Background: Kisspeptins, recently discovered hypothalamic neuropeptides encoded by the KISS1 gene, are essential for normal pubertal development and are modulated by diverse endocrine, metabolic and environmental signals. Exogenous kisspeptin administration potently stimulates LH secretion - by direct action on GnRH neurons while kisspeptin antagonists inhibit pulsatile LH secretion. Human studies of kisspeptin had hitherto used kisspeptin-54 that is cleaved further and the smallest bioactive form is a decapeptide (kisspeptin-10) with a shorter half-life. Kisspeptin-10 is thus putatively more attractive in studies assessing LH pulsatility and is also the basis for the development of antagonists. Unmet clinical needs: Decreased LH pulse frequency is the central pathology in pubertal delay, late-onset male hypogonadism and hypothalamic amenorrhoea. Manipulation of LH pulse frequency also has therapeutic potential in contraception, PCOS and sex-steroid dependant diseases such as endometriosis and prostatic hyperplasia. Hypothesis: That exogenous kisspeptin-10 enhances pulsatile LH secretion in healthy men and in patients with reproductive disorders associated with decreased pulse frequency. Research strategy: A first-in-human dose escalation study of kisspeptin-10 was performed in men and subsequently replicated in women. An intravenous infusion regime was optimised in healthy men and subsequently applied to hypogonadal patients. Specific questions were addressed sequentially as summarised below with key results. Dose escalation study: Question: Does kisspeptin-10 stimulate LH secretion in men? Findings: Six iv bolus doses (0.01 to 3 μg/kg) of GMP kisspeptin-10 and vehicle were administered at least a week apart to six healthy men. Rapid increase in LH, with peak concentrations was seen by 45 min post injection in all volunteers. There was a clear dose-dependent increase in LH concentrations in response to kisspeptin- 10 (P <0.0001). Area-Under-Curve analysis over 60 min following kisspeptin-10 administration showed 0.3 and 1μg/kg doses to be maximally stimulatory (P <0.01) with a reduced response at 3 μg/kg. Assessing the effect of steroid milieu: Question: Steroid feedback is central to the regulation of LH secretion: what effect does the steroid milieu have on LH responses to kisspeptin-10? Findings: The response to iv kisspeptin-10 (0.3μg/kg,) in the normal follicular phase (n=10) was compared with that in the presence of low endogenous sex steroids/high LH secretion (6 postmenopausal women) and in women taking combined contraceptive therapy (n=8) with suppressed LH secretion. Despite widely varying baseline secretion, LH increased significantly following kisspeptin-10 administration in the follicular phase (6.3±1.2 to 9.4±1.3 IU/L P=0.006), postmenopausal (35.3±2.8 to 44.7±3.4 IU/L P=0.005), etonogestrel (4.6±0.2 to 7.5±0.9 IU/L, P=0.02), and COCP groups (2.2±0.9 to 3.7±1.4 IU/L P<0.001). Pulse frequency study: Question: GnRH and LH secretion are pulsatile: can kisspeptin-10 enhance LH pulsatility? Findings: Four healthy men attended our clinical research facility for two visits five days apart for 10-min blood sampling. At the first visit, baseline LH pulsatility was assessed over a 9-hour period. During the second visit, an infusion of kisspeptin-10 was administered for 9 