ++ [START_REF] Petit | Clostridium perfringens epsilon-toxin induces a rapid change in cell membrane permeability to ions and forms channels in artificial lipid bilayers[END_REF][START_REF] Chassin | Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells[END_REF]. These cell membrane permeability changes are also shared by other pore forming toxins such as aerolysin and C. septicum alpha-toxin [START_REF] Knapp | Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis[END_REF][START_REF] Gonzalez | Pore-forming toxins induce multiple cellular responses promoting survival[END_REF][START_REF] Kennedy | Pore-forming activity of alpha-toxin is essential for Clostridium septicum-mediated myonecrosis[END_REF]. The signaling pathway from channel activity on plasma membrane to the ETX cytotoxicity remains to be unraveled.

While the activity of ETX mutants on cells showed comparable results between cell membrane permeability as monitored by PI entry and cytotoxicity, no strict correlation was observed between cell tests and ion selectivity as determined in lipid bilayers. As discussed above GST-ETX T175E is likely unstable in cells. In contrast to the other mutants which were cation selective, GST-ETX S156E-T175E was weakly anion selective compared to wt ETX and retained a low cytotoxic activity.

The most cation selective quadruple ETX mutant was inactive on cells, but two cation selective mutants GST-ETX S156E and to a lower extent GST-ETX S173E-T175E showed a cytotoxic activity.

This further supports that the passage of specific ions through the cell membrane instead of the ETX pore general diffusion activity is required to induce cytotoxicity. The functional particularity of ETX channels with the residues in the central position of each β-strand of the amphipathic β-hairpin loop controlling the size and ion selectivity of the channel might account for the cytotoxicity of ETX by modulating the passage of specific compounds involved in cell death pathway. It is noteworthy that the ETX mechanism of cytotoxicity is not fully understood. The interaction of ETX with its cell surface receptor, which is absent in artificial lipid bilayers, might induce a transmembrane signaling leading to cell death independently of pore formation. Indeed, MAL has been involved in carcinogenesis by a yet unknown mechanism [START_REF] Lara-Lemus | On The Role of Myelin and Lymphocyte Protein (MAL) In Cancer: A Puzzle With Two Faces[END_REF]. Moreover, the ETX demyelinating activity on rat cerebellum seemed independent of pore -formation in oligodendrocytes [START_REF] Wioland | Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination[END_REF]. The discrepancy between pore formation in lipid bilayers and cytotoxicity might also result from the differences between cell membrane

Figure 1 .

 1 Figure 1. Sequence comparison of transmembrane domain of ETX with those of aerolysin and the binding components Ib, and C2-II of the binary toxins Iota-and C2-toxin, respectively. Residues that were mutated are indicated above the arrows and are in red.The amino acids are given in the one letter code and residues facing the channel lumen are under-or upper-lined.

Figure 2 .

 2 Figure 2. Location of the mutated residues in the ETX channel structure according to the channel structure modelling of Savva et al. [27] (PDB ID: 6rb9). A) One ETX monomer is highlighted in green and its pore-forming domain (amino acids 151-181) in yellow. The mutated residues are in red. B) The mutated residues are located in the inner side of the channel. Residues of only one monomer are shown.

a

  The membranes were formed of DiPh-PC dissolved in n-decane. The aqueous solutions were unbuffered and had a pH of 6.0. The applied voltage was 50 mV, and the temperature was 20°C. The average single-channel conductance (±SD) was obtained from at least 100 single events. Amino acids are indicated in the single letter code. Zero-current membrane potentials, Vm, were measured in the presence of the different GST-ETX mutants for 5-fold KCl (0.1 M versus 0.5 M) gradients. Vm is defined as the difference between the potential at the dilute side and the potential at the concentrated side (T = 20°C) The permeability ratio PK/PCl was calculated with the Goldman-Hodgkin-Katz equation[START_REF] Benz | Ion selectivity of gram-negative bacterial porins[END_REF] on the basis of at least 3 individual experiments.

Figure 3 .

 3 Figure 3. Current recordings of DiPh-PC/n-decane membranes after addition of recombinant wild type GST-ETX and its mutants to the cis side of different membranes. The aqueous phase contained 1 M KCl, (pH 6) and about 1 nM wild type GST-ETX and GST-ETX mutants added to the cis side of the membrane. The applied membrane potential was 50 mV; T = 20°C.

Figure 4 .

 4 Figure 4. Histograms of the probability of the occurrence of certain conductivity units observed with membranes formed of DiPh-PC/n-decane in the presence of recombinant single mutant GST-ETX T175E (A) and the quadruple mutant GST-ETX S156EQ158E-S173E-T175E (B). The aqueous phase contained 1 M KCl, pH 6. The applied membrane potential was 50 mV; T = 20°C. (A) 1 nM GST-ETX T175E was added to the cis side of the membranes. The solid line represents a fit of the histogram with a Gaussian distribution. The maximum of the distribution is at a probability for single-channel occurrence of 0.497 ± 0.02 and the

Figure 5 .

