Testosterone Positively Regulates Hypothalamic Gonadotropin-Inhibitory Hormone Expression in the adult Male Rhesus Monkey (Macaca mulatta)

Muhammad Shahab^1,3; Nazar Hussain¹; Hira Zubair¹; Muhammad Saqib¹; Muhammad N. Khan¹; Sarwat Jahan²

Abstract Text:

Physiological effects of intrinsic sex steroids on regulation of hypothalamic-pituitary-gonadal (HPG)-axis in higher primates are not fully known yet. Recent reports have suggested an important role of gonadotropin-inhibitory hormone (GnIH) in down regulation of the reproductive axis under unfavorable conditions and GnIH neurons are reported to express cognate steroid receptors. Present study was conducted to further characterize the effect of varying internal testosterone milieu on the hypothalamic immune- and gene expression of GnIH in adult male rhesus macaques. Moreover, changes in mRNA levels of gonadotropin-releasing hormone and kisspeptin, important mediators of the reproductive axis were also evaluated.

Nine healthy laboratory-kept intact adult male rhesus monkeys divided into three main groups i.e., intact control (n=3); castrated (n=3) and castrated+Testosterone (T)-replaced (n=3) were used for the present study. Their body weights and testicular dimensions were noted, and blood samples were collected at the start of the study. Six animals were subjected to bilateral surgical castration while, control animals were kept intact. After the castrated animals had fully recovered, three were euthanised, and three were given four weeks of T-replacement therapy. These animals were administered with 400mg/kg/week testosterone enanthate via intramuscular injections. Every other day, all animals had their blood samples drawn for hormonal analyses. Plasma testosterone levels were measured using a commercially available competitive-type ELISA kit. The assay's sensitivity was 0.05 ng/ml, and the intra- and inter assay coefficients of variation were less than 10% and 9%, respectively. Hypothalami were collected from all nine monkeys and were subjected to immunohistochemical and qPCR analyses. Four, 30µm thick hemi-hypothalamic free floating frozen sections from each animal were processed for single label fluorescence immunohistochemistry using specific antibody against GnIH. Variation in GnIH cell number and its nerve terminals’ expression in various hypothalamic regions was analyzed. Other hemi-hypothalamic block from each animal was processed for gene expression of GnIH, GnRH and kisspeptin by qPCR.

Testosterone assays demonstrated that T-replacement therapy restored physiological (pre-castration) plasma testosterone levels in these animals. Relative mRNA expression of GnIH and mean number of GnIH positive cell bodies and fibers in hypothalamus were significantly decreased (P < 0.05) in castrated monkeys as compared to control monkeys. However, hypothalamic mRNA expression of GnIH and mean number of GnIH positive cell bodies and fibers were significantly increased (P < 0.05) in testosterone replaced animals implying that the testosterone replacement re-established the GnIH expression equivalent to that in intact/control animals. Additionally, a significant negative correlation was observed between GnIH-Kiss1 and GnIH-GnRH mRNA expression in castrated and testosterone-replaced groups.

Findings of the present study imply that GnIH is an important part of the circuitry propagating the inhibitory effect of circulating testosterone to the central GnRH pulse generator in the adult male monkey.  Most important finding of the current study is the stimulatory effect of plasma testosterone on hypothalamic GnIH expression. Based on present results it is concluded that inhibition of negative feedback from gonads to hypothalamus decreases the expression of GnIH while expression of GnRH and kisspeptin increases. Restoration of gonadal negative feedback via steroid replacement results in an increase in the expression of GnIH thus normalizing the activity of GnRH pulse generator in adult male monkeys.  Further studies on steroid regulation of GnIH expression might help in development of a novel therapeutic approach to treat fertility related disorders.