Please use this identifier to cite or link to this item:
http://hdl.handle.net/123456789/27771
Title: | Study of effect of castration and testosterone replacement on immuno- and mRNA-expression of GnIH in the hypothalamus of adult male rhesus monkey (Macaca mulatta) |
Authors: | Nazar Hussain |
Keywords: | Animal Sciences Zoology |
Issue Date: | 2022 |
Publisher: | Quaid I Azam university Islamabad |
Abstract: | Background: The major regulator of reproductive function is hypothalamic-pituitary-gonadal (HPG) axis and its activity is not only regulated by gonadotropin-releasing hormone (GnRH), but Tsutsui’s group found that gonadotropin-inhibitory hormone (GnIH) is also a major regulator of HPG axis. GnIH is a new RF amide peptide that has been identified in all vertebrate classes and share an LPXRF (X = L or Q) amide motif at the C-terminal. GnIH neurons exist in the paraventricular nucleus (PVN) in birds and the dorsomedial hypothalamic area (DMH) in mammals. GnIH reduces gonadotropin release and synthesis via its effect mediated through the GnIH receptor, GPR147, on gonadotrophs and GnRH neurons. Most of the literature focuses on how castration affects the HPG axis through changes in the expression of neurohormones and other steroid hormones. Although several studies have investigated the effect of castration associated with changes in the expression of GnIH in various species but up till no work has been reported that investigates the consequences of castration along with steroid replacement on HPG axis in higher primates at hypothalamic GnIH expression. Aims and Objectives: The present study was undertaken to evaluate the regulation of hypothalamic GnIH expression by sex steroids in male rhesus monkeys via gene and protein expression analysis. Another objective of this study was to determine the correlative changes in mRNA expressions of GnIH with Kiss1, GnRH and plasma testosterone level. Materials and Methods: Eleven intact adult male rhesus monkeys weighing 9-12 kg were used for this study. They were divided into three groups i.e., control (control for RT-qPCR (n=3); control for ICC (n=2), castrated (n=3) and castrated plus testosterone replaced (T replaced) (n=3). Surgical castration of six adult monkeys was carried out by the excision of both testicles, while the control monkeys were kept intact until euthanization. The blood sample of each monkey was collected for testosterone analysis. After proper recovery of all castrated animals, three animals were subjected to euthanization to obtain required tissues, while other three animals were subjected to testosterone replacement therapy for about four weeks. After steroid replacement they were also subjected to euthanization and required tissues were collected. Each hypothalamic block of dissected animals was cut into two hemi-hypothalamic blocks. For evaluating mRNA expression of GnIH, hemi-hypothalamic blocks were flash frozen in liquid nitrogen and then stored at -80°C until mRNA extraction. To evaluate immuno expression of GnIH, hemi hypothalamic blocks were processed and cryostat was used to DRSML QAU Abstract 2 coronally section the hemi-hypothalamic blocks at 25μm thickness. For each animal four random sections containing the MBH were processed for fluorescent immunocytochemistry using specific antibodies. The total number of GnIH-like immunoreactive (ir) terminals and fibers were manually counted in the entire section of experimental and control group of animals by using florescent microscope. The variations in GnIH-like immunoreactivity and mRNA expression in the hypothalamus of all three group of monkeys was evaluated statistically by using one-way analysis of variance (ANOVA) followed by post hoc test (Tukey’s multiple comparison). Results: Relative mRNA expression of GnIH and mean number of GnIH-like ir terminals and fibers were significantly decreased (P<0.05) in castrated monkeys as compared to control group. However, relative mRNA expression of GnIH and mean number of GnIH-like ir terminals and fibers were significantly increased in T-replaced group. Additionally, a significant linear negative correlation was observed between mRNA expression of GnIH with Kiss1 and GnRH, while a significant linear positive correlation was observed between mRNA expression of GnIH with plasma testosterone level in castrated and T-replaced group of animals. Conclusion: Castration causes decrease while steroid replacement results in an increase in the relative mRNA expression of GnIH and mean number of GnIH-like ir terminals and fibers in the hypothalamus of adult male rhesus monkeys. This possible effect of castration in lowering the relative mRNA expression of GnIH and GnIH-like immunoreactivity in the hypothalamus of adult male rhesus monkeys might be due to the loss of negative feedback effect of sex steroids that also results in an increased mRNA expressions of Kiss1 and GnRH. However, further studies are required to understand the cellular and molecular mechanisms by which sex steroids affect GnIH expression in higher primates. |
URI: | http://hdl.handle.net/123456789/27771 |
Appears in Collections: | M.Phil |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
BIO 7146.pdf | BIO 7146 | 2.76 MB | Adobe PDF | View/Open |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.