Study of the hypothalamic GnIH pathways in association with neurobiological mechanisms regulating the initiation of puberty in rodents and primates

Abstract

Background A hike in pulsatile discharge of GnRH is mandatory for puberty onset. However, the mechanisms governing the pubertal rise in GnRH secretion are yet ambiguous. During neonatal life, GnRH neuroendocrine system is active in rodents and primates, but shows subsequent dormancy during the juvenile/prepubertal period. Annihilation of GnRH release through prepubertal age is suggested to be carried out by an inhibitory neuronal system and resumption of pulsatile GnRH release at puberty onset is thought to be due to removal of this inhibition. Recently a new neuronal system, named GnIH secreting neurons, was detected in the brain of quail bird. GnIH, the peptide product of these neurons is an inhibitor of reproductive function, acting within the brain and at the level of the pituitary gonadotrophs. In the last years, a few studies have suspected GnIH as an imperative negative regulator of the reproductive function in mammals including rodents and primates. As a result, the purpose of this study was to investigate the GnIH regulation of reproductive plexus activity in rodents and higher primates at various developmental stages using gene and protein expression analyses. Additionally, interaction of GnIH neurons with GnRH neuronal elements and correlative changes between GnIH, GnRH and Kiss1 expression at various stages of pubertal development are also assessed. 1.2. Materials and Methods In rodent study, sixty-day old male (n=8) and female (n=16) C57BL/6J mice were used. One-night timed breeding protocol was used to collect fetuses and pups at specific embryonic (E15.5, E17.5 and E18.5) and postnatal (PND12, PND18, PND40) days (n=5 9/time point). Whole brains were collected from male and female fetuses and pups and every fourth 30µm thick coronal section was used for dual label fluorescence immunocytochemistry by using primary antibodies 1 General Abstract against GnIH and GnRH. Variation in GnIH cell number, its nerve terminals’ expression/density in various hypothalamic regions and their interaction with GnRH neuronal system across prenatal and postnatal development was analyzed. In the primate study, POA and MBH containing hypothalamic blocks were collected from fifteen male rhesus macaques (Macaca mulatta). Monkeys were categorized into distinct developmental groups i.e., infant, juvenile, prepubertal and adult/pubertal based on somatometric and hormonal parameters. All animals' hemi-hypothalamic blocks were fixed in paraformaldehyde solution for immunofluorescence and liquid nitrogen was used for flash freezing the other half of the hypothalamus which was then kept at -80˚C until RNA isolation. For plasma testosterone measurement, blood was collected before euthanization from every monkey. From each animal, three randomly selected 20µm thick, horizontal, hemi-hypothalamic sections were processed for single label fluorescence immunohistochemistry using specific antibodies directed against GnIH to elucidate the alteration in GnIH neuronal elements’ expression in MBH of male rhesus monkeys at various developmental stages. Real time polymerase chain reaction was utilized for quantification of the GnRH, Kiss1 and GnIH mRNA during different pubertal stages, using specific primers. 1.3. Results Our results delineated that the expression of GnIH varied significantly across development in male and female mice and male rhesus monkeys. In mice, DMH expression of GnIH cells showed a significant main effect of age (P<0.0001), sex (P<0.0001) and their interaction (P<0.05) during in utero and postnatal growth. In both male and female mice, GnIH cells increased significantly just before term (P<0.001) and declined after birth. Again, a significant rise was noted in GnIH cells number at PND18 (P<0.001) followed by a precipitous decline (P<0.01) at PND40. 2 General Abstract Number/density of GnIH immunoreactive nerve terminals expressed in ARC, PVN and in whole brain also increased analogously till PND18 (P<0.0001), followed by a precipitous decrease at PND40 (P<0.0001). Number of GnIH-GnRH axosomatic contacts increased significantly at PND18 compared to all other ages (P<0.0001), falling significantly at PND40 (P<0.001). Number of GnIH fibers contacting GnRH fibers also increased significantly at PND18 (P<0.0001) compared to other ages, followed by a significant decline at PND40 (P<0.0001). Bodyweight, testicular volume, testosterone level and testicular histological parameters of monkeys correlated with the reproductive state of the animal, being significantly higher in adults compared to all other stages. GnIH immunoreactivity in the MBH and ARC region was seen to be significantly higher (P<0.01) at prepubertal stage which then declined significantly (P<0.001) in the adult animals. Similarly, number of GnIH-ir fibers also increased significantly (P<0.001) at the prepubertal stage, declining significantly (P<0.001) in adult animals. GnIH mRNA levels were statistically (P<0.01) higher in monkeys of the prepubertal age whereas GnRH and Kiss1 mRNA levels were significantly (P<0.05) lower at this stage. Inverse situation was noticed at the adult stage, where GnIH mRNA level was significantly reduced (P<0.001). A significant inverse correlation between GnIH-GnRH (P<0.01) and GnIH-Kiss1 (P<0.001) mRNA was also noticed. 1.4. Conclusions In summary, the findings of this research work propose that reproductive axis activity during prenatal and pubertal development in rodents and primates is regulated by GnIH signaling. Present work reinforces our understanding about the inhibitory neuropeptide (GnIH) that might be of assistance in the imposition of the prepubertal brake on the reproductive axis activity. In addition, present study also suggests that fading away of this prepubertal brake might activate the GnRH pulse generation 3 General Abstract system, triggering onset of puberty in rodents and primates. Foregoing research envisages to generate developmental maturation of a hypothalamic neuropeptide function with respect to its role on key substrates driving the reproductive system and provides a strong rationale for GnIH in therapeutics as a contraceptive tool and as a remedy for the fertility related disorders in human.