Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 6 uppsatser.

Made available in DSpace on 2014-10-09T12:23:19Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:35Z (GMT). No. of bitstreams: 1 01276.pdf: 2214172 bytes, checksum: e105537b579f510e386e4b8e27eb1ef6 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP

Background: Hypothalamic kisspeptin and neurokinin B (NKB) are central regulators of GnRH and thus gonadotropin (LH and FSH) secretion. Men and women with loss-of-function mutations in NKB-kisspeptin pathway show hypogonadotropic pubertal delay with reduced GnRH/LH pulsatility. Studies in patients with defects in NKB signalling suggest that kisspeptin is functionally downstream of NKB, although there are very limited data on the relevance of the NKB pathway in normal men or women, and no hierarchical data on this. The studies described in this thesis have investigated the interaction between these neuropeptides in the control of human reproduction in conditions of varying sex-steroid environment, and in states of fast and slow LH secretion (men, menopause, various stages across the menstrual cycle). Overall hypothesis: Pharmacological blockade of NKB signalling will decrease LH secretion by modulating GnRH/LH pulsatility, indicating the involvement of the NKB pathway in normal human reproductive function. It is also hypothesised that this will not abrogate the stimulatory kisspeptin response, revealing a functional hierarchy whereby NKB signalling is upstream of kisspeptin. Research strategy: A specific neurokinin-3 receptor antagonist (NK3R antagonist, AZD4901) was administered 40 mg twice daily orally for 7 days with and without kisspeptin-10 (KP-10) challenge. Response of reproductive hormones (serum and urinary where applicable) was measured. LH was sampled every 10 minutes for 8 hours to assess LH pulsatility by blinded deconvolution. Results: Role of neurokinin B and kisspeptin in healthy men Six healthy men underwent LH pulsatility study pre-treatment and on day 7 of NK3R antagonist administration with iv KP-10 bolus (0.3 μg/kg) at 6 hours. NK3R antagonist reduced LH and testosterone secretion, whilst stimulatory LH response to KP-10 was unaffected. LH pulse frequency was unchanged by the NK3R antagonist but basal (nonpulsatile) and pulsatile LH secretion was markedly reduced. Role of neurokinin B and kisspeptin in postmenopausal women Eleven postmenopausal women underwent LH pulsatility study pre-treatment and on day 7 of NK3R antagonist administration with iv KP-10 bolus (0.3 μg/kg) at 6 hours. NK3R antagonist decreased LH secretion. Basal (nonpulsatile) LH secretion also fell and while LH pulse frequency did not change in a group as a whole, it did fall in the 8 of 11 postmenopausal womenwith hot flushes. These women reported a reduction in hot flush frequency (3.4±1.2 vs 1.0± 0.6 flushes/day with NK3Ra, p=0.008) and severity whilst on NK3R antagonist. LH response to KP-10 was minimal and unaffected by the NK3R antagonist. Role of neurokinin B across different phases of menstrual cycle The effect of NK3R antagonist on ovarian function was compared in early follicular (n=13), late follicular (n=6) and luteal phase (n=6) to no treatment control cycle. Early follicular: NK3R antagonist was commenced from cycle day 5-6. The diameter of the leading follicle was smaller than in controls at the end of treatment (9.3±0.4 vs 15.1±0.9 mm, p < 0.0001). Serum estradiol was also reduced and the endometrium was thinner. Although NK3R antagonist had no effect on LH pulse frequency, basal (nonpulsatile) LH secretion was decreased, suggesting that NKB modulates GnRH secretion. After stopping treatment, follicle development resumed and estradiol secretion increased thereby delaying the LH surge in 11/13 women (LH surge cycle day 22±1 vs 15±1, p=0.0006). The delayed LH surge and ovulation were confirmed by a similarly delayed rise in urinary progesterone and prolonged cycle length. NK3R antagonist did not affect luteal function. Late follicular: NK3R antagonist was administered from the emergence of a dominant follicle (≥12mm). Whilst there was an LH surge in all treated cycles, estrogen feedback was perturbed by the NK3R antagonist, as there was increased variation in the timing of LH surge compared to control cycle. NK3R antagonist had no effect on the growth of a dominant follicle and luteal function was unaffected. Luteal: NK3R antagonist was administered from day +2-3 of the disappearance of the dominant follicle. NK3R antagonist reduced the variation in the timing of peak estradiol secretion. Estradiol and progesterone concentrations remained unchanged, suggesting that luteal function was overall unaffected by this treatment. No difference in mean LH was observed, although LH pulsatility was not assessed. Role of neurokinin B and kisspeptin in the mid-cycle LH surge A model of follicular phase (cycle day 9-11) administration of estradiol (200μg/day) to induce LH secretion at 48 hours was used in twenty women, mimicking LH surge. In this model, KP-10 infusion (4μg/kg/hr for 7 hours) enhanced LH secretion, the response of which was directly correlated with estrogen concentration, indicating a role of kisspeptin in estrogen feedback. Pre-treatment with NK3R antagonist decreased LH pulse frequency and whilst the immediate LH response to KP-10 was unaffected, it blunted the duration of this response and abolished the relationship between estradiol and kisspeptin-induced LH secretion. Conclusions: These data indicate the role of NKB-KP pathway in regulating human reproductive function and that this is via the modulation of pulsatile GnRH secretion. Whilst NKB is predominantly proximal to kisspeptin, the hierarchy is more complex than simply linear in the control of human HPG axis. Manipulation of NKB-KP signalling has therapeutic potential in regulating GnRH/LH secretion in wide range of clinical settings, including contraception, sex-steroid dependent disorders and in the treatment of hot flushes.

