Luteinizing hormone (LH), one of the two gonadotropin hormones released from the pituitary gland, binds its receptor (LHR) in the gonads to initiate steroid hormone production, as well as gametogenesis and ovulation. Mutations of amino acid sequence within the receptor can render it either inactive or constitutively active. All activating mutations result in male-limited precocious puberty. Males afflicted with this condition undergo puberty around 4 years of age, with elevated testosterone levels and premature skeletal development. In order to better understand how chronic ligand-mediated activation of the LHR affects gonadal development and function, a mouse model expressing a yoked hormone-receptor (YHR) complex, engineered by covalently linking the hormone human chorionic gonadotropin to the rat LHR, has been studied. YHR+ males have prepubertally elevated testosterone and decreased gonadotropin levels. Histological evaluation of the testes of these animals show significantly smaller seminiferous tubules and Leydig cell clusters. Finally, testis gene expression analysis revealed a significant decrease in the relative mRNA expression of three Leydig cell specific genes. Based on these results, it was hypothesized that premature activation of the LHR impairs postnatal Leydig cell development. In the testis there are two morphologically and developmentally distinct populations of Leydig cells, the fetal and the adult. The first objective of this study was to quantify the populations of cells in the adult Leydig cell lineage in both the YHR+ and the WT controls. Real-time RT-PCR, for markers of the immature and adult Leydig cell populations, as well as Leydig cell quantification, suggested a delay in adult Leydig cell development. Interestingly, there was a significant increase in the fetal Leydig cell population in the YHR+ mice. The second objective was to determine if the decrease in the adult population is due to either a decrease in proliferation or an increase in apoptosis in the YHR+ animal. There was not a difference in apoptosis between the WT and the YHR+ at any age examined, however, there was a decrease in progenitor Leydig cell proliferation in the YHR+ animals at 2 weeks of age. The final objective was to determine if elevated neonatal testosterone levels impairs the development of the adult Leydig cell population. Seven-day old WT pups were subjected to testosterone supplementation via subdermal implant. Quantification of the total Leydig cell population revealed a significant decrease in the number of adult Leydig cells in the testosterone-treated group similar to that seen in the YHR+ animal. Taken together, these data suggest that elevated neonatal testosterone levels resulting from premature LHR activation inhibits the proliferation of progenitor Leydig cells, resulting in fewer adult Leydig cells in the YHR+ animals.

There is increasing evidence to suggest that fetal events can predetermine reproductive health and general wellbeing in adulthood, a process termed 'fetal programming'. This refers to the association between altered fetal growth/development and health disorders in adulthood e.g. the metabolic syndrome, which is linked to low male testosterone levels. Studies from both Europe and the USA have shown that adult male testosterone levels have been declining, independent of age. As low testosterone levels in aging men are associated with increased morbidity and mortality, this highlights the importance of investigating how testosterone levels are determined or potentially ‘programmed’ during fetal development. Evidence from human and rodent studies have shown that reduced fetal androgen exposure results in lower adult testosterone levels, although the mechanism(s) is unknown, to date. One way to explain how a fetal insult (e.g. androgen deficiency) could affect (testosterone producing) adult Leydig cells in adulthood, is if their progenitor cells were present during fetal life and were thus affected by such an insult. This hypothesis has been unexplored to date, due to the lack of a unifying/defining marker for adult Leydig progenitor cells. An earlier study promoted the hypothesis for the studies in this thesis, namely that chicken ovalbumin upstream promoter transcription factor-II (COUP-TFII) might constitute such a marker, as inducible knockout of COUP-TFII in pre-pubertal male mice results in failure of adult Leydig cells to develop. Therefore, the hypothesis which was explored in this thesis was that 'fetal programming' of COUP-TFII+ adult Leydig progenitor cells prior to their differentiation into adult Leydig cells, would explain how fetal events could predetermine adult testosterone levels. To investigate whether adult Leydig cells (ALC) develop from COUP-TFII+ interstitial