Les aclipocytokines (aclipo), produites par le tissu adipeux jouent un rôle clé dans la régulation des fonctions métaboliques mais qu'en est-il pour les fonctions de reproduction? Nous montrons que la chemerine et ses récepteurs, la visfatine et la résistine sont présents dans les cellules ovariennes humaines et bovines. ln vitro nous observons que la chemerine et la résistine diminuent la stéroïdogenèse des cellules de la granulosa (CG) humaine induite par IGF-1 alors que la visfatine l'augmente. Des résultats similaires sont observés chez la vache pour la chemerine et la visfatine. Dans les deux espèces, les aclipo influencent la prolifération des CG, et les voies de signalisation AKT, MAPK-ERKl/2 et P38 ou l'AMPK. Chez le bovin, la chemerine bloque la maturation ovocytaire in vitro. Nous observons aussi dans cette espèce que la concentration plasmatique de résistine et son expression dans les aclipocytes est augmentée après vêlage lorsque la lipomobilisation est élevée. Ces travaux confirment le rôle de la résistine dans la régulation métabolique chez la vache et montrent l'importance des adipo dans les cellules ovariennes humaine et bovine. Il reste à élargir leur rôle au niveau central dans les fonctions de reproduction. / The aclipokines (aclipo ), produced by the adipose tissue play a key role in the regulation of metabolic functions, but what about for reproductive functions? We show that chemerin and its receptors, visfatin and resistin are present in human and bovine ovary cells. ln vitro we observe that chemerin and resistin decrease steroidogenesis in granulosa cells (GC) in response to IGF-1 while visfatin increases it. Similar results are observed for chemerin and visfatin in cows. In both species, chemerin, visfatin and resistin affect the proliferation of CG and signaling pathways inclucling AKT, MAPKERKl I 2 and P38 or AMPK. In cattle, chemerin blocks in vitro oocyte maturation. In this species, we also observe that the plasma resistin and its expression in aclipocytes are increased after calving when the fatty acid mobilization is high. This work confirms the role of resistin in the metabolic regulations in cow and shows the importance of aclipo in the human and bovine ovary cells. It remains to investigate their role at the central level in the reproductive functions.

Steroid hormones act through two mechanisms. The first is the classical genomic level, which acts at a relatively slow pace from hours to days. The second non-genomic mechanism of steroid activity can influence cellular function during seconds or even milliseconds. During pregnancy both mechanisms take place. Steroid hormones that act non-genomically and influence neuronal excitability are called neuroactive steroids. The aim of this study was to measure the levels of steroid hormones and examine their relationship to the physiology and pathophysiology of pregnancy. The physiological importance of increased placental progesterone production, lowered production of 5B-pregnanone steroids and increased activity of steroid sulfotransferase with gestational age have been intensely discussed. On the basis of our results, we have proposed an alternative mechanism of maternal progesterone synthesis during pregnancy (progesterone, or the gestagen stabilizing activity of the myometrium = continuation of pregnancy). In contrast to previous results our observations indicate that gestagens arise from precursors in the fetus (from pregnenolone sulfate). This mechanism is closely connected with the distribution of placental oxido-reductase. Data from this study show an increasing trend in the conversion of...

