Studies of the Role of Kisspeptin-GPRS4 Signaling in the Testis

Abstract

Kisspeptin is an important neuropeptide involved in the regulation of reproductive axis. Its expression has been reported in reproductive axis but its complete role has not been deciphered in testicular tissue. The objective of this study was to find the expression and role of Kiss I-Kiss 1 r signalling in the testicular tissue of non-human primate. Three . different ways have been used to find this role: firstly, to localize the Kissl and Kisslr within specific cells in the testis by using single and double label immunohistochemistry and reverse transcriptase (RT-PCR). Secondly, to find any testicular effect of kisspeptin. This was done by measuring the effect of kisspeptin on testicular hormones I.e. testosterone and inhibin, from testicular fragments in incubation medium. Finally, the levels of kisspeptin were determined in testicular homogenate, testicular vein, spermatic vein and peripheral venous blood. This study was therefore based on the hypothesis that if kisspeptin is expressed in the testis then its levels should be high in testicular homogenate and it should show a concentration gradient in different testicular fluids In the expression studies, single and double label immunohistochemistry and RT-PCR were used to localize the kisspeptin in testis. Immunohistochemistry was carried out on formaldehyde fixed tissues obtained from 6 adult rhesus monkeys by using 5 J! thick . sections. Double label immunohistochemistry was used to localize the kiss 1 with germ cell marker T4 and kisslr with inhibin (a Sertoli cell marker) by using specific antibodies for them. The gene specific primers were used for carrying out the RT-PCR for kisspeptin and kisspeptin receptor. For in vitro study, testicular tissue fragments were obtained through testicular biopsy from five adult rhesus monkeys. The tissues were incubated in dulbecco's modified eagle's medium (DMEM) medium and treated with different kisspeptin doses for 30, 60 and 120 minutes. Kisspeptin effect was noted on testosteronem and inhibin released 1 General Abstract in the medium. At the end of kisspeptin dose time. heG and FSH were added to the medium for testing the competence of the Leydig and Sertoli cells in in vitro system. For determining the concentration gradient of kisspeptin in testicular fluid. blood plasma was collected from testis vein. spermatic vein and peripheral vein. Kisspeptin was then measured in these fluids using enzyme linked immunoabsorbent assay (EIA). Kisspeptin and kisspeptin receptor were found to be localized in testicular tissue of adult rhesus monkey. Immunohistochemistry showed the kisspeptin expression in spermatocytes and spermatids and kisspeptin receptor in spermatocytes and Sertoli cells. Kisspeptin expression was confirmed in the spennatids using double label Immunohistochemistry while kisspeptin receptor expression was confirmed in the Sertoli cells. In incubation study. no effect of kisspeptin was found on testosterone and inhibin release. In concentration gradient study of kisspeptin. an expected concentration gradient pattern of . kisspeptin was not found in testis homogenate. testicular vein. spermatic vein and peripheral blood. Thus, our results demonstrated the expression of kisspeptin receptor in the testis. Kisspeptin has no effect on testosterone and inhibin which suggests that kisspeptin has no direct endocrine effect on testis. We conclude that kisspeptin is present within testicular tissue but it may not have direct effects on testicular functions related to testosterone and inhibin release but it certainly has some other paracrine effects that may be more related to spermatogenesis.