cells, firstly an adult Leydig cell ablation/regeneration model was used, which involved a single injection of ethane dimethane sulphonate (EDS). This identified that in rats, ALC derive from COUP-TFII+ interstitial cells which do not express any other phenotypical adult Leydig or interstitial cell markers prior to differentiation. Secondly, COUP-TFII+ adult Leydig progenitor cells are abundant in the fetal testis and conserved across species, including man. Thirdly, fetal interstitial cells which differentiated into ALC, as evident from an ALC lineage tracer model, also expressed COUP-TFII. Overall, these findings suggest that the COUP-TFII+ interstitial cells which differentiate into ALC are 'adult Leydig progenitor cells'. The findings from this thesis also show that the identified adult Leydig progenitor cells express the androgen receptor (AR) in fetal life. Furthermore, experimental reduction of androgen action in fetal life in transgenic mice (AR knockout) or chemical manipulations to reduce fetal testosterone levels (di(n-butyl) phthalate; DBP exposure) resulted in a similar reduction (~40%) in progenitor cell numbers from birth through to adulthood. A parallel reduction of adult Leydig cell numbers across postnatal development was found in mice, but not rats, but as a result of altered fetal androgen action, both models showed evidence for compensated adult Leydig cell failure. This is defined as normal/low testosterone and elevated luteinising hormone (LH) levels. Cell-selective knockout of AR in peritubular myoid (PTM) cells (PTM-ARKO) or Sertoli cells (SC-ARKO) did not affect the numerical development of adult Leydig progenitor cells. To manipulate testicular testosterone action in postnatal life, rats were exposed to a potent AR antagonist, flutamide, which reduced the number of adult Leydig progenitor cells but did not affect ALC number/function. However, the combination of fetal DBP+postnatal flutamide exposure reduced adult Leydig progenitor cells and resulted in compensated ALC failure. Overall, these studies highlight the importance of fetal androgens for the normal development of adult Leydig progenitor cells and for the subsequent development of normally functioning adult Leydig cells. As fetal deficits in androgen exposure resulted in adult Leydig cell dysfunction, this thesis also investigated three separate models to determine whether increased fetal androgen exposure could increase/enhance adult Leydig progenitor cell development, resulting in a 'gain of adult Leydig cell function'. In the first model to increase fetal androgen exposure, pregnant dams injected with testosterone propionate (TP; 20mg/kg/day e14-21.5) were discarded, due to confounding factors including fetal growth restriction and aromatisation of TP. The second model utilised dihydrotestosterone (DHT; 10mg/kg/day), administered to pregnant dams, but there were no effects found in adulthood to male offspring. It was concluded that the administered dose was not sufficient to increase intratesticular testosterone levels in the fetus. The third model utilised an inducible nitric oxide synthase knockout (iNOS-/-) mouse model, for which previous evidence showed increased testis weight, Leydig and Sertoli cell number (~50%), and normal testosterone but low LH levels in adulthood. Stereological quantification showed an increase in the number of adult Leydig progenitor cells in postnatal, but not fetal life, which resulted in the conclusion that the observed changes were a consequence of postnatal effects. Finally, a potential mechanism to explain how DBP-induced androgen deficiency in fetal life, could result in adult Leydig cell dysfunction in adulthood was investigated. Analysis of testicular genes in adulthood, involved in the steroidogenic pathway, showed a reduction in 3b-hsd and StAR. The reduced StAR expression was associated with increased repressive histone methylation (H3K27me3) in its proximal promoter region, as demonstrated by a chromatin immunoprecipitation (ChIP) assay, qPCR, and densitometrical analysis. Accordingly, adult Leydig cells were shown to express increased H3K27me3 by immunohistochemistry, a change also evident in adult Leydig progenitor cells in the fetal testis. This would provide a potential mechanism to explain how fetal events can 'programme' adult Leydig cell testosterone production, namely via an epigenetic change to adult Leydig progenitor cells. In summary, the results in this thesis show how fetal events, including androgen action on progenitor cells, can potentially programme adult Leydig cell function and thus determine testosterone levels. As testosterone is crucial to man, the findings reported in this thesis may have important implications for the general health and longevity of man.

