SF-1 e LRH-1 são fatores de transcrição que exercem um papel fundamental na produção de esteroides nas gônadas e na suprarrenal, além de estarem envolvidos no processo tumorigênico desses órgãos. Por outro lado, POD-1 apresenta menor expressão em carcinomas adrenocorticais, e parece regular Sf-1. Nesse trabalho foi analisado o papel de POD-1 na regulação de SF-1 e de LRH-1 em células de tumores adrenocorticais. A hiperexpressão de POD-1 resultou em redução da expressão SF-1/SF-1. Em contraste, houve um aumento da expressão gênica de LRH-1, devido à diminuição da expressão de SHP, um regulador negativo de LRH-1. Nas células transfectadas com siRNA-POD-1, os níveis de POD-1 foram reduzidos e de SF-1 aumentado, reforçando o mecanismo regulatório entre os fatores. No ChIP assay, POD-1 se ligou a sequência E-box do promotor de SF-1. Por outro lado, não foi caracterizado a ligação de POD-1 no promotor LRH-1, embora POD-1 tenha se ligado ao E-box do promotor SHP. A redução de SF-1 diminuiu a expressão de StAR, mas não modulou a proliferação das células tumorais. Em resumo, POD-1 pode ter um papel mais amplo como regulador da transcrição de fatores que controlam o processo tumorigênico, e é um candidato a gene supressor de tumor nas células adrenocorticais. / SF-1 and LRH-1 have played a critical role in steroid production, adrenal and gonads. Moreover, there are evidences that they have acted in tumorigenesis process in these organs. POD-1 is downregulated in adrenocortical carcinoma (ACC) it seems to regulate Sf-1. In this work, it has been to analyse the role of POD-1 in SF-1 and LRH-1 regulation in adrenocortical tumor cells. The POD-1 overexpression has reduced SF-1/SF-1 expression. However, there was an increase of LRH-1 gene expression due to SHP expression decrease which is negative regulate of LRH-1. The POD-1 and SF-1 gene expression in transfected cells with siRNA-POD-1 has shown POD-1 decrease and SF-1 increase emphasizing a regulatory mechanism between POD-1 and SF-1. By ChIP assay it was shown that POD-1 binded in SF-1 promoter E-box sequence. It was not characterized that POD-1 binded in LRH-1 promoter, although POD-1 can bind in SHP promoter E-box sequence. The reduction of SF-1 expression by POD-1 has decreased the StAR expression, however, it was not enough to change tumor cell proliferation. In summary, POD-1 must have a wider role as regulator of fator transcription which controls tumorigenese process being a possible candidate as tumor supressor gene in adrenocortical cells.

SF-1 e LRH-1 são fatores de transcrição que exercem um papel fundamental na produção de esteroides nas gônadas e na suprarrenal, além de estarem envolvidos no processo tumorigênico desses órgãos. Por outro lado, POD-1 apresenta menor expressão em carcinomas adrenocorticais, e parece regular Sf-1. Nesse trabalho foi analisado o papel de POD-1 na regulação de SF-1 e de LRH-1 em células de tumores adrenocorticais. A hiperexpressão de POD-1 resultou em redução da expressão SF-1/SF-1. Em contraste, houve um aumento da expressão gênica de LRH-1, devido à diminuição da expressão de SHP, um regulador negativo de LRH-1. Nas células transfectadas com siRNA-POD-1, os níveis de POD-1 foram reduzidos e de SF-1 aumentado, reforçando o mecanismo regulatório entre os fatores. No ChIP assay, POD-1 se ligou a sequência E-box do promotor de SF-1. Por outro lado, não foi caracterizado a ligação de POD-1 no promotor LRH-1, embora POD-1 tenha se ligado ao E-box do promotor SHP. A redução de SF-1 diminuiu a expressão de StAR, mas não modulou a proliferação das células tumorais. Em resumo, POD-1 pode ter um papel mais amplo como regulador da transcrição de fatores que controlam o processo tumorigênico, e é um candidato a gene supressor de tumor nas células adrenocorticais. / SF-1 and LRH-1 have played a critical role in steroid production, adrenal and gonads. Moreover, there are evidences that they have acted in tumorigenesis process in these organs. POD-1 is downregulated in