Effect of castration and testosterone replacement on neuropeptide Y and its receptor (NPYY1R) mRNA in the hypothalamus of adult male rhesus monkey

Abstract

Neuropeptide Y, a prominent neuropeptide in the brain, binds with G-protein coupled receptors family including Y1 subtype, regulating various behavioral and physiological functions. Its expression is influenced by neuronal activity and peripheral hormones. Sex steroid-driven differences in NPY and NPYY1 receptor gene expression have been observed in many experimental models but not the higher primates. The present study was designed to determine the regulation of hypothalamic NPY and NPYY1 receptor expression by testosterone in male rhesus monkeys via gene expression analysis. Another objective of this study was to evaluate the correlative changes in mRNA expressions of NPY and NPYY1 receptor with Kiss1, GnRH and plasma testosterone level. For this study, nine intact adult male rhesus monkeys weighing 9-15 kg were used. They were divided into three groups i.e., control (n=3), castrated (n=3) and castrated plus testosterone-replaced (n=3). Before the start of the experiment, body weight and testicular volume of all monkeys were noted, and blood samples were also collected for hormonal analysis. Surgical castration was carried out by the excision of both testicles, of all the animals in the castrated and T-replaced groups, while animals in the control group were kept undisturbed until euthanization. After proper recovery of all castrated animals, three animals were subjected to euthanization, while the other three animals were subjected to testosterone enanthate replacement therapy regimen (400mg/week) for three weeks and (200µg) for fourth week, intra-muscularly. Blood samples were collected from these animals every other day throughout the replacement therapy for plasma testosterone estimation. Then they were also subjected to euthanization and their hypothalami was collected. The hypothalamus of each dissected monkey was cut into two hemi-hypothalamic blocks. One hemi-hypothalamic block from each animal was flash frozen in liquid nitrogen and was then stored at -80°C until mRNA extraction. Excised testicular tissue was fixed in sera and were later stained with eosin & hematoxylin to study the morphological parameters. Total RNA was extracted from hemi-hypothalami of all nine monkeys using manufacturer’s protocol, and then cDNA was synthesized using a commercially available synthesis kit. Then, SYBR green RT qPCR was performed to evaluate NPY and NPYY1 receptor mRNA expression. The variations in mRNA expression in the hypothalamus of all three group of monkeys was compared statistically by using one-way analysis of variance followed by Tukey’s multiple comparison post hoc analysis. Body weight, testicular volume, and testosterone levels at the start of the experiment were comparable between all experimental groups. While, plasma testosterone 1 Abstract levels were minimal in the castrated animals, and were comparable between the control and the castrated + T-replaced animals at the time of euthanization. Using unpaired t-test seminiferous tubular diameter and epithelial height was found to be comparable among control and T replaced groups. Fold change in mRNA expression of NPY was found to be significantly decreased (p<0.05) in T-replaced monkeys as compared to control and castrated group. Fold change in mRNA expression of NPYY1 receptor was seen to be significantly decreased (p<0.05) in castrated group and was comparable between control and T-replaced groups (p>0.05). A significant (p<0.05) positive correlation was observed between mRNA expression of NPY with Kiss1 and GnRH, while significant (p<0.05) negative correlation was observed between NPY mRNA expression with plasma testosterone level in castrated and T-replaced group of animals. However, significant (p<0.05) inverse association between mRNA expression of NPYY1 receptor with Kiss1 and GnRH was observed, while significant linear positive correlation between mRNA expression of NPYY1 receptor with plasma testosterone level in castrated and T-replaced group of animals was observed. In conclusion, castration does not affect the expression of NPY while testosterone replacement resulted in decreased expression of NPY. However, contrasting effects were seen in the case of NPYY1 receptor’s expression where it significantly declined in castrated animals and increased significantly after T-replacement therapy. These possible effects of castration in lowering the mRNA expression of NPYY1 receptor in the hypothalamus of adult male rhesus monkeys might be due to the loss of negative feedback effect of testosterone that also results in an increased mRNA expression of Kiss1 and GnRH. However, further studies are required to understand the cellular and molecular mechanisms by which sex steroids affect NPY and its receptor’s expression in higher primates, which might be of utility in the development of new therapeutic strategies for reproductive disorders in human