Phthalate esters (phthalates) such as dibutylphthalate (DBP) are commonly used as plasticisers and pesticides in a variety of products such as children‟s plastic toys, food packaging, cosmetics, medical equipment (including surgical equipment), and acaricides. Because of their widespread use phthalates are ubiquitous environmental contaminants that humans are commonly exposed to. Phthalates are known endocrine-disrupting chemicals (EDCs) that are well known to cause male reproductive defects such as cryptorchidism (failed descent of the testes) and hypospadias (malformations in the urethra) in a range of different species if they are exposed in utero. They do this by reducing testosterone production in Leydig cells, which are the primary site of testosterone biosynthesis in the male. Because phthalates are dose-additive they are considered to share the same mechanism of toxicity. However, the details of phthalates mechanism of toxicity are not fully understood. The aim of this research was to investigate the effects of DBP on the steroidogenesis pathway using the cultured rat Leydig cell cancer line R2C as a Leydig cell model. R2C cells were exposed to a range of DBP concentrations (10 μg/mL, 5 μg/mL, 1 μg/mL, and 0.1 μg/mL) and their steroid hormone production was analysed using reverse phase HPLC. R2C cells did not synthesise testosterone at detectable levels. However, DBP exposure stimulated cortisol biosynthesis at all concentrations but caused no change in progesterone biosynthesis. This cortisol stimulation in Leydig cells has not been observed before. Because cortisol and testosterone compete for precursors an increase in cortisol synthesis could starve testosterone synthesis of precursors. On top of this it has been shown that glucocorticoids including cortisol have an adverse effect on Leydig cell development reducing steroid production and even causing apoptosis. This could explain how DBP and other phthalates can cause male developmental defects such as cryptorchidism and hypospadias.

Philosophiae Doctor - PhD / The testes play a central role in the male reproductive system, as they represent the sites of male sex steroidogenesis and of spermatogenesis. Leydig cells, located at the testicular interstitium, produce predominantly testosterone upon stimulation with either chorionic gonadotropin (CG) or luteinising hormone by a series of enzymatic modifications of cholesterol. Sertoli cells respond to testosterone and follicle stimulating hormone to secrete inhibin and facilitate spermatogenesis by additional activities like maintaining Sertoli cell barrier (SCB) integrity and lactate secretion. Ultimately the Leydig cells, Sertoli cells etc. all work together to confer male fertility. However, infertility occurs globally; leading to the pursuit of treatment, including herbal medicines.

A number of investigations during the last two decades describe adverse trends in male reproductive health, which have been proposed to be caused by environmental factors with endocrine disrupting properties. In contrast to many other toxicants, endocrine disruptors often do not show linear dose-response relationships typical of those found in traditional toxicological studies. For many compounds, low-dose exposure causes effects opposite to the ones seen after high-dose exposure. In addition, the timing of exposure has been found to be critical. Hence, to correctly assess the impact of endocrine disruptors on reproductive health requires in-depth knowledge of their mechanisms of action. This thesis aimed at identifying the mechanisms underlying the effects of cadmium (Cd), phthalates and phytoestrogens on testicular steroidogenesis. For this purpose, in vitro as well as in vivo models were used. Cd was found to inhibit testosterone synthesis in vivo by down-regulating LH receptor gene expression and reducing the testicular levels of cAMP and StAR protein. In addition, Cd caused a pronounced increase in testicular prostaglandin F2ɑ (PGF2ɑ), suggesting that Cd exerts its suppressive effect on steroidogenesis also by inducing the inhibitory PKC pathway. Pre-treatment with zinc (Zn) protected completely against Cd-induced effects on testosterone and PGF2ɑ. Furthermore, we observed that Cd exposure increased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in the testis. GAPDH is a potent coactivator of androgen receptor-mediated transcription and the up-regulation found in our study is probably a compensatory response to reduced testosterone concentrations. This finding is interesting since GAPDH has been proposed to have an important role in the regulation of apoptosis as well as sperm motility. We discovered that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used phthalate di-(2-ethylhexyl) phthalate (DEHP), stimulates Leydig cell steroidogenesis in vitro, by a cAMP- and StAR-independent mechanism. MEHP exposure caused a similar effect in granulosa cells. Gene expression analysis revealed that MEHP is likely to stimulate steroidogenesis by increasing the amount of cholesterol available for steroid synthesis. In the last investigation, we examined the effects of low-dose phytoestrogen exposure on testosterone synthesis during puberty in male goats. Isoflavones present in clover increased plasma concentrations of testosterone and free as well as total triiodothyronine (T3). T3 has previously been shown to induce testosterone synthesis and it is possible that an elevated T3 secretion underlies the increased plasma testosterone levels. Reduced fertility and reproductive tract malformations affect both the individual and the society. Hence, a sound knowledge of reproductive toxicants is of crucial importance. The findings presented in this thesis provide new insights into the reproductive toxicology of endocrine disruptors and may be valuable for risk assessment purposes.

