Expression of GnIH and its receptor in the pituitary gland of rhesus macaque during pubertal development

Abstract

Background: Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide reported to control the secretion of pituitary gonadotropins and hence regulates reproduction. GPR-147 has been touted as a GnIH receptor identified in different animals, including chicken and humans. Recently, GnIH expression has been observed in the pituitary of Indian major carp and frog during early developmental stages. However, in the pituitary of higher primates the expression of GnIH and GPR-147 has not been reported during the pubertal development. Aims and Objectives: Current study was designed to explore the presence of GnIH and GPR-147 in the anterior pituitary of male rhesus monkeys and to compare variations in expression during prepubertal and adult stages. Materials and Methods: A total of eight male rhesus monkeys in the prepubertal and adult stage (n=4/group) were used. Body weight and testicular volume of each animal was measured after sedation. Testosterone was measured in extracted plasma of a single blood sample from each animal by standard ELISA testosterone kit. Paraffin-embedded testicular tissue sections (7 μm thick) were processed for hematoxylin and eosin staining to determine the animals' spermatogenic status. Anterior pituitary tissues were collected after euthanization of animals and fixed in 4 % paraformaldehyde solution. Sucrose treated dehydrated tissues were cut into 7μm and 20μm thick sections on freezing cryotome to carry out anterior pituitary histology and immunocytochemistry, respectively. Three random sections from each animal were processed for standard immunocytochemistry protocol to check the variations in the expression of GnIH-like immunoreactivity (IR) and GPR-147-like IR using highly specific antibodies for GnIH and GPR-147. Results: Body weight, total testicular volume and plasma testosterone levels were significantly elevated (t=6.107, df=6; p<0.001; t=8.518, df=6; p<0.001 and t=3.574, df=6; p<0.05, respectively) in adult male rhesus monkeys in comparison to prepubertal male rhesus monkeys. Testicular histology demonstrated active spermatogenesis in adult animals compared to prepubertal animals, while advanced germ cells were absent in the prepubertal testis, ensuring two different developmental stages. Anterior pituitary histology showed cords and trabeculae of polygonal epithelial cells however, lacking unmyelinated axons and any acellular content ensured the likelihood presence of anterior pituitary in all sections. In general, both GnIH-like IR and GPR- Abstract 2 147-like IR cells were evident to be distributed throughout the anterior pituitary of prepubertal and adult animals. GnIH-like IR expression was found to be located within the cells, while GPR-147-like IR was visible both at the periphery and inside the cells. Comparison of both GnIH-like IR cells and GPR-147-like IR cells showed that total number of cells expressing peptide and receptor were elevated (t=4.01, df=6; p<0.01 and t=11.94, df=6; p<0.0001, respectively) in prepubertal male monkeys in comparison to adult male monkeys in the anterior pituitary gland. Conclusion: Present findings of decreased expression of GnIH-like IR and GPR-147-like IR in anterior pituitary of adult male rhesus monkeys as compared to prepubertal animals suggest that GnIH pathway, contribute to initiation of puberty in primate males. However, further systematic studies are required using neuro molecular tools to confirm a physiological role of GnIH in regulating pubertal development in higher primates.