adrenocortical carcinoma (ACC) it seems to regulate Sf-1. In this work, it has been to analyse the role of POD-1 in SF-1 and LRH-1 regulation in adrenocortical tumor cells. The POD-1 overexpression has reduced SF-1/SF-1 expression. However, there was an increase of LRH-1 gene expression due to SHP expression decrease which is negative regulate of LRH-1. The POD-1 and SF-1 gene expression in transfected cells with siRNA-POD-1 has shown POD-1 decrease and SF-1 increase emphasizing a regulatory mechanism between POD-1 and SF-1. By ChIP assay it was shown that POD-1 binded in SF-1 promoter E-box sequence. It was not characterized that POD-1 binded in LRH-1 promoter, although POD-1 can bind in SHP promoter E-box sequence. The reduction of SF-1 expression by POD-1 has decreased the StAR expression, however, it was not enough to change tumor cell proliferation. In summary, POD-1 must have a wider role as regulator of fator transcription which controls tumorigenese process being a possible candidate as tumor supressor gene in adrenocortical cells.

<p>In mammals sex chromosomes direct and initiate the development of male and female gonads and subsequently secondary sex characteristics. In most vertebrates each individual is pre-destined to either become male or female. The process by which this genetic decision is carried out takes place during the embryonic development and involves a wide range of genes. The <i>fushi tarazu</i> factor-1 (FTZ-F1) is a nuclear receptor and transcription factor, which in mammals has proven to be essential for gonad development and directs the differentiation of testicular Sertoli cells. A mammalian FTZ-F1 homologue subtype, steroidogenic factor-1 (SF-1), is a member of the nuclear receptor 5A1 (NR5A1) group and regulate several enzymes involved in steroid hormone synthesis. It also regulates the expression of the gonadotropin releasing hormone receptor GnRHr and the β-subunit of the luteinizing hormone (LH), indicating that it functions at all levels of the reproductive axis. Another mammalian FTZ-F1 subtype, NR5A2, is in contrast to SF-1, not linked to steroidogenesis or sex determination. Rather, NR5A2 is involved in cholesterol metabolism and bile acid synthesis in liver. Hormones and environmental factors such as temperature and pH can influence teleost development and reproductive traits, rendering them vulnerable to pollutants and climate changes. Very little is known about teleost FTZ-F1 expression, regulation and function. In this thesis, expression patterns of four zebrafish FTZ-F1 genes (ff1a, b, c and d) and two Arctic char genes (acFF1α and β) were studied during development, displaying complex embryonic expression patterns. Ff1a expression was in part congruent with expression of both mammalian NR5A1 and NR5A2 genes but also displayed novel expression domains. The complexity of the expression pattern of ff1a led to the conclusion that the gene may be involved in several developmental processes, including gonad development, which also was indicated by its transcriptional regulation via Sox9a. Two ff1a homologues were also cloned in Arctic char and were shown to be involved in the reproductive cycle, as the expression displayed seasonal cyclicity and preceded that of the down stream steroidogenic genes StAR and CYP11A. High levels were correlated to elevated plasma levels of 11-ketotestosterone (11KT) in males and 17β-estradiol (E2) in females respectively. Treatment with 11KT did not affect FTZ-F1 expression directly but was indicated to alter expression of CYP11A and 3β-hydroxysteroid dehydrogenase. E2 treatment was indicated to down-regulate the expression of testicular FTZ-F1, which may contribute to the feminising effect previously observed in E2 treated salmonids. Ff1d is a novel FTZ-F1 gene, expressed in pituitary and interrenal cells during development, suggesting steroidogenic functions. In adult testis and ovary ff1d was co-expressed with anti-Mullerian hormone (AMH), a gene connected to sex determination in mammals and previously not characterised in teleost fish. The co-expression between ff1d and AMH was found in Sertoli and granulosa cells, which is congruent with the co-expression of mammalian SF-1 and AMH. This suggests that ff1d and AMH may have similar functions in teleost sex differentiation and reproduction, as their mammalian homologues. In conclusion, this study present data that connects members of the teleost FTZ-F1 family to reproduction, cholesterol metabolism and sex determination and differentiation.</p>