 5 Figure 5. Activity of GST-ETX and its mutants on MDCK cells. Cytotoxic activity (A and B). Confluent MDCK cells were treated for five hours with a decreasing amount of GST-ETX and its mutants (1:1 dilutions). Starting toxin concentration was 4.3 µM. Control cells were incubated only with DMEM. Thereafter MTT-solution was added and incubated for 1 h at 37C. MTT transformation was stopped by adding 100µl of lysis buffer and measured after 18 h at 540 nm. Data were collected from three experiments and means and standard deviation were calculated. (A) Experiments where the effect of wt GST-ETX is compared to the GST-ETX mutants GST-ETX S156E, GST-ETX S156E-QS158E, GST-ETX S156E-T175E, and GST-ETX S173E-TS175E. Starting toxin concentration was 4.3 µM. (B) Experiments where the effect of wt GST-ETX is compared to the GST-ETX mutants GST-ETX S156E-Q158E-T175E, GST-ETX S156E-S173E-T175E, and GST-ETX S156E-Q158E-S173E-T175E. Starting toxin concentration was again 4.3 µM. Entry of propidium iodide (PI) (C and D). MDCK cells in DMEM medium containing 5 µg•ml -1 PI were incubated with 10 -8 M GST-ETX or its mutants for 20 to 260 min at 37°C. Entry of PI was monitored by spectrofluorometry (excitation 380 nm and emission 620 nm). The data are expressed as percentages of fluorescence compared to the control consisting of cells treated with 0.5% Triton X100. Data were collected from three experiments and means and standard deviation were calculated.

Figure 6 .

 6 Figure 6. Binding of GST-ETX, GST-ETX T175E, and GST-ETX S173E-T175E tMDCK cell membranes. MDCK cell membranes were prepared by incubating MDCK cells in hypertonic buffer (PBS containing 30% sucrose) for 15 min, sonicated (10 s) and centrifuged (14,000 rpm, 20 min),washed with PBS and centrifuged. 30 µg of MDCK cell membranes as assayed by the BioRad protein assay were incubated with 1, 0.5, and 0.2 GST-proteins for 30 min at 37°C in PBS. The preparations were centrifuged (14,000 rpm, 20 min), washed in PBS, and centrifuged. The pellets were dissolved in Laemmli buffer, electrophoresed in SDS-PAGE, transferred on nitrocellulose and blotted with rabbit anti ETX. GST-ETX and GST mutants showed bands to the size of GST-ETX monomer. In contrast to GST-ETX and GST-ETX S173E-T175E which were cytotoxic for MDCK cells, GST-ETX T175E did not show or only poorly oligomer formation.

  Introduction of two additional negative charged residues in the central part of the ETX channel lumen by double mutations (GST-ETX S156E-T175E, GST-ETX S156E-Q158E, GST-ETX S173E-T175E) yielded mutants with a slightly reduced conductance and variable change in ion selectivity. GST-ETX S156E-Q158E and to a lower extent GST-ETX S173E-T175E were cation selective, whereas GST-ETX S156E-T175E remained anion selective, but weaker than the ETX wt. GST-ETX S173E-T175E showed a slight decrease in PI entry and cytotoxicity activities, and the two other mutants were drastically impaired in their activity on cells. Introduction of charged residues on the 151-161 β-strand induced more perturbations in channel and cytotoxic activities than on the 170-181 β-strand. This indicates that the two β-strands forming the architecture of the ETX pore are not symmetrical and that residues on one β-strand facing the channel lumen have a prominent role in controlling the channel characteristics. S156 and Q158 show a more central localization in the ETX channel lumen than S173 and T175, which can modulate their functionality (Fig.2). Additional charged residues in the channel lumen by triple and quadruple mutations increased the cation selectivity and dramatically prevented the pore and cytotoxic activities on MDCK cells.

Table 1 .

 1 Primers for the generation of GST-ETX point and deletion mutants.

	
	Mutation	Primer
	
	S156E	5´-CTGTGGGAACTGAGATACAAGCAACTGCT
		5´-AGCAGTTGCTTGTATCTCAGTTCCCACAG

Table 2 .

 2 Black Lipid Bilayer Data of the channel formed by epsilon-toxin and its mutants:

	Average single-channel conductance, G, in 0.1 and 1M KCl, selectivity, and channel
	forming activity a .
		Single-channel conductance G [pS]	Selectivity	Channel forming activity b
		0.1M KCl	1 M KCl	KCl pH6, 5-fold gradient (0.1 versus 0.5 M)	1M KCl pH6
	Epsilon mutant	G [pS]	V m /mV	P K /P Cl
	Wild type ETX*	60	550	-19.0	0.30	+++
	Wild type GST-ETX *	60	550	-17.9	0.33	+++
	GST-ETX S156E*	40	320	+6.9	1.50	+++
	GST-ETX T175E	45 ± 5.0	510 ± 75	-15.0 ± 1.9	0.40	+++
	GST-ETX S156E-Q158E	35 ± 4.5	385 ± 40	+11.5 ± 1.8	2.0	++
	GST-ETX S156E-T175E	38 ± 4.0	350 ± 43	-1.2 ± 0.3	0.90	++
	GST-ETX S173E-T175E	18 ± 1.9	370 ± 39	+6.6 ± 0.8	1.50	+++
	GST-ETX S156E-Q158E-T175E	37 ± 3.2	360 ± 30	+4.0 ± 0.4	1.25	+++
	GST-ETX S156E-S173E-T175E	42 ± 5.3	340 ± 55	+7.3 ± 0.7	1.53	++
	GST-ETX
	S156E-Q158E-S173E-T175	45 ± 7.2	400 ± 72	+25.4 ± 0.
	E

experiments and cell assays

ACKNOWLEDGEMENTS

The authors would like to thank Stephanie Lamer and Andreas Schlosser from the Rudolf Virchow Center of the University of Würzburg for the mass spectra of the GST-ETX mutants.

