The role of the hypothalamic kisspeptin/GPR54 system in the control of GnRH neurons

Summary : The hypothalamus represents less than 1 % of the total volume of the brain tissue, yet it plays a crucial role in endocrine regulations. Puberty is defined as a process leading to physical, sexual and psychosocial maturation. The hypothalamus is central to this process, via the activation of GnRH neurons. Pulsatile GnRH secretion, minimal during childhood, increases with the onset of puberty. The primary function of GnRH is to regulate the growth, development and function of testes in boys and ovaries in girls, by stimulating the pituitary gland secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several factors contribute to the timing of puberty, including sex and ethnicity, genetics, dietary intake and energy expenditure. Kisspeptins constitute a family of small peptides arising from the proteolytic cleavage of metastin, a peptide with 54 amino acids initially purified from human placenta. These kisspeptins were the subject of much attention following their discovery because of their antimetastatic properties, but it was more recently that their determining role in the reproductive function was demonstrated. It was shown that kisspeptins are ligands of a receptor, GPR54, whose natural inactivating mutation in humans, or knockout in the mouse, lead to infertility. GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH release and GnRH neuron firing activity, but the neurobiological mechanisms for these actions are unknown. Gprotein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting that both direct and indirect effects are possible. In the first part of my thesis, we investigated a possible connection between the acceleration of sexual development induced by leptin and hypothalamic metastin neurons. However, the data generated by our preliminary experiments confirmed that the commercially available antibodies are non-specific. This finding constituted a major drawback for our studies, which relied heavily upon the neuroanatomical study of the hypothalamic metastinergic pathways to elucidate their sensitivity to exogenous leptin. Therefore, we decided to postpone any further in vivo experiment until a better antibody becomes available, and focused on in vitro studies to better understand the mechanisms of action of kisspeptins in the modulation of the activity of GnRH neurons. We used two GnRH-expressing neuronal cell lines to investigate the cellular and molecular mechanisms of action of metastin in GnRH neurons. We demonstrated that kisspeptin induces an early activation of the MAP kinase intracellular signaling pathway in both cell lines, whereas the SAP/JNK or the Akt pathways were unaffected. Moreover, we found an increase in GnRH mRNA levels after 6h of metastin stimulation. Thus, we can conclude that kisspeptin regulates GnRH neurons both at the secretion and the gene expression levels. The MAPK pathway is the major pathway activated by metastin in GnRH expressing neurons. Taken together, these data provide the first mechanism of action of kisspeptin on GnRH neurons. Résumé : L'hypothalamus est une zone située au centre du cerveau, dont il représente moins de 1 du volume total. La puberté est la période de transition entre l'enfance et l'age adulte, qui s'accompagne de transformations somatiques, psychologiques, métaboliques et hormonales conduisant à la possibilité de procréer. La fonction principale de la GnRH est la régulation de la croissance, du développement et de la fonction des testicules chez les hommes, et des ovaires chez les femmes en stimulant la sécrétion de l'hormone lutéinisante (LH) et de l'hormone folliculostimulante (FSH) par la glande hypophysaire. Plusieurs facteurs contribuent au déclanchement de la puberté, y compris le sexe et l'appartenance ethnique, la génétique, l'apport alimentaire et la dépense énergétique. Les Kisspeptines constituent une famille de peptides résultant de la dissociation proteolytique de la métastine, un peptide de 54 acides aminés initialement purifié à partir de placenta humain. Ces kisspeptines ont fait l'objet de beaucoup d'attention à la suite de leur découverte en raison de leurs propriétés anti-metastatiques, et c'est plus récemment que leur rôle déterminant dans la fonction reproductive a été démontré. Les kisspeptines sont des ligands du récepteur GPR54, dont la mutation inactivatrice chez l'homme, ou le knockout chez la souris, conduisent à l'infertilité par hypogonadisme hypogonadotrope. Les neurones à GnRH jouent un rôle central dans le règlement des fonctions reproductrices et la kisspeptine stimule l'activité des neurones à GnRH et la libération de GnRH par ces neurones. Toutefois, les mécanismes neurobiologiques de ces actions ne sont pas connus. Dans la première partie de ma thèse, nous avons étudié le lien potentiel entre l'accélération du développement sexuel induite par la leptine et les neurones hypothalamiques à metastine. Les données générées dans cette première série d'expériences ont malheureusement confirmé que les anticorps anti-metastine disponibles dans le commerce sont aspécifiques. Ceci a constitué un inconvénient majeur pour nos études, qui devaient fortement s'appuyer sur l' étude neuroanatomique des neurones hypothalamiques à metastine pour évaluer leur sensibilité à la leptine exogène. Nous avons donc décidé de focaliser nos travaux sur une étude in vitro des mécanismes d'action de la kisspeptine pour moduler l'activité des neurones à GnRH. Nous avons utilisé deux lignées de cellules neuronales exprimant la GnRH pour étudier les mécanismes d'action cellulaires et moléculaires de la metastine dans des neurones. Nous avons ainsi pu démontrer que la kisspeptine induit une activation précoce de la voie f de signalisation de la MAP kinase dans les deux lignées cellulaires, alors que nous n'avons observé aucune activation de la voie de signalisation de la P13 Kinase et de la SAP/JNK. Nous avons en outre démontré une augmentation de l'expression de la GnRH par la stimulation avec la Kisspeptine. L'ensemble de ces données contribue à élucider le mécanisme d'action avec lequel la kisspeptine agit dans les neurones à GnRH, en démontrant un effet sur l'expression génique de la GnRH. Nous pouvons également conclure que la voie de la MAPK est la voie principale activée par la metastine dans les neurones exprimant la GnRH.

