Chez les mammifères, le contrôle de la reproduction est médié par un réseau hypothalamique qui intègre divers stimuli pour réguler la sécrétion de la Gonadotropin Releasing Hormone (GnRH). Ces neurones à GnRH naissent dans la placode olfactive et migrent vers le cerveau le long des axones vomeronasaux et terminaux au cours du développement embryonnaire. Bien que ce processus a bien été étudié chez les rongeurs, sa caractérisation complète chez l'homme reste inachevée. Il est largement admis que des perturbations dans le développement ou dans la sécrétion de GnRH sont associées chez l’homme à un hypogonadisme hypogonadotrope congénital (CHH), qui est un trouble caractérisé par un retard ou une absence de la puberté conduisant à l'infertilité.Les systèmes GnRH et olfactif ont des liens complexes au cours du développement, le syndrome de Kallmann (KS) représente un trouble qui associe l'hypogonadisme dû à une déficience en GnRH et l'anosmie. Le CHH et le KS sont des troubles oligogéniques, les mutations génétiques sous-jacentes n’expliquent que 50% des cas cliniques.Dans cette étude, nous avons entrepris une caractérisation complète du processus migratoire des neurones à GnRH au cours du premier trimestre de gestation sur une grande série d'embryons et de fœtus humains, ce qui nous a permis d’élaborer le premier atlas chronologique et quantitatif de la distribution de GnRH. En effet, l'utilisation d’une nouvelle approche de transparisation des tissus embryologiques humains par de solvants organiques, a permis d’établir pour la première fois, une véritable représentation des neurones dans leur contexte natal in vivo.De plus, les résultats de cette étude ont non seulement révélé que le nombre de neurones GnRH chez l'homme était significativement plus élevé que prévu, mais aussi que ces derniers migrent vers plusieurs régions du cerveau extra-hypothalamique, en plus de l'hypothalamus. Leur présence dans ces régions soulève l'hypothèse qu’ils pourraient exercer des rôles non reproductifs, créant de nouvelles pistes pour la recherche sur les fonctions du système GnRH dans les processus cognitifs, comportementaux et physiologiques.Le second objectif de ce travail a visé à caractériser un nouveau gène candidat impliqué dans le développement du système GnRH: L'hormone Anti-Müllerienne (AMH), connue pour son rôle dans la différenciation de la gonade bipotentielle chez les mâles. Néanmoins, une récente étude menée par notre équipe a mis en évidence son rôle extragonadique sur les neurones à GnRH en période post-natale.Le séquençage complet d'une large cohorte de patients européens a révélé plusieurs nouvelles mutations faux-sens dans le gène de l’AMH chez les patients atteints de CHH et KS, non retrouvés dans la cohorte des témoins. L’évaluation de la pertinence fonctionnelle de ces mutations a ensuite été effectuée par diverses analyses biochimiques in vitro de la bioactivité des mutations, ainsi que par la caractérisation d'une lignée de souris transgénique. Ce qui a entraîné une diminution de la sécrétion de l'AMH et une diminution de la bioactivité de la protéine sécrétée dans les études in vitro; Conduisant à une éventuelle réduction de la capacité migratoire. Cela suggère fortement que ces mutations pourraient avoir un effet pathogène.En outre,nous montrons que le récepteur AMHR2 est exprimé le long des fibres olfactives et par les neurones à GnRH pendant le processus migratoire GnRH. L'analyse pathohistologique des souris Amhr2 -/- a révélé une altération de la migration embryonnaire des neurones à GnRH vers le cerveau antérieur basal, entraînant une réduction significative du nombre total de neurones GnRH dans les cerveaux adultes de ces animaux, conduisant à une fertilité réduite. L’ensemble de ces travaux indiquent que l'insuffisance de signalisation AMH contribuerait à la pathogenèse des troubles de CHH chez l’homme, et met en évidence un nouveau rôle de l'AMH dans développement et la fonction des neurones GnRH. / The control of mammalian reproduction is mediated by a hypothalamic network that integrates various stimuli to regulate the periodic secretion of gonadotropin-releasing hormone (GnRH). GnRH neurons, originate in the olfactory placode and enter the brain along vomeronasal and terminal axons during embryonic