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In mammals, the NR5A1 nuclear receptor (Steroidogenic Factor 1) is essential for the testis development, in which it is specifically expressed by the somatic supporting cells called Sertoli cells (SC). The establishment of the testicular architecture and more globally of the urogenital tract, depends on signals emanating from the SC. The goal of my project is to study the role of Nr5a1 gene in SC during testicular development. For this I analyzed the phenotype of male mice in which Nr5a1 gene is deleted in SC after sexual differentiation. This work shows that in the absence of Nr5a1, SC are no longer able to perform their functions effectively and that their transcriptomic signature reflects a change in their identity. Eventually, all of the SC invalidated for Nr5a1 die, which allowed us to obtain a unique model to analyze the development of the testis and the urogenital tract in the absence of SC. Taken together, these results allowed us to decipher the role of Nr5a1 in maintaining the identity and the survival of SC, as well as to understand the influence of SC on other cell types of the testis and on the development of the urogenital tract., Chez les mammifères le récepteur nucléaire NR5A1 (Steroidogenic Factor 1) est essentiel pour développement du testicule, dans lequel il est notamment exprimé par les cellules somatiques de soutien appelées cellules de Sertoli (CS). La mise en place de l’architecture testiculaire et plus globalement du tractus-urogénital, dépendent de signaux émanant des CS. Mon projet consiste à étudier le rôle de Nr5a1 dans les CS au cours du développement testiculaire. Pour cela j'ai analysé le phénotype des souris mâles dont le gène Nr5a1 est invalidé dans les CS après la différenciation sexuelle. Ces travaux ont montré qu’en l’absence de Nr5a1 les CS ne sont plus en mesure d’assurer leurs fonctions et que leur signature transcriptomique traduit un changement de leur identité. A terme, la totalité des CS invalidées pour Nr5a1 meurent ce qui nous a permis d’obtenir un modèle d’étude unique pour analyser le développement du testicule et du tractus-urogénital en l’absence de CS. Ensemble mes résultats permettent de mieux appréhender d’une part, le rôle de Nr5a1 dans le maintien de l’identité des CS et leur survie, et d’autre part de comprendre l’influence des CS sur les autres types cellulaires du testicule et sur le développement du tractusurogénital.

In mammals, the NR5A1 nuclear receptor (Steroidogenic Factor 1) is essential for the testis development, in which it is specifically expressed by the somatic supporting cells called Sertoli cells (SC). The establishment of the testicular architecture and more globally of the urogenital tract, depends on signals emanating from the SC. The goal of my project is to study the role of Nr5a1 gene in SC during testicular development. For this I analyzed the phenotype of male mice in which Nr5a1 gene is deleted in SC after sexual differentiation. This work shows that in the absence of Nr5a1, SC are no longer able to perform their functions effectively and that their transcriptomic signature reflects a change in their identity. Eventually, all of the SC invalidated for Nr5a1 die, which allowed us to obtain a unique model to analyze the development of the testis and the urogenital tract in the absence of SC. Taken together, these results allowed us to decipher the role of Nr5a1 in maintaining the identity and the survival of SC, as well as to understand the influence of SC on other cell types of the testis and on the development of the urogenital tract., Chez les mammifères le récepteur nucléaire NR5A1 (Steroidogenic Factor 1) est essentiel pour développement du testicule, dans lequel il est notamment exprimé par les cellules somatiques de soutien appelées cellules de Sertoli (CS). La mise en place de l’architecture testiculaire et plus globalement du tractus-urogénital, dépendent de signaux émanant des CS. Mon projet consiste à étudier le rôle de Nr5a1 dans les CS au cours du développement testiculaire. Pour cela j'ai analysé le phénotype des souris mâles dont le gène Nr5a1 est invalidé dans les CS après la différenciation sexuelle. Ces travaux ont montré qu’en l’absence de Nr5a1 les CS ne sont plus en mesure d’assurer leurs fonctions et que leur signature transcriptomique traduit un changement de leur identité. A terme, la totalité des CS invalidées pour Nr5a1 meurent ce qui nous a permis d’obtenir un modèle d’étude unique pour analyser le développement du testicule et du tractus-urogénital en l’absence de CS. Ensemble mes résultats permettent de mieux appréhender d’une part, le rôle de Nr5a1 dans le maintien de l’identité des CS et leur survie, et d’autre part de comprendre l’influence des CS sur les autres types cellulaires du testicule et sur le développement du tractusurogénital.