Maintenance of ancestral sex chromosomes in Palearctic tree frogs: direct evidence from Hyla orientalis.

Contrasting with the situation found in birds and mammals, sex chromosomes are generally homomorphic in poikilothermic vertebrates. This homomorphy was recently shown to result from occasional X-Y recombinations (not from turnovers) in several European species of tree frogs (Hyla arborea, H. intermedia and H. molleri). Because of recombination, however, alleles at sex-linked loci were rarely diagnostic at the population level; support for sex linkage had to rely on multilocus associations, combined with occasional sex differences in allelic frequencies. Here, we use direct evidence, obtained from anatomical and histological analyses of offspring with known pedigrees, to show that the Eastern tree frog (H. orientalis) shares the same pair of sex chromosomes, with identical patterns of male heterogamety and complete absence of X-Y recombination in males. Conservation of an ancestral pair of sex chromosomes, regularly rejuvenated via occasional X-Y recombination, seems thus a widespread pattern among Hyla species. Sibship analyses also identified discrepancies between genotypic and phenotypic sex among offspring, associated with abnormal gonadal development, suggesting a role for sexually antagonistic genes on the sex chromosomes.

46, XY gonadal dysgenesis: new SRY point mutation in two siblings with paternal germ line mosaicism.

Stoppa-Vaucher S, Ayabe T, Paquette J, Patey N, Francoeur D, Vuissoz J-M, Deladoëy J, Samuels ME, Ogata T, Deal CL. 46, XY gonadal dysgenesis: new SRY point mutation in two siblings with paternal germ line mosaicism. Familial recurrence risks are poorly understood in cases of de novo mutations. In the event of parental germ line mosaicism, recurrence risks can be higher than generally appreciated, with implications for genetic counseling and clinical practice. In the course of treating a female with pubertal delay and hypergonadotropic hypogonadism, we identified a new missense mutation in the SRY gene, leading to somatic feminization of this karyotypically normal XY individual. We tested a younger sister despite a normal onset of puberty, who also possessed an XY karyotype and the same SRY mutation. Imaging studies in the sister revealed an ovarian tumor, which was removed. DNA from the father's blood possessed the wild type SRY sequence, and paternity testing was consistent with the given family structure. A brother was 46, XY with a wild type SRY sequence strongly suggesting paternal Y-chromosome germline mosaicism for the mutation. In disorders of sexual development (DSDs), early diagnosis is critical for optimal psychological development of the affected patients. In this case, preventive karyotypic screening allowed early diagnosis of a gonadal tumor in the sibling prior to the age of normal puberty. Our results suggest that cytological or molecular diagnosis should be applied for siblings of an affected DSD individual.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis.

Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.

Manipulation of arthropod sex determination by endosymbionts: diversity and molecular mechanisms.

Arthropods exhibit a large variety of sex determination systems both at the chromosomal and molecular level. Male heterogamety, female heterogamety, and haplodiploidy occur frequently, but partially different genes are involved. Endosymbionts, such as Wolbachia, Cardinium,Rickettsia, and Spiroplasma, can manipulate host reproduction and sex determination. Four major reproductive manipulation types are distinguished: cytoplasmic incompatibility, thelytokous parthenogenesis, male killing, and feminization. In this review, the effects of these manipulation types and how they interfere with arthropod sex determination in terms of host developmental timing, alteration of sex determination, and modification of sexual differentiation pathways are summarized. Transitions between different manipulation types occur frequently which suggests that they are based on similar molecular processes. It is also discussed how mechanisms of reproductive manipulation and host sex determination can be informative on each other, with a special focus on haplodiploidy. Future directions on how the study of endosymbiotic manipulation of host reproduction can be key to further studies of arthropod sex determination are shown.

