Study of interaction between NPY and kisspeptin neuronal system in the hypothalamus during pubertal development in the male rhesus monkey (Macaca mulatta)

Abstract

Introduction: The neuroendocrine mechanism underlying intermittent pattern of GnRH secretion by gonadotropin-releasing hormone (GnRH) pulse generator activity during postnatal development mimicking sinusoidal wave nature, the crest of which experience at infantile and adult stage characterizing mini and true puberty intervene by a trough at juvenile stage in which pulse generator brought into check resembling hypogonadotropic state, still needs to be meticulously defined. Particularly, the quest to find neurobiological central restrain that keeps GnRH pulse generator activity at nadir during juvenile period is still an intriguing mystery. Before the advent of kisspeptinocentric era, the role of neuropeptide Y (NPY) has been contemplated as potent neuroinhibitory factor in restricting GnRH/LH release in various species including non-human higher primates. Furthermore, owing to the heterogeneity of NPY receptors, pharmacological intervention with various antagonists partially supports NPY inhibition of GnRH/LH release via NPY 1 receptor (NPY1R). Since the discovery of kisspeptin different physiological, pharmacological, clinical, and genetic approaches have been implemented to decipher the prime importance of kisspeptin as component of the GnRH pulse generator to trigger pubertal onset in primate and non-primate mammals. Albeit kisspeptin has been shown to be an integral component of the GnRH pulse generator but not the trigger for pubertal onset. Close proximity between the two neuropeptides and NPY's direct regulation of kisspeptin neurons reaffirm the significance of these two neuropeptides in regulating reproductive axis activity. These observations harken back to the concept that diminishing activity of GnRH pulse generator before pubertal onset may be attributed by NPY which may act as a central brake for kisspeptin neurons. At the time of puberty, the attenuation of NPY brake may cause instigation of hypothalamic-pituitary-gonadal (HPG) axis activity via kisspeptin dependent pathway. The current investigations were designed to examine the interaction between NPY and kisspeptin neuronal system in the hypothalamus during pubertal development in the male rhesus monkey by performing three experiments. First experiment was to see correlative fluctuations in the relative mRNA expression of key neuropeptides (NPY and KISS1) and their receptors from hypothalamus encompassing mediobasal area. Second experiment was concerned about assessment of developmental variations in NPY 1 General Abstract and NPY1R expression and their interaction with kisspeptin neurons during pubertal development. Third study designed to delineate the role of NPY1R in mediating the putative inhibitory action of NPY on the HPG axis after intravenous (iv) administration of highly selective NPY1R antagonist (BIBO 3304) in GnRH primed juvenile male rhesus monkey. Materials and Methods: Fifteen intact male rhesus monkeys (Macaca mulatta) were used to conduct first two experiments. These animals were assigned into four different developmental groups [infant, (4-7 months), n=3], [juvenile, (10-14 months), n=4], [pre pubertal, (21-24 months), n=4], [adult, (7-10 years), n=4] based upon physical (body weight and testicular volume), morphometric parameters (epithelial height and seminiferous tubule diameter) of testicular histology, and hormonal (testosterone) data. Hemi-hypothalamic blocks from each animal, snapped frozen in liquid-nitrogen then stored at -80 º C until processed for RNA extraction, were used to study differential expression of KISS1 and NPY along with KISS1R and NPY1R through real-time quantitative PCR (RT-qPCR) by using gene specific primers. Other hemi-hypothalamic blocks from all fifteen animals after fixation (4% PFA) and dehydration (20% and 30% sucrose) were processed for cryosectioning to obtain 20 µm thick sections. These sections were used to assess morphological interactions and variations in NPY and NPY1R expression on kisspeptin neurons during pubertal progression through double label fluorescent immunocytochemistry using specific antibodies against kisspeptin, NPY and NPY1R. In addition, four GnRH primed juvenile intact male rhesus monkeys (Macaca mulatta) 10-14 months old were used to decipher the effect of intravenous (iv) administration of NPY1R specific antagonist BIBO 3304 on the HPG axis activity indirectly via testosterone measurement as a surrogate of LH. Sequential blood samples (~ 0.5-0.7 ml) at 30 min interval, three samples at -60, -30, 0 min before and nine samples from 30 min up to 270 min after vehicle or NPY1R antagonist (BIBO 3304) administration