La voie de signalisation Hippo est impliquée dans de nombreux processus au cours du développement embryonnaire ainsi que chez l’adulte afin d’assurer, entre autres, la prolifération et la différenciation cellulaire, et le maintien de l’homéostasie tissulaire. Les principaux effecteurs qui agissent en aval de cette voie de signalisation sont les cofacteurs transcriptionnels YAP (Yes associated protein) et TAZ (Transcriptionnal coactivator with PDZ-binding motif). La voie Hippo a été étudiée dans de nombreux tissus pour son rôle dans la physiologie et certaines pathologies, cependant très peu d’études ont été menées sur le rôle de cette voie dans le testicule et la glande surrénale. L’hypothèse émise est que cette voie de signalisation pourrait jouer un rôle important dans le développement (déterminisme sexuel mâle) et la fonction (synthèse de testostérone) des testicules ainsi que dans la physiologie surrénalienne. L’objectif de ce travail de doctorat est d’élucider le rôle des effecteurs de la voie Hippo, YAP et TAZ, dans le développement et la fonction des cellules de Sertoli, des cellules de Leydig, ainsi que dans les cellules du cortex surrénalien. Nos travaux ont mis en évidence le rôle de YAP/TAZ dans le maintien de l’identité des cellules de Sertoli. Nous avons constaté que l’inactivation de Yap/Taz dans un modèle de culture primaire de cellules immatures de Sertoli entraine une diminution de l’expression des gènes impliqués dans le déterminisme sexuel mâle au profit des gènes de différenciation femelle. Nous avons ensuite procédé à la création d’un modèle murin permettant l’inactivation conditionnelle de Yap/Taz dans les cellules de Sertoli (Yap(flox/flox);Taz(flox/flox);Amh(cre/+)). Malgré une légère modification de l’expression de certains gènes associés au déterminisme sexuel, l’inactivation de Yap/Taz n’entrainait pas de changement de sexe chez les adultes. Afin d’évaluer le rôle de la voie Hippo dans le développement des gonades (et des glandes surrénales) nous avons créé un modèle de souris permettant l’inactivation de Yap/Taz dans les précurseurs des cellules de Sertoli, des cellules de Leydig et des cellules de l’adrénocortex au niveau du primordium adrenogonadique (Yap(flox/flox);Taz(flox/flox); Nr5a1(cre/+)). Si la recombinaison dans les cellules de Sertoli et de Leydig n’a pas été efficace, la recombinaison au niveau du cortex surrénalien a été hautement efficace. La caractérisation du modèle de souris Yap(flox/flox);Taz(flox/flox);Nr5a1(cre/+) nous a donc permis de définir un rôle pour YAP/TAZ dans le maintien de la glande surrénale adulte. Enfin, afin de déterminer le rôle de la voie Hippo dans les cellules de Leydig, nous avons évalué le rôle de YAP dans les cellules de Leydig MLTC-1. Par l’utilisation d’un inhibiteur pharmacologique de YAP et des ARNs interférants (siARNs) dirigés contre Yap, nous avons démontré que YAP régulait la stéroïdogenèse de manière négative dans ces modèles de cellules de Leydig. Nous avons aussi démontré que YAP pouvait interagir avec le promoteur proximal de Star (Steroidogenic acute regulatory protein) et que cette liaison était perturbée par la voie PKA (Protéine kinase A) qui entraine la phosphorylation de YAP suggérant un mécanisme d’action par lequel YAP régule la stéroïdogenèse. En résumé, à l’aide d’approches in vitro et in vivo, nous avons mis en évidence pour la première fois le rôle de la voie Hippo dans le maintien de l’identité « mâle » des cellules de Sertoli, dans la régulation de la stéroïdogenèse dans les cellules de Leydig dans des cellules immortalisées ainsi que dans le maintien de l’homéostasie du cortex surrénalien., Hippo is an evolutionary conserved signaling pathway with well-established roles in cell fate determination, differentiation, and proliferation during embryonic and postnatal development, as well as tissue homeostasis throughout adulthood. The main effectors of the Hippo pathway are the transcriptional coregulators YAP (Yes associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif). The Hippo pathway has been studied in many tissues for its diverse roles in physiology and pathology, however few studies have been done to elucidate its role in the testis and the adrenal gland. We hypothesized that the Hippo pathway could play a major role in testicular development (male sexual determination) and function (testosterone production) as well as adrenal gland physiology. The objectives of the present thesis are to elucidate the function of the Hippo effectors YAP and TAZ in the development and function of the Sertoli and Leydig cells, as well as in adrenocortical cells. The results of this thesis highlighted the role of YAP/TAZ for the maintenance of the male identity of the Sertoli cells. We found that Yap/Taz inactivation in primary cultured immature Sertoli cells leads to a downregulation of male sex determination genes and an upregulation of genes involved in female differentiation. Then, we generated a mouse model in which Yap and Taz were conditionally deleted in Sertoli cells (Yap(flox/flox);Taz(flox/flox);Amh(cre/+)). Despite slight modifications in the expression of genes associated with sex determination, Yap/Taz inactivation didn’t cause sex reversal in adults. In order to evaluate the role of Hippo in gonadal (and adrenal gland) development, we generated a new mouse model to direct Yap/Taz inactivation in Sertoli, Leydig and adrenocortical progenitors cells (Yap(flox/flox);Taz(flox/flox);Nr5a1(cre/+)). Recombination wasn’t efficient in Sertoli and Leydig cells but was highly efficient in the adrenocortical cells. Characterization of Yap(flox/flox);Taz(flox/flox);Nr5a1(cre/+) the mouse model allowed us to define a role for YAP/TAZ in adult adrenal gland maintenance. Finally, to determine the role of the Hippo pathway in Leydig cells, we evaluated the YAP’s role in the Leydig cell lines MLTC-1. Using a pharmacological inhibitor of YAP and siRNAs targeting Yap, we showed that YAP negatively regulates steroidogenesis in these Leydig cell line models. We also demonstrated that YAP can recruit the proximal promoter of Star, and that this recruitment decreases following PKA-dependent YAP phosphorylation, suggesting a mechanism of action whereby YAP regulates steroidogenesis. To resume, combining in vitro and in vivo approaches, we elucidated for the first time a role of the Hippo signaling pathway in Sertoli cell differentiation, in steroidogenesis regulation in Leydig cells, as well as in adrenocortical homeostasis.