Posttraumatic stress avoidance symptoms as mediators in the development of alcohol use disorders after exposure to childhood sexual abuse in a Swiss community sample.

This study examined the role of posttraumatic stress disorder (PTSD) symptoms of re-experience, avoidance, and hyperarousal in the relationship between different types of trauma and alcohol use disorders (AUD). We used data from 731 trauma-exposed individuals who participated in the first wave of the PsyCoLaus-study. Trauma characteristics were assessed relatively to the occurrence of lifetime PTSD symptoms and AUD. The results suggest that lifetime and childhood sexual abuse as well as overall childhood trauma were directly linked to AUD and PTSD symptoms, in particular to avoidance symptoms. From single symptom clusters PTSD avoidance was found to specifically mediate the trauma-AUD pathway. Both childhood and sexual trauma strongly contribute to the comorbidity of PTSD and AUD and avoidance-type symptoms appear to play a central role in maintaining this association. Hence, the alleviation of avoidance symptoms might be an important target for therapeutic intervention among victims of sexual abuse before specific addiction treatment is initiated.

Posttraumatic stress avoidance symptoms as mediators in the development of alcohol use disorders after exposure to childhood sexual abuse in a Swiss community sample.

This study examined the role of posttraumatic stress disorder (PTSD) symptoms of re-experience, avoidance, and hyperarousal in the relationship between different types of trauma and alcohol use disorders (AUD). We used data from 731 trauma-exposed individuals who participated in the first wave of the PsyCoLaus-study. Trauma characteristics were assessed relatively to the occurrence of lifetime PTSD symptoms and AUD. The results suggest that lifetime and childhood sexual abuse as well as overall childhood trauma were directly linked to AUD and PTSD symptoms, in particular to avoidance symptoms. From single symptom clusters PTSD avoidance was found to specifically mediate the trauma-AUD pathway. Both childhood and sexual trauma strongly contribute to the comorbidity of PTSD and AUD and avoidance-type symptoms appear to play a central role in maintaining this association. Hence, the alleviation of avoidance symptoms might be an important target for therapeutic intervention among victims of sexual abuse before specific addiction treatment is initiated.

Sexual dimorphism in resource acquisition and deployment: both size and timing matter.

Background and Aims The males and females of many dioecious plant species differ from one another in important life-history traits, such as their size. If male and female reproductive functions draw on different resources, for example, one should expect males and females to display different allocation strategies as they grow. Importantly, these strategies may differ not only between the two sexes, but also between plants of different age and therefore size. Results are presented from an experiment that asks whether males and females of Mercurialis annua, an annual plant with indeterminate growth, differ over time in their allocation of two potentially limiting resources (carbon and nitrogen) to vegetative (below-and above-ground) and reproductive tissues.Methods Comparisons were made of the temporal patterns of biomass allocation to shoots, roots and reproduction and the nitrogen content in the leaves between the sexes of M. annua by harvesting plants of each sex after growth over different periods of time.Key Results and Conclusions Males and females differed in their temporal patterns of allocation. Males allocated more to reproduction than females at early stages, but this trend was reversed at later stages. Importantly, males allocated proportionally more of their biomass towards roots at later stages, but the roots of females were larger in absolute terms. The study points to the important role played by both the timing of resource deployment and the relative versus absolute sizes of the sinks and sources in sexual dimorphism of an annual plant.

Increased condom use without other major changes in sexual behavior among the general population in Switzerland.

The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.

Genetic causes of transitions from sexual reproduction to asexuality in plants and animals.

The persistence of sexual reproduction in the face of competition from asexual invaders is more likely if asexual lineages are produced infrequently or have low fitness. The generation rate and success of new asexual lineages will be influenced by the proximate mechanisms underlying transitions to asexuality. As such, characterization of these mechanisms can help explain the distribution of reproductive modes among natural populations. Here, we synthesize the literature addressing proximate causes of transitions from sexual to asexual reproduction in plants and animals. In cyclical and facultatively asexual taxa, individual mutations can cause obligate asexuality. The evolution of asexuality in obligately sexual groups is more complex, requiring the simultaneous acquisition of two traits generally controlled by different genetic factors: unreduced gamete formation and spontaneous development of unfertilized gametes. At least three 'pre-adaptations' could favour transitions to obligate asexuality in obligate sexuals. First, linkage among loci affecting separate key components of asexuality facilitates its spread, with evidence for these linkage blocks in plants. Second, asexuality should evolve more readily in haplodiploids; support for this hypothesis comes from two examples where a single locus causes transitions to asexuality. Third, standing genetic variation for the production of unreduced gametes could facilitate transitions to asexuality, but whether the ability to produce unreduced gametes contributes to the evolution of obligate asexuality remains unclear. We close by reviewing the associations between asexuality, hybridization and polyploidy, and argue that current data suggest that hybridization is more likely to play a causal role in transitions to asexuality than polyploidy.