No description available.

Atrial natriuretic peptide (ANP) of atrial myocyte origin, has been shown to play a role in the diuresis, natriuresis, and antagonism of angiotensin and vasopressin. However, it is now apparent that in addition to the production of the peptide in the heart and in its role in fluid and electrolyte homeostasis, it is also produced in the central nervous system participating in the regulation of pituitary hormone secretion. Administration of ANP through both central and peripheral routes has been shown to inhibit secretion of luteinizing hormone (LH) in the gonadectomized rat model. A better understanding of the modulatory role of ANP on LH secretion and its possible mechanisms will add to our knowledge of the effects of neuropeptides on reproductive function. Brain natriuretic peptide (BNP) is a bioactive peptide of 26 amino acid residues recently identified in porcine brain. The peptide exerts potent diuretic-natriuretic and vasorelaxant effects, in a manner similar to that of ANP. BNP has a remarkable high sequence homology to ANP, especially in the 17 amino acid ring formed by an intramolecular disulfide linkage which is required for biological activity. The presence of BNP with ANP in the mammalian brain and remarkable resemblance in their molecular structures and physiological functions implies that BNP may also exert an inhibitory effect on LH secretion like ANP. This research focused on the effects of centrally administered ANP and BNP on pulsatile LH secretion and their possible mechanisms of action in ovariectomized rats. After third ventricle infusion of ANP or BNP, inhibition of mean plasma LH level, LH pulse amplitude and pulse frequency was observed. In searching for the possible mechanisms of inhibitory effect of ANP or BNP on pulsatile LH secretion, the effect of inhibiting the endogenous opiate system with naloxone on the action of centrally administered ANP or BNP was tested. Application of naloxone reversed the inhibitory effect of ANP and BNP on mean plasma LH level and LH pulse amplitude, but in terms of pulse frequency, naloxone treatment failed to reverse the inhibitory effect of ANP or BNP. In separate experiments, pretreatment with pimozide, a dopaminergic receptor blocker, prevented the inhibitory action of ANP and BNP on LH secretion. After infusion of ANP or BNP, there were no significant decrease in mean plasma LH level, pulse amplitude and pulse frequency in the pimozide-pretreated rats. In summary, the present study shows that both ANP and BNP inhibit pulsatile LH secretion, suggesting that the inhibitory effects on LH secretion once thought to be mediated by ANP alone may be regulated through a dual mechanism involving both ANP and BNP. Furthermore, the inhibitory mechanisms may involve the interactions of ANP and BNP with central opiate system and dopaminergic system on LH secretion. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

No description available.