In mammals sex chromosomes direct and initiate the development of male and female gonads and subsequently secondary sex characteristics. In most vertebrates each individual is pre-destined to either become male or female. The process by which this genetic decision is carried out takes place during the embryonic development and involves a wide range of genes. The fushi tarazu factor-1 (FTZ-F1) is a nuclear receptor and transcription factor, which in mammals has proven to be essential for gonad development and directs the differentiation of testicular Sertoli cells. A mammalian FTZ-F1 homologue subtype, steroidogenic factor-1 (SF-1), is a member of the nuclear receptor 5A1 (NR5A1) group and regulate several enzymes involved in steroid hormone synthesis. It also regulates the expression of the gonadotropin releasing hormone receptor GnRHr and the β-subunit of the luteinizing hormone (LH), indicating that it functions at all levels of the reproductive axis. Another mammalian FTZ-F1 subtype, NR5A2, is in contrast to SF-1, not linked to steroidogenesis or sex determination. Rather, NR5A2 is involved in cholesterol metabolism and bile acid synthesis in liver. Hormones and environmental factors such as temperature and pH can influence teleost development and reproductive traits, rendering them vulnerable to pollutants and climate changes. Very little is known about teleost FTZ-F1 expression, regulation and function. In this thesis, expression patterns of four zebrafish FTZ-F1 genes (ff1a, b, c and d) and two Arctic char genes (acFF1α and β) were studied during development, displaying complex embryonic expression patterns. Ff1a expression was in part congruent with expression of both mammalian NR5A1 and NR5A2 genes but also displayed novel expression domains. The complexity of the expression pattern of ff1a led to the conclusion that the gene may be involved in several developmental processes, including gonad development, which also was indicated by its transcriptional regulation via Sox9a. Two ff1a homologues were also cloned in Arctic char and were shown to be involved in the reproductive cycle, as the expression displayed seasonal cyclicity and preceded that of the down stream steroidogenic genes StAR and CYP11A. High levels were correlated to elevated plasma levels of 11-ketotestosterone (11KT) in males and 17β-estradiol (E2) in females respectively. Treatment with 11KT did not affect FTZ-F1 expression directly but was indicated to alter expression of CYP11A and 3β-hydroxysteroid dehydrogenase. E2 treatment was indicated to down-regulate the expression of testicular FTZ-F1, which may contribute to the feminising effect previously observed in E2 treated salmonids. Ff1d is a novel FTZ-F1 gene, expressed in pituitary and interrenal cells during development, suggesting steroidogenic functions. In adult testis and ovary ff1d was co-expressed with anti-Mullerian hormone (AMH), a gene connected to sex determination in mammals and previously not characterised in teleost fish. The co-expression between ff1d and AMH was found in Sertoli and granulosa cells, which is congruent with the co-expression of mammalian SF-1 and AMH. This suggests that ff1d and AMH may have similar functions in teleost sex differentiation and reproduction, as their mammalian homologues. In conclusion, this study present data that connects members of the teleost FTZ-F1 family to reproduction, cholesterol metabolism and sex determination and differentiation.

