Assessment of mRNA expression of hypothalamic neuropeptide Y and its receptor (NPYY1) and its correlation with Kiss1 after 48 hours of immobilization stress in adult male rhesus monkeys (Macaca mulatta)

Abstract

Background: Stress signifies the organism’s response to environmental and internal circumstances. Acute immobilization is well known to trigger a stress response in animals that is mediated by various endocrine markers. Neuropeptide Y is one such marker, which is released from different parts of the hypothalamus, including the arcuate nucleus, and plays a significant role in energy metabolism, food intake, stress and regulation of reproductive function. Numerous studies in different animal models have documented observations concerning the effects of acute restraint stress on the expression of NPY and its receptor NPYY1 in the arcuate nucleus. However, the role of NPY in mediating stress and regulation of reproductive axis activity in higher primates has not been studied to date. The present study was designed to elucidate the variation in expression of NPY and its receptor in the hypothalamus of male rhesus macaque (a representative higher primate), and their association with Kiss1, via gene expression analysis. Materials and Methods: A total of six intact, healthy, adult male intact rhesus monkeys (Macaca mulatta) were used for this study, categorized into two main groups: control (n=4) and stressed (n=2). All animals were captured from the wild and were acclimatized for two weeks under laboratory conditions. Subsequently, the stressed group endured a 48-hour immobilization stress combined with isolation stress, followed by immediate euthanization. Control animals were undisturbed and were dissected at the same time. Before dissection, the body weight and testicular volume of all animals were noted and compared. Blood samples were also collected from all animals during the stress protocol and at the time of dissection for hormonal analyses, using commercially available ELISA kits. Hypothalamic and testicular tissues of control and stressed monkeys were collected under deep sedation. Hypothalamic tissues were promptly frozen in liquid nitrogen and were stored at -80ºC. Later, mRNA was extracted from the hemi-hypothalamic blocks of six animals and the quality of mRNA was determined by using nanodrop. After this, cDNA was made by following the instructions given with a commercially available cDNA synthesis kit and was stored at -20ºC until further analysis. Testicular tissues were fixed in sera and after 1 Abstract microtomy, Hemotoxylin and eosin staining was done on 5µm thick sections. Later, RT-qPCR was performed and the fold-change in mRNA expression of NPY, NPYY1 receptor and Kiss1 was calculated and correlated. Statistical analyses were done by using GraphPad Prism. Results: Expression of mRNA of NPY and NPYY1 receptor in hypothalamus was significantly increased and that of Kiss1 was significantly decreased in stressed animals as compared to the control group. Plasma testosterone level (P<0.05) was significantly decreased in stressed monkeys as compared to control monkeys after 48 hours of stress. A significant increase in plasma cortisol level (P<0.05) was observed in stressed monkeys as compared to control monkeys. Seminiferous epithelial height (P<0.05), seminiferous tubular diameter (P<0.05), and seminiferous luminal diameter (P<0.05) of stressed monkeys were significantly reduced as compared to those in control monkeys. NPY and Kiss1 expression (P=0.0512) exhibited a negative correlation. Similarly, an inverse correlation was observed between NPYY1 receptor and Kiss1 (P=0.0512) expression. Conclusions: Our results indicate that immobilization stress, combined with isolation stress triggers an increase in hypothalamic NPY and its receptor’s expression to mediate stress response to the reproductive axis in higher primates. Present results reinforce our understanding of the inhibitory action of NPY on the reproductive axis activity during unfavorable conditions. Foregoing research envisages generating data on the modulation of reproductive activity mediated by variation in the expression of hypothalamic NPY during untoward conditions, suggesting its role as a potential therapeutic for fertility-related disorders in humans.