Leydig cells produce testosterone in response to luteinizing hormone (LH) via the cyclic adenosine monophosphate (cAMP)/protein kinase A pathway. Additionally, these cells are responsible for producing insulin-like peptide 3 (INSL3), a peptide hormone that is essential for testicular descent. The insl3 promoter in Leydig cells can be activated by cAMP through the transcription factor Nur77, which has also been shown to regulate the promoters of the steroidogenic enzymes, cyp17 and 3b-hsd. While the mechanism of LH action on testosterone production is well characterized, the effect of LH on insl3 abundance has yet to be shown directly. The MA-10 Leydig cells treated with hCG exhibited a transient and robust increase in nur77 mRNA, while insl3 mRNA abundance remained unchanged. Further, cAMP failed to affect insl3 mRNA, though nur77 mRNA abundance was significantly increased. Inhibition of LH-receptor-linked signal transduction pathways in the presence of hCG implicated multiple signaling networks in the regulation of both insl3 and nur77. Treatment with hCG or cAMP did not affect the abundance of 3b-hsd mRNA. Interestingly, though the MA-10 cell line has been reported to lack CYP17 activity and mRNA and so produce progesterone instead of testosterone, cyp17 mRNA was present and inducible by hCG and cAMP. The addition of hCG, testosterone, nor the combination of hCG and testosterone affected insl3 mRNA abundance. Though hCG consistently increased nur77 mRNA abundance, the addition of testosterone did not enhance the effects of hCG. Collectively, these results indicate that insl3 is regulated by factors other than LH/CG and cAMP in the MA-10 cell line.

The accumulation of androstenone in the fat of mature boars results in boar taint; the conjugation of androstenone would decrease this important meat quality problem by decreasing the accumulation and increasing the excretion of androstenone. Leydig cells and testis microsomes from mature boars were incubated with radiolabeled pregnenolone, and the free and conjugated metabolites were examined by HPLC. Sulfated androstenone with a mass of 367 m/z was directly identified by MS, with a novel tentative structure of 3-keto-4- sulfoxy-androstenone. Addition of enolase to the microsomal incubations increased the formation of 3-keto-4-sulfoxy-androstenone. Overexpression of SULT2A1 in HEK cells resulted in the sulfoconjugation of dehydroepiandrosterone, but not androstenone, suggesting that SULT2A1 may not be involved in sulfoconjugation of androstenone. This thesis describes the novel direct characterization of androstenone sulfate and the importance of enolase in its formation. The relevance to boar taint metabolism is discussed.

Introdução: Hiperandrogenemia associada a sinais clínicos de virilização na mulher após a menopausa é uma condição rara e pouco estudada. Os tumores ovarianos secretores de andrógenos (TOSA) e a hipertecose do estroma ovariano (HPT) são as etiologias mais frequentes de hiperandrogenismo nesta faixa etária. A diferenciação entre estas duas condições é difícil, pois as manifestações clínicas são semelhantes e caracterizadas por hirsutismo, alopecia androgênica, clitoromegalia, hipertrofia muscular e agravamento da voz. O perfil hormonal das mulheres pós-menopausadas com TOSA e HPT pode não ser um parâmetro ideal para discriminar estas duas condições. Além disso, os estudos de imagem podem não caracterizar com precisão estas lesões ovarianas. Devido às dificuldades, em estabelecer o diagnóstico diferencial entre TOSA e HPT, a ooforectomia bilateral é a terapêutica indicada para as mulheres menopausadas com diagnóstico de hiperandrogenismo de origem ovariana; embora na HPT o tratamento clínico com análogo do hormônio liberador de gonadotrofinas (aGnRH) possa ser uma opção terapêutica eficaz. Objetivos: O nosso objetivo foi avaliar a contribuição das características clínicas, do perfil hormonal e dos exames radiológicos para o diagnóstico diferencial entre TOSA e HPT em mulheres na pós-menopausa. Métodos: Trinta e quatro mulheres pós-menopausadas, na faixa etária de 52 a 80 anos de idade, que foram encaminhadas à Unidade de Endocrinologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, entre 1999 e 2013 por hiperandrogenismo clínico e com diagnóstico histológico de TOSA (13 mulheres) e HPT (21 mulheres) foram avaliadas retrospectivamente. Os diagnósticos