development. This process has been well studied in rodents, however, a full characterisation in humans was lacking. Alterations either in the development of this system or in the secretion of GnRH are associated with congenital hypogonadotropic hypogonadism (CHH) in humans, a condition characterized by failure of sexual competence. Due to the inextricable links in the development of the olfactory and GnRH systems, there also exists the developmental condition, Kallmann syndrome (KS), associating hypogonadism due to GnRH deficiency and anosmia. Both CHH and KS are oligogenic disorders, with the underlying genetic mutations only explained in 50% of the clinical cases.At the heart of this study, we have undertaken a full characterisation of the GnRH migratory process during the first trimester of gestation in a large series of human embryos and foetuses and provide the first chronological and quantitative atlas of GnRH distribution. This has been aided by the novel application of organic solvent based tissue clearing techniques to human embryological tissue. This allows, for the first time, a true representation and appreciation of cells in their native, in vivo context. The results of this study have revealed not only that the number of GnRH neurons in humans is significantly higher than previously thought, but that GnRH cells migrate into several extrahypothalamic brain regions in addition to the hypothalamus. Their presence in these areas raises the possibility that GnRH has non-reproductive roles, creating new avenues for research on GnRH functions in cognitive, behavioural and physiological processes.The second aim of this work has been to characterise a novel candidate gene responsible for the development of the GnRH system. Anti-Müllerian hormone (AMH), best known for its role in facilitating the differentiation of the bipotential gonad in males, has recently been shown by our lab to exert significant effects on GnRH neurons postnatally. Here we have undertaken whole exome sequencing of a large cohort of European CHH and KS patients, identifying several novel missense mutations in the Amh gene that are not present in the control cohort. Various in vitro and biochemical analyses of mutations bioactivity as well as analysis of a transgenic mouse line have been used to assess the functional relevance of these mutations.Mutations in Amh resulted in impaired secretion of AMH and reduced bioactivity of the secreted protein in in vitro studies; eventually leading to reduced migratory capacity. This strongly suggests that these mutations could have a pathogenic effect. We also show that its receptor AMHR2 is expressed along the olfactory fibres and by GnRH neurons during the GnRH migratory process. Pathohistological analysis of Amhr2-/- mice revealed defective embryonic migration of GnRH cells to the basal forebrain, leading to a significant reduction in the total number of GnRH neurons in the adult brains of these animals resulting in reduced fertility. We therefore propose that AMH signalling insufficiency contributes to the pathogenesis of human CHH conditions and highlights a novel role for AMH in the correct development and function of GnRH neurons.

Introdução: O processo de envelhecimento reprodutivo ocorre em virtude do declínio progressivo na quantidade e qualidade de óvulos que se inicia após a puberdade, se mantém ao longo da menacme, com redução gradual da fertilidade, e termina na menopausa, caracterizada pelo esgotamento do número de folículos e, consequentemente, da reserva ovariana funcional (ROF). A vida pré-natal constitui um importante período para o desenvolvimento dos órgãos genitais internos femininos e mudanças nessa fase podem ter repercussões futuras: quando o feto é submetido a condições adversas intrauterinas, mecanismos metabólicos e endócrinos de adaptação podem mudar o eixo metabólico pós-natal predispondo a certas doenças na vida adulta. Com base nesses dados, postulou-se que condições desfavoráveis de vida intrauterina que poderiam se refletir com alterações do peso ao nascer (PN) poderiam levar à reprogramação de genes envolvidos no controle da ROF e que talvez nascer pequeno para idade gestacional (PIG) ou grande (GIG) possibilitaria a interferência com a ROF estimada por meio das concentrações