Search

Hypothalamic control of reproduction relies on GnRH and kisspeptin (KP) secretions. KP neurons are sensitive to sex steroids and metabolic status and their distribution overlaps with neurons producing apelin, a metabolic hormone known to decrease LH secretion in rats. Here, we observed neuroanatomical contacts between apelin fibers and both KP and GnRH neurons in the hypothalamus of male rodents. Intracerebroventricular apelin infusion for 2 weeks in male mice did not decrease LH levels nor did it affect gene expression for KP, neurokinin B and dynorphin. Finally, increasing apelin concentrations did not modulate Ca

International audience; Kisspeptin (Kp), a family of peptides comprising products of the Kiss1 gene, was discovered 20 years ago; it is recognised as the major factor controlling the activity of the gonadotrophin-releasing hormone (GnRH) neurones and thus the activation of the reproductive axis in mammals. It has been widely documented that the effects of Kp on reproduction through its action on GnRH neurones are mediated by the GPR54 receptor. Kp controls the activation of the reproductive axis at puberty, maintains reproductive axis activity in adults and is involved in triggering ovulation in some species. Although there is ample evidence coming from both conditional knockout models and conditional-induced Kp neurone death implicating the Kp/GPR54 pathway in the control of reproduction, the mechanism(s) underlying this process may be more complex than a sole direct control of GnRH neuronal activity by Kp. In this review, we provide an overview of the recent advances made in elucidating the interplay between Kp- and GnRH- neuronal networks with respect to regulating the reproductive axis. We highlight the existence of a possible mutual regulation between GnRH and Kp neurones, as well as the implication of Kp-dependent volume transmission in this process. We also discuss the capacity of heterodimerisation between GPR54 and GnRH receptor (GnRH-R) and its consequences on signalling. Finally, we illustrate the role of mathematical modelling that accounts for the synergy between GnRH-R and GPR54 in explaining the role of these two receptors when defining GnRH neuronal activity and GnRH pulsatile release.

The control of ovulation helps guarantee the success of reproduction and as such, contributes to the fitness of a species. In mammals, two types of ovulation are observed: induced and spontaneous ovulation. Recent work on camelids, that are induced ovulators, highlighted the role of a factor present in seminal plasma, beta Nerve Growth Factor (β-NGF), as the factor that triggers ovulation in a GnRH dependent manner. In the present work, we characterized alpaca β-NGF (aβ-NGF) and its 3D structure and compared it with human recombinant β-NGF (hβ-NGF). We showed that the β-NGF enriched fraction of alpaca semen and the human recombinant protein, both stimulated spontaneous electrical activity of primary GnRH neurons derived from mouse embryonic olfactory placodes. This effect was dose-dependent and mediated by p75 receptor signaling. P75 receptors were found expressed in vitro by olfactory ensheathing cells (OEC) in close association with GnRH neurons and in vivo by tanycytes in close vicinity to GnRH fibers in adult mouse. Altogether, these results suggested that β-NGF induced ovulation through an increase in GnRH secretion provoked by a glial dependent P75 mediated mechanism.

We present in this paper a global methodology for the spike detection in a biological context of fluorescence recording of GnRH-neurons calcium activity. For this purpose we first propose a simple stochastic model that could mimic experimental time series by considering an autoregressive AR(1) process with a linear trend and specific innovations involving spiking times. Estimators of parameters with asymptotic normality are established and used to set up a statistical test on estimated innovations in order to detect spikes. We compare several procedures and illustrate on biological data the performance of our procedure.