No Estado do Maranhão, na região da Baixada Maranhense, presenta na fauna silvestre o réptil Kinosternon scorpioides, um quelônio de água doce popularmente conhecido como jurará e que possui valor social, econômico e ambiental para os ribeirinhos da cidade de São Bento. Este estudo contempla suas características biológicas reprodutivas baseadas em seu ambiente natural, com o intuito de permitir a preservação e o estabelecimento de planos de manejo reprodutivo e de uso sustentável da espécie. Recentemente poucos estudos em todo o mundo tratam sobre os aspectos do ciclo reprodutivo de tartarugas em face das características endócrinas, na América do Sul estudos desse tipo são recentes e escassos, sendo assim este é o primeiro estudo, que se tem conhecimento, que elucida um padrão sazonal reprodutivo da espécie K. scorpioides, associando hormônios gonadais com aspectos comportamentais. Trinta e oito animais adultos tiveram seus órgãos reprodutivos caracterizados para as enzimas esteroidogênicas P450 aromatase, P450c17 e PNADPH redutase através de imunomarcação e blotting, além de índice gonadossomático, morfometria e concentração de testosterona, corticosterona e estradiol pela técnica de radioimunoensaio. As mudanças biométricas, morfometria celular e a esteroidogênese testicular entre os períodos chuvoso e seco sugerem que o estrógeno produzido pelas células de leydig podem afetar a produção e a apoptose de células germinais durante o processo de espermatogênese, e a presença das enzimas P450aromatase e P450c17 em células de leydig acompanhados com a recrudescência testicular também reforça a ideia, de que elas podem desempenhar um papel na quiescência testicular. Esse trabalho evidenciou que as enzimas citocromo P450, citocromo P450c17 e NADPH-citocromo P450-redutase estão presentes no testículo e epidídimo nos diferentes períodos climáticos e podem estar relacionados à síntese de testosterona em tartarugas concordando com os diferentes achados para biometria e espermiogênese nos períodos chuvoso e seco, o que, nos leva a sugerir um estado de quiescência durante o período seco e atividade espermatogênica no período chuvoso, semelhante ao que ocorre com as espécies que apresentam comportamento reprodutivo sazonal / In the state of Maranhão, in the lowland region, the reptile Kinosternon scorpioides, a freshwater chelonian, popularly known as swear has social, economic and environmental value to the riparian of the city of São Bento. This study includes reproductive biological characteristics based on their natural environment in order to enable the preservation and the establishment of reproductive management plans and sustainable use of the species. To date, few studies around the world address the aspects of the reproductive cycle of the turtles in the face of the endocrine features. In South America, these studies are recent and scarce. So this is the first study that elucidates a reproductive seasonal pattern of the species K. scorpioides, by associating gonadal hormones with behavioral aspects. Thirty-eight adult animals had their reproductive organs characterized for P450 steroidogenic enzyme aromatase, P450c17 and NADPH reductase by immunostaining and immunoblotting, and gonadossomatic index, morphology and concentration of testosterone, corticosterone and estradiol by radioimmunoassay. Biometric changes, cell morphology and testicular steroidogenesis between the rainy and dry seasons suggests that the estrogen produced by the leydig cells can affect the production and apoptosis of germ cells during spermatogenesis process; and the presence of P450aromatase and P450c17 enzymes in the leydig cells accompanied with testicular recrudescence also reinforces the idea that they may play a role in testicular quiescence. This work showed that the cytochrome P450 enzymes, P450c17 and NADPH-cytochrome P450 reductase are present in the testis and epididymis at different climatic periods and may be related to testosterone synthesis in turtles agreeing to the different findings for biometrics and spermiogenesis in and dry rainy periods. These findings suggest a state of quiescence during the dry season and spermatogenic activity during the rainy season, similar to those found in species with seasonal reproductive behavior

Perdas embrionárias e alterações gestacionais são frequentemente observadas em prenhezes de embriões bovinos manipulados in vitro. Sabe-se que tais anormalidades são resultantes de alterações epigenéticas ocasionadas pela manipulação dos gametas e/ou do embrião durante as técnicas de reprodução assistida (ARTs), com destaque para as técnicas de fecundação in vitro (FIV) e da clonagem por transferência nuclear de células somáticas (SCNT). Tais alterações resultam em distúrbios no desenvolvimento do concepto em algum momento crítico entre a fertilização e o parto, fornecendo bons modelos de estudos sobre a fisiopatologia de perdas embrionárias e dos distúrbios de desenvolvimento. Caracterizam-se como momentos críticos após a transferência do embrião (TE) o desenvolvimento embrionário no útero, o reconhecimento materno da gestação, a placentação e o desenvolvimento da placenta e do feto, os quais tem de ser transpassados sem