hours at 1.5μg/kg/hr after an hour of baseline sampling. LH pulse frequency increased in all subjects, with a mean increase from 0.7±0.1 to 1.0±0.2 pulses/hr (P = 0.01), with resultant increase in mean LH from 5.2±0.8 IU/L at baseline to 14.1±1.7 IU/L (P <0.01). High dose, longer duration infusion study: Question: Can kisspeptin-10 enhance testosterone secretion? Findings: Four healthy men attended our clinical research facility for a 34-hour supervised stay. Blood samples were collected at 10 min intervals for two 12 hour periods on consecutive days and hourly overnight. After 10.5 hours of baseline sampling a continuous intravenous infusion of kisspeptin-10 (4μg/kg/hr) was maintained for 22.5 hrs. Mean LH increased from 5.5±0.8 at baseline to 20.9±4.9 IU/L (P <0.05) and serum testosterone increased from 16.6±2.4 to 24.0±2.5 nmol/L (P <0.001). Translational studies in hypogonadal men with type 2 diabetes Question: Can kisspeptin-10 normalise testosterone secretion in hypogonadal men? Findings: Five hypogonadal men with T2DM (age 33.6±3 yrs, BMI 40.6±6.3, testosterone 8.5±1.0 nmol/L, LH 4.7±0.7 IU/L, HbA1c <8 %, duration of diabetes <5 yrs) and seven age matched healthy men were studied. Kisspeptin-10 was administered intravenous (0.3 μg/kg) with frequent (10-min) blood sampling. Mean LH increased in controls (5.5±0.8 to 13.9±1.7 IU/L P <0.001) and in T2DM (4.7±0.7 to 10.7±1.2 IU/L P=0.02) with comparable ΔLH (P=0.18). Baseline serum sampling for LH at 10-min intervals and hourly testosterone measurements were performed subsequently in four T2DM men for 12 hours. An intravenous infusion of kisspeptin-10 (4 μg/kg/hr) was administered 5 days later for 11 hours, with increases in serum LH (3.9±0.1 IU/L to 20.7±1.1 IU/L (P=0.03,) and testosterone (8.5±1.0 to 11.4±0.9 nmol/L, P=0.002). LH pulse frequency at baseline was lower in hypogonadal men with diabetes (0.6±0.1 vs. 0.8±0.1 pulses/hr, P=0.03) and increased to 0.9±0 pulses/hr (P=0.05). Translational studies in pubertal delay: Question: Defective Neurokinin B activity is associated with pubertal delay and the hierarchical interactions between kisspeptins and Neurokinin B remain to be elucidated: can kisspeptin-10 stimulate LH secretion with impaired Neurokinin B signalling? Findings: Four patients with TAC3 or TACR3 inactivating mutations presenting with delayed puberty were admitted for two 12 hr blocks of blood sampling every 10 min with vehicle (saline) or kisspeptin-10 (1.5 μg/kg/hour) infused intravenously. Mean LH and LH pulses frequency increased with kisspeptin-10 (P<0.05). However, four patients with Kallmann syndrome (with defective GnRH neuron migration), studied in parallel, did not respond, suggesting a potential diagnostic application for kisspeptin-10 in pubertal dysfunction. Conclusions In first-in-man studies of kisspeptin-10, it was demonstrated that endogenous LH pulse frequency can be enhanced in healthy men. The therapeutic potential of this finding in common reproductive endocrine disorders associated with decreased LH pulse frequency, i.e., late-onset male hypogonadism and pubertal dysfunction, was suggested in subsequent studies. Furthermore, kisspeptin signalling occurs upstream of GnRH neurons and is independent of Neurokinin B signalling in the central regulation of the hypothalamic-pituitary-gonadal axis.