séricas do hormônio antimülleriano (AMH). Objetivos: O objetivo deste estudo foi avaliar a relação do PN com a ROF, estimada por meio da concentração sérica do AMH em mulheres na menacme com 34 a 35 anos de idade. Pacientes e Métodos: Trata-se de um estudo transversal aninhado a uma coorte de mulheres que nasceram no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP-USP), no período de 01 de junho de 1978 e 31 de maio de 1979. O desfecho primário avaliado foi a concentração sérica de AMH, um marcador da ROF, e sua correlação com o PN, divididos em três grupos: PIG, adequado para a idade gestacional (AIG) e GIG. Resultados: Das 274 pacientes incluídas no estudo, 19 foram classificadas como PIG, 238 como AIG e 17 como GIG. As concentrações médias de AMH não foram significativamente diferentes (p=0,11) entre mulheres na menacme nascidas PIG, AIG e GIG (2,14 ng/mL, 2,13 ng/mLe 2,57 ng/mL, respectivamente). Conclusão: Não se observou diferença nas concentrações séricas de AMH entre mulheres nascidas PIG, AIG e GIG avaliadas entre 34 e 35 anos de idade. A casuística avaliada permitiu detectar ou descartar uma grande diferença entre os grupos (effectsize de 0,7). Dessa forma, evidenciou-se que o PN não apresenta grande influência sobre a ROF, estimada pelas concentrações séricas do AMH, em mulheres na menacme, entre 34 e 35 anos de idade. Caso novos estudos evidenciem que diferenças pequenas ou moderadas nas concentrações séricas do AMH possam apresentar relevantes repercussões clínicas em mulheres nesta faixa etária, outras pesquisas serão necessárias, sendo os dados do presente estudo úteis para o cálculo amostral. / Background: The reproductive aging process occurs by a progressive decline in the quantity and quality of oocyte, starting after pubertal onset, remaining through menacme with gradual reduction of fertility and ends with menopause, which is the depletion of ovarian follicles and hence the depletion of functional ovarian reserve (FOR). Prenatal life corresponds to a critical window for the development of female internal genitalia and changes at this stage may have future repercussions: when the fetus is submitted to intra uterine adverse conditions, adaptive metabolic and endocrine mechanisms will change the metabolic axis in the postnatal period thereafter predisposing to several diseases in adulthood. Based on this correlation, we postulate that unfavorable conditions of intrauterine life that could reflect on birth weight (BW) could lead to the reprogramming of genes involved in the control of FOR and that maybe being born small for gestational age (SGA) or large for gestational age (LGA) could interfere with the FOR estimated through serum concentrations of Anti-Müllerian hormone (AMH). Objective: To investigate the relationship between BW and ROF estimated through AMH serum concentration in menacme women with 34-35 years old. Patients and Methods: This is a prospective birth cohort assessing all women who were born in Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP-USP) between June 1, 1978 and May 31, 1979. The primary endpoint was to evaluate serum AMH concentration, a marker of FOR and its correlation with BW divided into three groups: SGA, adequate for gestational age (AGA) and LGA. Results: Out of the 274 patients included in the study: 19 were classified as SGA, 238 as AGA, and 17 as LGA. The average of AMH concentrationwas not significantly different (p=0.11) among women in reproductive age born SGA, AGA and LGA (2.14 ng/mL, 2.13 ng/mL, and 2.57 ng/mL respectively. A variance analysis between the three groups and OR did not find a significant different between them (p=0.11). Conclusion: There was no difference in serum AMH concentration in women born SGA, AGA and LGA with 34 to 35 years old. This sample also allowed to detect or rule out a major difference between the groups (effect size of 0.7). In conclusion, BW does not have a great influence on FOR, estimated through serum AMH concentration in menacmewomen, between 34 and 35 years old. If new studies show clearly that small or moderate differences in serum AMH concentration could impact clinical outcomes in women at this age, further studies will be needed, and the data of this study could be useful for sample size calculation.

