The aim of the present research was to investigate the expression profiles of GnRH-R and neuropeptide Y (NPY) genes in brain and pituitary of Orange-spotted Grouper, Epinephelus coioides and also to understand the regulatory mechanism by administering different sex steroids. GnRH-R (TMD2 to TMD6) was partially involved cloned in this study. Tissue distribution analysis revealed a significant expression of GnRH-R in pituitary compared to others tissues. The expression of GnRH-R in brain and pituitary of groupers at different ages showed a significant increased during the fourth year, probably indicating the time of maturation. However, there was no significant difference in the expression of GnRH-R during different seasons. Treatment of two and three year old groupers with different sex steroids revealed an increase in the expression of GnRH-R in pituitary by E2 in both the age groups tested, while T could induce the expression of GnRH-R only in three year old groupers. The result, thus, indicates that the sensitivity of grouper to sex steroid is dependent on the age and the kind of steroid adminstered. In different sections of brain, the GnRH-R expression was in general lower in the group treated with E2 or T compared to the control group. The expression of the gene was more or less the same in two year and three year old control groups. This result may have been caused by suppression of GnRH-R expression in forebrain and midbrain after sex steroid injection. Administration of T induced a significant increase in the expression of GnRH-R in forebrain and midbrain, while E2 treatment did not have a similar effect. In hindbrain, the expression profile GnRH-R was not affected by sex steroid treatment in both two year and three year old groupers. The results suggest that sex steroids can only regulate the expression of GnRH-R in the forebrain and midbrain, probably due to the wide distribution of steroid¡¦s receptor in these regions. LHRH and pimozide injections to two year old groupers showed an increase in the expression of GnRH-R in pituitary after LHRH treatment while there was no stimulatory effect on other sections of the brain. In contrast, treating the fish with pimozide alone or pimozide together with LHRH did not stimulate GnRH-R expression in brain. Thus, the study suggests that LHRH can significantly increase the expression of GnRH-R in pituitary while dopamine has no stimulatory effect. Studies on NPY showed that the gene was distributed in different sections of brain especially in the forebrain but it was also present in gills, liver, intestine ¡K etc. The presence of NPY in gills, though less compared to that in brain, suggests that NPY might play an important role in osmosis regulation. The expression of NPY decreased with increase in age which may be due to the effect of other regulatory factors. Treatment of two and three year old groupers with different sex steroids did not effect the expression of NPY significantly in brain, which is different from other published reports. This may be due to the difference in the zoning of brain regions. In the present study, forebrain and midbrain were sampled together for analyses. The expression of NPY in brain did not change by treating the fish with LHRH or pimozide.

Variant GnRH ligand and receptor subtypes have been identified in a number of non-mammalian vertebrate species, however research into avian species GnRH systems is lacking. Two isoforms of GnRH are present in the domestic chicken, the evolutionarily conserved GnRH-II and diverged cGnRH-I. The expression of two GnRH ligands parallels the expression of two chicken GnRH receptor subtypes; cGnRH-R-I and the novel cGnRH-R-III. The occurrence of two isoforms of the receptor in the chicken raises questions about their specific biological functions and interactions with the two ligands. Differential roles for these molecules in regulating gonadotrophin secretion or other functions are currently unclear. To investigate this, cGnRH-R-III cDNA was cloned from a broiler chicken anterior pituitary gland and its structure and expression was compared with cGnRH-R-I. Expression profiling of cGnRH-R-III cDNA showed that it is predominantly expressed in the anterior pituitary, approximately 1400 times more abundantly than cGnRH-R-I suggesting that cGnRH-R-III is the predominant regulator of chicken gonadotrophin synthesis and secretion. Additionally, pronounced sex and age differences existed, with higher pituitary cGnRH-R-III mRNA levels in sexually mature females versus juvenile females. In contrast, higher mRNA expression levels occurred in juvenile males compared to sexually mature males. Determination of ligand-binding selectivity and the level of cGnRH-R-III activation in response to the endogenous ligands, cGnRH-I and GnRH-II, was anticipated as facilitating