Menopause is a natural transition heralded by the cessation of menstrual cycles and ovulation, and it occurs in all women at an average of about 50 years of age. While not a disease, menopause is often accompanied by symptoms that interfere with the quality of life and these symptoms are due to the relatively abrupt deprivation of E2 and P4 experienced during reproductive aging. Reproductive aging consists of changes in the synthesis and release of hormones from the hypothalamus, pituitary and gonad, which make up the HPG axis. Because gonadal hormones play critical roles in many systems throughout the body and brain, not just reproduction, treatment of menopausal symptoms to date largely involves hormone replacement therapy (HRT) with E2, P4 or their combination. While not intended to treat other neurobiological symptoms beyond hot flushes, HRT has the potential to exert widespread actions due to the abundance of hormone receptors throughout the nervous system. Thus, a fuller understanding of the neurobiology of menopause is badly needed. Although much of the research into the mechanisms that underlie reproductive aging focuses on ovarian failure and follicular atresia (cell death), there is evidence that there are significant alterations in the function of the neuroendocrine levels - the hypothalamus and pituitary - that also contribute to this process. As the mean age of the population increases, the number of post-menopausal women continues to grow with broad economic, healthcare and social costs. It is increasingly important to understand the complex mechanisms underlying reproductive aging and the effects of HRT. In this dissertation, I focus on the question of how the female non-human primate hypothalamus changes both with aging and in response to steroid hormone treatments. / text

L’alpha-foetoprotéine (AFP) est la protéine majoritaire du sérum fœtal de mammifère. C’est une glycoprotéine produite et sécrétée par l’endoderme viscéral du sac vitellin, les hépatocytes fœtaux et dans une moindre mesure, par l’intestin fœtal. Son profil d’expression est onco-fœtal : la synthèse de cette protéine chute fortement après la naissance mais peut reprendre en cas de régénération hépatique ou en cas de tumeurs diverses. Cette protéine est capable de fixer les oestrogènes et jouerait un rôle dans la différenciation sexuelle du cerveau femelle. Deux invalidations du gène de l’AFP ont été réalisées au Laboratoire de Biologie du Développement afin de comprendre la fonction de cette protéine. Dans les deux cas, des souris homozygotes pour l’allèle invalidé (souris AFP KO) ont été obtenues. Elles sont viables et apparemment phénotypiquement normales, mais les femelles sont stériles, suite à une anovulation. Il a été démontré que l’absence d’ovulation provient d’un mauvais fonctionnement de l’axe hypothalamo-hypopysaire. Nous avons démontré au cours de cette thèse que l’ARN messager d’une série de gènes était sous-représenté au sein de l’hypophyse des souris femelles invalidées pour le gène Afp. Nous trouvons parmi ces gènes, ceux d’une cascade particulièrement importante pour l’ovulation, la cascade du récepteur de la GnRH. La fertilité des souris femelles AFP KO, ainsi que le taux d’expression des gènes testés dans l’hypophyse, sont restaurés si ces souris se développent dans un environnement appauvri en oestrogènes. Nous avons donc pu corriger le phénotype des souris femelles invalidées pour le gène de l’AFP. Nous avons en ce faisant démontré que l’AFP, par sa capacité de fixer les oestrogènes, protégeait le cerveau femelle en développement des effets masculinisants de ces hormones

Castration of male lambs is performed in all major sheep producing counties as a standard management practice. The reasons to castrate may be different and will depend on the size and type of the farm. Castration gives more control over genetics of the flock, stops inbreeding, unwanted pregnancies and behaviours. It also gives improved carcass characteristics. However, it has been shown that castration is painful and distressing to the animals. Different techniques are used to castrate sheep at the present time such as rubber ring, Burdizzo, combined, short scrotum, and surgical castration. Due to changing attitudes towards animal pain and unnecessary suffering there is a need for further development and implementation of new castration methods, efficient pain assessment techniques, animal welfare codes of practice and legislative requirements to improve lamb well-being. Recent increase of public concern regarding animal welfare is putting pressure on many government bodies to strengthen research in this area and increase attempts to regulate by law unnecessary suffering during standard livestock management practices. Immunocastration with an anti-GnRF vaccine has the potential to be an alternative to common physical castration methods. Nonetheless there is little or no information about the impact of vaccination against GnRF on the physiology of lambs (rams’ reproductive tract, endocrine regulation), emotionality (possible changes to normal behavioural patterns like increased aggression, anxiety) and health (is the vaccine safe to be used and if there are any adverse effects of vaccination like tissue damage, swelling, lesions etc.). There is also little or no information on how the vaccine affects sheep at the time of injection. This study investigates three main questions: Is Immunocastration a pain free alternative to traditional physical methods of castration? Is Immunocastration safe and practical to use? Does Immunocastration influence the male reproductive system in a way to achieve sterility without any negative impact on the ram natural behaviours, wellbeing and health?