COMPETING INTERESTS

The authors declare no competing interests.

SUPPORTING INFORMATION

The paper contains supporting information in the form of 5 supplemental figures S1 to S5.


Sophie Lamothe

Valérie Bernard

Sophie Christin-Maitre
email: sophie.christin-maitre@aphp.fr

GONAD DIFFERENTIATION TOWARD OVARY La différenciation gonadique vers l'ovaire

Keywords: différenciation gonadique, ovaire, gonade, développement ovarien, phénotype, facteur SRY gonad differentiation, ovary, gonad, ovarian development, phenotype, SRY factor

La différenciation gonadique dépend d'un ensemble de signaux cellulaires et hormonaux qui interagissent entre eux dans un ordre défini, avec des fenêtres d'action très précises, pour contribuer à la mise en place de l'appareil génital et à l'établissement d'un phénotype masculin ou féminin. La recherche a initialement progressé sur les étapes de la différenciation de la gonade en testicule, notamment suite à l'identification en 1990 du facteur SRY sur le chromosome Y. Les mécanismes impliqués dans la détermination de la gonade en ovaire ont été plus longs à identifier. Grâce à des patients porteurs de développement sexuel différent (DSD) ou de modèles animaux d'invalidation de gènes, les cascades impliquées dans l'activation et le maintien du développement ovarien ont beaucoup progressé ces dernières années.

Introduction

Gonad determination is a concept that emerged, scientifically, mainly since the late 1950s. The decisive role of the Y chromosome in the transformation of the gonad into the testis represented a major advance in the understanding of gonad determination. In 1990, the SRY factor (sex-determining region of the Y chromosome) was identified. The mechanisms involved in the transformation of the gonad into an ovary took longer to identify. In recent years, knowledge of the factors necessary for differentiation of the gonad toward ovary has progressed markedly. Thanks to patients with different sexual development (DSD) and animal models of gene knock-out, the cascades involved in the activation and maintenance of ovarian development are improving. After a historical review of the theories of sex determination, this chapter will address the different stages and the main factors involved in the formation of the undifferentiated gonad and its transformation toward the ovary, and the factors involved in the maintenance of the ovary.in the adult gonad.

I. HISTORY OF GONAD DETERMINATION

Theories of sexual determination have existed since ancient times. According to Anaxagoras (500-428 BC), if the sperm comes from the right testicle it will produce a boy, and if it comes from the left testicle, a girl. Others, such as Empedocles (496-435 BC), believed that sperm that enter a warm uterus induce males and females when entering in a cold uterus become females, these temperature differences being attributed to colder or warmer menstrual flow. A century later, Aristotle described new theories of sexual determination, according to which the male is the bearer of the divine principle and transmits human nature. The woman, devoid of seed, contributes only raw material to generation. "The very first genetic deviation is the birth of a female instead of a male" (1). Aristotle admitted that the female is necessary to conserve sexual difference; but women are nonetheless a failure of human nature [START_REF] Badinter | L'un est l'autre: des relations entre hommes et femmes[END_REF]. New explanations later refer to mechanisms of the animal world: depending on the species, the bi-potential gonad engages in male or female differentiation according to environmental factors such as temperature or behavior, and not according to genetic heritage. This is called environmental sex determination. In lizards, for example, an egg incubation temperature of eggs below 30°C induces male sex and a higher temperature female sex. The opposite is true for turtles [START_REF] Angelopoulou | Sex determination strategies in 2012: towards a common regulatory model?[END_REF]. In fish, different types of social factor can determine sex. For example, in clownfish, sex is determined by the social rank of the individual. These fish live in groups in anemones. A female in a monogamous couple dominates the colony alongside an adult male, with infertile males and juveniles around this couple. The aggressive behavior of the dominants toward infertile males prevents their sexual maturation and the aggressive behavior of the female prevents the adult male from becoming a female. If the female dies, the male of the couple becomes the dominant female while one of the infertile males becomes the fertile male (4). This hypothesis of sex determination sex by the environment persisted until the late 19 th century, when new theories came to be proposed. The "metagam" theory was based on an external influence: the fertilized egg is asexual and sex is determined more or less late in the embryo. In "progame" theory, on the other hand, the ovum itself is sexually differentiated: there are male and female ova; the sperm plays no role in the determination of sex, but only gives the egg an incentive to develop; sex thus preexists fertilization. In "syngamous" theory, sex is determined at the very moment of fertilization; it results from the combination of the properties of the egg and the sperm which combine to form the fertilized egg [START_REF] Kollman | Le déterminisme du sexe chez l'homme : Discussion de quelques théories[END_REF]. The theory put forward by the Austrian embryologist Schenk (1840Schenk ( -1902) ) later made a big splash. Schenk had noticed a relationship between maternal nutritional status and the sex of the fetus. Pregnancy is accompanied by varying levels of glycosuria; if glycosuria is very high, miscarriage occurs and, when it is low, a girl is born; a perfectly healthy woman gives birth to a boy. According to Schenk, providing women who have only daughters with an appropriate diet, starting two or three months before conception and continuing for three months after, will obtain a boy [START_REF] Schenk | Ein flussauf das geschlecht sverhàltniss[END_REF]. Even today, attempts to control sex determination through nutrition continue: according to certain theories, the diet to obtain a boy requires unstinting consumption of meat, salted, smoked or canned fish, salty aperitif crackers, and pastries, while having a girl requires eating vegetables, milk and dairy products [START_REF] Papa | Choisissez le sexe de votre enfants[END_REF]. Scientifically, Alfred Jost in 1947 showed that removing the testes in rabbit embryos induced a female phenotype at birth. This type of experiment gave rise to the hypothesis that the female phenotype was the consequence of non-differentiation of the gonad into testicle: differentiation "by default ". However, around 1961, genetic data highlighted the existence of sex chromosomes X and Y in mammals, as well as the major impact of the Y chromosome in testicular differentiation [START_REF] Jacobs | A case of human intersexuality having a possible XXY sexdetermining mechanism[END_REF][START_REF] Russell | Genetics of mammalian sex chromosomes[END_REF]. In 1990, the SRY factor (Sex-determining Region on the Y chromosome) was identified. It is now known that there is an undifferentiated gonadal stage and then a positive determination stage from gonad to ovary.