histológicos foram revisados e confirmados por um único patologista com experiência em patologia ginecológica. Os dados clínicos de hiperandrogenismo, o perfil hormonal (T, E2, LH, FSH) e as imagens radiológicas pélvicas (Ultrassom transvaginal e Ressonância Magnética, RM) foram obtidos a partir da revisão de prontuários médicos. Resultados: Em relação aos dados da história clínica, não houve diferença significativa entre os dois grupos de pacientes para nenhuma das variáveis clínicas analisadas, exceto para o número de gestações, que foi significantemente maior no grupo com TOSA. Os sinais clínicos de hiperandrogenismo, especialmente agravamento da voz (p < 0,001) e hipertrofia muscular (p = 0,01), foram mais prevalentes no grupo de pacientes com TOSA do que o grupo de HPT. Embora na análise dos parâmetros hormonais, os pacientes do grupo com TOSA tenham apresentado níveis mais elevados de T e E2 e níveis mais baixos de gonadotrofinas (p < 0,01 e p <0,01, respectivamente) do que o grupo de pacientes com HPT, uma grande sobreposição nos níveis hormonais foi observada entre os pacientes dos dois grupos. A RM de pelve apresentou uma boa acurácia para diferenciar os TOSAs da HPT em mulheres pós-menopausadas com hiperandrogenismo. Conclusão: Neste grupo de pacientes, as características que mais contribuíram para o diagnóstico diferencial entre TOSA e HPT foram o agravamento da voz e a hipertrofia muscular, os níveis séricos de testosterona e gonadotrofinas e a presença de nódulo ovariano na RM de pelve. Embora a associação das características clínicas, hormonais e radiológicas contribua para a elaboração de uma hipótese diagnóstica fundamentada, a análise histopatológica continua a ser o padrão ouro para o diagnóstico diferencial de hiperandrogenismo de origem ovariana em mulheres na pós-menopausa / Introduction: The presence of virilizing signs associated to high serum of androgen levels in postmenopausal women is a rare and poorly understood condition. Virilizing ovarian tumors (VOT) and ovarian stromal hyperthecosis (OH) are the most common hyperandrogenism etiologies in the postmenopausal women. The differential diagnosis between the two conditions is often difficult, because they present similar clinical features such as hirsutism, androgenic alopecia, clitoromegaly, muscle hypertrophy and deepening of the voice. The hormonal profile of postmenopausal women with VOT and OH may not be the optimal discriminating factor between these two conditions. Moreover, imaging may not accurately characterize these ovarian lesions. Due to the difficulties in establishing the differential diagnosis between VOT and OH, bilateral oophorectomy is the treatment of choice in postmenopausal women with hyperandrogenism of ovarian origin. However, the treatment with gonadotropin-releasing hormone analogue (GnRHa) might be an effective therapy in women with OH. Objectives: Our aim was to evaluate the contribution of clinical features, hormonal profile and radiological studies in the differential diagnosis between VOT and OH in postmenopausal women. Methods: Thirty-four postmenopausal women ranging from 52 to 80 years of age with clinical hyperandrogenism referred to the Endocrinology Unity of Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, between 1999 and 2013, with diagnosis of VOT (13 women) and OH (21 women) were evaluated retrospectively. Histological diagnoses were reviewed and confirmed by a single pathologist with expertise in gynecologic pathology. Clinical hyperandrogenism data, hormonal status (T, E2, LH, FSH) and the pelvic images (Transvaginal sonography and Magnetic Resonance Image- MRI) findings were obtained from medical records. Results: No clinical data evaluated in the study was significantly different between the two groups of patients. A higher number of pregnancies in the VOT group was observed, which was statistically different from the OH group. The clinical signs of hyperandrogenism, especially deepening of the voice (p < 0.001) and muscle hypertrophy (p = 0.01), were more prevalent in the VOT\'s than OH\'s group. Although, the VOT\'s group showed higher T and E2 levels and lower gonadotropins levels than the OH\'s group (p < 0.01 and p < 0.01, respectively), a great overlap in the hormone levels occur between VOT and OH patients. Pelvic MRI presented a good accuracy to differentiate these two conditions in hyperandrogenic postmenopausal women. Conclusion: In this group of patients, the main features to the differential diagnosis between VOT and OH were deepening of the voice and muscle hypertrophy, serum levels of testosterone and gonadotropins and presence of ovarian nodule in the pelvic MRI. Although the association of clinical, hormonal and radiological features contributes to the differential diagnosis between these two conditions, histopathological analysis remains the gold standard for the differential diagnosis of ovarian hyperandrogenism in post menopausal women