Anti-müllerian hormone (AMH) is expressed in male embryos and represses development of müllerian ducts during testis differentiation in mammals, birds and reptiles. Amh orthologues have been identified in teleosts despite them lacking müllerian ducts. Previously we found sexually dimorphic aromatase activity in tilapia brains before ovarian differentiation. This prompted us to search for further dimorphisms in tilapia brains during sex differentiation and see whether amh is expressed. We cloned the tilapia amh gene and found that it contains 7 exons but no spliced forms. The putative protein presents highest homologies with Amh proteins of pejerrey and medaka as compared to other Perciformes. We analysed amh expression in adult tissues and found elevated levels in testes, ovary and brain. Amh expression was dimorphic with higher levels in XY male brains at 10–15 dpf, when the gonads were still undifferentiated and gonadal amh was not dimorphic. Male brains had 2.7-fold higher amh expression than gonads. Thereafter, amh levels decreased in the brain while they were up-regulated in differentiating testes. Our study indicates that amh is transcribed in male brains already at 10 dpf, suggesting that sexual differentiation may be occurring earlier in tilapia brain than in gonads. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Anti-müllerian hormone (AMH) is expressed in male embryos and represses development of müllerian ducts during testis differentiation in mammals, birds and reptiles. Amh orthologues have been identified in teleosts despite them lacking müllerian ducts. Previously we found sexually dimorphic aromatase activity in tilapia brains before ovarian differentiation. This prompted us to search for further dimorphisms in tilapia brains during sex differentiation and see whether amh is expressed. We cloned the tilapia amh gene and found that it contains 7 exons but no spliced forms. The putative protein presents highest homologies with Amh proteins of pejerrey and medaka as compared to other Perciformes. We analysed amh expression in adult tissues and found elevated levels in testes, ovary and brain. Amh expression was dimorphic with higher levels in XY male brains at 10–15 dpf, when the gonads were still undifferentiated and gonadal amh was not dimorphic. Male brains had 2.7-fold higher amh expression than gonads. Thereafter, amh levels decreased in the brain while they were up-regulated in differentiating testes. Our study indicates that amh is transcribed in male brains already at 10 dpf, suggesting that sexual differentiation may be occurring earlier in tilapia brain than in gonads. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

<p>An up-regulation of gene expression of the gonadotropin receptors FSHR and LHR, and a down-regulation of anti-Müllerian hormone (AMH), in the gonads of early maturing male Atlantic salmon parr (<em>Salmo salar</em>) have been shown to take place during the process of sexual maturation. It has however not been determined if this happens prior to or after the onset of spermatogenesis. The aim of this study was to see if such an up-regulation of the gene expression of FSHR and LHR mRNA, and down-regulation of AMH in the gonad tissue could be seen prior to the onset of spermatogenesis in early maturing salmon. For this study gonad tissues were sampled before and after the onset of spermatogenesis. Small pieces of testis tissue were removed using surgery from individually tagged one year old male Atlantic salmon parr in April prior to the onset of spermatogenesis. The same salmon were sampled again in July and maturing and non-maturing fish could be distinguished by differences in GSI. Each tissue sample from April could be matched with the July sample from the same individual by pit-tag numbers. This made it possible to separate the April samples into a maturing and non-maturing group. Gene expression levels were analysed using real-time PCR. The findings of this study showed that all three genes were expressed in the gonads in April but no significant difference in expression levels between maturing and non-maturing salmon was seen for any of the genes, which indicate that no up- or down-regulations had taken place in early maturing fish at this time. In July however, total FSHR and LHR expression levels/testis were significantly higher in maturing salmon which is in accordance with previous studies. AMH expression levels/unit RNA in July were found to be on average 25 times higher in the non-maturing group. A 100-fold drop in AMH from April through July was seen in the maturing fish, while only a 4-fold drop was seen in the non-maturing group which may indicate that a down-regulation of AMH expression took place as spermatogenesis was initiated in the maturing males.