Sterility affects 15 % of French population and its prevalence is propagating since four or five decades. Many human physiological and epidemiological arguments support the impact of lipid homeostasis on the gonads; indeed, cholesterol is a key regulator of steroidogenesis and gametogenesis. Nevertheless, the molecular mechanisms remain hidden. Liver X receptors alpha and beta (LXRα and β) belong to the superfamily of nuclear receptors and are activated upon binding to oxysterols. LXRs are mainly implicated in cholesterol homeostasis. Increasing bulk of literature identified these non-steroid nuclear receptors as major regulators of the gonad physiology. This work uncovers previously unidentified putative roles of LXRs and ability of cholecterol excess to alter male and female germ cell maturation. Herein, we analyse a new mouse strain (Lxrαϐ-/-:AMHLxrϐ) re-expressing LXRβ under control of AMH promoter (specific to Sertoli in testis and granulosa cells in ovary) in a background of Lxrαϐ-/- mouse. Our results identify LXRs as primordial to maintain male and female fertility. They have pleotropic « non-classical » roles ranging from lipid homeostasis to the regulation of germ cell maturation and bi-directional control of steroid synthesis.If the cellular defects in the absence of LXRs within the testis are significant, they are generally compensated and consequently, single cell compartment is tolerated.Unlike the testis, LXRβ in the granulosa cells is « the regulator » of multiple mechanisms essential for follicle maturation, ovocyte survival and for controlled ovulation. LXRβ is therefore a potnetial target to regulate female fertility and to prevent ovarian hyperstimulation syndrome.Our results open the perspectives for the identification of new diagnostic and/or prognostic markers in both male and female fertility., La stérilité affecte à l’heure actuelle près de 15-20 % des couples et sa prévalence est en progression depuis quatre à cinq décennies. Cette progression évolue parallèlement à la prévalence de l’épidémie d’obésité et de syndrome métabolique dans le monde. De multiples arguments physiologiques et épidémiologiques chez l’Homme soutiennent l’hypothèse de l’influence de l’homéostasie des lipides sur la fonction gonadique. En particulier, le cholestérol est un facteur clef dans la régulation de la stéroïdogenèse et de la gamétogenèse. Bien que les atteintes gonadiques semblent multifactorielles, les mécanismes moléculaires restent méconnus dans la majorité des cas. Les Liver X receptors (LXRα et β) sont des récepteurs nucléaires activés par les oxystérols. Ce sont classiquement des régulateurs du métabolisme lipidique. Plusieurs études ont démontré l’importance de ces récepteurs dans la physiologie des gonades.Ce travail identifie les rôles multiples des LXRs dans le maintien de la fertilité masculine et féminine, et décrit l’effet de l’homéostasie du cholestérol sur la maturation des cellules germinales dans le testicule et l’ovaire. Ce travail se concentre sur l’analyse comparative d’une lignée de souris ré-exprimant LXRβ (Lxrαϐ-/-:AMHLxrϐ) sous contrôle de promoteur d’AMH humain (expression spécifique dans les cellules de Sertoli dans le testicule et les cellules de granulosa dans l’ovaire) sur un fond génétique de souris Lxrαϐ-/-.L’absence d’un isoforme d LXR aboutit à des défauts spécifiques dans un type cellulaire du testicule. Néanmoins, le dysfonctionnement d’un type cellulaire est compensé. En effet, de multiples défauts sont nécessaires pour aboutir à la stérilité.LXR dans les cellules de granulosa est critique pour la maturation et la survie des ovocytes, l’ovulation, et par conséquence pour la fertilité. Ainsi, LXRβ est une cible potentielle pour réguler la fertilité féminine et la prévention de syndrome d’hyperstimulation ovarienne.Nos résultats ouvrent des perspectives pour des nouvelles cibles diagnostiques et pronostiques dans la fertilité.

Normal spermatogenesis results from a balance between process of cell proliferation, cell differentiation and apoptosis that concern somatic cells and germ cells. Dysfunction of spermatogenesis may be the result of constitutional or acquired abnormalities of spermatogonia stem cells or somatic cells. To overcome these problems, it seems necessary to implement preventive measures for germ stem cell preservation or substitute measures to replace them, the objective being to replicate in vivo or in vitro the process of spermatozoa production. This article will discuss the different experimental strategies for considering the in vivo or in vitro production of spermatozoa, outside the physiological process.

The production of fertilizable ova is the consequence of multiple events that start as soon as ovarian development and culminate at the time of ovulation. Throughout their development, germ cells are associated with companion somatic cells, which ensure germ cell survival, growth and maturation. Data obtained in vitro and in vivo on several animal models of germ cell depletion have led to uncover the many roles of germ cells on both ovarian development and folliculogenesis. During ovarian development, germ cells become progressively enclosed within epithelial structures called "ovigerous cords" constituted by pregranulosa cells, lined by a basement membrane. At the end of ovarian development, ovigerous cords fragment into primordial follicles, which are epithelial units constituted by an oocyte surrounded by a single layer of granulosa cells. Germ cells are necessary for the fragmentation of ovigerous cords into follicles, since in their absence, no follicle will form. Germ cells also ensure the differentiation of the ovarian somatic lineage, and they may inhibit the testis-differentiating pathway by preventing the conversion of pregranulosa cells into Sertoli cells, their counterpart in the testis. Regularly, primordial follicles are recruited into the growing follicle pool and initiate their growth. They develop through primary, preantral, antral and preovulatory stages before being ovulated. Interestingly, the action of the oocyte on companion somatic cells tightly depends on the follicular stage. In primordial follicles, the oocyte prevents the transdifferentiation of granulosa cells into cells resembling Sertoli cells. By contrast, as soon as the follicle enters growth, the oocyte regulates the functional differentiation of granulosa cells and at the latest stages, it prevents their premature maturation into luteal cells. Overall, these data demonstrate that the female germ cell act on companion somatic cells to regulate ovarian development and folliculogenesis, thereby actively supporting its own maturation.