nenhuma falha, permitindo um desenvolvimento normal do concepto até o termo. Sendo assim, o presente trabalho abordou três fases distintas do amplo período gestacional em prenhezes por ARTs. O Estudo 1 foi realizado durante o período peri-reconhecimento materno da gestação e objetivou descrever a abundância de expressão de genes estimulados pelo interferon tau (ISGs) de células mononucleares do sangue periférico (PBMCs) maternas em gestações oriundas de ARTs no primeiro mês de gestação; o Estudo 2 compreendeu os primeiros 35 dias de gestação e objetivou descrever as mudanças morfológicas e vasculares do complexo útero-concepto-ovário e o estímulo à expressão de ISGs em PBMCs em gestações de conceptos clonados por SCNT com diferentes fenótipos de desenvolvimento, sendo esses denominados gestação anembrionada e CL persistente; e o Estudo 3 foi conduzido durante o período pré-parto e objetivou descrever as alterações na produção de esteroides sexuais e corticosteroides em gestações produzidas por ARTs. Três hipóteses foram testadas: (1) Gestações de conceptos clonados por SCNT apresentam uma baixa e mais tardia estimulação de ISGs em PBMCs maternas quando comparadas com gestações de conceptos produzidos por FIV e IA; (2) O concepto clonado por SCNT apresenta um menor estímulo sobre mudanças morfológicas e vasculares do complexo útero-ovário e ISGs em PBMCs maternas durante os primeiros 35 dias de gestação, quando comparado com conceptos oriundos de IA; e (3) Gestações de embriões oriundos de ARTs apresentam alterações na dinâmica esteroidogênica no pré-parto quando comparados com gestações de IA. No estudo 1 foram coletadas amostras de sangue de gestações produzidas por inseminação artificial (IA), FIV e clonagem por SCNT, nos dias 15, 18, 20, 22, 24, 26, 28 e 31 pós-ovulação e foi realizada mensuração da abundância de transcritos de ISGs (OAS1 e ISG15) em PBMCs maternas. No estudo 2, gestações produzidas por IA e clonagem por SCNT, foram submetidas a escaneamentos ultrassonográficos dos ovários, útero e concepto a cada 3 dias do dia 14 ao 35 (dia 0 = ovulação) e amostras de sangue foram coletadas nos dias 15, 18, 20, 22, 24, 26, 28 e 31 para mensuração da abundância de transcritos de ISGs (OAS1 e ISG15) em PBMCs maternas. No estudo 3, foram coletadas amostras de sangue no último mês em gestações naturais, oriundas de FIV e de clonagem por SCNT para análise hormonal de 10 esteroides utilizando o método de espectrometria de massas multi-hormonal de alta resolução LC-MS/MS. O primeiro estudo mostrou semelhanças na expressão de genes estimulados pelo IFNT em gestações oriundas de ARTs e produzidas por IA. Entretanto, a estimulação nas gestações oriundas de ARTs aparentou ser quatro dias mais prolongada, sugerindo uma maior funcionalidade do trofectoderma em conceptos oriundos de ARTs. O segundo estudo demonstrou um aumento na expressão de ISGs em PBMCs maternas tanto em gestações de conceptos normais quanto em anormais, justificando a manutenção da função luteal mesmo na ausência de detecção do concepto por ultrasonografia. No terceiro estudo, demonstrou-se alterações na esteroidogênese nas gestações de embriões FIV e clonados no último mês de gestação, sendo essas compatíveis com a hiperativação da enzima aromatase durante todo o último mês de gestações oriundas de FIV e hiperativação das enzimas P450C11 e P450C21 trinta dias antes do parto em gestações oriundas de clonagem por SCNT. O presente estudo concluiu que conceptos oriundos de FIV e clonagem por SCNT apresentam um prolongamento no estímulo de ISGs pelo IFNT, conceptos clonados anormalos apresentam estímulo de ISGs, o que justifica a manutenção da função luteal, e, por fim, a cascata esteroidonênica que culmina com o parto apresenta-se alterada em gestações oriundas de FIV e clonagem por SCNT. / Pregnancy losses and gestational abnormalities are frequently observed in pregnancies from in vitro produced embryos in bovines. It is known that these abnormalities are due to epigenetic changes from the manipulation of gametes and/or embryo during the use of assisted reproduction techniques (ARTs), especially for the in vitro fertilization (IFV) and cloning by somatic cells nuclear transfer (SCNT). These changes results in disturbances of conceptus development in any critical stage between the fertilization and parturition, which provides good models for the study of physiopathology of embryo losses and disturbances of development. Critical stages after the embryo transfer (ET) to the uterus are characterized as the maternal recognition of pregnancy, placentation, and fetal-placental development, which needs to be surpassed without failures, in order to develop a normal conceptus until term. Therefore, the present work approached three distinct phases of the wide gestational period in pregnancies from ARTs. The Study 1 was conducted during the maternal peri-recongnition of pregnancy period and aimed to describe the expression of interferon stimulated genes (ISGs) in maternal peripheral blood mononuclear cells (PBMCs) in pregnancies derived of ARTs; the Study 2 comprise the first 35 days of pregnancy and aimed to describe morphological and vascular changes of the complex uterus-ovaries-conceptus, as