FUNCTIONAL AND DEVELOPMENTAL STUDIES OF THE PRIMATE TESTIS

Abstract

Primate testis acts as the main source for androgen and sperm under the functional regulation of pituitary gonadotropins. The understanding of the functional regulation of adult primate testis as well as the developmental aspects of maturation during puberty in primates is still not complete. The present thesis aimed to assess the localization and functional significance of kisspeptin signaling in adult non human primate testis as well as to characterize the pubertal and testicular development in a New World monkey. Rhesus macaque (Macaca mulatta) and common marmoset (Callithrix jacchus) were employed as primate models. To sort out the functional significance of kisspeptin signaling in the primate testis in vivo pharmacological approach was used by employing chemical hypophysectomy by using acyline (a GnRH receptor antagonist) in adult rhesus monkeys (n=4). Animals were given iv boluses of kisspeptin (50μg) and kisspeptin (50μg) with hCG (50IU). Blood samples were taken and testosterone and LH was analyzed in the plasma by specific RIAs. To find out the testicular localization of kisspeptin receptor immunocytochemistry was performed on paraffin embedded testis tissue from adult rhesus (n=2) and marmoset monkey (n=2) whereas for testicular localization of kisspeptin immunocytochemistry was performed on paraffin embedded testis tissue from adult rhesus monkey (n=2). 12 sections from each testis were used for immunocytochemical analysis. To characterize the pubertal testicular development, immature common marmoset (n=48) were observed for a period of 13 months for monthly changes in body weight, testis volume and serum testosterone. At the end of the study animals were sacrificed and testis tissue was collected and processed for paraffin embedding. PAS staining was conducted. Histological and morphometric data were determined. Results of the experiments demonstrated that kisspeptin, given as an intravenous bolus enhanced stimulated plasma testosterone in pituitary clamped adult male rhesus monkeys whereas the same bolus had no effect on the basal levels of plasma testosterone. The immunocytochemical localization revealed that kiss1r positive areas were present at the periphery of the seminiferous tubules in the adult rhesus testis. Same pattern of localization for kiss1r was found in adult marmoset testis tissue. Whereas the kisspeptin like immunoreactivity was found to be present in the interstitial area of the adult rhesus testis tissue. The detailed examination revealed that the kiss1r localization was present around the spermatogonial stem cells (A spermatogonia) which are present at the periphery of the seminiferous tubules. While the kisspeptin like immunoreactivity was observed in the peritubular myoid cells, Leydig cells and underlying layers of basement membrane. Developmental studies showed that the start of pubertal activation in common marmoset occur around 7 months of age and was characterized by sudden peaks of serum testosterone accompanied by a rapid increase in the testis volume. Morphometric analysis revealed that the pubertal activation created a lumen in the center of seminiferous tubules. The germ cell compliment was observed to divide mitotically after the initial increase in serum testosterone depicted by presence of B spermatogonia in the seminiferous tubules after 7 months of age. The complete spermatogenesis characterized by the presence of sperm in epididymis was first observed around 12 month of age in the common marmoset testis. Although testis volume continued to grow after 12 months of age, the qualitative maturity of testicular functions was achieved at one year of age in the male common marmoset. The present findings implicate that an active paracrine kisspeptin-kisspeptin receptor signaling is present in the adult primate testis; where the kisspeptin can enhance stimulated testosterone thus hinting towards an indirect action on the Leydig cell. The finding of the tubular immunolocalization of the kisspeptin receptor in primate testis where Sertoli cells were found to be positive further extends our in-vivo results and affirms the assumption of an indirect action of kisspeptin on Leydig cell via the Sertoli cells. However to understand the true nature of this signaling cascade further investigations at the cellular level are required. The developmental kinetics of the process of puberty including pubertal activation and testicular development in common marmoset mimics higher primate like pattern. Although the endocrine correlates for these developmental events need to be further substantiated in common marmoset.