The corpus luteum (CL) is a transient endocrine structure formed from the remnants of an ovulated follicle with the primary purpose of producing progesterone (P4), a hormone vital for the establishment and maintenance of pregnancy. The precise regulation of CL function is essential for normal reproductive cycles and maintenance of early pregnancy. In mammals, the pituitary hormones prolactin (PRL) and luteinizing hormone (LH) function as luteotrophic factors during pregnancy, and these two hormones form a functional luteotrophic complex to control CL function in rodents. The mechanistic underpinnings of the luteotrophic actions of PRL and LH, as well as the interplay between the two hormones are poorly understood, and has been the focus of the current investigation. There are several limitations involved in studying luteal function under cell culture conditions. Hence, in vivo animal models employing dopaminergic receptor agonist, 2-Bromo-α-Ergocryptine Mesylate (CB-154; inhibits pituitary PRL secretion) and GnRH receptor antagonist, cetrorelix (CET; inhibits pituitary LH secretion) have been standardized for purposes of examining the roles of PRL and LH in the regulation of CL structure and function in rats. Administration of CB-154 or CET to pregnant rats caused inhibition of CL function and concomitant loss of conceptuses. The CB-154 treatment induced loss of implants was determined to be the result of inhibition of luteal function, rather than the non-specific effects of CB-154 or requirement of PRL for uterine maintenance of implants. To understand how PRL and LH regulate luteal function, targets of PRL and LH in the rat CL needs to be established; however, this has not been well defined by previous studies. The present study observed that CB-154 induced inhibition of luteal function was gradual in its onset; hence, transcriptional changes of genes involved in steroid genesis were examined. mRNA expression of genes involved in P4 production were found to be down regulated, while 20α-hydroxysteroid dehydrogenate (20α-HSD), a P4 catabolizing enzyme was unregulated by CB-154 treatment. CET treatment also had a similar effect on mRNA expression of steroidogenic genes. Interestingly, mRNA expression of the steroidogenic acute regulatory protein (StAR), a key regulator of steroid genesis was not regulated by CB-154 or CET treatment. The luteolytic factor PGF2α also inhibited CL function in pregnant rats but did not down regulate mRNA expression of StAR. However, examination of phospho-StAR (Ser-195), the activated form of StAR, during CET and PGF2α-induced luteolysis suggested that regulation of StAR in the CL of pregnant rats might primarily be at the level of phosphorylation. PRL has been implicated in maintaining luteal expression of LH/choriogonadotrophin receptor (LH/CGR), the cognate receptor for LH. Hence, the luteotrophic actions of PRL may be indirect, by way of regulating LH signalling. Hence, the importance of the LH/CGR pathway and its regulation were examined. LH/CGR mRNA expression was found to correlate with CL function, with CET and CB-154 treatments resulting in down regulation of LH/CGR mRNA expression. Further, CB-154 treatment down regulated LH/CGR pre-mRNA levels, suggesting a role for PRL in the regulation of LH/CGR transcription. mRNA expression of LRH-1, a constitutively active transcription factor previously reported to be important in CL function was down regulated by both CB-154 and CET treatments and hence correlated with LH/CGR mRNA expression. Further, luciferase assays in HeLA cells transiently expressing LRH-1 suggests its involvement in activating the LH/CGR promoter. Estrogen receptor (ER)-α and ER-β also appear to correlate with LH/CGR expression and may play a role along with LRH-1 in the regulation of LH/CGR mRNA expression in the CL of pregnant rats. To examine mechanisms by which PRL may regulate its downstream targets, pathways employed by PRL in the CL of pregnant rats were analysed. The Akt pathway including downstream targets were down regulated by CB-154 treatment. The pathway was found to be regulated at the level of Akt1 mRNA expression. Hence, actions of PRL may regulate the survival of CL. This study has also made observations of LH playing a similar role in survival of the CL. The results of these studies taken together, shed light on the regulation of CL structure and function by PRL and LH, and provide molecular evidence for the two hormones having similar downstream targets and functioning as a luteotrophic complex in pregnant rats, which could only mean a robust interaction between the signalling pathways employed by the two hormones.