well as the expression of ISGs in maternal PBMCs in pregnancies of conceptus cloned by SCNT with different phenotypes of development, denominated as anembryonic gestation and persistent CL; the Study 3 was conducted during the pre-partum period and aimed to describe changes in the production of sexual steroids and corticosteroids during the last month of pregnancies derived of ARTs. Three hypothesis were tested: (1) Pregnancies of conceptus cloned by SCNT presented a decrease and delay in the stimulation of ISGs in maternal PBMCs when compared with conceptuses produced by IFV and AI; (2) Stimulus from the conceptus for changes in the morphology and vasculature of the the uterus-ovarian complex, detected by ultrasonography in B and Doppler modes, and the stimulation of ISGs in maternal PBMCs during the first 35 days of pregnancy of conceptus cloned by SCNT are less intense when compared with conceptus derived from AI; and (3) Pregnancies derived of ARTs present changes in the steroidogenic dynamics in the pre-partum, when compared with pregnancies derived from AI. In Study 1 blood samples were collected from pregnancies produced by AI, IVF, and cloning by SCNT, at days 15, 18, 20, 22, 24, 26, 28, and 31 post-ovulation for the measurement of abundance of transcripts of ISGs (OAS1 and ISG15) in maternal PBMCs. In Study 2, pregnancies derived of AI and cloning by SCNT, were submitted to ultrasonographic scans for the evaluation and description of morphological and vascular changes in ovaries, uterus, and conceptus every 3 days from day 14 to 35 (day 0 = ovulation) and blood samples were collected on days 15, 18, 20, 22, 24, 26, 28 e 31 for the measurement of the abundance of transcripts of ISGs (OAS1 and ISG15) in maternal PBMCs. In Study 3, blood samples were collected during the last month of pregnancies naturally conceived, derived of IVF, and cloned by SCNT for the analysis of 10 steroids using the method of mass spectrometry high resolution LC-MS/MS. The first study showed similarities in the ISGs expression stimulation in pregnancies derived of ARTs and AI. However, the stimulation in the ART derived pregnancies was apparently 4 days longer, suggesting a greater placental function in conceptus derived of ARTs. The second study showed an increase in ISG expression in both normal and abnormal conceptus development, which justifies the maintenance of CL in the absence of a conceptus structure detected by ultrasonography. In the third study, was detected changes in the steroidogenesis of pregnancies derived of IFV and cloning by SCNT during the last month of pregnancy, which are compatible with the hyperactivation of the aromatase enzyme during the last month of IFV derived pregnancies, and hyperactivation of the enzymes P450C11 and P450C21 thirty days before parturition in pregnancies derived of cloning by SCNT. The present study concludes that conceptus derived of IFV and cloning by SCNT present a prolonged stimulus of ISGs, cloned conceptus with anomalous development presents a stimulus of ISGs, which justifies the CL function maintenance, and, ultimately, the steroidogenic cascade that culminates with the term is altered in pregnancies derived from IFV and cloning by SCNT.

L’embryologie de la surrénale humaine et la pathogénie de l’hypoplasie congénitale des surrénales sont mal connues et complexes. L’une pouvant expliquer l’autre, nous présentons, à partir de 119 fœtus humains normaux de12 à 36 semaines de développement (SD), une étude cinétique histologique et moléculaire de la surrénale fœtale et nous décrivons 3 cas d’hypoplasie surrénalienne. Après un rappel des mécanismes moléculaires connus, régulant ensemble l’embryologie surrénalienne, gonadique et hypophysaire et ceux de l’hypoplasie surrénale congénitale, nous présentons nos résultats. Dans la corticosurrénale humaine fœtale normale, les cellules du cortex permanent prolifèrent et, dès la 12ème SD, expriment la NCAM, la 3β-HSD et la P450 c21. Elles ont la capacité de synthétiser des minéralocorticoïdes et/ou du cortisol. Les cellules du cortex fœtal ne prolifèrent pas et expriment ni la 3β-HSD, ni la NCAM. La médullosurrénale est formée par des neuroblastes immatures (CgA-, NCAM+) qui migrent et prolifèrent de la périphérie vers le centre de la glande, où ils se différencient en neuroblastes matures (CgA+). Dans les deux cas d’hypoplasie surrénale de type anencéphalique, avec absence de mutation de DAX-1 et de SF-1, la dysembryoplasie surrénalienne est probablement d’origine hypophysaire, par absence de cellules gonadotropes. Dans le 3ème cas, jamais décrit, associant un RCIU, une hypoplasie surrénalienne congénitale, une ambiguïté sexuelle, une absence de différenciation des cellules antéhypophysaires, l’absence de mutation de gènes connus (DAX-1, SF-1, SRY, FGF9, SOX2, SOX3, SOX5 et SOX9) suggère l’existence de nouveaux gènes régulant la différenciation précoce de ces trois glandes endocrines. / The embryology of the human adrenal and the pathogenesis of the hypoplasia adrenal congenital are complex and not well known. We studied the histological and functional characteristics of the fetal human adrenal in normal fetuses aged 12 to 36 weeks development (WD) and