La maîtrise du moment de l’ovulation chez les mammifères d’élevage permet d’améliorer la fertilité, la gestion des mises bas, mais les méthodes actuelles sont basées sur l’utilisation de fortes doses d’hormones. Cette revue présente de nouveaux paradigmes pour maitriser le moment de l’ovulation, respectueux de la physiologie de l’animal, sans supplémentation hormonale basés sur la connaissance des mécanismes physiologiques.[br/] [br/] Le déclenchement de l’ovulation peut se faire selon deux modes chez les mammifères : spontané et provoqué. L’ovulation spontanée intervient au cours du cycle œstral sous l’effet de facteurs internes hormonaux. L’ovulation provoquée est déclenchée par l’accouplement avec un mâle. Dans les deux cas, c’est une augmentation de la sécrétion de GnRH qui conduit à l’augmentation de LH qui provoque l’ovulation. Les facteurs qui conduisent à l’augmentation de sécrétion de GnRH sont différents selon les deux modalités : principalement la kisspeptine (ovulation spontanée) et le b-NGF (ovulation provoquée). Les protocoles d’induction de l’ovulation actuels reposent sur une action directe sur l’ovaire, grâce à l’utilisation de gonadotropines hétérologues, ou bien par une action sur l’hypophyse grâce aux agonistes du GnRH. Ces protocoles présentent différents inconvénients : perte d’efficacité dans le temps, stimulation supra-physiologique qui peut être délétère, problème éthique posé par l’obtention de certaines molécules. De nouveaux paradigmes d’induction de l’ovulation ciblant l’hypothalamus, et favorisant un déclenchement physiologique de l’ovulation sans supplémentation en hormones sont en cours de développement. Cette revue a pour objectif de présenter au lecteur les mécanismes intimes impliqués dans la régulation et le déclenchement de l’ovulation ainsi que deux approches nouvelles respectueuses de la physiologie de l’animal et de l’environnement., The triggering of ovulation in mammals can be done according to two modes: spontaneous and provoked. Spontaneous ovulation occurs during the estrous cycle as a result of internal endocrine factors. Induced ovulation is triggered by mating. In both cases, it is the increase in GnRH secretion that leads to an increase in LH that causes ovulation. The factors involved in the stimulation of GnRH secretion are different according to the two modalities, mainly kisspeptin for spontaneous ovulators and beta-NGF for provoked ovulators. Current protocols used to induce ovulation rely on a direct action on the ovary, through the use of heterologous gonadotropins, or by an action on the pituitary gland with GnRH agonists. These protocols present different disadvantages: loss of activity with time, supra-physiological stimulation, ethic questioning about the production of some of these products. New paradigms for triggering ovulation by targeting the hypothalamus, respectful of animal welfare and the environment are presented in this review. This review aims to introduce the reader to the cellular and molecular mechanisms involved in the regulation and triggering of ovulation as well as two new approaches that are being developed and that are respectful of the animal and of its environment.

Intracellular cytoplasmic calcium ([Ca(2+) ]i ) has an important regulatory role in gamete functions. However, the biochemical components involved in Ca(2+) transport are still unknown in birds, an animal class that has lost functional sperm-specific CatSper channels. Here, we provide evidence for the presence and expression of various Ca(2+) channels in chicken sperm, including high voltage-activated channels (L and R types), the store-operated Ca(2+) channel (SOC) component Orai1, the transient receptor potential channel (TRPC1) and inositol-1,4,5-trisphosphate receptors (IP3 R1). L- and R-type channels were mainly localized in the acrosome and the midpiece, and T-type channels were not detected in chicken sperm. Orai1 was found in all compartments, but with a weak, diffuse signal in the flagellum. TRCP1 was mainly localized in the acrosome and the midpiece, but a weak diffuse signal was also observed in the nucleus and the flagellum. IP3 R1 was mainly detected in the nucleus. The L-type channel inhibitor nifedipine, the R-type channel inhibitor SNX-482 and the SOC inhibitors MRS-1845, 2-APB and YM-58483 decreased [Ca(2+) ]i sperm motility and acrosome reaction capability, with the SOC inhibitors inhibiting these functions most efficiently. Furthermore, we showed that Ca(2+) -mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOC inhibition. Our identification of important regulators of Ca(2+) signaling in avian sperm suggests that SOCs play a predominant role in gamete function, whereas T-type channels may not be involved. In addition, Ca(2+) entry via SOCs appears to be the most likely pathway for AMPK activation and energy-requiring sperm functions such as motility and the acrosome reaction.