the elucidation of the physiological roles of the receptor subtypes. Additionally, the development of analogs that differentially promote or inhibit activation of the receptor subtypes may be valuable tools for determining the role of receptor types in the regulation of gonadotrophin production. To investigate this, pharmacological profiling of cGnRH-R-III in terms of ligand-binding selectivity and inositol phosphate production in response to GnRH analogs was determined in comparison with the pharmacological profile of cGnRH-R-I. Functional studies in COS-7 cells indicated that cGnRH-R-III has a higher binding affinity for GnRH-II than cGnRH-I (IC50: 0.57 v 19.8 nM) and more potent stimulation of inositol phosphate production (EC50: 0.8 v 4.38 nM). Similar results were found for cGnRH-R-I, (IC50: 0.51 v 10.8 nM) and (EC50: 0.7 v 2.8 nM). Mammalian receptor antagonist 27 distinguished between cGnRH-R-I and cGnRH-R-III (IC50: 2.3 v 351 nM), and application of this synthetic peptide may facilitate delineation of receptor subtype function either in-vitro or in-vivo. The length of the C-terminal tail of cGnRH-R-III is 8 residues longer than that of cGnRH-R-I and this observation stimulated investigation of differences in ligand-induced internalisation between the two receptor subtypes. The initial rate of receptor internalisation was faster for cGnRH-R-III than for cGnRH-R-I (26%.min-1 v 15.8%.min-1). Although proteins encoded by cGnRH-R-III splice variants do not bind GnRH ligands independently and mRNAs were not detectable by Northern blot analysis, cGnRH-R-III_SV2 significantly reduced maximum ligand-binding of cGnRH-R-III, suggesting that it may impair the function of the full-length type III cGnRH receptor. It was anticipated that the two cGnRH-R subtypes may have differential roles in the regulation of luteinising hormone (LH) and follicle stimulating hormone (FSH) gene transcription through the activation of differential second messenger pathways. Three putative Src homology domain 3 (SH3) binding motifs were identified in the type III cGnRH receptor cytoplasmic C-terminal tail domain which are not present in the type I cGnRH-R and suggested the potential for differential coupling to the Mitogen Activated Protein Kinase (MAPK) cascade. To investigate this possibility, activation of the MAPK cascade via cGnRH-R-III and cGnRH-R-I was determined by quantifying elevation of phosphorylated ERK (pERK 1/2) in response to GnRH. Studies performed in COS-7 cells showed a 4-6 fold increase in ERK 1/2 phosphorylation via the type I and type III receptors within 10 minutes of GnRH-I or GnRH-II stimulation, indicating that both receptors signal through the ERK 1/2 pathway in response to cGnRH-I or GnRH-II. The responses were dose-dependent at cGnRH-R-I and cGnRH-R-III. Effects of pre-treatment with PLC and c-Src inhibitors showed that both cGnRH-Rs may activate pERK 1/2 independently of PLC but dependently upon c-Src. However, it must be noted that 100% of the PLC activity was not inhibited by PLC inhibitor as measured by inositol phosphate production at 60 minutes, and the PLC inhibitor has not been shown to inhibit PLC in the same time frame used for the pERK experiments. Mutagenesis of the individual SH3 binding motifs of cGnRH-R-III were performed and the effects on pERK 1/2 levels quantified. The results indicated that the SH3 binding motifs of cGnRH-R-III do not contribute to additional MAPK activation when compared to the native cGnRH-R-III. Both cGnRH-R-I and cGnRH-R-III were HA epitope-tagged (HA-cGnRH-R-I and HA-cGnRH-R-III) and the methodology was optimised for HA-cGnRH-R-III immuno-precipitation. Several size forms of HA-cGnRH-R-III were detectable by immuno-precipitation, facilitating characterisation of the composition of the receptor protein-protein complexes formed using a western blot approach. In summary, the abundance of cGnRH-R-III expression compared to cGnRH-R-I suggests it is probably the major mediator of pituitary gonadotroph function, and that tissue-specific recruitment of cGnRH-R-isoforms has occurred in the avian pituitary during evolution. Pharmacological profiling demonstrated that cGnRH-R-III, like cGnRH-R-I, has a higher ligand-binding selectivity and induction of inositol phosphate production in response to GnRH-II than with cGnRH-I, although cGnRH-I is established as the physiological regulator of gonadotroph function. These results suggest that evolutionary recruitment of ligand-receptor pairing for particular physiological processes does not correlate with in-vitro properties such as highest ligand-binding affinity or efficacy of inositol phosphate production. Therefore evolutionary plasticity has occurred in the tissue-specific adoption of GnRH ligand and receptor subtypes for regulation of particular physiological functions in birds.