</p> / <p>En uppreglering av genuttrycket av gonadotropinreceptorerna (FSHR och LHR) samt en nedreglering av anti-Müllerian hormon (AMH) har observerats i gonaderna under spermatogenesen hos tidigt mognande atlantlaxhanar (<em>Salmo salar</em>). Man har dock inte kunnat visa huruvida detta sker före eller efter spermatogenesen inletts. Syftet med denna studie var att undersöka om en uppreglering av FSHR och LHR samt en nedreglering av AMH kunde ses hos tidigt mognande laxar redan före spermatogenesen inletts. I den här studien användes gonadprover tagna före och efter spermatogenesen inletts. Små gonadvävnadsprover togs från individuellt märkta ettåriga atlantlaxhanar i april, före spermatogenesen inletts, genom ett operativt ingrepp. I juli togs sedan gonadprover från samma individer igen och mognande och icke-mognande individer kunde nu särskiljas genom deras GSI-värden. Varje gonadprov från april kunde matchas med proverna från juli för samtliga individer genom att jämföra pit-tag nummer och möjliggjorde att även aprilproverna kunde sorteras i en mognande och en icke-mognande grupp. Nivåerna av genuttryck av mRNA analyserades med real-time PCR. Resultaten från denna studie visade att alla tre generna var uttryckta i de omogna gonaderna i april men det var ingen signifikant skillnad mellan de mognande och icke-mognande fiskarna för någon av generna, vilket betyder att det ännu inte skett någon upp- eller nedreglering vid denna tidpunkt hos de tidigt mognande fiskarna. I juli var dock de totala FSHR och LHR mRNA nivåerna/testikel signifikant högre hos mognande fiskar vilket överrensstämmer med tidigare studier. AMH mRNA/enhet RNA var uttryckt 25 gånger högre i de omogna gonaderna jämfört med de mognande. Mellan april och juli föll AMH nivåerna hos mognande fiskar nästan 100 gånger, medan de hos de omogna minskade endast 4 gånger vilket kan indikera att en nedreglering av genuttrycket för AMH skett i de individer där spermatogenesen inletts.</p>

An up-regulation of gene expression of the gonadotropin receptors FSHR and LHR, and a down-regulation of anti-Müllerian hormone (AMH), in the gonads of early maturing male Atlantic salmon parr (Salmo salar) have been shown to take place during the process of sexual maturation. It has however not been determined if this happens prior to or after the onset of spermatogenesis. The aim of this study was to see if such an up-regulation of the gene expression of FSHR and LHR mRNA, and down-regulation of AMH in the gonad tissue could be seen prior to the onset of spermatogenesis in early maturing salmon. For this study gonad tissues were sampled before and after the onset of spermatogenesis. Small pieces of testis tissue were removed using surgery from individually tagged one year old male Atlantic salmon parr in April prior to the onset of spermatogenesis. The same salmon were sampled again in July and maturing and non-maturing fish could be distinguished by differences in GSI. Each tissue sample from April could be matched with the July sample from the same individual by pit-tag numbers. This made it possible to separate the April samples into a maturing and non-maturing group. Gene expression levels were analysed using real-time PCR. The findings of this study showed that all three genes were expressed in the gonads in April but no significant difference in expression levels between maturing and non-maturing salmon was seen for any of the genes, which indicate that no up- or down-regulations had taken place in early maturing fish at this time. In July however, total FSHR and LHR expression levels/testis were significantly higher in maturing salmon which is in accordance with previous studies. AMH expression levels/unit RNA in July were found to be on average 25 times higher in the non-maturing group. A 100-fold drop in AMH from April through July was seen in the maturing fish, while only a 4-fold drop was seen in the non-maturing group which may indicate that a down-regulation of AMH expression took place as spermatogenesis was initiated in the maturing males. / En uppreglering av genuttrycket av gonadotropinreceptorerna (FSHR och LHR) samt en nedreglering av anti-Müllerian hormon (AMH) har observerats i gonaderna under spermatogenesen hos tidigt mognande atlantlaxhanar (Salmo salar). Man har dock inte kunnat visa huruvida detta sker före eller efter spermatogenesen inletts. Syftet med denna studie var att undersöka om en uppreglering av FSHR och LHR samt en nedreglering av AMH kunde ses hos tidigt mognande laxar redan före spermatogenesen inletts. I den här studien användes gonadprover tagna före och efter spermatogenesen inletts. Små gonadvävnadsprover togs från individuellt märkta ettåriga atlantlaxhanar i april, före spermatogenesen inletts, genom ett operativt ingrepp. I juli togs sedan gonadprover från samma individer igen och mognande och icke-mognande individer kunde nu särskiljas genom deras GSI-värden. Varje gonadprov från april kunde matchas med proverna från juli för samtliga individer genom att jämföra pit-tag nummer och möjliggjorde att även aprilproverna kunde sorteras i en mognande och en icke-mognande grupp. Nivåerna av genuttryck av mRNA analyserades med real-time PCR. Resultaten från denna studie visade att alla tre generna var uttryckta i de omogna gonaderna i april men det var ingen signifikant skillnad mellan de mognande och icke-mognande fiskarna för någon av generna, vilket betyder att det ännu inte skett någon upp- eller nedreglering vid denna tidpunkt hos de tidigt mognande fiskarna. I juli var dock de totala FSHR och LHR mRNA nivåerna/testikel signifikant högre hos mognande fiskar vilket överrensstämmer med tidigare studier. AMH mRNA/enhet RNA var uttryckt 25 gånger högre i de omogna gonaderna jämfört med de mognande. Mellan april och juli föll AMH nivåerna hos mognande fiskar nästan 100 gånger, medan de hos de omogna minskade endast 4 gånger vilket kan indikera att en nedreglering av genuttrycket för AMH skett i de individer där spermatogenesen inletts.