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Many studies have been developed to characterize the function of angiotensin-renin system (ARS) in the female reproductive organs. Evidences from the literature have pointed a relevant role of angiotensin II (Ang II) in mammals, through its type 2 receptor (AT2) in oocyte maturation as in ovulation. Nevertheless, the participation of Ang II in other important reproductive features such as steroidogenesis has not been fully clarified. Therefore, the main objective of this work was to detect in vitro the steroidogenic effects of Ang II in theca cells. For that, bovine theca cells were obtained from follicles (larger than 8mm of diameter) collected from a local abattoir and submitted to different treatments in a sequence of experiments. In Experiment 1, Ang II was added to LH-treated (10 ng/ml) theca cells. Experiment 2 employed Ang II, in different concentrations, in addition to insulin (100 ng ̸ml) and LH (100 ng ̸ml). Experiment 3 explored the effects of an Ang II antagonist (saralasin) in theca cells co-stimulated by insulin and LH (both at 100 ng ̸ml). After 24 hours, culture media were collected and evaluated for testosterone and androstenedione levels measured by high performance liquid chromatography (HPLC). In parallel, gene expression of key steroidogenic enzymes and proteins, respectively, HSD3B2, CYP11A1 e CYP17A1 and STAR were accessed by qRT-PCR, with exception of experiment 1, in which only CYP17A1 was evaluated. Overall, absence of Ang II action was observed in all Ang II doses evaluated. Despite the difference in gene expression for CYP17A1 against controls in experiment 1, neither an increase in androgens levels nor a negative impact of saralasin were detected. Although very important for oocyte maturation and the ovulation, Ang II seems not influence androgen production by theca cells in vitro. In conclusion our results do not support the role for Ang II in thecal steroidogenesis, at least in bovine, as the primary hypothesis of the study. / Diversos estudos vêm sendo desenvolvidos para caracterizar o sistema renina angiotensina (RAS) no aparelho reprodutivo feminino. Evidências da literatura apontam um importante papel da angiotensina II (Ang II), via receptor tipo 2 (AT2), tanto na maturação dos oócitos quanto na ovulação em mamíferos. No entanto, a participação da Ang II em outros aspectos reprodutivos importantes, como a esteroidogênese, ainda não foi completamente elucidada. Desta forma, o objetivo deste trabalho foi verificar o efeito in vitro da Ang II nas células da teca cultivadas. Para isso, células da teca bovina foram obtidas de folículos com mais de 8mm de diâmetro de ovários oriundos de abatedouro local e submetidas a diferentes tratamentos em uma sequência de experimentos. No experimento 1, Ang II foi adicionada a células da teca tratadas com LH na dose de 10 ng/ml. No experimento 2, foi utilizada Ang II em diferentes concentrações em adição ao tratamento com insulina (100 ng ̸ml) e LH (100 ng ̸ml). O experimento 3, explorou o possível efeito de um antagonista da Ang II (saralasina) em células da teca co-estimuladas com insulina e LH (ambos em 100 ng ̸ml). Após 24 horas, o meio de cultura foi coletado e avaliado para verificação dos níveis de testosterona e androstenediona aferidos pela técnica de cromatografia líquida de alta performance (HPLC). Em paralelo, a expressão gênica de enzimas e proteínas chaves na esteroidogênese, respectivamente, HSD3B2, CYP11A1 e CYP17A1 e STAR, foram avaliadas por qRT-PCR, com exceção do experimento 1 onde somente a CYP17A1 foi estudada. De maneira geral, não foi observada uma ação da Ang II nas doses utilizadas. Apesar de uma diferença na expressão de CYP17A1 ter sido verificada em relação aos controles, nem o aumento dos níveis de androgênios ou um impacto negativo pelo uso de saralasina foram detectados. Embora reconhecida como muito importante para maturação oocitária e ovulação, a Ang II parece não influenciar a produção de androgênios in vitro. Em conclusão, nossos resultados não demonstraram um papel da Ang II na esteroidogênese tecal, pelo menos na espécie bovina, ao contrário da hipótese original deste estudo.