presented three cases of congenital adrenal hypoplasia with pituitary abnormalities. In the human fetal adrenal cortex, only the cells of the definitive zone proliferate from the 12th WD. We observe the expression of an adherence protein (NCAM) and of two steroidogenesis enzymes (3β-HSD and P450 c21) in the definitive zone cells, attesting to the capacity of these cells to synthesize mineralocorticoids and/or cortisol. In the fetal zone, only P450 c21 immunoreactivity is detected. The adrenal medulla is formed by immature neuroblasts (CgA -, NCAM+) which migrate and proliferate from the periphery toward the center of the gland, where they differentiate in mature neuroblasts (CgA+). In the two cases of congenital adrenal hypoplasia of anencephalic type without DAX 1 and SF-1 mutation, the cause is probably from pituitary origin and related to absence of gonadotrope cells. In the third case of congenital adrenal hypoplasia, never described, combining a retard intrauterine development, a congenital adrenal hypoplasia, sexual ambiguity, pituitary abnormalities. The absence of mutation of known genes (DAX -1, SF -1, SRY, FGF9, SOX2, SOX3, SOX5 and SOX9) suggests the existence of new genes regulating early differentiation of the adrenal, the gonad and the pituitary.

Para a metabolização de hormônios esteroides sexuais é essencial a participação de enzimas esteroidogênicas. A enzima citocromo P450c17 é responsável pela produção de andrógenos e a enzima citocromo P450 aromatase é responsável pela produção de estrógenos, sendo que, ambas as enzimas necessitam formar um complexo com uma enzima parceira, denominada NADPH citocromo P450 redutase, para realizar a metabolização destes hormônios essenciais para a diferenciação sexual. Objetivou-se imunolocalisar as três enzimas acima citadas no tecido vaginal de fêmeas de roedores Galea spixii. Para tanto, o experimento foi desenvolvido utilizando técnicas de citologia esfoliativa vaginal para definição das fases do ciclo estral, técnicas de microscopia de Luz, Eletrônica de Varredura e testes de imunohistoquímica para detecção das enzimas: citocromo P450c17, citocromo P450 aromatase, e NADPH citocromo P450 redutase. Constatou-se no presente estudo que o ciclo estral das fêmeas de preás silvestres do semiárido apresenta ciclo estral com duração de 15,85 ± 1,4 dias, com a formação e ruptura da membrana de oclusão vaginal durante cada ciclo estral. A vagina apresenta um clitóris hipertrofiado com o óstio uretral que se abre no topo do mesmo, e ausência de um vestíbulo vaginal. O epitélio da mucosa vaginal sofre modificações proliferativas, se espessando e se adelgando de acordo com a respectiva fase do ciclo estral. As enzimas citocromo P450 aromatase e NADPH redutase estão imunolocalizadas durante todo o ciclo estral no epitélio e tecido conjuntivo da vagina de Galea spixii. Por outro lado, a enzima P450c17 está imunolocalizada no epitélio vaginal durante todas as fases do ciclo estral, porém não está presente no estro. Com estes dados pode-se sugerir que ocorre uma metabolização local de hormônios estrógenos e andrógenos no tecido vaginal o que podem estar relacionado com a proliferação celular, variação na vascularização e inervação e ruptura da membrana de oclusão vaginal. / To the metabolism of sex steroid hormones is essential the participation of esteroidogenic enzymes. The enzyme cytochrome P450c17 is responsible for androgen production; and the enzyme aromatase cytochrome P450 is responsible for estrogen production, however, both enzymes require forming a complex with an enzyme partner called NADPH cytochrome P450 reductase to perform the metabolism of these hormones, which are essential for sexual differentiation. This study aimed to immunolocalization of the three enzymes above mentioned in the vaginal tissue from female rodents Galea spixii. Therefore, the experiment was carried out using vaginal cytology techniques for definition of the estrous cycle phases, techniques for light microscopy, scanning electron and immunohistochemical tests for the detection of enzymes: cytochrome P450c17, cytochrome P450 aromatase, and NADPH cytochrome P450 reductase. It was found that the estrus cycle of Galea spixii female lasts 15.85 ± 1.4 days, with the formation and rupture of vaginal closure membrane during each oestrous cycle. The vagina has a hypertrophied clitoris with the urethral orifice that opens on top of it, and it is observed the absence of a vaginal vestibule. The vaginal mucosal epithelium undergoes proliferative changes, with thickening and thinning according to the respective estrous cycle phase. The enzymes cytochrome P450 aromatase and NADPH reductase are immunolocated throughout the oestrous cycle in the epithelium and connective tissue of the vagina from Galea spixii. On the other hand, the enzyme cytochrome P450c17 is immune located in the vaginal epithelium during all stages of the estrous cycle, except at estrous. With these data may be suggested that there is a local estrogens and androgens metabolism in vaginal tissue which may be associated with cell proliferation, vascularization and innervation variation besides the formation and rupture of the vaginal closure membrane.