Introdução: Hiperandrogenemia associada a sinais clínicos de virilização na mulher após a menopausa é uma condição rara e pouco estudada. Os tumores ovarianos secretores de andrógenos (TOSA) e a hipertecose do estroma ovariano (HPT) são as etiologias mais frequentes de hiperandrogenismo nesta faixa etária. A diferenciação entre estas duas condições é difícil, pois as manifestações clínicas são semelhantes e caracterizadas por hirsutismo, alopecia androgênica, clitoromegalia, hipertrofia muscular e agravamento da voz. O perfil hormonal das mulheres pós-menopausadas com TOSA e HPT pode não ser um parâmetro ideal para discriminar estas duas condições. Além disso, os estudos de imagem podem não caracterizar com precisão estas lesões ovarianas. Devido às dificuldades, em estabelecer o diagnóstico diferencial entre TOSA e HPT, a ooforectomia bilateral é a terapêutica indicada para as mulheres menopausadas com diagnóstico de hiperandrogenismo de origem ovariana; embora na HPT o tratamento clínico com análogo do hormônio liberador de gonadotrofinas (aGnRH) possa ser uma opção terapêutica eficaz. Objetivos: O nosso objetivo foi avaliar a contribuição das características clínicas, do perfil hormonal e dos exames radiológicos para o diagnóstico diferencial entre TOSA e HPT em mulheres na pós-menopausa. Métodos: Trinta e quatro mulheres pós-menopausadas, na faixa etária de 52 a 80 anos de idade, que foram encaminhadas à Unidade de Endocrinologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, entre 1999 e 2013 por hiperandrogenismo clínico e com diagnóstico histológico de TOSA (13 mulheres) e HPT (21 mulheres) foram avaliadas retrospectivamente. Os diagnósticos histológicos foram revisados e confirmados por um único patologista com experiência em patologia ginecológica. Os dados clínicos de hiperandrogenismo, o perfil hormonal (T, E2, LH, FSH) e as imagens radiológicas pélvicas (Ultrassom transvaginal e Ressonância Magnética, RM) foram obtidos a partir da revisão de prontuários médicos. Resultados: Em relação aos dados da história clínica, não houve diferença significativa entre os dois grupos de pacientes para nenhuma das variáveis clínicas analisadas, exceto para o número de gestações, que foi significantemente maior no grupo com TOSA. Os sinais clínicos de hiperandrogenismo, especialmente agravamento da voz (p < 0,001) e hipertrofia muscular (p = 0,01), foram mais prevalentes no grupo de pacientes com TOSA do que o grupo de HPT. Embora na análise dos parâmetros hormonais, os pacientes do grupo com TOSA tenham apresentado níveis mais elevados de T e E2 e níveis mais baixos de gonadotrofinas (p < 0,01 e p <0,01, respectivamente) do que o grupo de pacientes com HPT, uma grande sobreposição nos níveis hormonais foi observada entre os pacientes dos dois grupos. A RM de pelve apresentou uma boa acurácia para diferenciar os TOSAs da HPT em mulheres pós-menopausadas com hiperandrogenismo. Conclusão: Neste grupo de pacientes, as características que mais contribuíram para o diagnóstico diferencial entre TOSA e HPT foram o agravamento da voz e a hipertrofia muscular, os níveis séricos de testosterona e gonadotrofinas e a presença de nódulo ovariano na RM de pelve. Embora a associação das características clínicas, hormonais e radiológicas contribua para a elaboração de uma hipótese diagnóstica fundamentada, a análise histopatológica continua a ser o padrão ouro para o diagnóstico diferencial de hiperandrogenismo de origem ovariana em mulheres na pós-menopausa / Introduction: The presence of virilizing signs associated to high serum of androgen levels in postmenopausal women is a rare and poorly understood condition. Virilizing ovarian tumors (VOT) and ovarian stromal hyperthecosis (OH) are the most common hyperandrogenism etiologies in the postmenopausal women. The differential diagnosis between