II. FROM GENITAL CRESTS TO DIFFERENTIATED GONADE

II.a The undifferentiated or bi-potential gonad

The development of the gonad is the first step in organizing the reproductive system. It begins very early, from the 3rd or 4th week of embryo development, with the establishment of an identical undifferentiated structure in XX and XY embryos. At the start of their formation, the gonads are said to be "bi-potential" because they have the capacity to follow a path of male or of female differentiation. These undifferentiated gonads are composed of distinct cell populations: primordial germ cells (PGCs), from which originate gametes, and somatic cells. Each somatic line has a double potential: the support cell line, from which granulosa cells originate, and the steroid cell line, which evolve into theca cells [START_REF] Bowles | Sex determination in mammalian germ cells: extrinsic versus intrinsic factors[END_REF]. The gonads and adrenals share a common developmental origin: the adreno-gonadal primordium [START_REF] Stévant | Genetic control of gonadal sex determination and development[END_REF]. Initial gonads differentiate from the mesonephros (transient embryonic kidneys). The PGCs then colonize the gonadal territory called the genital crest. The arrival of PGCs at the genital crests leads to proliferation of deep coelomic epithelial cells, which are able to surround the germ cells and thus form the primitive sexual cords. At this stage, the gonad still has a strictly identical morphology between the two sexes, with primitive sexual cords in both cortical and medulla regions [START_REF] Stévant | Genetic control of gonadal sex determination and development[END_REF]. Progression toward one sex or the other depends on the activation and repression of a large number of genes and a sufficient local concentration of specific proteins encoded by these genes: gonadal determination is a "dose-dependent" phenomenon. Several genes play a crucial role in the formation of genital crests, in particular, WT1 (Wilm's Tumor gene 1) and SF1 (Steroidogenic Factor 1). The proteins encoded by these genes are involved in the proliferation, differentiation and survival of somatic cells in the gonad.

The WT1 gene, located on chromosome 13, plays a major role in the development of the urogenital system, with expression in the mesenchyme of the renal vesicles, the glomerular epithelium of the kidney and the genital crests from the 6th week of development, and then in the fetal gonad. WT1 binding to the promoter of the Nr5a1 gene (nuclear receptor subfamily 5 group A member 1) enables expression and action during gonad development [START_REF] Kreidberg | WT-1 is required for early kidney development[END_REF][START_REF] Biason-Lauber | Ovarian development and disease: The known and the unexpected[END_REF]. Studies in mice confirmed the role of WT1 in urogenital development: mice mutated for WT1 showed absence of development of the kidneys and gonads [START_REF] Kreidberg | WT-1 is required for early kidney development[END_REF]. In humans, mutations or deletions of the WT1 gene are responsible for various syndromes, the prevalence of which is very low. They can induce full or partial 46, XY DSD and also impair the urogenital system. For example, Frasier syndrome combines 46, XY DSD and glomerular nephropathy. Denys-Drash syndrome combines 46, XY DSD, diffuse mesangial sclerosis and nephroblastoma. WAGR syndrome (Wilms's tumor, Aniridia and Genital anomalies with mental retardation) associates Wilm's renal tumor and total or partial aniridia, with DSD ranging from sexual ambiguity to testicular ectopy, and intellectual deficiency of varying severity.