Background: The first-line treatment for patients with advanced prostate cancer (PC) is androgen deprivation therapy. This therapy is initially effective, but after some time tumors relapse, predominantly within the bone, and are then termed castration-resistant prostate cancer (CRPC). The majority of CRPC tumors show androgen receptor (AR) activity despite castrate levels of circulating testosterone. AR activity could be caused by several mechanisms including; intratumoral androgen synthesis, AR amplification, AR mutations and expression of AR splice variants. The mechanisms controlling CRPC growth in the clinically most relevant metastatic site, the bone, are not fully identified. The purpose of this thesis was therefore to explore AR expression and possible mechanisms behind CRPC growth in PC bone metastases in order to find mechanisms that could be targeted for treatment and/or predict response to certain therapies. Materials and Methods: We have examined hormone-naïve and CRPC bone metastases samples obtained from patients at metastasis surgery, non-malignant and malignant prostate samples obtained from patients at radical prostatectomy, and PC cell lines cultured in vitro. Analysis has been performed using RT-PCR, whole-genome expression arrays, immunohistochemistry, western blotting, FISH, copy number assays and gene ontology analysis. Functional studies have been made by protein overexpression and knock-down in PC cells in vitro and effects studied by evaluation of cell viability, migration, and invasion. Results: We found that high nuclear AR immunostaining (presumed to reflect high AR activity) in bone metastases from CRPC patients was associated with a particularly poor prognosis, while no difference in AR staining was observed between hormone-naïve and CRPC metastases. Further, expression of AR splice variants (AR-V7, AR-V567es) was associated with a high nuclear AR immunostaining score and shown to be increased in CRPC compared to hormone-naïve bone metastases. High levels (levels in the upper quartile) of AR splice variants in CRPC bone metastases was related to disturbed cell cycle regulation and short patients survival. No differences in steroidogenic enzyme levels were detected between CRPC and hormone-naïve bone metastases. Higher levels of enzymes involved in late steps of androgen synthesis (adrenal gland steroid conversion) were observed in bone metastases than in non-malignant and/or malignant prostate tissue, while the enzyme levels in earlier steps (de novo steroidogenesis) were lower in bone metastases. A subgroup of metastases expressed very high levels of AKR1C3, indicating that this group may have an induced capacity of converting adrenal-gland derived steroids into more potent androgens. This was not associated to CRPC but merely with the advanced stage of metastasis. High protein levels of AR splice variants were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR variant levels. Furthermore, about half of the CRPC bone metastases showed androgen receptor gene amplification which was associated with co-amplification of YIPF6, and a gene expression pattern that pointed at decreased osteoclast activity, and consequently decreased bone resorption. Conclusions: The majority of CRPC bone metastases show high nuclear AR immunostaining that seems to be associated with a particularly unfavorable outcome after metastasis surgery. Subgroups of CRPC bone metastases could be identified according to presence of AR amplification and expression levels of AKR1C3 or AR splice variants, which might have clinical relevance for treatment of PC patients. / <p>Författaren är även publicerad med efternamnet Hörnberg.</p>