Neuroendocrine research on the horse has mostly focused on the neuroendocrine control of reproduction and more specifically on the control of ovulation, which represents an economically important matter in horse breeding. Knowing the timing of ovulation is economically important in the equine industry, as it can be used to improve reproductive success during management of brood mares (i.e. insemination close to the time of ovulation) particularly when semen is valuable. Mares display a seasonal (long days) mono‐ovulatory polyestrus cycle. During the reproductive season in spring and summer, the estrus cycle length is about 22 days, with 5‐7 days of estrus. There are significant differences in estrus length between individuals and also within a given animal, according to the timing within the reproductive season. Ovarian cyclicity is controlled by the pituitary gonadotrophins: Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH). LH and FSH are themselves under the control of the hypothalamic neurohormone: Gonadotropin Releasing Hormone (GnRH). One of the most successful research approaches in this field relies on a non‐invasive methodology allowing the collection of blood from the pituitary efferent vein and the measurement the pulsatile release GnRH, LH and FSH. During the luteal phase, when the progesterone concentration is high, GnRH pulse frequency is low (mean inter‐pulse interval (IPI) 120min). Whereas during the pre‐ovulatory period, when estradiol concentration is high, GnRH pulses are high (IPI 30 min). In contrast to other mammals studied, there is no obvious GnRH surge preceding ovulation but an increase in pulse frequency, and the LH peak lasts for approximately 6 days, with the concentration peak occurring approximately 24h post‐ovulation. In sheep, primates and rodents, GnRH secretion is stimulated by a neuropeptide, Kisspeptin, which is also responsible for triggering the GnRH and LH preovulatory peaks. In the mare, despite stimulating LH and FSH secretions, Kisspeptin does not induce ovulation.

International audience; During mammalian embryonic development, GnRH neurones differentiate from the nasal placode and migrate through the nasal septum towards the forebrain. We previously showed that a category of glial cells, the olfactory ensheathing cells (OEC), forms the microenvironment of migrating GnRH neurones. Here, to characterize the quantitative and qualitative importance of this glial, we investigated the spatiotemporal maturation of glial cells in situ and the role of maturing glia in GnRH neurones development ex vivo. More than 90% of migrating GnRH neurones were found to be associated with glial cells. There was no change in the cellular microenvironment of GnRH neurones in the regions crossed during embryonic development as glial cells formed the main microenvironment of these neurones (53.4%). However, the phenotype of OEC associated with GnRH neurones changed across regions. The OEC progenitors immunoreactive to brain lipid binding protein formed the microenvironment of migrating GnRH neurones from the vomeronasal organ to the telencephalon and were also present in the diencephalon. However, during GnRH neurone migration, maturation of OEC to [GFAP+] state (glial fibrillary acid protein) was only observed in the nasal septum. Inducing depletion of OEC in maturation, using transgenic mice expressing herpes simplex virus thymidine kinase driven by the GFAP promoter, had no impact on neurogenesis or on triggering GnRH neurones migration in nasal explant culture. Nevertheless, depletion of [GFAP+] cells decreased GnRH neurites outgrowth by 57.4%. This study suggests that specific maturation of OEC in the nasal septum plays a role in morphological differentiation of GnRH neurones.

International audience; Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 mu M 18-alpha-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-alpha-glycyrrhetinic acid washout.

Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+), or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+) but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+) than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+). Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+) entry in sperm through the Ca(2+)/CaM/CaMKKs/CaMKI pathway. The Ca(2+)/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2+) entry in the cells.

Proceedings of the 18th ICAR, 26-30 June 2016, Tours, France; During adulthood, the mammalian brain retains the capacity to generate new cells and new neurons in particular. It is now well established that the birth of these new neurons occurs in well described sites: the hippocampus and the subventricular zone of the lateral ventricle, as well as in other brain regions including the hypothalamus. In this review, we describe the canonical neurogenic niches and we illustrate the functional relevance of adult-born neurons of each neurogenic niche in the reproductive physiology. More specifically we highlight the effect of reproductive social stimuli on the neurogenic processes and conversely, the contributions of adult-born neurons to the reproductive physiology and behavior. We next review the recent discovery of a novel neurogenic niche located in the hypothalamus and the median eminence (ME) and the compelling evidence of the link existing between the new-born hypothalamic neurons and the regulation of metabolism. Additionally, new perspectives on the possible involvement of hypothalamic neurogenesis in the control of photoperiodic reproductive physiology in seasonal mammals are discussed. Altogether the studies highlighted in this review demonstrate the potential role of neurogenesis in reproductive function and emphasize the importance of increasing our knowledge on the regulation processes and the physiological relevance of these adult-born neurons. This constitutes a necessary step towards a potential manipulation of these plasticity mechanisms.

International audience