the two conditions is often difficult, because they present similar clinical features such as hirsutism, androgenic alopecia, clitoromegaly, muscle hypertrophy and deepening of the voice. The hormonal profile of postmenopausal women with VOT and OH may not be the optimal discriminating factor between these two conditions. Moreover, imaging may not accurately characterize these ovarian lesions. Due to the difficulties in establishing the differential diagnosis between VOT and OH, bilateral oophorectomy is the treatment of choice in postmenopausal women with hyperandrogenism of ovarian origin. However, the treatment with gonadotropin-releasing hormone analogue (GnRHa) might be an effective therapy in women with OH. Objectives: Our aim was to evaluate the contribution of clinical features, hormonal profile and radiological studies in the differential diagnosis between VOT and OH in postmenopausal women. Methods: Thirty-four postmenopausal women ranging from 52 to 80 years of age with clinical hyperandrogenism referred to the Endocrinology Unity of Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, between 1999 and 2013, with diagnosis of VOT (13 women) and OH (21 women) were evaluated retrospectively. Histological diagnoses were reviewed and confirmed by a single pathologist with expertise in gynecologic pathology. Clinical hyperandrogenism data, hormonal status (T, E2, LH, FSH) and the pelvic images (Transvaginal sonography and Magnetic Resonance Image- MRI) findings were obtained from medical records. Results: No clinical data evaluated in the study was significantly different between the two groups of patients. A higher number of pregnancies in the VOT group was observed, which was statistically different from the OH group. The clinical signs of hyperandrogenism, especially deepening of the voice (p < 0.001) and muscle hypertrophy (p = 0.01), were more prevalent in the VOT\'s than OH\'s group. Although, the VOT\'s group showed higher T and E2 levels and lower gonadotropins levels than the OH\'s group (p < 0.01 and p < 0.01, respectively), a great overlap in the hormone levels occur between VOT and OH patients. Pelvic MRI presented a good accuracy to differentiate these two conditions in hyperandrogenic postmenopausal women. Conclusion: In this group of patients, the main features to the differential diagnosis between VOT and OH were deepening of the voice and muscle hypertrophy, serum levels of testosterone and gonadotropins and presence of ovarian nodule in the pelvic MRI. Although the association of clinical, hormonal and radiological features contributes to the differential diagnosis between these two conditions, histopathological analysis remains the gold standard for the differential diagnosis of ovarian hyperandrogenism in post menopausal women

Depuis plusieurs années, un nombre conséquent d’études décrivent une augmentation de l’incidence de pathologies liées à la fonction de reproduction masculine. Ces anomalies ont été regroupées sous le terme de « syndrome de dysgénésie testiculaire ». Ce syndrome aurait pour origine les effets délétères de polluants environnementaux sur le développement du testicule en période fœtale. Parmi ces polluants environnementaux, les phtalates et le bisphénol A (BPA) sont les plastifiants les plus produits et les plus répandus dans les objets de consommation courante. De nombreuses études leur sont consacrées et ont permis de les classer au rang de perturbateurs endocriniens en mettant notamment en cause leurs effets reprotoxiques. Mon travail de thèse est une étude des effets de ces deux perturbateurs endocriniens sur le développement du testicule fœtal.Nous avons réalisé une première étude concernant les effets du BPA sur le développement du testicule fœtal. Grâce au modèle de culture organotypique, nous avons développé notre étude dans trois espèces : le rat, la souris et l’Homme. Nous démontrons que le BPA diminue la sécrétion de testostérone dans le testicule fœtal humain à partir d’une concentration de 10-8M, alors que chez le rat et la souris, la sécrétion de testostérone n’est affectée qu’à partir de 10-5M de BPA. Nous avons également démontré une diminution de l’expression du gène de l’Insl-3, dans ces mêmes conditions. Ceci nous a permis de mettre en évidence une différence de sensibilité entre les espèces. Pour tenter de comprendre les mécanismes par lesquels le BPA exerce