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objectives of the present study was to determine the concentration of angiotensin II (AngII) in follicular fluid, to characterize the expression of renin-angiotensin system (RAS) genes in follicular cells and to verify the role of AngII in the follicular wave, using an in vivo model with intrafollicular injection and in vitro model with granulosa cells culture. AngII concentration in follicular fluid increased on dominant follicle during and after deviation. Saralasin inhibited follicular growth in all treated cows (4/4), suggesting that AngII is essential for follicular growth in 7-8mm follicles. However, intrafollicular injection of AngII affected neither follicular growth nor the pattern of follicular dynamics, which were similar to control cows. These results imply that bovine ovarian follicles contain sufficient AngII for follicle development. In another experiment, saralasin inhibitory effect was overcome by systemic FSH supplementation, suggesting that AngII is essential to follicular growth during FSH-low dependence stages. The injection of AngII or angiotensin receptor type 2 (AGTR2) agonist in second largest follicle prevented the expected atresia of subordinate follicle and the treated follicle grew at the same rate as the dominant during 24h. To understand why a single intrafollicular injection of AngII antagonist induces follicular atresia, dominant follicle was injected with saralasin or saline and the cows were ovariectomized 24h later. The inhibition of AngII action decreased estradiol concentration in follicular fluid and abundance of mRNA encoding aromatase (CYP19), 3!-hydroxysteroid dehydrogenase (3!HSD), LH receptor, SerpinE2 and cyclinD2 in granulosa cells. On granulosa cell culture, AngII increased CYP19 expression just in the presence of FSH. Taken together, these results show that AngII is essential for follicular growth, and plays important role in regulating genes involved in granulosa cell proliferation (cyclinD2) and differentiation (LHr, aromatase, 3!HSD), which are necessary for development of the dominant follicle. In conclusion, these data suggest that AngII signaling is involved in follicle growth and dominance in cattle. / Os objetivos do presente trabalho foram determinar a concentração de angiotensina II (AngII) no fluido folicular, caracterizar a expressão dos genes do sistema renina-angiotensina (RAS) nas células foliculares e verificar o papel da AngII na onda folicular, utilizando um modelo in vivo de injeção intrafolicular e in vitro de cultivo de células da granulosa. A concentração de AngII no fluido folicular aumentou no folículo de maior diâmetro, durante e após a divergência folicular. A administração intrafolicular de saralasina, um bloqueador competitivo dos receptores de AngII, inibiu o crescimento folicular em todas as vacas injetadas (4/4), demonstrando que a AngII é essencial para o crescimento de folículos de 7-8mm de diâmetro. Contudo, a injeção de AngII não afetou o crescimento folicular, sugerindo que os folículos contém AngII suficiente para o seu desenvolvimento. Em um outro experimento, a administração sistêmica de FSH reverteu o efeito inibitório da saralasina, sugerindo que a AngII é indispensável para o desenvolvimento folicular após o período de dependência ao FSH. A injeção, no segundo maior folículo, de AngII ou CGP42122 (agonista AGTR2) preveniu a regressão esperada do folículo subordinado, demonstrando que a AngII desempenha um papel fundamental na seleção do folículo dominante. Para determinar o mecanismo de atresia induzido por saralasina, o folículo dominante de cada vaca foi injetado com saralasina ou solução salina e os animais foram ovariectomizados após 24 horas. A inibição de AngII diminuiu a concentração de estradiol no fluido folicular e a abundância de mRNA que codifica para os genes aromatase (CYP19), 3! HSD, LHr, Serpin E2 e ciclina D2 nas células da granulosa. Além disso, em cultivo primário de células da granulosa, a AngII, somente na presença do FSH, induziu um aumento na expressão de aromatase. Em resumo, os resultados demonstram que a sinalização da AngII é essencial para o crescimento folicular, regulando genes envolvidos com a proliferação (ciclina D2) e diferenciação (LHr, aromatase, 3!HSD) das células da granulosa os quais são necessários para o desenvolvimento do folículo dominante. Em conclusão, os resultados sugerem que a AngII está envolvida no desenvolvimento e dominância folicular em bovinos.