Σκοπός της μελέτης ήταν η διερεύνηση ύπαρξης μεταλλάξεων στα γονίδια KAL, της GnRH, του υποκινητή της GnRH, του υποδοχέα της GnRH, και του υποκινητή του υποδοχέα της GnRH, σε ασθενείς με ανεπάρκεια GnRH, με στόχο την εξαγωγή συμπερασμάτων σχετικά με τη συχνότητα των διαφόρων μορφών μετάδοσης της νόσου στον ελληνικό χώρο, καθώς και η συσχέτιση μεταξύ του γονότυπου των ασθενών και ειδικών κλινικών φαινοτύπων. Μελετήθηκαν συνολικά τριάντα οκτώ (38) ασθενείς με ανεπάρκεια GnRH, δώδεκα (12) ασθενείς με σύνδρομο Kallmann και είκοσι έξι (26) ασθενείς (13 άνδρες και 13 γυναίκες) με ιδιοπαθή υπογοναδοτροφικόΤο σύνδρομο ανεπάρκειας της GnRH περιλαμβάνει ετερογενή, αλλά συναφή προς το κλινικό φαινότυπο πληθυσμό ασθενών οι οποίοι παρουσιάζουν πλήρη ή μερική απώλεια της ικανότητας του οργανισμού τους να προάγει την κατά ώσεις έκκριση της GnRH. Η ανεπάρκεια αυτή οδηγεί στην πλήρη ή μερική αναστολή της σεξουαλικής ωρίμανσης και σε στειρότητα του ασθενούς. Η παρουσία συνοδού ανοσμίας αναφέρεται ως σύνδρομο Kallmann, ενώ η απουσία άλλων συνοδών ανωμαλιών ως ιδιοπαθής υπογοναδοτροφικός υπογοναδισμός (ΙΥΥ). Σκοπός της μελέτης ήταν η διερεύνηση ύπαρξης μεταλλάξεων στα γονίδια KAL, της GnRH, του υποκινητή της GnRH, του υποδοχέα της GnRH, και του υποκινητή του υποδοχέα της GnRH, σε ασθενείς με ανεπάρκεια GnRH, με στόχο την εξαγωγή συμπερασμάτων σχετικά με τη συχνότητα των διαφόρων μορφών μετάδοσης της νόσου στον ελληνικό χώρο, καθώς και η συσχέτιση μεταξύ του γονότυπου των ασθενών και ειδικών κλινικών φαινοτύπων. Μελετήθηκαν συνολικά τριάντα οκτώ (38) ασθενείς με ανεπάρκεια GnRH, δώδεκα (12) ασθενείς με σύνδρομο Kallmann και είκοσι έξι (26) ασθενείς (13 άνδρες και 13 γυναίκες) με ιδιοπαθή υπογοναδοτροφικό υπογοναδισμό (ΙΥΥ). Η μεθοδολογία της εργαστηριακής έρευνας περιέλαβε απομόνωση DNA γονιδιώματος από τους ασθενείς εκλεκτικό πολλαπλασιασμό της κωδικοποιησης με την μέθοδο PCR και τέλος προσδιορισμό της αλληλουχίας του DNA στα προϊόντα της PCR. Στους ασθενείς με σύνδρομο Kallmann δεν ανευρέθη αλλαγή βάσεων του γονιδίου KAL. Επισης, στους ασθενείς με ιδιοπαθή υπογοναδοτροφικό υπογοναδισμό δεν εντοπίστηκαν μεταλλάξεις στους υποκινητές των γονιδίων της GnRH και του υποδοχέα της. Σε πέντε ασθενείς με ιδιοπαθή υπογοναδοτροφικό υπογοναδισμό που αφορούσαν σποραδικές περιπτώσεις, εντοπίστηκε η μετάλλαξη (gc στο κωδικόνιο 16 Trp16Ser ) στο γονίδιο της GnRH η οποία αποτελεί φυσικό πολυμορφισμό. Στο γονίδιο του υποδοχέα της GnRH εντοπίστηκαν δύο μεταλλάξεις. Η μετάλλαξη (ct στο κωδικόνιο 146), ανιχνεύτηκε σε δύο ασθενείς με οικογενή κληρονομικότητα. Η μετάλλαξη αυτή προκαλεί αλλαγή του αμινοξέως στη θέση 146 της πρωτείνης από προλίνη σε σερίνη την οποία φέρουν και οι δύο ασθενείς σε ετεροζυγωτία. Στους δύο ασθενείς με ΙΥΥ και τη μετάλλαξη Pro146Ser, καθώς και σε ένα άνδρα με ΙΥΥ και φυσιολογική αλληλουχία του υποδοχέα της GnRH παρατηρήθηκε αντίσταση στη δράση της GnRH. Ασθενείς με ΙΥΥ και αντίσταση στη δράση της GnRH αποτελούν φυσικά πρότυπα για τη μελέτη και τον εντοπισμό των πολλαπλών γονοτυπικών συνδυασμών οι οποίοι έχουν ως κατάληξη την εμφάνιση του συγκεκριμένου φαινοτύπου. Ο μη εντοπισμός νοσογόνων μεταλλάξεων ομοζυγωτίας ή διπλής ετεροζυγωτίας στους συγκεκριμένους ασθενείς συνηγορεί στην ύπαρξη διαταραχών στην έκφραση γονιδίων τα οποία επηρεάζουν ή την ενδοκυττάρια μετάδοση του μηνύματος ή την ίδια την έκφραση του υποδοχέα της GnRH. Αν και το γονίδιο KAL έχει ενοχοποιηθεί