Müllerian inhibiting substance (MIS) has been discovered by Alfred Jost at the beginning of the fifties. MIS is a glycoprotein belonging to the TGF-beta family. Its various functions differ between males and females and according to the age of the individual. In male, the protein is synthesized by Sertoli's cells and induces the disappearance of Müllerian's ducts, the development of the male genital tract. Its role in adult males remains quite unknown. In female, the protein is secreted by granulosa cells and plays a role during folliculogenesis as it regulates the initial and cyclic recruitment of ovarian follicles. MIS is also a good marker of follicular reserve and ovarian function. Therefore, it plays a role in different areas such as assisted medical reproduction and oncology. This protein represents a potential major diagnosis as well as prognostic tool in reproduction.

Two major functions are assumed by the testis: the production of male gametes (that is, spermatozoa) and the production of steroid hormones. Both two functions are established during fetal life and are essential to the adult fertility and the masculinization of the internal tract and genitalia. For many years, our laboratory has been interested in the ontogeny of those two functions in rodents and, since 2003, in collaboration with gynecology and obstetrics service of professor R. Frydman in Antoine-Béclère hospital, we have studied them in human. The first aim of this work was to improve the global knowledge of the human fetal testis development by using both our experimental data and the literature. Then, we focused on the different defects that can occur during the fetal testis development. Indeed, male reproductive abnormalities have been steadily increasing since the last decades and are thought to be related to the concomitant increase of the concentration of contaminants and particularly of endocrine disruptors in the environment. Thus, we decided to study the effect of endocrine disruptors on human fetal testis and, more particularly, the effect of phthalates, by using an organ culture system developed for human. In contrast to the data obtained in rat, mono (ethylhexyl)-phthalate (MEHP), an active metabolite of the most widespread phthalate in the environment, does not disturb the steroidogenic function. On the other hand, it has a negative effect on the male germ cells number. This study is the first experimental demonstration of a negative effect of phthalates directly on human fetal testis.

The aim of the present work is to investigate the role of genetic and epigenetic anomalies in severe spermatogenesis impairment. First, we looked for somatic mosaicisms for the Y microdeletion in 44 infertile patients, and no mosaicism was observed. We have also studied the Y chromosome of 3 infertile patients with a Y rearrangement. This investigation allowed a more precise description of the abnormal Y and shed light on how Y chromosomal rearrangements could be involved in the infertility phenotype. Second, the sperm aneuploidy rates for chromosome X, Y, 18, 13 and 21, were assessed by FISH in 31 infertile patients. Nearly half of the infertile patients had a significantly increased disomy rate. Moreover, we have identified four clinical and/or biological factors associated with higher numbers of aneuploid sperm. Finally, the presence of highly acetylated histone H4 was detected on testicular sections from 33 men with Sertoli cell only syndrome and/or a testicular tumour. In tubules lacking meiotic and post-meiotic germ cells, the Sertoli cells' nuclei showed a global increase in H4 acetylation. This result suggests that histone acetylation could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be deregulated in SCO syndromes and testicular cancers. Finally, this work could contribute to the improvement of the clinical care of severe male infertility cases.; L'objectif de ce travail est d'approfondir l'exploration du génome et de l'épigénome somatique et germinal chez des hommes présentant une atteinte sévère de la spermatogenèse. Concernant le génome somatique, nous avons recherché la présence de mosaïques somatiques pour le chromosome Y microdélété chez 44 hommes infertiles, et aucune mosaïque n'a été détectée. Nous avons également analysé des réarrangements complexes du chromosome Y chez 3 patients infertiles. Cette étude nous a permis d'affiner leur caryotype et de mieux définir l'implication de l'anomalie du Y dans le phénotype d'infertilité. La ségrégation méiotique des chromosomes X, Y, 18, 13 et 21 a été étudiée par FISH multi-couleurs sur les spermatozoïdes de 31 patients infertiles. Nous avons montré des taux de disomies spermatiques augmentés chez la moitié de ces patients et identifié 4 facteurs de risques cliniques ou biologiques associés à l'augmentation des anomalies chromosomiques spermatiques. Enfin, concernant l'exploration de l'épigénome germinal, nous avons caractérisé le profil d'acétylation des histones par immunohistochimie sur les biopsies testiculaires de 33 patients atteints d'un syndrome des cellules de Sertoli isolées et/ou d'une tumeur testiculaire. Nous avons mis en évidence une hyperacétylation globale massive du noyau des cellules de Sertoli lorsque les tubes séminifères sont dépourvus de cellules méiotiques et post-méiotiques. Cette étude a révélé que l'acétylation des histones pourrait être impliquée dans le dialogue entre cellules germinales et cellules de Sertoli, et que sa dérégulation pourrait être associée à la genèse des cancers testiculaires. Ce travail ouvre des perspectives intéressantes pour la prise en charge des infertilités masculines sévères.