Steroid hormones in testis play an important role in spermatogenesis, maintenance of the male reproductive tract, production of semen and the maintenance of secondary sex characteristics and libido. They are also discussed as a target for substances called endocrine disruptors (EDs). No complex study was conducted on evaluation of relationships between EDs and steroid spectrum in 2 biological fluids; seminal plasma and plasma. The aim of the PhD. thesis was to develop and validate a method for determination of bisphenol A (BPA) and steroid spectrum in plasma and seminal plasma and to shed more light into mechanisms of ED action and effects of BPA and polychlorinated biphenyls (PCBs) on human spermatogenesis and steroidogenesis. Two new liquid-chromatography mass spectrometry methods for determination of BPA and 11 steroids in plasma and seminal plasma were developed and validated. The methods were used for estimation of analyte concentrations in 191 men with a different degree of fertility. Concurrently, the levels of six congeners of PCBs, gonadotropins, selenium and zinc in plasma were estimated. Partial correlations adjusted for age and BMI were calculated to evaluate relationships between these analytes. Seminal BPA, but not plasma BPA, was negatively associated with sperm concentration...

Para a metabolização de hormônios esteroides sexuais é essencial a participação de enzimas esteroidogênicas. A enzima citocromo P450c17 é responsável pela produção de andrógenos e a enzima citocromo P450 aromatase é responsável pela produção de estrógenos, sendo que, ambas as enzimas necessitam formar um complexo com uma enzima parceira, denominada NADPH citocromo P450 redutase, para realizar a metabolização destes hormônios essenciais para a diferenciação sexual. Objetivou-se imunolocalisar as três enzimas acima citadas no tecido vaginal de fêmeas de roedores Galea spixii. Para tanto, o experimento foi desenvolvido utilizando técnicas de citologia esfoliativa vaginal para definição das fases do ciclo estral, técnicas de microscopia de Luz, Eletrônica de Varredura e testes de imunohistoquímica para detecção das enzimas: citocromo P450c17, citocromo P450 aromatase, e NADPH citocromo P450 redutase. Constatou-se no presente estudo que o ciclo estral das fêmeas de preás silvestres do semiárido apresenta ciclo estral com duração de 15,85 ± 1,4 dias, com a formação e ruptura da membrana de oclusão vaginal durante cada ciclo estral. A vagina apresenta um clitóris hipertrofiado com o óstio uretral que se abre no topo do mesmo, e ausência de um vestíbulo vaginal. O epitélio da mucosa vaginal sofre modificações proliferativas, se espessando e se adelgando de acordo com a respectiva fase do ciclo estral. As enzimas citocromo P450 aromatase e NADPH redutase estão imunolocalizadas durante todo o ciclo estral no epitélio e tecido conjuntivo da vagina de Galea spixii. Por outro lado, a enzima P450c17 está imunolocalizada no epitélio vaginal durante todas as fases do ciclo estral, porém não está presente no estro. Com estes dados pode-se sugerir que ocorre uma metabolização local de hormônios estrógenos e andrógenos no tecido vaginal o que podem estar relacionado com a proliferação celular, variação na vascularização e inervação e ruptura da membrana de oclusão vaginal. / To the metabolism of sex steroid hormones is essential the participation of esteroidogenic enzymes. The enzyme cytochrome P450c17 is responsible for androgen production; and the enzyme aromatase cytochrome P450 is responsible for estrogen production, however, both enzymes require forming a complex with an enzyme partner called NADPH cytochrome P450 reductase to perform the metabolism of these hormones, which are essential for sexual differentiation. This study aimed to immunolocalization of the three enzymes above mentioned in the vaginal tissue from female rodents Galea spixii. Therefore, the experiment was carried out using vaginal cytology techniques for definition of the estrous cycle phases, techniques for light microscopy, scanning electron and immunohistochemical tests for the detection of enzymes: cytochrome P450c17, cytochrome P450 aromatase, and NADPH cytochrome P450 reductase. It was found that the estrus cycle of Galea spixii female lasts 15.85 ± 1.4 days, with the formation and rupture of vaginal closure membrane during each oestrous cycle. The vagina has a hypertrophied clitoris with the urethral orifice that opens on top of it, and it is observed the absence of a vaginal vestibule. The vaginal mucosal epithelium undergoes proliferative changes, with thickening and thinning according to the respective estrous cycle phase. The enzymes cytochrome P450 aromatase and NADPH reductase are immunolocated throughout the oestrous cycle in the epithelium and connective tissue of the vagina from Galea spixii. On the other hand, the enzyme cytochrome P450c17 is immune located in the vaginal epithelium during all stages of the estrous cycle, except at estrous. With these data may be suggested that there is a local estrogens and androgens metabolism in vaginal tissue which may be associated with cell proliferation, vascularization and innervation variation besides the formation and rupture of the vaginal closure membrane.