για την ανάπτυξη ψυχοπαθολογικών εκδηλώσεων, με κύριο εκπρόσωπο τη σχιζοφρένεια, οι εκδηλώσεις από τη ψυχική σφαίρα ασθενών με σύνδρομο Kallmann δεν έχουν μελετηθεί. Επιπλέον σκοπός της παρούσης μελέτης ήταν να περιγράψει αυτές τις διαταραχές και να τις συσχετίσει με το γονότυπο των ασθενών. Ένας εκ των ασθενών με σύνδρομο Kallmann παρουσίαζε και σχιζοφρένεια χωρίς όμως να αναδειχθούν μεταλλάξεις στο γονιδίωμά του. Ενώ παράλληλα οι ασθενείς με ανεπάρκεια GnRH δεν διαφέρουν σημαντικά στις κλίμακες ψυχοπαθολογίας ούτε από τις μέσες τιμές φυσιολογικού πληθυσμού, ούτε από τις τιμές που έδωσε η ομάδα ελέγχου με χρόνιες σωματικές παθήσεις, δίνουν σημαντικά χαμηλότερες τιμές σε όλες τις κλίμακες ψυχοπαθολογίας από ότι οι ψυχιατρικά ασθενείς, εκτός από την υποκλίμακα του «θυμού». / The syndrome of GnRH insufficiency is due to a functional deficit of GnRH production or secretion in the hypothalamus resulting in the loss of pulsatile secretion of GnRH. This deficiency leads to a complete or partial arrest of sexual maturation and infertility. Patients with no further anomalies are referred as having Idiopathic Hypogonadotropic Hypogonadism (IHH) and when accompanied with anosmia, it is called Kallmann syndrome. The aim of this study was to identify mutations in the KAL gene, the GnRH gene, the GnRH receptor gene and their promoters in patients with GnRH insufficiency, the prevalence in the Greek population and the relevance between the genotype and individual phenotype of these patients. The study included thirty eight (38) patients with GnRH insufficiency, twelve patients (12) with Kallmann syndrome and twenty six patients with IHH (13 male and 13 female). Detection was carried out by isolation of genomic DNA from whole blood, which was used as a template for PCR amplification and finally, cycle sequencing analysis of all exons spanning the entire coding regions of the genes. No mutations were found in the KAL gene, whereas in patients with IHH, no mutations were identified in transcription factor binding sites of the promoters of the GnRH and GnRH receptor gene. In the GnRH gene of five (5) patients with IHH a natural polymorphism was identified in codon 16 (gc Trp16Ser). In the GnRH receptor gene a novel mutation was found in codon 146, resulting in substitution of proline with serine identified in two sisters harboring this mutation in hetrozygosity. The two sisters and a male patient with IHH with normal gene sequencing were found to be resistant to GnRH action. Resistance to GnRH is particularly rare among IHH patients. One might hypothesize, that these patients ought to have inactivating mutations in their receptor gene. However such a defect was not found, therefore making these patients an ideal clinical phenotype of an aberration in signal transduction pathway or in transcriptional factors which regulate the expression of the GnRH receptor. A common pathogenesis for KS and schizophrenia had been proposed, based on shared pathologies of these two disorders. The gene for the X-linked form of KS (known as KAL) has been implicated in the genetic pathogenesis of schizophrenias, although no such clinical associations have ever been reported. An additional aim of this study was to identify any pshychopathologies in these patients. One of our patients with KS also developed schizophrenia but no mutations were identified in all 14 exons of the KAL gene.