The aim of the present work is to investigate the role of genetic and epigenetic anomalies in severe spermatogenesis impairment. First, we looked for somatic mosaicisms for the Y microdeletion in 44 infertile patients, and no mosaicism was observed. We have also studied the Y chromosome of 3 infertile patients with a Y rearrangement. This investigation allowed a more precise description of the abnormal Y and shed light on how Y chromosomal rearrangements could be involved in the infertility phenotype. Second, the sperm aneuploidy rates for chromosome X, Y, 18, 13 and 21, were assessed by FISH in 31 infertile patients. Nearly half of the infertile patients had a significantly increased disomy rate. Moreover, we have identified four clinical and/or biological factors associated with higher numbers of aneuploid sperm. Finally, the presence of highly acetylated histone H4 was detected on testicular sections from 33 men with Sertoli cell only syndrome and/or a testicular tumour. In tubules lacking meiotic and post-meiotic germ cells, the Sertoli cells' nuclei showed a global increase in H4 acetylation. This result suggests that histone acetylation could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be deregulated in SCO syndromes and testicular cancers. Finally, this work could contribute to the improvement of the clinical care of severe male infertility cases., L'objectif de ce travail est d'approfondir l'exploration du génome et de l'épigénome somatique et germinal chez des hommes présentant une atteinte sévère de la spermatogenèse. Concernant le génome somatique, nous avons recherché la présence de mosaïques somatiques pour le chromosome Y microdélété chez 44 hommes infertiles, et aucune mosaïque n'a été détectée. Nous avons également analysé des réarrangements complexes du chromosome Y chez 3 patients infertiles. Cette étude nous a permis d'affiner leur caryotype et de mieux définir l'implication de l'anomalie du Y dans le phénotype d'infertilité. La ségrégation méiotique des chromosomes X, Y, 18, 13 et 21 a été étudiée par FISH multi-couleurs sur les spermatozoïdes de 31 patients infertiles. Nous avons montré des taux de disomies spermatiques augmentés chez la moitié de ces patients et identifié 4 facteurs de risques cliniques ou biologiques associés à l'augmentation des anomalies chromosomiques spermatiques. Enfin, concernant l'exploration de l'épigénome germinal, nous avons caractérisé le profil d'acétylation des histones par immunohistochimie sur les biopsies testiculaires de 33 patients atteints d'un syndrome des cellules de Sertoli isolées et/ou d'une tumeur testiculaire. Nous avons mis en évidence une hyperacétylation globale massive du noyau des cellules de Sertoli lorsque les tubes séminifères sont dépourvus de cellules méiotiques et post-méiotiques. Cette étude a révélé que l'acétylation des histones pourrait être impliquée dans le dialogue entre cellules germinales et cellules de Sertoli, et que sa dérégulation pourrait être associée à la genèse des cancers testiculaires. Ce travail ouvre des perspectives intéressantes pour la prise en charge des infertilités masculines sévères.

We report the case of a 14-year-old girl with a testicular feminization syndrome. The inguinal cryptorchid testis contained plurifocal hamartomas ranging from 0.5 to 1 cm. They were composed of tubules lined by cylindrical Sertoli cells immunoreactive for alpha-inhibin and p30/32(MIC2). The stroma contained few Leydig cells. Ultrastructural study showed tubules with immature Sertoli cells. The testicular feminization syndrome is caused by mutations of the androgen receptor gene. Patients with male genotype 46, XY have a female morphotype with external sexual organs without ambiguity. They have neither uterus nor ovary but two cryptorchid testis in which sex-cord stromal tumors can develop. Their malignant transformation is rare but requires preventive bilateral orchidectomy.

We report one case of "sex-cord stromal tumor" composed by Leydig and Sertoli cells. An epidermoid cyst of the testicle will be described. Finally, a case of scrotal leiomyoma is reported.

Spermatogenesis is a complex process of cellular multiplication and differentiation, the regulation of which is only partially understood. This may be due; 1) to the redundancy of the mechanisms of local regulation; 2) to the ubiquitous character of those growth factors and cytokines present in the testis; 3) to the lack of long term culture systems allowing male germ cell development. We describe herein two culture systems associating Sertoli cells and germ cell in which part of the meiotic step can occur in vitro. In the first system, pachytene spermatocytes prepared by centrifugal elutriation are cultured on a layer of Sertoli cells. In the second system small fragments of seminiferous tubules are seeded in bicameral chambers. The cytological and immunocytochemical studies presented show that pachytene spermatocytes of stages IV-VI differentiate into spermatids of steps 1 to 5.

Within the male gonad, TNF alpha is secreted by macrophages in the interstitial tissu and by germ cells in seminiferous tubules. TNF alpha receptors (type I) have been detected in the somatic cells of the testis (i.e. Leydig and Sertoli cells). TNF alpha has two type of action within the testis: the cytokine has an anti-hormonal role and play a role in the interactions between Sertoli and germ cells. Anti-hormonal actions of TNF alpha are (i) inhibition of LH-induced testosterone production in Leydig cells and (ii) inhibition of FSH-induced inhibin production in Sertoli cells. Moreover, TNF alpha; produced by germ cells, enhanced expression of growth factors in Sertoli cells (IGFBP3, receptor type I of bFGF) and enhanced production of energetic metabolite important for germ cells such as lactate (via enhancement of lactate dehydrogenase enzyme, isoform 5). Moreover, Fas/Fas ligand system, proteins related to TNF alpha, seem to play a key role in the protection of germ cells against the immune system within the testis. In conclusion, TNF alpha and related proteins, could play a great role in testicular functions such as spermatogenesis (interactions between Sertoli and germ cells) and in the interactions between immune system and spermatogenesis (role of TNF alpha originating form blood/testicular macrophages, role of Fas/Fas ligand system).