The SF1 protein is coded by the Nr5a1 gene (nuclear receptor subfamily 5). It is expressed as early as the 6th week of development in the coelomic epithelium and the cells supporting the bi-potential gonad [START_REF] Luo | A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation[END_REF][START_REF] Martinerie | SF-1, a key player in adrenal and gonadal differentiation: implications in gonadal dysgenesis and primary ovarian insufficiency[END_REF]. This transcription factor is involved in the development and proliferation of cells supporting the bi-potential gonad [START_REF] Birk | The LIM homeobox gene Lhx9 is essential for mouse gonad formation[END_REF]. The first loss of function mutation in NR5A1 was described in humans in 1999 [START_REF] Achermann | A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans[END_REF]. It was identified in a 46,XY individual, phenotypically female, who developed postnatal adrenal insufficiency and bilateral cryptorchidism. Bilateral gonadectomy in adolescence identified two small dysgenic testes. In 2009, the involvement of SF1 in premature ovarian failure or primary ovarian insufficiency (POI) was described for the first time [START_REF] Lourenço | Mutations in NR5A1 associated with ovarian insufficiency[END_REF]. The NR5A1 gene was sequenced in 4 families affected by both 46,XY dysgenesis and POI, and in 25 sporadic cases of POI without associated adrenal insufficiency. This sequencing identified mutations in several 46,XX members of the 4 families and in 2 of the 25 sporadic cases of POI. The spectrum of damage linked to NR5A1 mutations is very wide. To summarize, SF1 plays a role in testicular development and in maintaining the ovarian follicle pool.

II.b Sex determination

Sex determination enables the initial gonad or genital crest to initiate a developmental path leading to the formation of the ovary or the testis: i.e., gonadal sex. Differentiation is the process that guarantees the development of a genital tract and external genitalia according to gonadal sex, thereby constituting the phenotypic sex. During ovarian determination, the primitive sex cords segment into clusters in which primordial gonocytes are found. These primitive sexual cords are connected to the mesonephrotic tubes by a network of cellular cords constituting the rete-ovari. The superficial epithelium of the female gonad continues to proliferate. It also gives birth to a second generation of cords, the cortical sex cords, that penetrate the underlying mesenchyme. The gonad determination process for the ovary is much longer than that for the testis. At the start of the 3rd month of pregnancy, the cortical area changes. The gonocytes turn into ovogonies and the cells of the sex cords organize around the germ cells, which results in the formation of primordial follicles [START_REF] Monniaux | The ovarian reserve of primordial follicles and the dynamic reserve of antral growing follicles: what is the link?[END_REF]. The mesenchymal elements give the ovarian stroma. In the center of the ovary, the spinal cords regress as welle as the rete-ovari and the mesonephronic remains. All of these remnants are at the origin of Rosenmüller's organ [START_REF] Siffroi | Embryologie de l'appareil géntital[END_REF].

II.c Factors determining the development of the ovary

II.c.1 Differentiation of ovarian somatic cells

In mammals, the formation of the testis or the ovary depends on a set of cellular and hormonal signals that interact in a specific order, with very precise windows of action, to contribute to the establishment of the genital apparatus and therefore a male or female phenotype. Comprehensive research progressed in depicting the chronological stages of the differentiation of the gonad into testis [START_REF] Morel | Genetics of gonadal determination[END_REF]. The SRY gene, discovered in 1990, located on the short arm of the Y chromosome, close to the pseudo-autosomal region, is considered the key gene for male determination [START_REF] Palmer | Genetic evidence that ZFY is not the testis-determining factor[END_REF][START_REF] Sinclair | A gene from the human sex-determining region encodes a protein with homology to a conserved DNAbinding motif[END_REF]. SRY protein is a transcription factor that modulates the expression of target genes essential for testicular differentiation, including SrY-box containing gene 9 (SOX9). Thus, the translocation of SRY on X chromosome induces, in 46,XX embryos carrying SRY, the development of testes, which can be normal [START_REF] Andersson | Chromosome Y-specific DNA is transferred to the short arm of X chromosome in human XX males[END_REF]. Conversely, a mutation of SRY in a 46,XY subject causes a defect in masculinization of the primitive gonads. This defect is often total, inducing streak gonads [START_REF] Jäger | A human XY female with a frame shift mutation in the candidate testis-determining gene SRY[END_REF][START_REF] Cameron | Mutations in SRY and SOX9: testis-determining genes[END_REF]. Thus, until recently, gonad differentiation toward ovary was considered to occur by default, due to the absence of the SRY gene and/or SOX9. It is now accepted that the formation of the ovary is the consequence of a brake on testicular development, but also of a positive determinism. There is indeed a cascade of events inducing gonad differentiation toward ovary (Figure 1).

The majority of "anti-testicular" genes induce repression of SOX9 [START_REF] Morel | Genetics of gonadal determination[END_REF]. The main players in female gonadal differentiation are the WNT4 / β catenin-pathway modulated by R-Spondin 1 (RSPO1) [START_REF] Chassot | Genetics of ovarian differentiation: Rspo1, a major player[END_REF] and the Forkead box L2 (FOXL2) transcription factor.