No description available.

The control of reproductive cycles in higher primates is largely dependent on negative and positive feedback mechanisms by both steroidal and non-steroidal substances of the ovaries which regulate the function of hypothalamo-pituitary system. To gain insights into the role of INH A, the non steroidal ovarian hormone in the feedback control of pituitary FSH secretion, studies were conducted to examine the interrelationships of hormones throughout the menstrual cycle of the bonnet macaque. The findings of chapter II provide a detailed description of endocrine hormone profile during the menstrual cycle of the bonnet macaques with special attention to the feedback role of INH A on pituitary FSH secretion. To characterize the endocrine profile of different hormones, both ovarian (E2, P4, INH A) and pituitary (FSH, LH) hormones were measured daily for more than 40 days. To further examine the site of secretion of INH A and its relationship with pituitary FSH dynamics, surgical lutectomy and pharmacological induction of luteolysis employing the third generation GnRH R antagonist, Cetrorelix (CET) studies were carried out in the subsequent experiments. The results obtained from these studies suggest that INH A and P4 secreted from the CL during luteal phase regulate pituitary FSH secretion. The selective rise in FSH observed during the late menstrual cycle and during menstruation (referred to as luteo-follicular transition), as has been reported previously in higher primates, considered necessary for initiation of follicular growth and recruitment of follicles for ensuing menstrual cycle was characterized in the monkey. Surgical lutectomy and induction of luteolysis by CET experiments suggested that increased GnRH secretion is essential for this selective rise in FSH following withdrawal of inhibition by P4 and INH A. In clinical cases of reproductive ageing, the shortened follicular phase in human females has been identified to be the result of occurrence of early onset of FSH rise during the luteal-follicular transition period. The cause(s) of declining fertility with age in women who still have regular menstrual cycles are not clear, but issues of relationship between dysregulation of selective FSH rise in the late luteal phase and associated infertility could be examined using bonnet monkey as a model system. INH A is secreted in significant quantities by CL in higher primates and the feto placental unit suggesting its importance during fertility and pregnancy. Apart from the negative feedback regulation of pituitary FSH secretion, the complete repertoire of actions of this hormone during pregnancy is yet to be fully understood. The data presented in this thesis is the first comprehensive report showing the endocrine hormone profile of gonadotropins and ovarian hormones including INH A throughout the menstrual cycle of the bonnet macaque. The characterization of INH A profile in bonnet monkey will be of significant value for studies examining the role of INH A in higher primates. Dimeric inhibin has been suggested to be important for regulation of fertility and reproductive functions. Also, inhibin-α (one of the subunits of the dimeric protein) knock out mice model has provided convincing evidence that it acts as a tumour suppressor. A great deal of new information has been generated in recent years regarding the potential clinical usefulness of monitoring inhibin levels in blood and biological fluids in gynaecological diseases, pathological pregnancies and other disorders. Emerging clinical roles of inhibin have made INH A an important candidate molecule to study its molecular regulation. The results presented in chapter II suggested that LH regulates luteal INH A secretion (induction of luteolysis by CET administration experiment). As a first step towards understanding molecular regulation of inhibin-α expression in the macaque CL, in silico promoter analysis of macaque inhibin-α was performed and it revealed several transcriptional factor binding sites that were conserved across species. In rats FSH up regulates while preovulatory LH surge suppresses inhibin-α mRNA expression in the ovary and this suppression has been suggested to be necessary for occurrence of secondary FSH surge during metestrus. To address differential regulation of inhibin-α by LH and FSH in rat ovary during the periovulatory period, studies employing immature rats were carried out and the results are presented in chapter III. The results suggest that immature rat ovaries respond to exogenous gonadotropins in terms of LH signaling (cAMP production), luteinization (P4 production) and as well induction of ICER expression required for repression of inhibin-α subunit expression. PDE4 inhibitor (rolipram) treatment enhanced the ovarian cAMP concentrations suggesting that PDE4 play a major role in controlling intraovarian cAMP concentrations in rat ovaries. However increased cAMP concentrations did not appear to up regulate the ICER expression at the time point examined in this study. In higher primates time course of second FSH surge and continued synthesis and secretion of INH A in the CL are different from non primate species. In the monkey, the second FSH rise occurs during the late luteal phase and experiments have been carried out to examine the regulation of inhibin-α subunit expression by ICER. Expressions of ICER (mRNA/protein) and