En dépit de l'abondance de données scientifiques, les mécanismes cellulaires régulant la sécrétion du pic pré-ovulatoire de LH lors du proestrus, restent encore mal compris.Afin de pouvoir étudier les mécanismes sous-jacents à cette sécrétion, j’ai tout d’abord adapté des techniques innovantes d’imagerie fonctionnelle en microscopie de fluorescence in vivo, d’injections de vecteurs viraux dans l’hypophyse, d’optogénétique sur animal éveillé et d’immunohistofluorescence sur organe entier.J’ai ensuite montré la plasticité structurelle des cellules gonadotropes et des péricytes (cellules « murales » péri-vasculaires) lors du proestrus sur des hypophyses transparisées. Ce remodelage a permis de proposer l’existence d’une unité Gonadotrope-Vasculaire (GVU) composée des cellules gonadotropes, des capillaires fenêtrés et des péricytes dans laquelle ces derniers moduleraient le pic pré-ovulatoire de LH.La contraction des péricytes via l’activation de la Channelrhodopsine-2 a permis de mettre en évidence leur rôle dans la potentialisation de la sécrétion de LH chez des animaux libres de leurs mouvements et implantés d’une fibre optique.Des expériences de microscopie à l’aide d’une lentille GRIN implantée au-dessus de l’hypophyse ont permis, chez l’animal éveillé en configuration « tête-fixée », d’étudier le flux sanguin et l’activité calcique de cellules de la GVU exprimant GCaMP6. Cette étude a également été menée sur la face ventrale de l’hypophyse sur souris anesthésiée. Les résultats montrent une activité calcique in vivo augmentée dans les cellules endocrines hypophysaires et diminuée dans les péricytes lors d’une sécrétion de la LH induite par la GnRH. / In spite of abundance of scientific data, cellular mechanisms regulating the secretion of the pre-ovulatory LH surge during proestrus are still poorly understood.In order to study the mechanisms underlying this secretion, I adapted innovative tech-niques for in vivo fluorescence functional imaging, injection of viral vectors in the pitui-tary gland, optogenetics in awake animals and immunohistofluorescence in the whole organ.I then showed structural plasticity of gonadotroph cells and pericytes (perivascular "mural" cells) during proestrus in cleared hypophyses. This suggested the existence of a Gonadotroph-Vascular Unit (GVU) composed of gonadotroph cells, fenestrated capil-laries and pericytes, in which the latter would modulate the pre-ovulatory LH surge.Pericytes contraction via Channelrhodopsine-2 activation permitted to demonstrate their role in the sensitization of LH secretion in freely moving animals implanted with an optical fiber.Finally, blood flow and calcium activity in GVU cells expressing GCaMP6 were performed in awake « head-fixed » animals in which visualization of the pituitary gland was achievable through an implanted GRIN lens. These experiments were also conduct-ed at the ventral side of the pituitary gland in anesthetized mice. Analysis showed that in vivo calcium activity increases in endocrine cells and decreases in pericytes during GnRH-induced LH secretion.

Chez les rongeurs, les facteurs sociaux sont connus pour pouvoir moduler la transition pubertaire. Ainsi une jeune souris femelle mise en contact avec un mâle adulte présentera une ouverture vaginale plus précoce qu’une femelle isolée du mâle. L’objectif de ma thèse a été de caractériser les conséquences d’une exposition précoce au mâle dans l’espèce caprine. Les caprins sont une espèce dont la reproduction est saisonnée et permettant, de par sa taille, une étude plus fine des sécrétions endocrines que les rongeurs. Nos résultats ont permis de mettre en évidence que la présence de boucs sexuellement actifs induit une puberté précoce chez les chevrettes, l’ovulation pouvant être induite dès l’âge de 3 mois et demi. Les femelles présentent suite à cette première ovulation une cyclicité régulière ainsi qu’une maturation précoce du tractus génital. Le niveau d’activité sexuelle du bouc est un facteur crucial à l’induction d’une puberté précoce chez la chèvre puisque la présence de mâles castrés n’a aucun effet et que les femelles sont toutes pubères dans le mois suivant l’entrée en saison sexuelle des mâles. Ce travail démontre, dans l’espèce caprine, un rôle crucial de l’environnement social dans la régulation de la maturation sexuelle. Plus particulièrement, cela met en évidence que la présence de boucs peut réactiver efficacement et de manière très précoce l’axe gonadotrope de jeunes chèvres immatures. / In rodents, social factors are known to modulate the pubertal transition. Hence, young female mice exposed to adult male exhibit an earlier vaginal opening than young females isolated from male. The aim of my thesis was to characterize the consequences of a precocious exposure to male in another specie, goats. Goats are seasonal breeders and due to their size the fine study of endocrine secretions is easier than in rodents. Our results highlighted that an early exposure to sexually active bucks induces a precocious puberty in young female goats. The first ovulation can be induced as early as 3.5 months old, following this induced first ovulation, goats remain cycling regularly. Females precociously exposed to bucks also exhibit an acceleration of the genital tract maturation. The level of sexual activity of the male is a crucial criteria to induce a precocious puberty in goats as exposure to castrated bucks had no effect on the age at puberty. Moreover, all females exposed to intact bucks ovulated for the first time within a month after buck started to exhibit sexual behaviors. This work revealed, in goats, a crucial role of the social environment on the regulation of sexual maturation. More precisely, it highlights that exposure to bucks is highly efficient to reactivate precociously the gonadotrope axis of youg immature goats.