The WNT/β-catenin pathway

When activated by WNTs (Wingless-type MMTV integration site), two transmembrane receptors form a heterodimer -which transmits a signal that causes β-catenin stabilization in the cell. β-catenin then acts as a transcriptional coregulator in the nucleus (28). Several WNT signaling pathways come into play when the undifferentiated gonad takes on a male or female destiny. Two main ligands have been identified, in particular WNT4 (Wnt family member 4) and RSPO1 (R-Spondin 1) (Figure 2). The WNT4 gene is located on chromosome 1 at 1p35. WNT4 is expressed in the undifferentiated gonad of XX and XY embryos. Its expression decreases in the male gonad at the differentiation, while it persists in the developing ovary. In the 46,XX fetus, between 12 and 18 weeks of gestation, moderate WNT4 expression is detected in the pre-granulosa and granulosa cells while expression is very low in the oocytes. At 19 weeks, WNT4 expression increases intensively in ovocytes [START_REF] Jääskeläinen | WNT4 is expressed in human fetal and adult ovaries and its signaling contributes to ovarian cell survival[END_REF]. Mice with a XX karyotype, invalidated for Wnt4 (Wnt4 -/ -), show partial sexual reversion, with a masculinization of the gonad. Support cells adopt characteristics of Sertoli cells and there is a decrease in the number of oocytes, suggesting that Wnt4 is necessary to maintain the development of female germ cells [START_REF] Vainio | Wnts as kidney tubule inducing factors[END_REF]. On the molecular level, Wnt4 inhibits expression of Sox9 in mice [START_REF] Bernard | Wnt signaling in ovarian development inhibits Sf1 activation of Sox9 via the Tesco enhancer[END_REF]. In humans, homozygous WNT4 "loss-of-function" mutations are responsible for SERKAL syndrome (SEx Reversion, Kidneys, Adrenal and Lung dysgenesis). This syndrome is extremely rare, with prevalence <1/1,000,000 (32). It is lethal, and associates testicular development in 46,XX fetus, as well as renal, adrenal and pulmonary abnormalities [START_REF] Mandel | SERKAL syndrome: an autosomal-recessive disorder caused by a loss-of-function mutation in WNT4[END_REF]. Heterozygous mutation of WNT4 was first described in 2012 in a 46,XX individual. This 18-year-old patient consulted for primary amenorrhea. Her thelarche had occurred at the age of 12. The etiology of her amenorrhea was uterine agenesis. The patient also had renal agenesis [START_REF] Biason-Lauber | WNT4, RSPO1, and FOXL2 in sex development[END_REF]. This phenotype is similar to that of Rokitansky-Kuster-Hauser syndrome but is accompanied by mild hyperandrogenism. This is a very rare disorder, and only a few cases have been reported [START_REF] Biason-Lauber | WNT4 deficiency--a clinical phenotype distinct from the classic Mayer-Rokitansky-Kuster-Hauser syndrome: a case report[END_REF][START_REF] Philibert | Identification and functional analysis of a new WNT4 gene mutation among 28 adolescent girls with primary amenorrhea and mullerian duct abnormalities: a French collaborative study[END_REF]. Conversely, overexpression of WNT4 by duplication of the gene was reported in a 46,XY individual [START_REF] Jordan | Upregulation of WNT-4 signaling and dosage-sensitive sex reversal in humans[END_REF]. This patient presented sexual ambiguity with severe hypospadias, fibrous gonads, rudimentary uterus and vagina. WNT4 thus appears to be a major player in ovarian development as well as the inhibition of testicular development. It acts by increasing the expression of DAX1, another anti-testicular actor [START_REF] Jordan | Upregulation of WNT-4 signaling and dosage-sensitive sex reversal in humans[END_REF]. In all, WNT4 has a brake role in the development of the gonad of the testis and decreases the synthesis of androgens by the gonad in women.

-RSPO1

The RSPO1 gene is located on chromosome 1 at 1p34.3. It is expressed early in ovarian development [START_REF] Tomaselli | Human RSPO1/R-Spondine 1 is expressed during early ovary development and augments β-catenin signaling[END_REF], and not expressed in testicular tissue. The R-Spondin 1 protein (RSPO1) plays a key role in gonad differentiation toward ovary, activating the WNT/β-catenin pathway. It is part of a large family of growth factors. At gonadal differentiation, RSPO1 is expressed in 46,XX but 46,XY gonads [START_REF] Parma | R-Spondine 1 is essential in sex determination, skin differentiation and malignancy[END_REF]. It is also expressed in fibroblasts and regulates the proliferation and differentiation of keratinocytes [START_REF] Parma | R-Spondine 1 is essential in sex determination, skin differentiation and malignancy[END_REF]. The phenotype of Rspo1 -/-mutant mice is close to that of Wnt4 -/-mice [START_REF] Chassot | Genetics of ovarian differentiation: Rspo1, a major player[END_REF]. In humans, the first "loss of function" homozygous mutations of RSPO1 were described in 2006 in an inbred Italian family. Within the family, the 4 mutated brothers, with a karyotype 46,XX (SRY-negative), presented sexual ambiguity. Their external genitals were more of a male type but with hypospadias or greater ambiguities. Gonadal biopsies performed in some patients mutated for the RSPO1 gene in some cases revealed testicular structures with Leydig cell hyperplasia and in other cases ovotestis with small areas of residual ovarian tissue [START_REF] Chassot | Genetics of ovarian differentiation: Rspo1, a major player[END_REF]. These patients also showed palmoplantar hyperkeratosis and a predisposition to squamous cell carcinoma of the skin [START_REF] Parma | R-Spondine 1 is essential in sex determination, skin differentiation and malignancy[END_REF]. This pathology is extremely rare, with fewer than 10 cases described to date. However, their description has shown that R-Spondin 1 plays a key role in differentiating the gonad toward the ovary. Chronologically, it is one of the first stages of this differentiation. It is interesting to note that the two factors WNT4 and RSPO1 are also involved in the early stages of gonadal establishment, playing a role in the proliferation of coelomic epithelium cells. Chassot et al. demonstrated this phenomenon in 2012 in a Wnt4 and/or Rspo1 knockout mouse model: whether mice were XX or XY, they exhibited decreased cell proliferation at the genital crests.