INH A were examined during different stages of CL and the results indicated no clear inverse relationship between the ICER and inhibin-α mRNAs. With no conclusive role for the ICER in regulating luteal inhibin-α observed in the study, the role of transcriptional activators in the regulation of inhibin-α like GATA4, SF-1, β-catenin were further examined. Since luteal INH A secretion was dependent on pituitary LH as determined earlier in chapter II, expressions of transcriptional activators were examined in CL of different stages and also during induced luteolysis and the results are described in chapter IV. In conclusion, our results indicate cross talk between WNT, cAMP and P38 MAP kinase signaling pathways in the regulation of luteal INH A secretion. The pituitary gonadotropin, LH, is the primary luteotropin in primate species acting to maintain the structure and function of the CL during the menstrual cycle. However whether the actions of LH are direct or mediated by local factors such as P4 remain unknown. Moreover, P4 secretion which is dominant during luteal phase has any role in regulating CL structure and function is not clearly defined. To address these and issues concerning P4 actions, initially, experiments were performed in the rat model to study the importance of P4 in the regulation of ovarian functions. An antiprogestin, RU486, was employed as a tool to uncover the PR regulated pathways during ovulation in rats and the findings are presented in the chapter V. The results indicated that blockade of PR action by RU486 during gonadotropin-induced superovulation resulted in inhibition of follicular rupture and ovulation in immature rats. Further to understand the downstream effectors of PR action, and to identify the candidate target genes of PR activation, semi-quantitative RT-PCR and western blot analyses were performed. The results obtained indicated that betacellulin, a member of EGF family and MMP-9 a proteolytic enzyme, were markedly repressed in response to RU486 treatment in rat ovaries. Also, the down stream pathway of EGF signaling leading to activation of ERK was markedly repressed in RU486 treated ovaries. It was next examined what role the P4/PR system has in the regulation of CL structure and function. Surprisingly, PR expression is absent in CL of rats, while it is present in higher primates. Experiments were carried out to examine intracrine actions of P4 in the regulation of CL structure and function in monkeys. The recently reported model system of induced luteolysis yet capable of responsive to trophic support from the laboratory provided an ideal opportunity to examine direct effects of P4 on structure and function of CL in the monkey. A series of pilot experiments were carried out in monkeys experiencing summer amenorrhea, to determine dose and mode of administration of exogeneous P4 to simulate mid luteal phase circulating P4 concentrations in monkeys subjected to induced luteolysis. Based on the results of pilot experiments, implantation of Alzet pumps containing 97.5mg of P4 was selected for maintaining mid luteal phase P4 concentrations. The microarray data of induced luteolysis previously deposited by the laboratory in NGBI’s gene expression omnibus were mined for identification and validation of differentially expressed genes of PR and its target genes following LH depletion and LH replacement experiments. Expressions of PR, PR cofactors and expressions of PR downstream target genes through out the luteal phase and in CL from day1 of menses were also examined. Analysis of expressions of genes revealed that of the 45 genes identified to be regulated by LH treatment, 4 genes were found to be responsive to P4, and 14 were identified to be responsive to both P4 and LH. Morphology of CL tissue sections revealed that P4 treatment appeared to have reversed the induced-luteolysis changes. In another experiment, implantation of P4 during late luteal phase (i.e., the period of declining P4 concentrations) for 24h caused changes in expressions of genes associated with tissue remodeling and morphology of luteal cells. Taken together, the results suggest that induced luteolysis plus P4 replacement model is suitable for assessing the effects of P4 on CL function. The results also suggest that CL could serve as target tissue for examining the genomic and non genomic actions of P4. In summary, studies carried out in the present thesis provides a comprehensive endocrine hormone profile throughout the menstrual cycle of the bonnet monkey with special emphasis on time course of INH A and FSH secretion which is very useful for future investigations. Studies have been carried out in rats and monkeys with different experimental model systems to address molecular mechanisms underlying inhibin-α regulation in the ovary in general and CL in particular. Experimental findings in monkeys could help elucidate the underlying molecular nature of CL functionality and extrapolate to understand luteal insufficiency and infertility producing conditions in humans. Also different model systems have been validated to examine the actions of P4/PR system in rats and monkeys and more importantly to address the direct effects of P4 upon monkey CL structure and function were established. Future investigations based on findings of these studies should help clarify relative roles for LH and P4 during maintenance of CL function and luteolysis.