In the present body of work two primary subjects have been addressed, both individually and in their correspondence, namely 1) the potential for Neanderthals to have contributed to the Modern Human population, and 2) the genetic diversity of one of the most prehistorically impactful human popuations, the Armenians. The first subject is addressed by assessing 1000 mutations in 384 current humans, particularly for those mutations which appear to derive from the Neanderthal lineage. Additionally, the validity of the Neanderthal sequences themselves is evaluated through alignment analysis of fragementary DNA derived from the Vindija Cave sample. Armenian genetic diversity is analyzed through the autosomal short tandem repeats, y-chromsome single nucleotide polymorphisms, and y-chromosome short tandem repeats. The diversity found indicates that Armenians are a diverse group which has been genetically influenced by the various migrations and invasions which have entered their historic lands. Further, we find evidence that Armenians may be closely associated with the peopling of Europe.

Environmental and anthropogenic stressors can affect wildlife populations in a number of ways. For marine mammals (e.g. the Florida manatee Trichechus manatus latirostris), certain stressors or conservation risk factors have been identified, but sublethal effects have been very difficult to assess using traditional methods. The development of 'biomarkers' allows us to correlate effects, such as impaired reproduction, with possible causes. A recently developed biomarker (anti-Müllerian hormone, AMH) provides an enzyme-linked immunosorbent assay of gonadal function. The study objective was to determine AMH levels in wild manatees. In total, 28 male and 17 female manatee serum samples were assayed. Animal demographics included collection date, body weight (kg) and total length (cm). In certain cases, age of individuals was also known. AMH levels ranged from 160 to 2451.85 ng ml -1 (mean = 844.65 ng ml -1) in males and 0.00 to 0.38 ng ml -1 (mean = 0.10 ng ml -1) in females. Linear regression analyses revealed a significant relationship between male AMH levels and body weight (R 2 = 0.452; p < 0.001) and length (R 2 = 0.338; p < 0.001). Due to the small sample size, regression analyses for female AMH and body weight and length were not significant. This represents the first report of AMH detection in a marine mammal. AMH levels in male manatees are the highest of any species observed to date, whereas levels in females are within reported ranges. Further studies will promote improved conservation decision by assessing AMH levels in the manatee as a function of various stressors including, but not limited to, nutritional status, serious injuries (e.g. watercraft collisions), exposure to biotoxins or contaminants, or disease.