-FOXL2

The third known factor in positive ovarian determination is FOXL2, which is encoded by the FOXL2 (Forkhead box L2) gene, located on chromosome 3 at 3q22.3. This transcription factor is expressed very early during ovarian development. An immunofluorescence study showed expression of FOXL2 in gonad supporting cells, and especially pre-granulosa and granulosa cells [START_REF] Duffin | The forkhead transcription factor FOXL2 is expressed in somatic cells of the human ovary prior to follicle formation[END_REF]. The role of FOXL2 in ovarian determination was initially highlighted in goats, where loss of function of Foxl2 had several impacts. Firstly, lack of feminization of the XX gonad with loss of aromatase expression and therefore absence of estrogen synthesis was observed. In addition, masculinization of the XX gonad occurs by overexpression of the male factor SOX9, resulting in differentiation of Sertoli cells and expression of anti-Müllerian hormone, causing Müller channels to regress [START_REF] Pannetier | FOXL2, the gatekeeper of ovarian identity[END_REF]. However, disabling the Foxl2 gene in XX mice did not result in sexual reversion [START_REF] Schmidt | The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance[END_REF][START_REF] Uda | Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development[END_REF]. The ovaries of Foxl2 -/-mice are small and severely disorganized, and primordial follicles do not form [START_REF] Uhlenhaut | Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation[END_REF]. Furthermore, overexpression of Fox12 in Sertoli XY cells was shown to induce disorganization of the seminiferous tubules and the development of an ovotestis-type gonad [START_REF] Ottolenghi | Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells[END_REF]. Thus, there is a phenomenon of mutual exclusion within the somatic cells of the gonads between FOXL2 for the ovary and SOX9 for the testicle, inhibiting each other and resulting in two dichotomous states: male or female. In humans, heterozygous mutations of FOXL2 were described in BPES syndrome (Blepharophimosis-Ptosis-Epicanthus inversus), which leads to eyelid abnormalities, with diminished palpebral cleft and widening of the distance between the eyes called epicanthus. BPES can be associated with premature ovarian failure (BPES type 1) or be isolated (BPES type 2) [START_REF] Bell | Two families with blepharophimosis/ptosis/epicanthus inversus syndrome have mutations in the putative forkhead transcription factor FOXL2[END_REF][START_REF] Crisponi | The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ ptosis/epicanthus inversus syndrome[END_REF][START_REF] De Baere | Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype-phenotype correlation[END_REF]. No abnormal sexual development has been described in individuals with FOXL2 mutation to date.

II.c.2 Differentiation of ovarian germ cells

On arrival in the gonads, PGCs continue to actively proliferate. In the ovary, the germ cells cease mitotic multiplication to enter meiosis, and more precisely prophase I of meiosis. Meiosis in the human species stops before birth. Prophase I is divided into five stages (leptotene, zygotene, pachytene, diplotene, diacinesis) characterized by changes in the chromosomes. The first four stages take place during fetal life, then the germ cells become blocked at the diplotene stage. The last step (diacinesis) resumes at puberty. Meiosis is eventually completed when the egg fertilizes. After entering meiosis, ovogonies are referred to as oocytes I.

II.c.3 Genetic factors involved in prophase I of meiosis

One of the first factors necessary to engage germ cells in the process of meiosis is DAZL (Deleted in azoospermia-like). In mice, Dazl is expressed by male and female germ cells (post-migration). Inactivation of Dazl leads to infertility in both sexes, with absence of expression of the genes involved at the start of prophase I, including Stra8 (Stimulated by retinoic acid gene 8), the gene that initiates prophase I [START_REF] Lin | Germ cell-intrinsic and -extrinsic factors govern meiotic initiation in mouse embryos[END_REF]. Early expression of Dazl upstream of Stra8 allows the germ cells to be "competent" to enter meiosis. Once the cells have become "competent", several studies showed that entry in meiosis by female mouse germ cells is dependent on retinoic acid (RA), the active metabolite of vitamin A [START_REF] Bowles | Retinoid signaling determines germ cell fate in mice[END_REF][START_REF] Koubova | Retinoic acid regulates sex-specific timing of meiotic initiation in mice[END_REF]. Induction of meiosis by RA has also been demonstrated in humans [START_REF] Bouffant | Meiosis initiation in the human ovary requires intrinsic retinoic acid synthesis[END_REF]. Retinoic acid binds to its RAR receptor and the receptor-ligand couple activates one of its target genes, Stra8 [START_REF] Oulad-Abdelghani | Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene[END_REF], which is required for pre-meiotic DNA synthesis, initiation of meiosis and progression of meiosis in germ cells.