Le récepteur hypophysaire de la GnRH (RGnRH) joue un rôle crucial dans le contrôle de la fonctionde reproduction. Dans le promoteur distal du Rgnrh, j’ai caractérisé un élément de réponsebifonctionnel répondant aux protéines LIM à homéodomaine ISL1/LHX3 et à GATA2. D’autre part,deux motifs TAAT situés dans la région plus proximale confèrent à ce gène la capacité de répondreaux facteurs Paired-like PROP1 et OTX2. Tous ces facteurs, exprimés précocement au cours del’ontogenèse hypophysaire, pourraient participer à l’émergence de l’expression du Rgnrh. Hors del’hypophyse, j’ai découvert que le Rgnrh est exprimé au cours du développement postnatal dansl’hippocampe de rat, où il module la plasticité synaptique. Par ailleurs, j’ai identifié deux nouveauxsites d’expression, la rétine et la glande pinéale. Ces résultats mettent en lumière l’importancefonctionnelle de ce récepteur et de son ligand et les rôles multiples qu’il ont acquis au cours del’évolution des Vertébrés. / In the pituitary, the GnRH receptor (GnRHR) plays a crucial role in the neuroendocrine control ofreproductive function. Within the distal region of the Gnrhr promoter, I have characterized abifunctional response element modulated by the LIM homeodomain proteins ISL1/LHX3 and byGATA2. Besides, in the proximal region of the promoter, two TAAT motifs conferred response toPaired-like factors PROP1 and OTX2. All these factors are expressed during pituitary ontogenesis andcould participate in the onset and regulation of Gnrhr expression. Outside of the pituitary, I havediscovered that the Gnrhr was expressed during postnatal development in the rat hippocampus, whereit modulated synaptic plasticity. Furthermore, I have identified two novel sites of Gnrhr expression, theretina and the pineal gland. Altogether, these data highlight the functional importance of this receptorand its ligand as well as the multiple roles they have acquired during vertebrate evolution.

La fonction de reproduction est sous le contrôle de la neurohormone hypothalamique GnRH qui régule la synthèse et la libération des gonadotropines hypophysaires. La GnRH agit par l’intermédiaire d’un récepteur couplé aux protéines G exprimé à la surface des cellules gonadotropes, le récepteur de la GnRH (RGnRH). Ce récepteur, chez les mammifères, a la particularité d’être dépourvu de queue C terminale ce qui le rend insensible aux systèmes classiques de désensibilisation. Ainsi, les mécanismes qui régulent l’efficacité et la spécificité de sa signalisation demeurent mal connus. Nous avons recherché des partenaires d’interaction du RGnRH, jusqu’alors inconnus, avec l’idée que ces protéines en interagissant avec les domaines intracellulaires du récepteur influenceraient son couplage aux voies de signalisation. Nos travaux ont permis d’identifier le premier partenaire d’interaction du RGnRH : la protéine SET. Par des expériences de « GST pull down », nous avons montré que SET interagit directement avec le RGnRH via le premier domaine intracellulaire du récepteur. Cette interaction implique des séquences riches en acides aminés basiques sur le récepteur et les domaines N- et C-terminaux de SET. Nous avons également montré, par co-immunoprécipitation, que le RGnRH dans sa conformation native interagit avec la protéine SET dans les cellules gonadotropes alphaT3-1 et, par immunocytochimie, que les deux protéines colocalisent à la membrane plasmique. En développant au laboratoire des outils biosenseurs permettant de mesurer avec une grande sensibilité et en temps réel les variations intracellulaires de calcium et d’AMPc, nous avons mis en évidence que le RGnRH se couple non seulement à la voie calcique mais aussi à la voie AMPc dans la lignée alphaT3-1, apportant pour l’AMPc la première démonstration d’un tel couplage. En utilisant différentes stratégies expérimentales visant à diminuer ou au contraire favoriser l’interaction du récepteur avec SET (ARN antisens, peptide correspondant à la première boucle intracellulaire du récepteur, surexpression de SET), nous avons montré que SET induit