The ability of the male gonad to convert androgens into estrogens is well known; the microsomal enzymatic complex involved in this transformation is named aromatase and is composed of a specific cytochrome P450 aromatase (P450arom) and a ubiquitous reductase. Using a highly specific RT-PCR method we have measured the amount of P450arom mRNA in purified Leydig and Sertoli cells prepared from 20, 40 and 70-80 day-old rats. The amount of P450arom mRNA in the Leydig cells is independent of age (40 x 10(-3) attomoles/micrograms of total RNA); in contrast, in the immature rat Sertoli cells, after 5 days of culture the amount of P450arom mRNA is 20-fold lower when compared to that of 20-day-old rat Sertoli cells (71 x 10(-3) attomoles/micrograms of total RNA). Nevertheless, irrespective of the age, the addition of either FSH or dbcAMP for 6 h increases the level of P450arom mRNA in the rat Sertoli cell preparations. Therefore, we evidenced that during testicular maturation not only the Leydig cells but also the Sertoli cells of the rat have the capacity to express the gene for cytochrome P450 aromatase.

In addition to the endocrine control of Leydig cell functions by LH, paracrine control of Leydig cell functions has been suspected from the indirect stimulatory effect of FSH on Leydig cells. Coculture experiments of Leydig and Sertoli cells and the effect of Sertoli cell conditioned media on Leydig cells confirmed the production by Sertoli cells of acute steroideogenic factor(s) and factors involved in the positive or negative control of Leydig cell differenciated functions. Characterization and purification of these paracrine factors has been until recently unsuccessful. Another approach has been to investigate whether compounds of known biological activities in other systems, were produced within the testis and act on leydig cells. IGF-I is produced by Sertoli and Leydig cells under the control of their respective gonadotropin, LH and FSH. IGF-I enhanced hCG responsiveness of Leydig cells by increasing both LH receptor and steroidogenic enzymes. On the contrary TGF-beta which is also produced by Sertoli and Leydig cells is a potent inhibitor of Leydig cell functions. Its production by Sertoli cells is inhibited by FSH. Inhibin enhanced Leydig cell differentiated cell functions. Activin has, conversely to what has been published in the rat, a stimulatory effect on Leydig cell functions in the immature porcine Leydig cell model. The effects of these growth factors or related molecules mainly consist in positive (IGF-I, Inhibin, Activin) or negative (TGF-beta, TGF-alpha/EGF, bFGF) trophic effects regulating LH/hCG receptor number and mRNAs and steroidogenic enzyme mRNAs and activites, allowing regulation of the responsiveness of Leydig cells to LH. Thus both gonadotropins contribute, directly for LH and indirectly, through paracrine mecanisms, for FSH, to testosterone production.

This study was performed in adult male goats in which seasonal variations were abolished by rapid alternations of long days and short days. These treatments have been shown previously to prevent seasonal changes in the hypothalamo-pituitary axis and to maintain testis weight and sperm production at a high level. The experimental groups were exposed for 3 years to an alternation of either a 1 month short (16 h dark; 8 h light) and 1 month long (16 L; 8 D) photoperiod (2 month cycle; n = 5) or of a 2 month short and 2 month long photoperiod (4 month cycle; n = 4). The control groups were maintained in natural photoperiodic conditions (45 degrees N) and goats were slaughtered in the non-breeding season (end of April RS; n = 5) at the same period as light-treated bucks, or in the breeding season (end of September BS; n = 6). The total weight of the testes, the length and mean diameter of the seminiferous tubules of light-treated goats were similar to those in the breeding season, and higher than those in the non-breeding season. The total number of A0 spermatogonia was increased by light treatments as compared to control goats in the breeding and non-breeding season. The daily production of A1 spermatogonia, leptonene primary spermatocytes and round spermatids in light-treated goats was maintained at the peak breeding season level. The intra-testicular concentration of testosterone, total volumes of intertubular tissue and of Leydig cells, and the number of Leydig cells per testis did not differ between groups. Although the mean cross-sectional area of Leydig cells in light-treated goats was similar to this area in non-breeding season goats, it was significantly lower than that of breeding season goats. In conclusion, the rapid alternation of short and long days allowed an increase in all the germ cells from the A0 spermatogonia onwards, which was responsible for the maintenance of high spermatogenetic activity of light-treated goats.

Many Sertoli functions are regulated by the receptor-mediated action of follicle-stimulating hormone (FSH). The interaction of FSH with its specific cell surface receptors leads to stimulation of a number of intracellular events, including the activation of guanine nucleotide binding protein (G protein), adenylate cyclase and the cAMP-dependent protein kinase (PKA) pathway. In addition to positive regulation of cell functions, a phenomenon of refractoriness occurs after primary exposure of target cells to the hormone. Different sites of lesion have been suggested including down-regulation of FSH receptor, uncoupling of the receptor and the G protein/adenylate cyclase complex, and stimulation of nucleotide phosphodiesterases or inhibition of PKA activity. Alterations of cell responsiveness are mediated by a combination of these different mechanisms occurring over different time-scales and hormonal concentrations.

Effects of epidermal growth factor (EGF) on pH transients in aggregates of Sertoli cells and germinal cells have been investigated with confocal microscopy using a fluorescent pH sensitive indicator. In some Sertoli cells EGF caused a rapid rise in the pH whereas other Sertoli cells did not respond to EGF. Some Sertoli cells showed a delayed response which coincidated with a similar pH change in neighbouring germinal cells. Since isolated germinal cells never showed a pH response after exposure to EGF, we have concluded that some Sertoli cells may communicate with germinal cells via gap junctions.