were withdrawn. Three iv boli (1ml/animal) at 0, 60 and 120 min of vehicle or BIBO 3304 were administered. Plasma was harvested and used to measure plasma testosterone concentration by commercially available human testosterone ELISA kit. 2 General Abstract Results: Significant uprise in both mRNA of KISS1 and its receptor (KISS1R) (p<0.05 0.01) was noted in adult animals in comparison to pre-pubertal animals. Contrasting to kisspeptin and its receptor, NPY and its receptor NPY1R showed significant decrease (p<0.05) in mRNA expression in adult group with respect to preceding age group. Inverse correlative age-associated fluctuations across pubertal development stages were also evident in pre-pubertal group with respect to infant animals. As pre-pubertal group manifested significantly higher (p<0.05) relative expression of NPY mRNA and insignificant (p>0.05) decrease in KISS1 relative mRNA. The same pattern of expression was also observed for NPY1R and KISS1R mRNA. Immunofluorescent data revealed significantly augmented (p<0.001 and p<0.05) percentage interaction of kisspeptin neurons with NPY in juvenile and pre-pubertal monkeys as compared to infant monkeys. Moreover, significant decrease (p<0.01 and p<0.05) percentage of dual labelled kisspeptin and NPY was evident in adult and pre-pubertal group in comparison to juvenile group. Percentage of kisspeptin neurons with NPY1R expression showed significant increase (p<0.01 and p<0.001) in juvenile and pre-pubertal animals in comparison to infant. In contrast, the percentage of dual label kisspeptin neurons with NPY1R showed significant decrease (p<0.001 and p<0.01) in adult group as compared to pre-pubertal and juvenile group, respectively. Intravenous (iv) administration of NPY1R specific antagonist showed that neither treatment nor time has a significant effect on testosterone secretion. Moreover, no significant difference in mean testosterone levels and area under the curve (AUC) for testosterone between vehicle or antagonist administered animals was noted. No significant difference in mean pre- and post- BIBO 3304 or vehicle testosterone secretion was also observed. Conclusions: In summary gene and protein expression analyses demonstrated that suppression of the kisspeptin neurons may be attributed due to enhanced activity of NPY neurons. Increased expression of NPY and NPY1R and decreased expression of kisspeptin and kisspeptin receptor during juvenile/pre-pubertal stage may be interpreted that enhanced NPY signaling may downregulate GnRH pulse generator activity by diminishing KISS1 KISS1R signaling. Developmentally increased expression of NPY1R on kisspeptin neurons during pre-pubertal/juvenile stage is also suggestive of NPY mediated inhibition 3 General Abstract of kisspeptin neurons via NPY1R. Thus, enhanced NPY signaling may act as upstream central neurobiological brake for dormancy of kisspeptin neurons keeping GnRH pulse generator in check during pre-pubertal period, while alleviation of NPY brake may likely to occasion kisspeptin dependent resurgence in the HPG axis activity at the onset of puberty. However, non-exclusive involvement of NPY1R in mediating the putative inhibitory action of NPY on the HPG axis during the juvenile period as evident from the pharmacological data is suggestive to reappraise the role of NPY via NPY1R signaling in occasioning pre-pubertal suppression of GnRH pulse generator in rhesus monkey. The results of the present data corroborated the notion that suppression of the HPG axis during pre-pubertal stage after transient stimulation in infant stage may be contributed, at least in part, by NPY mediated suppression of kisspeptinergic drive to GnRH neurons. However, an extensive exploration about the functionality of NPY1R in mediating NPY based inhibition of GnRH release during the juvenile period remains to be substantiated by different experimental paradigms to unwind twists and turns in the pubertal development story. In addition, it is pertinent to propose that NPY mediated inhibition of the kisspeptin neurons may involve different receptor subtype other than NPY1R. Furthermore, NPY may act in conjunction with some other neurobiological component, and the collaborative message of these may be translated to repress GnRH pulse generator activity before pubertal onset. The nature of this component that regulates NPY-kisspeptin-GnRH pathway still needs to be defined explicitly. Nonetheless, current findings amplify the role of NPY and kisspeptin neuronal system in the hypothalamus during pubertal development in non-human higher primates. These findings hitherto may be helpful to open new avenues in the development of novel clinical, pharmacological, and therapeutic strategies to cater fertility problems in humans.