une réorientation de la signalisation du RGnRH de la voie calcique vers la voie AMPc. Nos résultats concernant l’activité du promoteur du gène du Rgnrh nous conduisent à postuler que SET pourrait favoriser l’induction par la GnRH de gènes régulés via la voie AMPc et notamment celui codant le RGnRH. Nos travaux mettent également en évidence que la GnRH régule non seulement l’expression de la protéine SET dans les cellules gonadotropes mais aussi son degré de phosphorylation favorisant ainsi sa relocalisation dans le cytoplasme des cellules alphaT3-1. Ceci suggère que la GnRH exerce une boucle de régulation permettant d’amplifier l’action de SET sur la signalisation de son propre récepteur. Enfin, nous avons mis en évidence que l’expression de SET est fortement augmentée dans l’hypophyse au moment du prœstrus chez le rat, apportant ainsi la première démonstration d’une variation de SET dans un contexte physiologique. Étant donné que le couplage du RGnRH à la voie de signalisation AMPc est augmenté au moment du prœstrus, nos résultats suggèrent que SET pourrait jouer un rôle important in vivo en favorisant ce couplage à ce stade particulier du cycle œstrien. / Reproductive function is under the control of the hypothalamic neurohormone GnRH, which regulates the synthesis and the release of pituitary gonadotropins. GnRH acts on a G-protein coupled receptor expressed at the surface of pituitary gonadotrope cells, the GnRH receptor (GnRHR). This receptor, in mammals, is unique because it is devoided of the C terminal tail, which makes it insensitive to classical desensitization processes. Therefore, the mechanisms that regulate the efficacy and the specificity of its signaling are still poorly known. We searched for interacting partners of GnRHR with the idea that these proteins by interacting with the intracellular domains of the receptor could influence receptor coupling to its signaling pathways. Our work identified the first interacting partner of GnRHR: the protein SET. By GST pull down assays, we showed that SET interacts directly with GnRHR through the first intracellular loop of the receptor. This interaction involves sequences enriched in basic amino acids in the receptor and both N- and C terminal domains of SET. We also showed, by co-immunoprecipitation, that GnRHR in its native conformation interacts with the endogenous SET protein in gonadotrope alphaT3-1 cells and, by immunocytochemistry that the two proteins colocalize at the plasma membrane. By developing in the laboratory biosensors tools that allow to measure with high sensitivity and in real-time intracellular variations in calcium and cAMP concentrations, we demonstrated that GnRHR couples not only to the calcium pathway but also to the cAMP pathway in alphaT3-1 cell line, providing for cAMP the first demonstration of such coupling. Using several experimental strategies to reduce or increase receptor interaction with SET (small interfering RNA, peptide corresponding to the first intracellular loop of the receptor, overexpression of SET), we have shown that SET induces a switch of GnRHR signaling from calcium to cAMP pathway. Our results concerning the activity of the Gnrhr gene promoter led us to postulate that SET could favor the induction by GnRH of genes regulated through the cAMP pathway, notably those encoding the GnRHR. Our study also showed that GnRH regulates not only SET protein expression in gonadotropes, but also its phosphorylation level leading to its relocation in the cytoplasm of alphaT3-1 cells. This suggests that GnRH induces a regulatory loop to amplify SET action on signaling of its own receptor. Finally, we demonstrated that SET expression is markedly increased in the pituitary gland at prœstrus in female rats, providing the first demonstration of a variation of SET expression in a physiological context. Given that GnRHR coupling to the cAMP pathway is increased at prœstrus, our results suggest that SET may play an important role in vivo by promoting such coupling at this particular stage of the estrus cycle.