A major area of interest is the identification of proteins that play a role in hormone dependent cancers and in collaboration with the MRC Centre for Reproductive Health we studied the gonadotropin releasing hormone receptor (GnRH-R). Other targets described in the thesis are the SH3 domain of PSD-95 and the protein BLyS. In order to identify potential inhibitory small molecules we have used a variety of computational data base mining approaches as well as using and developing experimental binding assays. It has become increasingly challenging to evaluate the most representative drug like small molecule compounds when using traditional high throughput screening methods. This thesis assesses the use of in silico tools to probe key protein-protein and protein-peptide interactions. These tools provide a means to identify enriched compound datasets which can be purchased and tested in vitro in a time and cost efficient way. The transmembrane protein GnRH-R provides an interesting opportunity to identify small molecules that could inhibit the binding of its peptide ligand GnRH. This is a challenging project as there are few examples in the literature of drug-like molecules that bind to such protein-peptide interfaces. The first step involved receptor modelling using solved crystal structures of homologous proteins. The model was then validated by developing structure activity relationships for established high affinity ligands. We also performed crystallographic and biophysical studies on the native GnRH decapeptide. Two other protein-protein systems were also examined using the same virtual screening and experimental ligand binding methodology. SH3 domains play an important role in cell signalling and we used the PSD-95 protein as our target for study as a crystal structure has been published. As well as identifying potential ligands we characterised structural properties of PSD-95 fusion proteins and also developed the basis for compound assay. The third system studied was B Lymphocyte Stimulator (BLyS) which is a target for treatment of a number of autoimmune diseases. This presented an interesting target for study as the protein binds to multiple receptors depending on its multimeric state. BLyS protein was characterised using electron microscopy and other biophysical techniques.

The objective was to investigate the effects of progesterone (P4) on luteinizing hormone (LH) release, follicle development, and oocyte competence in cattle. We tested the general hypotheses that: 1) The suppressive effect of P4 on gonadotrophin releasing hormone (GnRH)-mediated LH release can be overcome by increasing GnRH dose or pre-treatment with estradiol (E2); and 2) a shorter period of P4 exposure during the growing phase of the ovulatory follicle improves oocyte competence and fertility after fixed-time artificial insemination or superstimulation in cattle. <p>In the first experiment, heifers (n=22) were treated with 100 or 200 µg of GnRH or pretreated with E2 prior to administration of GnRH during high or low circulating P4 concentrations to characterize LH release (Chapter 2). Increasing the dose of GnRH did not alter LH secretion; however, E2 pretreatment overcame the suppressive effect of high P4 on LH secretion. Cattle with lower (n=11) P4 concentrations had higher circulating LH concentrations than those with higher P4 concentrations (n=11), and tended to have higher ovulation rates. <p>Two experiments were conducted to determine the effect of the duration of P4 exposure during the ovulatory wave on fertility followed fixed-time artificial insemination or superstimulation. In the first experiment (Chapter 3), the dominant follicle was allowed to grow for 3 days (n=181) or 6 days (n=184). Six days of growth resulted in a larger dominant follicle, but in both groups, ovulatory follicles had similar capacities to ovulate and establish pregnancy. In the second experiment (Chapter 4), multiple follicles were allowed to grow for 3 or 6 days by 8 or 14 injections of FSH (at 12-hour intervals). There was no difference between groups for ovulation rate or total ova/embryo recovery rate. Although the 3-day group had higher embryo quality at slaughter (4 days after insemination), further development (7, 9, and 10 days after insemination) did not differ among groups. The effect of FSH starvation following 4 days of FSH treatment (Chapter 4) resulted in loss of ovulatory capability. Overall, a shorter duration of P4 exposure during ovulatory follicle growth did not improve fertility after fixed-time AI or oocyte competence after superstimulation.