II.d Formation of primordial follicles

Follicles form when the oocytes are blocked in prophase I of meiosis, from 14-15 weeks of gestation, in the human species. This process begins when the germ cells have colonized the gonads and divide to form clusters called cysts (or syncitia). These cysts are organized in ovigerous cords, where cytoplasmic bridges, surrounded by somatic ovarian cells, connect the oocytes to each other. The formation of primordial follicles results from the fragmentation of the ovigerous cords. These primordial follicles then consist of an oocyte surrounded by a few flattened somatic cells, the future granulosa cells. The different stages of follicle maturation are controlled by many factors and require a very precise dialogue between the oocyte and the somatic cells that surround it.

Among the important factors involved in the formation of primordial follicles are FIGLA factor, SOHLH factors, neurotrophins and FOXL2. In the ovaries of Foxl2 -/-mice, the growing oocytes are (and remain) surrounded by a single layer of granulosa cells. This indicates that granulosa cell differentiation has been altered from the early stages of folliculogenesis. In addition, the oocytes remain clustered (up to 10 oocytes) and are surrounded by a layer of support cells. These structures persist for up to 8 weeks after birth as clusters of unfragmented oocytes and ovarian cords. These data show that FOXL2 is necessary for the fragmentation of cysts and the formation of primordial follicles [START_REF] Uda | Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development[END_REF].

III. MAINTAINING THE OVARY FOXL2, guardian of ovarian identity

Many active mechanisms maintain the differentiated state of the ovary. The "testicular" genes must be constantly suppressed. A major player in this process is FOXL2. In adult female mice, it is interesting to note that targeted Fox12 gene knock-out in follicular cells induces expression of the male gene Sox9 in the cells; in such animals, follicular cells in the ovary then differentiate into Sertoli cells [START_REF] Uhlenhaut | Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation[END_REF].

FOXL2 is expressed by granulosa cells, where it directly controls expression of aromatase, the key enzyme in the production of estradiol [START_REF] Pannetier | FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development[END_REF]. It is by the joint action of FOXL2 and estrogens, via their receptors (ER), that the major gene of male identity, Sox9, is kept silent.

FOXL2 works synergistically with the alpha estradiol receptor (ERα) and prevents the transdifferentiation of support cells into testicular cells in the adult ovary [START_REF] Pannetier | Involvement of FOXL2 and RSPO1 in Ovarian Determination, Development, and Maintenance in Mammals[END_REF]. Lack of expression of FOXL2 or of both types of estrogen receptor (ERα and ERβ) induces a lack of Sox9 inhibition, inducing masculinization of somatic cells in the ovary [START_REF] Couse | Postnatal sex reversal of the ovaries in mice lacking estrogen receptors alpha and beta[END_REF]. This illustrates an important role for FOXL2, estrogens and their two receptors ERα and ERβ. These experiments showed that, in addition to its role in the morphogenesis of the female gonad, FOXL2, in adulthood, is a guardian of ovarian identity.

Another key factor in ovarian identity is the Rspo1 gene. On the one hand, as described above, it plays a role in the inhibition of the Sox9 gene by a molecular path distinct from that of FOXL2. On the other hand, in addition to its role in determining and differentiating PGCs in follicular cells [START_REF] Biason-Lauber | WNT4, RSPO1, and FOXL2 in sex development[END_REF], Rspo1 is inhibited after birth, allowing maintenance of homeostasis in the adult ovary [START_REF] De Cian | Amplification of R-Spondine 1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary[END_REF].

CONCLUSION

Different sexual developments (DSD) are rare pathologies, affecting between 1/4,500 and 1/5,000 births. Since the Chicago consensus conference, they have been classified as chromosomal, gonadal or phenotypic anomalies. There are 46,XX and 46,XY DSDs. Some etiologies are linked to an abnormality in the development of the gonad. Description of certain patients highlighted many factors involved in differentiating the undifferentiated gonad toward the ovary. It is now recognized that there is a positive gonad differentiation, with the action of anti-testicular and pro-ovarian factors, such as R-Spondin 1 and the WNT4/β catenin pathway. However, to date, many cases of DSD remain unresolved. New techniques in genetics, and particularly whole-genome study, should improve knowledge of ovarian determination. An interesting perspective is based on the fact that certain genes involved in gonad determination have been shown to be mutated in infertile patients, even in the absence of DSD. Therefore, research on gene differentiation in the ovarian gonad may in the near future elucidate certain so-called idiopathic etiologies of infertility.

Figure 2 .

 2 Figure 1. Factors involved in ovarian development over time before birth