Cortisol is a glucocorticoid produced by the zona fasciculata (ZF) of the adrenal cortex. Traditionally, cortisol production and release was seen as being regulated strictly by adrenocorticotropic hormone (ACTH). While this is true of baseline cortisol levels and in response to acute mental stress, the picture is somewhat more complicated in other situations.Interleukin-6 (IL-6) contributes to the maintenance of cortisol levels in situations of prolonged immune or inflammatory stress. AMP activated protein kinase (AMPK) was investigated as a possible mediator of the action of IL-6 or as an independent actor in raising cortisol levels in response to hypoxemic or hypoglycemic stress.5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside (AICAR) was used to activate AMPK. Bovine ZF tissue fragments were exposed to AICAR alone and together with a known AMPK inhibitor, compound C. Protein or mRNA was then extracted from these tissue fragments. As an indicator of overall steroidogenic activity, these extracts were tested using RT-PCR and western blot assays for relative protein and mRNA levels of steroidogenic acute regulatory (StAR) protein, steroidogenic factor-1 (SF-1), and dosage sensitive sex reversal adrenal hypoplasia congenita gene on the X chromosome, gene 1 (DAX-1). Also a reporter gene assay was performed on H295R cells with a transfected StAR promoter.In bovine ZF tissue fragments, AICAR caused a significant increase of StAR protein and mRNA and SF-1 protein with a decrease of DAX-1 protein in a dose and time dependant manner. DAX-1 mRNA was shown to decrease in response to AICAR administration in a dose dependant manner. AICAR induced increases in StAR protein and SF-1 protein, and the attendant decrease in DAX-1 protein were all shown to be reduced by administration of compound C. This demonstrated that in this situation AICAR is acting through AMPK. When IL-6 was given with compound C the levels of StAR, SF-1, and DAX-1 were significantly reduced from samples treated with IL-6 alone. AICAR exposure also increased StAR promoter activity in a dose and time dependant manner. This AMPK induced increase in steroidogenic activity provides a possible mechanism for increased cortisol during hypoxia and hypoglycemia, and a possible mediator for IL-6 in the ZF.

The adrenal cortex is a crucial endocrine gland in the mammalian stress response. In chronic inflammatory stress, cortisol is elevated whereas adrenal androgens are decreased. Furthermore, ACTH levels have poor correlation with the plasma cortisol in these conditions, thus suggesting that other factors are driving the stress response during chronic inflammatory stress. Interleukin-6 (IL-6), a cytokine which is released during chronic inflammatory stress, is assumed to be one such factor. Thus the biochemical pathways by which IL-6 increases cortisol release from the zona fasciculata (ZF), and decreases adrenal androgen release from the zona reticularis (ZR) were investigated. Since IL-6 activates AMP-activated kinase (AMPK) in skeletal muscle, AMPK was investigated for IL-6- induced effects in ZF and ZR tissue. The effects of AMPK activation and IL-6 exposure on the expression of the steroidogenic proteins, steroidogenic acute regulatory protein (StAR) and cholesterol side chain cleavage enzyme (P450scc), and on the steroidogenic nuclear factors steroidogenic factor-1 (SF-1) and adrenal hypoplasia congenita, critical region on the X chromosome, gene-1 (DAX-1) were investigated. AMPK activation and IL-6 exposure increased the expression of StAR, P450scc, and SF-1, and decreased DAX-1 in the ZF. Meanwhile, AMPK activation and IL-6 exposure decreased the expression of StAR, P450scc, and SF-1, and increased DAX-1 in the ZR. AMPK inhibition blocked the effects of AMPK activation and IL-6 on the ZF and ZR. Activator Protein-1 (AP-1) was the second biochemical intermediate studied since in other tissues AMPK activation increases the expression and phosphorylation of AP-1 subunits. IL-6 stimulation and AMPK activation increased the expression of the AP-1 subunits cFOS, cJUN, JUN B, and JUN D, while increasing the phosphorylation of cJUN in both the ZF and the ZR. These effects were blocked by AMPK inhibition. Inhibition of AP-1 leads to decreased StAR, P450scc, and SF-1, and increased DAX-1 in the ZF. Meanwhile, AP-1 inhibition leads to increased StAR, P450scc, SF-1, and decreased DAX-1 in the ZR. Therefore the AP-1 complex functions as a biochemical intermediate in the IL-6 and AMPK regulation of steroidogenic enzymes in the ZF and ZR. Overall, the results suggest that IL-6 activates AMPK, which increases the expression and phosphorylation of AP-1 subunits in the ZF and the ZR. However, increased AP-1 activation leads to increased StAR and P450scc in the ZF, but decreased StAR and P450scc in the ZR.