Recent data on TGF beta (and related peptides) expression and action in the testis are briefly reviewed. At least, three isoforms of TGF beta are expressed during the male gonadal development. TGF betas are expressed in somatic and germ cells. The expression of the different isoforms varies during gonadal development and is cell specific. Both types I, II and III TGF beta receptors have been localized in somatic cells. TGF beta inhibits gonadotropin action both in Sertoli cells and Leydig cells as well as proliferation of germ cells and Leydig cells. Inhibins and activins (in terms of mRNA and proteins) are expressed in somatic cells and germ cells. Inhibin production in Sertoli cells is under the control of FSH, local factors (ex: EGF, insulin, opiods) and probably of germ cells. Activins and inhibins regulate Leydig and Sertoli cells activities and germ cells development. The existence of activin receptors in somatic and germ cells has been suggested by the presence of type II receptor mRNA (ActRII and ActRIIB).

The existence of multiple interactions between somatic cells in the testis, has been suspected in vivo, and demonstrated in vitro by cocultures and cultures in the presence of conditioned media. Long term Sertoli-Leydig cell interactions results in a local regulation of the specific differentiated functions of these cells, and more precisely in a control of their responsiveness to their respective gonadotropin. Among the paracrine and/or autocrine factors, growth factors are involved in the control of differentiated functions. It is the case for IGF-I and TGF-beta, which respectively exhibit a stimulatory and inhibitory effect on Leydig cell functions and more specifically on LH responsiveness.

In Percoll purified Leydig cells from mature rat incubated in Ham F12/DME medium, we have demonstrated that the testosterone production (1 ng/10(6) Leydig cells/3 h) is increased 9 fold in presence of a saturating amount of oLH but diminished when larger doses of gonadotropin are used. However, if SCM or STM is added together with oLH to the Leydig cell incubation medium, a further increase (69%) of testosterone output is noticed. Obviously, when the testosterone production is low consecutive to a desensitizing dose of oLH, the STM or SCM improves at least 2 fold the steroid synthesis. Therefore, the paracrine factor (or factors) presents in SCM (or STM) which is testis specific and acts in synergy with dbcAMP and 22R-hydroxycholesterol, uses an alternative pathway to improve rat Leydig cell testosterone production since its biological effect is highly potent in absence and in presence of oLH. Therefore, these data bring new insights in the regulation of rat Leydig cell steroidogenesis through cAMP-independent channel suggesting either an increase of the cholesterol side chain cleavage activity and/or availability of the cholesterol to mitochondria.

In the mammalian testis, the Sertoli cell plays a key role in the development and maintenance of spermatogenesis. Indeed, within the seminiferous tubule, the structure of the Sertoli cells and the specialized junctions between them and the neighbouring germ cells, contribute to create the sophisticated microenvironment and to bring all the nutriments require for a full development of germ cells. In this review, we have compared the main Sertoli cell functions in the rat and the pig to the available data concerning the human. We have included our recent results obtained from testicular tissues of 15 young men (mean age: 29 years) from which we have prepared the Sertoli cells. In addition to the lactate and estradiol-17 beta syntheses, the human Sertoli cell produces several proteins, namely transferrin, ferritin and inhibin under the control of germ cells.

Previous studies have suggested that FSH promotes the intercellular coupling of Sertoli cells from immature rat testis in primary culture ([1], [2]). In order to test this hypothesis, we have investigated the diffusional coupling between Sertoli cells in primary culture with the FRAP technique. The coupling is low in unstimulated cells but increases in the presence of FSH. This effect is not reversed by returning to the control medium. Testosterone decreases this coupling, an effect which is reversed by a new exposure to FSH. Taken together these data show that FSH initiates diffusional coupling in Sertoli cells and that testosterone antagonizes this effect.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.

Anti-Müllerian hormone, responsible for Müllerian regression in male fetuses, is a glycoprotein dimer with two 72 kD subunits. The AMH gene is a small (2,800 bp) gene with 5 exons, localized on the tip of the short arm of chromosome 19, band p 133, and transcribed in a 2,000 kbp mRNA. Persistent Müllerian duct syndrome, a rare form of male pseudohermaphroditism characterized by the presence of uterus and Fallopian tubes in patients with normally virilized genitalia, may result from defective AMH gene or from target-organ insensitivity. Four mutations were identified in the AMH gene, 3 are point mutations (2 stop codons, the third altering the secondary structure of the molecule), the last is a 14 bp deletion, leading to alteration of the reading frame of the mRNA.

Testicular function is regulated not only by circulating hormones, among which the gonadotrophins play the main role, but also by local factors originating in multiple and complex interactions among cells. In this review, the example of gonadotrophins (LH and FSH) and Transforming Growth Factor beta (TGF beta) was chosen to illustrate the role of interactions between circulating hormones and gonadal growth factors in testicular function control; TGF beta-like activity has been found in the male gonad and we have used a model of cultured purified testicular cells to show that the action of TGF beta on testicular function mainly involves antagonism of the effect of gonadotrophins. Conversely, TGF beta promotes differentiated Leydig and Sertoli cell function. The example of interactions between TGF beta and gonadotrophins reported here shows that locally produced growth factors can regulate the response of testicular cells to gonadotrophins, a finding that extends our concept of reproductive endocrinology to cell-cell interactions.