son effet toxique, nous avons comparé ses effets à ceux du DES, autre perturbateur endocrinien œstrogénomimétique. Contrairement au BPA, le DES diminue la sécrétion de testostérone fœtale chez les rongeurs, et non chez l’Homme. Ce résultat suggère l’implication de deux voies de signalisation différentes pour ces deux xéno-œstrogènes. Cette hypothèse est d’ailleurs renforcée par l’étude que nous avons SourceMécanismes d’action des perturbateurs endocriniens bisphénol A et phtalates sur le développement du testicule fœtal / par Thierry N’Tumba-Byn ; sous la direction de Virginie Rouiller-Fabre, Université Paris Sud, 2013 [Thèse de Biologie de la Reproduction et du Développement]réalisée sur des souris invalidées pour le récepteur des œstrogènes ERα, dans lesquelles l’effet anti-androgénique du BPA persiste, contrairement à celui du DES.Parallèlement, nous avons recherché les mécanismes d’action des phtalates et de leur métabolite actif le plus répandu, le MEHP (mono-2-éthyl-hexyl phtalate). Dans la continuité de plusieurs travaux réalisés dans notre laboratoire sur les effets du MEHP, nous avons tenté de comprendre les mécanismes par lesquels le MEHP induit un effet pro-apoptotique dans les cellules germinales mâles. Nous avons mis en évidence une augmentation de l’expression du gène Stra8 dans les cellules germinales traitées au MEHP. Ce résultat nous suggère que le MEHP pourrait induire une différenciation erronée des cellules germinales mâles. De plus, nous avons recherché les récepteurs et la voie de signalisation activée par le MEHP. Nous observons que les agonistes des récepteurs PPARα et de PPARγ entrainent dans les cellules germinales les mêmes phénotypes que le MEHP, à savoir une augmentation du taux d’apoptose et de l’expression du gène Stra8. / For several years, an increase in the incidence of pathologies connected to the male reproductive functions has been described in numerous studies. These anomalies are classified under the term “testicular dysgenesis syndrome”. This syndrome might find its origins in the deleterious effects of environmental pollutants on the testis development in fetal period. Among theses environmental pollutants, phthalates and bisphenol A (BPA) are the most produced plasticizers found in products of common use. Many studies were performed in order to determine their effects, and allowed to classify them as endocrine disruptors because of their reprotoxic effects. My thesis work is a study of the effects of these two endocrine disruptors on the fetal testis development.Our first study focuses on the effects of BPA on the fetal testis development. Using the organotypic culture model, we developed our study in three species: rat, mouse and human. We demonstrated that BPA decreases the testosterone secretion in the human fetal testis from a 10-8M concentration, while in rat and mouse the testosterone secretion is only affected by 10-5M BPA. We also demonstrated a decreased Insl-3 gene expression, in the same conditions. These results allowed us to evidence a difference of sensibility between species. To understand the mechanisms involved in the BPA toxic effect, we compared it with the effect of DES, another endocrine disruptor with estrogenic activity. Unlike BPA, DES decreases the fetal testosterone secretion in rodents and not in human. This result suggests the involvement of two different signalisation pathways for these two xenoestrogens. This hypothesis is reinforced by the study that we performed in mice invalidated for the estrogen receptor ERα. In those mice, the anti-androgenic effect of BPA is maintained, unlike DES effect.In parallel, we investigated the mechanisms of action of phtalates and particularly of their most prevalent active metabolite, the MEHP (mono-2-ethyl-hexyl phthalate). Following previous studies performed in our laboratory concerning the effects of MEHP, we intended to understand the mechanisms by which MEHP induces the apoptosis in male germ cells. We evidenced an increase in Stra8 gene expression in MEHP treated germ cells. This result suggests that MEHP might induce a wrong differentiation in male germ cells. Furthermore, we investigated the receptors and the signalisation pathway activated by MEHP. We observe that PPARα and PPARγ receptors agonists induce the same phenotypes as MEHP, namely an increase in the apoptosis and in Stra8 gene expression in germ cells.