Le récepteur nucléaire orphelin facteur stéroïdogénique 1 (SF-1 ou NR5A1) est un modulateur indispensable du développement surrénal et gonadique et qui joue un rôle dans la détermination du sexe, le développent hypothalamique, la fonction hypophysaire et la stéroïdogénèse. Toutefois, les études sur SF-1 dans le milieu de la biologie de la reproduction portent majoritairement sur des modèles embryonnaires ou de mammifères immatures. L’objectif principal de cette thèse était de déterminer le rôle de SF-1 dans les évènements clés de la fonction reproductrice chez les mâles et femelles matures. Ce facteur de transcription est exprimé dans différents organes, principalement ceux de l’axe hypothalamo-hypophyso-gonadique, et dans divers types cellulaires des gonades. Nous avons donc généré 4 modèles de souris knockout conditionnels (cKO) en utilisant les allèles Cre-recombinase et flox de SF-1 (SF-1f/f) afin d’identifier son rôle dans les différentes populations cellulaires des testicules et ovaires de souris matures. Dans la première étude, nous avons présenté une analyse des souris femelles du modèle cKO du récepteur de la progestérone (PRCre/+;Nr5a1f/f), où la suppression de SF-1 est spécifique aux cellules gonadotropes de l’hypophyse et aux cellules ovariennes de type granulosa suite au déclenchement du signal ovulatoire ainsi que les cellules lutéales du corps jaune. Cette étude a révélé de nouveaux rôles in vivo de SF-1 durant l’ovulation et la lutéinisation, tout en suggérant que SF-1 est un médiateur de la synthèse et sécrétion des gonadotrophines. Les femelles PRCre/+;Nr5a1f/f cKO étaient infertiles principalement en raison de l’importante réduction de FSH et LH sécrété dans la circulation, causé par le phénotype hypophysaire. Afin de contourner cette dysfonction hypophysaire, des traitements de gonadotrophines exogènes ainsi que des transplantations d’ovaires nous ont permis de démontrer que SF-1 régule la transcription de gènes impliqués dans l’expansion du cumulus ainsi que la rupture de follicules pour induire l’ovulation. De plus, nous avons montré que l’absence de SF-1 dans les ovaires de souris matures peut mener à l’infertilité, indépendamment du phénotype hypophysaire. D’autre part, nos trouvailles indiquent que, malgré la basse expression de SF-1 dans le corpus luteum chez la souris, sa déplétion dans les cellules lutéales conditionnée par recombinase Cre inhibe la production de progestérone. Aucun phénotype reproductif a été observé chez les souris PRCre/+;Nr5a1f/f cKO mâles. La deuxième étude a démontré le rôle essentiel de SF-1 dans la fonction du testicule mature. La souris mâle P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f) cKO, où la suppression de SF-1 est spécifique aux cellules de Leydig, était fertile malgré la taille réduite de ses testicules, la malformation de ses tubes séminifères, la perturbation de la spermiogénèse, ainsi que la réduction d’expression de gènes de la stéroïdogénèse. Bien que les mâles aromatase (Cyp19Cre/+;Nr5a1f/f) cKO, supprimant SF-1 dans les cellules de Sertoli, étaient fertiles et démontraient des capacités reproductives similaires aux mâles contrôle, les souris Cyp17Cre/++Cyp19Cre/+; Nr5a1f/f cKO (dKO) étaient soit infertiles ou montrait une fertilité affaiblie. La dysgénésie sévère du cordon testiculaire ainsi que la spermatogénèse perturbée chez la souris dKO étaient causées par la déplétion simultanée de SF-1 chez les cellules de Leydig et Sertoli, suggérant que les cellules de Sertoli peuvent compenser pour l’absence de SF-1 dans les cellules de Leydig et vice versa. Ces données démontrent que SF-1 est requis pour une stéroïdogénèse testiculaire et une spermatogénèse ainsi qu’une fertilité normale, bien que savoir si la régulation de ces fonctions par SF-1 est directe ou indirecte reste à élucider. De façon intéressante, les femelles des trois lignées cKO étudié dans ce deuxième article étaient fertiles et la sous expression de SF-1 dans les cellules ovariennes de type granulosa ou de la thèque a produit des effets mineurs sur leur fonction reproductive. En somme, la recherche présentée dans cette thèse contribue à l’avancement des connaissances sur SF-1 et son rôle dans la régulation d’événements reproductifs