Using intracellular microelectrode recordings, it was observed that FSH caused the hyperpolarization of cultured Sertoli cells from the testis of 17-day old rats; this hyperpolarization was related to the activation of a Na/K pump and an increase in Na/Ca influx and K efflux. The effect was irreversible and mediated by cAMP. Some other results suggested an initial effect of FSH on the junctional complex between the Sertoli cells and then its calcium dependence. Voltage-clamped membrane currents were investigated from whole-cell patch clamp recordings performed on single Leydig cells isolated from adult rat testis. Two outward currents were evoked by depolarizing voltage steps: a potassium current recorded in cells dialyzed with low-calcium media (10(-9)-10(-8) M) and a chloride current recorded in cells dialyzed with high-calcium media (10(-7)-10(-6) M). These responses to modifications of intracellular calcium were not affected by hCG. However, this hormone induced an early change of the K current to a Cl current when the cells were dialyzed by a low-buffered EGTA-calcium medium. These results suggest that hCG stimulation involved the blockade of a potassium current and the activation of a chloride current through an increase of intracellular calcium.

H-Y (histocompatibility Y) antigen plays a role in gonadal organogenesis, which is poorly understood. Indeed, it is not immunologically well-defined. The use of cytotoxicity tests cannot lead to a right quantification of its expression. In vertebrates, it is a marker of the heterogametic sex. As it is also detected in invertebrates, H-Y antigen is therefore ubiquitous and has a high phylogenic conservation. Its role, as an organizer of mammal testes, was carried out by controversial experiments of dissociation-reorganization, in gonadal cells. In gonads. H-Y antigen can be studied under 3 diverse aspects : secretion, fixation and expression, Recently, Ohno proposed a very attractive pattern of testicular and ovarian organogenesis. In males, organization of seminiferous tubules is the result of the interaction between H-Y antigen and its specific gonadal receptor. In females, primordial follicle formation is induced by the interaction between an hypothetic ovary-organizing antigen (similar to H-Y antigen) and the same specific receptor as in testis. These different hypotheses are discussed. In addition, it is underlined how expression of H-Y antigen (anchorage site of H-Y antigen on plasma membrane associated with beta 2-m) can be distinguished from its fixation on gonadal specific receptors. In view of controversial data, the masculinisation of bovine free-martin gonads by H-Y antigen is discussed. In XX males and XY females, H-Y expression which is variable is correlated with its gonadal organizing role. Finally, at present time location of H-Y structural and control genes remains unsolved. In view of all hypotheses postulated, it is not clearly demonstrated how H-Y antigen can act on gonadal organogenesis. Nevertheless, priority of cell-cell recognition, via H-Y antigen, has to be emphasized.

The blood-testis barrier in man is mainly due to the tight junctions of Sertoli cells. It creates favorable conditions for the spermatogenesis and sperm survival in the testicular fluid. It prevents the occurrence of autoimmunity after puberty. The alteration of the blood-testis barrier allows the production of antisperm antibodies and may lead to infertility.

Electron microscopic study of Sertoli cells from normal and Misulban-treated rats has been realized during foetal and postnatal life. From 15th day of foetal life, morphological aspects of Sertoli cells plead for a steroidogen activity and protein synthesis. Ultrastructural organels of Sertoli cells are the same in sterile and normal seminiferous tubule, except the Sertoli junctions which are less numerous in sterile tubules and appear later.

Spermatogenesis and spermiogenesis are controlled by FSH and testosterone but need also the participation of several paracrine and autocrine mechanisms of regulations. The relationships between peritubular, Sertoli and Leydig cells are currently investigated. High intratesticular testosterone levels are maintained by a binding to a protein called ABP which is synthetized by Sertoli cell and regulated by pituitary FSH. Leydig cell testosterone, peritubular cell P-Mod-S (protein modulating Sertoli function) and Sertoli cell FRP (follicle regulatory protein). Accumulation of testosterone results to aromatase activity modulation. Aromatization is stimulated by FSH, activin, alpha-MSH but is inhibited by aromatase inhibitor, inhibin, FSHBI (FSH binding inhibitor). Other molecules, growing factors, mitogenic factors, energetic substrates are synthetized in the testis under the control of germ cells. Understanding of these mechanisms of intratesticular regulation will permit to discover therapies capable of correcting certain fertility dysfunctions.

The reproductive function of both the male and female is under the control of several pituitary hormones including lutropin (LH) and follitropin (FSH). These hormones are highly homologous glycoprotein dimers sharing a common subunit. Their polymorphism mainly resides in the microheterogeneity of their carbohydrate chains that is also responsible for the variations in biological activity. In the gonads, these hormones bind to specific receptors and regulate steroidogenesis and gametogenesis. LH receptors also bind the choriogonadotropin hormone and are located in Leydig cells in testis, interstitial and medulla cells in ovary. FSH receptors are present in Sertoli cells in the male while they control follicular maturation in the female. In both sexes, their activity is coupled to that of other receptors such as that of prolactin or one able to recognize GnRH. The purification of LH receptor has been successfully achieved in several animal species and showed a significant structural homology between the receptors of both sexes.

The ultrastructure of the Sertoli cell studied in sexually active control animals during May-June and experimental animals sexually activated by light in winter, presents the commonly described ramified aspect with an infolded nucleus, well developed Golgi complexes, rough and smooth endoplasmic reticulum, numerous microfilaments, few liposomes and lysosomal formations; In the regressed testes of hibernating animals or blinded spring animals, the Sertoli cells are more round shaped with a significant increase in number and size of liposomes, lysosomes and various necrotic bodies. These morphological changes may be in favour of the possibility of a synchronization of the function of the Sertoli cell with the spermatogenic cycle to coordinate locally the proliferation and maturation of the germ cell.