Introducción: Desde su primera descripción en 1984 las indicaciones de donación de óvulos han ido aumentando, lo que ha provocado un incremento progresivo en el número de ciclos realizados a nivel mundial. Aunque esta técnica de reproducción garantiza una elevada tasa de embarazo en la receptora, los profesionales también se esfuerzan en convertir el tratamiento para la donante en un proceso sencillo y seguro. Durante los últimos diez años los antagonistas de la GnRH, por sus características farmacodinámicas, ha sido el fármaco utilizado para desarrollar protocolos de estimulaciones ováricas sencillos y seguros para las donantes de óvulos. Materiales: La presente tesis doctoral resume las conclusiones de dos artículos publicados recientemente (2010) sobre la utilización de antagonistas de GnRH en la estimulación ovárica de donantes de óvulos. Además se discuten las conclusiones de otros cuatro artículos (publicados en revistas científicas de impacto durante los años 2006 y 2009) estrechamente relacionados a los aquí publicados. Los objetivos de esta tesis son: 1. comparar la eficacia de protocolos de estimulación basados en antagonistas de GnRH en comparación con protocolos basados en agonistas de GnRH a través de una revisión sistemática de la literatura y meta-análisis. 2. ilustrar que los protocolos de estimulación basados en antagonistas de GnRH aumentan la seguridad de la estimulación ovárica para la donante a través de una eliminación completa del riesgo del síndrome de hiperestimulación ovárica (SHO). Resultados: 1. El meta-análisis de de ocho ensayos clínicos llevados al cabo en donantes de óvulos estimuladas con antagonistas de GnRH no han demostrado diferencia significativa en el número de ovocitos obtenidos y las tasas de embarazo evolutivos en las receptoras correspondientes en comparación con agonistas de GnRH. 2. El estudio observacional, prospectivo realizado en donantes de óvulos de alto riesgo ha demostrado la eliminación completa de SHO moderado/severo tras la descarga con bolo de agonista de GnRH. Además se discuten las conclusiones de cuatro otros estudios apoyando las conclusiones mencionados arriba. Conclusiones: En el contexto de la donación de óvulos los protocolos de estimulación ovárica basados en antagonistas de la GnRH son igual de eficaces que los protocolos con agonistas de la GnRH. La inducción final de la maduración ovocitaria se puede llevar a cabo satisfactoriamente con un bolo de agonista de GnRH en vez de hCG, lo que prácticamente elimina el riesgo de SHO moderado/severo. La utilización preferencial de este protocolo de estimulación ovárica es muy aconsejable porque permite un tratamiento más sencillo y aumenta considerablemente la seguridad de la estimulación ovárica en donantes de óvulos. / Background: Since its first description in 1984 the indications of oocyte donation (OD) has widened considerably which has led to a continuous increase in the number of OD treatment cycles performed worldwide. Although this treatment option secured the highest pregnancy rates for the recipients of donor oocytes increased efforts were also made to achieve safer and simpler treament protocols for the oocyte donor. During the last decade with the advent and increased use of the GnRH antagonists this new pharmacological agent was also explored in ovarian stimulation protocols specifically tailored for oocyte donors. Materials: The present doctoral thesis summarizes the findings of two recently published articles (2010) on the application of GnRH antagonists in the ovarian stimulation of oocyte donors. Furthermore the findings of another four strictly related articles (published in high-impact international journals between 2006 and 2009) are also discussed. The primary objectives were: 1. to compare efficiency of GnRH antagonist protocols in comparison with GnRH agonist-based protocols in the context of oocyte donation by means of a systematic review and meta-analysis and 2. to illustrate that GnRH antagonist protocols substantially increase the safety of ovarian stimulation for oocyte donors by reducing or even eliminating the incidence of moderate/severe ovarian hyperstimulation syndrome (OHSS). Results: 1. A meta-analysis of eight randomized clinical trials (RCTs) performed in oocyte donors undergoing stimulation with GnRH antagonists showed no significant difference in the number of retrieved oocytes or recipient ongoing pregnancy rate when compared with GnRH agonists. 2. A prospective, follow-up study of a group of high risk oocyte donors showed that early onset moderate/severe OHSS was completely eliminated after triggering with a GnRH agonist. Furthermore the findings of four studies supporting the above conclusions are also presented. Conclusions: In the context of oocyte donation the GnRH antagonist based ovarian stimulation protocols are equally efficient compared to down regulation by GnRH agonists. The induction of final oocyte maturation can be successfully achieved by a GnRH agonist instead of hCG which practically eliminates early-onset moderate/severe OHSS. The proposed ovarian stimulation protocol should be preferentially used because it permits the simplification and considerably increases the safety of ovarian stimulation for oocyte donors.