Immune-brain-endocrine communication influences behaviour and contributes to the development of the central nervous system (CNS) in a sexually dimorphic manner. The bed nucleus of the stria terminalis (BST) is a highly sexually dimorphic brain region; in most mammalian species the male BST is larger than the female BST. Previously, our lab has shown that male and female mice lacking T cells due to knock out of the beta (b) and delta (d) chains of the T cell receptor (TCRb-/-d-/-) have reduced anxiety-like behaviour. This was shown with increased time spent in the open arms of the elevated plus maze by TCRb-/-d-/- mice compared to wild type (WT) mice of both sexes. T cell deficient mice also show differences in brain volume compared to WT, including a lack of sexual dimorphism in volume of the BST. The present study explored the impact of T cell deficiency on immune and endocrine factors implicated in sex differences of the CNS. The first analysis was of serum Anti-Müllerian hormone (AMH). AMH is a key determinant of the male phenotype during fetal development. It has also been shown by others to contribute to sexual dimorphic development of the BST. Our postnatal analysis of serum AMH using ELISA demonstrated an age and genotype effect, where a peak in serum AMH levels in WT mice of both sexes was absent in both male and female TCRb-/-d-/- mice at postnatal day (P) 7. These results suggest that T cells have an impact on the endocrine system in early life but the process does not appear to be sexually dimorphic. The present study also explored the impact of TCR knockout on microglia, the resident immune cells of the brain. Other have shown microglia contribute to sexual dimorphic brain development. This contribution occurs through interaction with endocrine factors, making them a key player in the immune-brain-endocrine crosstalk. Using immunohistochemistry and the microglial marker, anti-Iba1, microglia were examined in adult and P7 WT and TCRb-/-d-/ mice. To quantify microglia, soma were traced using AxioVision microscope software, and microglia cell number, perimeter, radius, feret ratio, and area in dorsal and ventral BST were assessed. Our results show sex differences in microglia number in dorsal BST in adult WT mice, where female WT mice had a lower number of microglia compared to WT males, however this difference was absent in TCRb-/-d-/- adult mice. There were no effects on microglia number in the ventral BST and morphology analysis did not reveal any effects in the dorsal or ventral BST. Furthermore, the difference in microglia number was absent in all groups of P7 mice and analysis of soma morphology did not reveal any significant effects. This study explored the impact of TCR knockout on the BST by exploring the immune and endocrine factors shown to contribute to its sexual dimorphic development. The results suggest a non-dimorphic impact on the endocrine system in the postnatal period and a dimorphic impact on microglia that is age and region-specific. The findings reveal a complex network emphasizing the importance of a systems-wide approach to the study of sex differences in the CNS. / Thesis / Master of Science (MSc)

L’hormone anti-Müllerienne (AMH), une glycopréotéine exclusivement produite par les cellules de la granulosa (CG) des follicules ovariens de la femme, est un marqueur unique de du statut folliculaire ovarien. Contrairement à l’inhibine B, l’estradiol et la FSH, l’AMH est produite par un large éventail de follicules allant des follicules primaires aux follicules à petit antrum. Cependant, les mécanismes précis régulant la production d’AMH par les CG restent mal connus. Nous avons montré que la sélection folliculaire précoce au cours de la phase de transition lutéo-folliculaire, un phénomène fréquemment retrouvé chez les femmes ayant un vieillissement ovarien, caractérisé par la présence d’au moins un follicule surdéveloppé au cours de la phase folliculaire précoce, n’altérait pas la puissance de la relation entre le compte folliculaire antral et les concentrations sériques d’AMH. En revanche, cette situation perturbait significativement celle entre le nombre de follicules antraux et les taux sériques de FSH, d’inhibine B et d’estradiol. Nous avons par la suite mis en évidence, en utilisant un nouvel outil, nommé Follicular Output RaTe (FORT), que le pourcentage de follicules qui répondent effectivement à la FSH exogène en atteignant la maturation pré-ovulatoire, était négativement et indépendamment lié aux taux sériques d’AMH, ce qui va dans le sens de l’hypothèse d’un effet inhibiteur de l’AMH sur la sensibilité des follicules à la FSH. Ensuite, nous avons regardé si la production d’AMH par ovaire et par follicule était altérée chez les femmes n’ayant plus qu’un seul ovaire suite à une ovariecomie unilatérale. En effet, tout indique que chez ces femmes, des réarrangements majeurs de la folliculogenèse sont mis en place pour maintenir une fonction ovarienne malgré la perte brutale d’une partie du pool folliculaire. Ainsi, par une analyse extensive et comparative de la