The ontogeny of sexual size dimorphism of a moth: when do males and females grow apart?

Sexual dimorphism in body size (sexual size dimorphism) is common in many species. The sources of selection that generate the independent evolution of adult male and female size have been investigated extensively by evolutionary biologists, but how and when females and males grow apart during ontogeny is poorly understood. Here we use the hawkmoth, Manduca sexta, to examine when sexual size dimorphism arises by measuring body mass every day during development. We further investigated whether environmental variables influence the ontogeny of sexual size dimorphism by raising moths on three different diet qualities (poor, medium and high). We found that size dimorphism arose during early larval development on the highest quality food treatment but it arose late in larval development when raised on the medium quality food. This female-biased dimorphism (females larger) increased substantially from the pupal-to-adult stage in both treatments, a pattern that appears to be common in Lepidopterans. Although dimorphism appeared in a few stages when individuals were raised on the poorest quality diet, it did not persist such that male and female adults were the same size. This demonstrates that the environmental conditions that insects are raised in can affect the growth trajectories of males and females differently and thus when dimorphism arises or disappears during development. We conclude that the development of sexual size dimorphism in M. sexta occurs during larval development and continues to accumulate during the pupal/adult stages, and that environmental variables such as diet quality can influence patterns of dimorphism in adults.

Melanin-based coloration is a nondirectionally selected sex-specific signal of offspring development in the Alpine swift

Two mutually exclusive hypotheses have been put forward to explain the evolution and adaptive function of melanin-based color traits. According to sexual selection theory melanism is a directionally selected signal of individual quality, whereas theory on the maintenance of genetic polymorphism proposes that alternative melanin-based variants achieve equal fitness. Alpine swift (Apus melba) males and females have a conspicuous patch of white feathers on the breast with their rachis varying continuously from white to black, and hence the breast varies from white to striated. If this trait is a sexually selected signal of quality, its expression should be condition dependent and the degree of melanism directionally selected. If variation in melanism is a polymorphism, its expression should be genetically determined and fitness of melanin-based variants equal. We experimentally tested these predictions by exchanging eggs or hatchlings between randomly chosen nests and by estimating survival and reproduction in relation to melanism. We found that breast melanism is heritable and that the environment and body condition do not significantly influence its expression. Between 5 and 50 days of age nestlings were heavier and their wings longer when breast feathers of their biological father were blacker, and they also fledged at a younger age. This shows that aspects of offspring quality covary positively with the degree of melanism. However, this did not result in directional selection because nestling survival and recruitment in the local breeding population were not associated with father breast melanism. Furthermore, adult survival, age at first reproduction and probability of skipping reproduction did not covary with the degree of melanism. Genetic variation in breast melanism is therefore maintained either because nonmelanic males achieve fitness similar to melanic males via a different route than producing fast-growing offspring, or because the advantage of producing fast-growing offspring is not sufficiently pronounced to result in directional selection.

Morphology, testes development and behaviour of unusual triploid males in microchromosome-carrying clones of Poecilia formosa.

In a microchromosome-carrying laboratory stock of the normally all-female Amazon molly Poecilia formosa triploid individuals were obtained, all of which spontaneously developed into males. A comparison of morphology of the external and internal insemination apparatus and the gonads, sperm ploidy and behaviour, to laboratory-bred F(1) hybrids revealed that the triploid P. formosa males, though producing mostly aneuploid sperm, are partly functional males that differ mainly in sperm maturation and sexual motivation from gonochoristic P. formosa males.

The development and pilot-testing of a training curriculum in adolescent medicine and health.

PURPOSE: To select and propose a set of knowledge, attitudes, and skills essential for the care of adolescents; to encourage the development of adolescent health multidisciplinary networks; and to set up training programs in as many European countries as possible. METHODS: The curriculum was developed by 16 physicians from 11 European countries with various professional specializations. In line with modern guidelines in medical education, it is a modular, flexible instrument which covers the main teaching areas in the field, such as basic skills (i.e. setting, rights and confidentiality, gender and cultural issues) as well as specific themes (i.e. sexual and reproductive health, eating disorders, chronic conditions). It consists of 17 thematic modules, each containing detailed objectives, learning approaches, examples, and evaluation methods. RESULT: Two international one-week summer schools were used to assess the feasibility and appropriateness of the curriculum. The overall evaluation was good, with most of the items surpassing three on a four-point Likert scale. However, it pointed to several aspects (process and content) which will need to be refined in the future, such as an increase in interactive sessions (role playing), and a better mix of clinical and public health issues.