The objective was to investigate the effects of progesterone (P4) on luteinizing hormone (LH) release, follicle development, and oocyte competence in cattle. We tested the general hypotheses that: 1) The suppressive effect of P4 on gonadotrophin releasing hormone (GnRH)-mediated LH release can be overcome by increasing GnRH dose or pre-treatment with estradiol (E2); and 2) a shorter period of P4 exposure during the growing phase of the ovulatory follicle improves oocyte competence and fertility after fixed-time artificial insemination or superstimulation in cattle. <p>In the first experiment, heifers (n=22) were treated with 100 or 200 µg of GnRH or pretreated with E2 prior to administration of GnRH during high or low circulating P4 concentrations to characterize LH release (Chapter 2). Increasing the dose of GnRH did not alter LH secretion; however, E2 pretreatment overcame the suppressive effect of high P4 on LH secretion. Cattle with lower (n=11) P4 concentrations had higher circulating LH concentrations than those with higher P4 concentrations (n=11), and tended to have higher ovulation rates. <p>Two experiments were conducted to determine the effect of the duration of P4 exposure during the ovulatory wave on fertility followed fixed-time artificial insemination or superstimulation. In the first experiment (Chapter 3), the dominant follicle was allowed to grow for 3 days (n=181) or 6 days (n=184). Six days of growth resulted in a larger dominant follicle, but in both groups, ovulatory follicles had similar capacities to ovulate and establish pregnancy. In the second experiment (Chapter 4), multiple follicles were allowed to grow for 3 or 6 days by 8 or 14 injections of FSH (at 12-hour intervals). There was no difference between groups for ovulation rate or total ova/embryo recovery rate. Although the 3-day group had higher embryo quality at slaughter (4 days after insemination), further development (7, 9, and 10 days after insemination) did not differ among groups. The effect of FSH starvation following 4 days of FSH treatment (Chapter 4) resulted in loss of ovulatory capability. Overall, a shorter duration of P4 exposure during ovulatory follicle growth did not improve fertility after fixed-time AI or oocyte competence after superstimulation.

No description available.

Peptide hormones are critical modulators of physiology and development in humans and have been well characterized for their effects on humans and other mammals. The question of the origin of the many families of peptide hormones in mammals is pressing, as it gives us a window into the evolution of important systems in all extant animals and their common ancestors. The focus of this thesis was to examine the origin of a select group of peptide hormone families including the secretin superfamily, reproductive neuropeptides, insulin and the insulin-like peptides, and stanniocalcin. The evolution of the secretin superfamily was found to have originated with the vertebrates, and new information from the genomes of basal vertebrates like the lamprey Petromyon marinus and elephant shark Callorhinchus milii allows us to better piece together the gene duplications that produced the current hormone family in humans and fish. The reproductive hormones, including gonadotropin-releasing hormone (GnRH), vasopressin/oxytocin, and kisspeptin were examined, with a focus on the evolution of their G protein-coupled receptors. GnRH was found to have originated in the early bilaterians, and its receptors clearly belong to a superfamily also containing receptors of the related neuropeptides adipokinetic hormone and corazonin, which have only been found in protostome invertebrates. Vasopressin/oxytocin receptors share a common ancestor with the GnRH receptors, although their peptides are not structurally related, and evolved at a similar time. Kisspeptin evolved later, within the vertebrates, however its receptors are closely related to an orphan receptor in protostome invertebrates, GPR54, with an unknown ligand. Insulin family members from the tunicate Ciona intestinalis and the amphioxus Branchiostoma floridae were identified, isolated and characterized to determine the nature of the insulin superfamily at the origin of the chordates, and it appears this family was well-developed already. Finally, the calcium-regulator stanniocalcin was identified, isolated and characterized in C. intestinalis and compared with the vertebrate and amphioxus stanniocalcins. A group of stanniocalcins were also discovered in a wide range of both protostomes and unicellular eukaryotes, indicating this ancient group of neurohormones appeared early in eukaryotic evolution. / Graduate