cruciaux dans l’hypophyse, l’ovaire et le testicule de souris matures. Les lignes de souris produites dans ce projet vont servir d’outil indispensable pour élucider les mécanismes de régulation de SF-1 sur la fonction gonadique and présenter de nouvelles avenues de recherches pour ce récepteur orphelin. / The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable modulator of adrenal and gonadal development, playing key roles in sex determination, hypothalamic development, pituitary function and steroidogenesis. Yet, studies to date of SF-1 in reproductive biology mostly focus on embryonic and immature mammalian models. The overall objective of this thesis was to determine the role of SF-1 in key events of mature male and female mouse reproductive function. This transcription factor is expressed in a variety of organs, mainly those of the hypothalamic-pituitary-gonadal axis, as well as in multiple cell types of the gonads. Therefore, we generated four conditional KO (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (SF-1f/f) to identify its role in different cell types of the testes and ovaries of mature mice. Our first study presents an analysis of female mice from the progesterone receptor (PRCre/+;Nr5a1f/f) cKO model, where SF-1 depletion is specific to gonadotropes in the pituitary gland as well as granulosa cells of the peri-ovulatory follicle and luteal cells of the corpus luteum. This research highlighted new in vivo roles for SF-1 in ovulation and luteinization, and provided further evidence that SF-1 is a mediator of gonadotropin synthesis and secretion. PRCre/+;Nr5a1f/f cKO females were infertile, due in large part to the reduced secretion of FSH and LH, caused by the pituitary phenotype. Exogenous gonadotropin treatments and ovarian transplantation experiments allowed us to circumvent the pituitary dysfunction to demonstrate that SF-1 in granulosa cells regulates the transcription of cumulus expansion and follicle rupture genes to induce ovulation. In addition, we showed that the absence of SF-1 in ovaries of mature mice can lead to female infertility, independent of the pituitary phenotype. Moreover, the data showed that, though SF-1 expression is reduced in mouse corpus luteum, its Cre-mediated depletion in luteal cells abrogates progesterone production. No reproductive phenotype was observed in PRCre/+;Nr5a1f/f cKO males. Results from our second study demonstrated that SF-1 plays an essential role in mature testicular function. The P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f) cKO male mouse, where the SF-1 depletion is specific to Leydig cells, were fertile, though showed reduced testis size with disrupted seminiferous tubules and impaired spermiogenesis, in addition to reduced expression of steroidogenic genes. While the aromatase (Cyp19Cre/+;Nr5a1f/f) cKO males were fertile and showed reproductive capacities comparable to control males, the Cyp17Cre/++Cyp19Cre/+; Nr5a1f/f cKO (dKO) model were either infertile or showed significantly impaired fertility. The dKO males displayed severe testis cord dysgenesis and impaired spermatogenesis caused by the depletion of SF-1 in both Leydig and Sertoli cells, suggesting that Sertoli cells can compensate for the absence of SF-1 in Leydig cells and vice versa. These data provide strong evidence that SF-1 is required for normal testicular steroidogenesis, spermatogenesis and male fertility, though whether the regulation of these functions is direct or indirect remains to be elucidated. Interestingly, the females of the three cKO mouse lines studied in this second article were fertile and the depletion of SF-1 in granulosa cells of antral follicles or in theca cells produced minor effects on their steroidogenic capacities. Collectively, the research presented in this thesis contributes to advance our understanding of the role of SF-1 in the regulation of essential reproductive events in the pituitary, ovary and testis of mature mouse gonads. The mouse lines generated for this project will serve as valuable tools to elucidate the mechanisms underlying SF-1 regulation of gonad function and present novel directions for the investigation of this nuclear receptor.

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