folliculogenèse utilisant des marqueurs hormono-folliculiares, nous n’avons pu mettre aucune modification significative chez les femmes avec un ovaire unique, comparativement aux contrôles. A l’aide du modèle précédemment utilisé, nous avons constaté une augmentation de la sensibilité des follicules antraux à la FSH exogène, évaluée par le FORT, chez des femmes avec un seul ovaire, comparativement aux femmes avec 2 ovaires. Ces résultats supportent l’hypothèse d’une augmentation de la sensibilité folliculaire à la FSH, qui pourrait faire partie des possibles mécanismes compensatoires en jeu dans le maintien d’une folliculogenèse efficace chez les femmes ayant eu une ovariectomie unilatérale.Finalement, à l’aide de 2 approches complémentaires, in vitro and in vivo, nous avons montré que la FSH et l’AMPc stimulaient la transcription de l’AMH, et que la LH avait un effet additif. Nous avons montré que les gonadotrophines et l’AMPc agissaient à travers la protéine kinase A et la P38 MAP Kinase, impliquant notamment les facteurs de transcription GATA binding factor-4 et le steroidogenic factor-1. Par ailleurs, nous avons également mis en évidence que l’expression d’AMH pouvait être régulée de manière différentielle par l’estradiol, en fonction du type de récepteur aux estrogènes exprimés par les CG. Ainsi, la chute d’expression de l’AMH au sein des CG des follicules matures, qui expriment essentiellement ERβ, est probablement liée à un effet de l’estradiol. En résumé, ces travaux de thèse ont permis d’apporter de nouvelles données sur la régulation de la croissance folliculaire et sur la production d’AMH chez la femme. / Anti-Müllerian hormone (AMH), a glycoprotein that is exclusively produced by the granulosa cells (GC) of ovarian follicles in the adult female, is a unique biomarker of ovarian follicular status. In contrast with inhibin B, estradiol and FSH, AMH is produced in a wide range of follicles that goes from the primary to the small antral stages of folliculogenesis. However, the precise mechanisms that drive AMH expression by GC remain poorly understood.We showed that untimely and/or accelerated antral follicle growth during the luteal–follicular transition, a phenomenon that is frequent in ovarian-aged women and that is characterized by the presence of at least one overdeveloped antral follicle during the first days of the follicular phase does not alter the strength of the relationship between antral follicle count and serum AMH levels but does affect the relationship between serum FSH, inhibin B and estradiol levels and the number of antral follicles. The heftiness of AMH in relation to advanced antral follicle growth provides a further explanation for the reported stronger association between serum AMH levels and antral follicle counts as compared with the other hormonal markers of the ovarian fertility status. We subsequently demonstrated, using an innovative tool, the Follicular Output RaTe (FORT), that the percentage of follicles that effectively respond to exogenous FSH by reaching pre-ovulatory maturation is negatively and independently related to serum AMH levels, which is in keeping with the hypothesis that AMH inhibits follicle sensitivity to FSH. Given this hypothesis, we wondered if per-ovary and per-follicle AMH production could be altered in patients having a single ovary as a result of unilateral oophorectomy. Indeed, all indicate that major rearrangements of folliculogenesis occur to preserve and maintain ovarian function despite the abrupt halving of follicular stockpile in these patients. We performed an extensive and comparative evaluation of the folliculogenesis using homono-follicular markers failing to show major changes in unilaterally oophorectomized when compared with control women. Using the same model, we demonstrated an increased antral follicle responsiveness to exogenous FSH, as assessed by FORT, in normo-ovulating unilaterally oophorectomized women undergoing controlled ovarian hyperstimulation. These results support the hypothesis that increased FSH sensitivity ranks among the possible compensating mechanisms at stake in the maintenance of successful folliculogenesis after unilateral oophorectomy.Finally, using complementary approaches, in vitro and in vivo, we showed that FSH and cAMP enhance AMH transcription, and LH has an additive effect. Gonadotropins and cAMP act through protein kinase A and p38 MAPK signaling pathways and involve the GATA binding factor-4 and steroidogenic factor-1 transcription factors, among others. The expression profile of AMH and the dynamics of serum AMH after gonadotropin stimulation have been interpreted as a down-regulating effect of FSH upon AMH production by GC. We also demonstrated that AMH expression can be differentially regulated by estradiol depending on the estradiol receptors by GC. Therefore the decrease in AMH expression by GC of mature follicles, which mainly express ERβ, is likely due to the effect of estradiol.In short, this Ph.D. work offers new insight into the regulation of the follicular growth and AMH production in woman.