Effects of osteocalcin on synthesis of testosteron and insl3 during adult leydig cell differentation

Testosterone is an important steroid hormone that plays a role in the functions of various organs and tissues, especially the reproductive system. Testosterone is known to be essential for skeletal development and bone health throughout human life. Insulin Like Factor-3 (INSL3) is an another hormone that plays a critical role in bone metabolism, produced by prenatal and postnatal Leydig cells such as testosterone. It is known that INSL3 expression reflects the development process and functional capacities of Leydig cells. However, there is not enough evidence in the literature about its synthesis and regulation in the testis. Osteocalcin (OC) released from osteoblasts responsible for stimulating pancreatic B cell proliferation and insulin secretion, induces testosterone production in Leydig cells independent from HPG (Hypothalamo-Pituitary-Gonad) except for bone formation. Leydig cells that develop from 2 different generations during fetal and adulthood, complete their proliferation and differentiation mainly in puberty. In many studies, it has been reported that the adult Leydig cell population is eliminated and regenerated by Ethane Dimethane Sulphonate (EDS) administration, except for the postnatal normal developmental process. Although there are many studies supporting the relationship between bone and testis in the literature, any studies have been encountered about the possible effects of OC on testosterone and/or INSL3 synthesis during Leydig cell development. In this study, it was aimed to investigate OC-mediated regulation of testosterone and INSL3 synthesis and compare the possible effects of OC and LH (Luteinizing Hormone) during differentiation of adult Leydig cells. Rats were divided into 2 groups: 32 prepubertal rats and 40 adult EDS injected rats. Each group was divided into 4 subgroups. Gonadotropin Releasing Hormone (GnRH) antagonist and/or OC were applied to these subgroups between prepubertal 35-63 days and between 35-63 days following EDS administration. At the end of the 63th day when adult Leydig cells completed their development, testicular tissue samples obtained from the sacrificed rats were examined by light and electron microscopic, immunohistochemical and biochemical methods. Significant degeneration in seminiferous tubules and Leydig cells in light microscopic and electron microscopic levels, down-regulation in 3βHSD, INSL3 and GPRC6A expressions, reduction in Leydig cell counts and serum LH, testosterone, ucOC levels were observed in testicular tissue sections of GnRH antagonist treated groups. Slight up-regulation in 3βHSD, INSL3 and GPRC6A expressions, increase in serum testosterone levels with a partial improvement in light and electron microscopic levels were observed in the testis tissue sections of the groups treated with GnRH antagonist and osteocalcin. Light and electron microscopic findings of testicular tissue sections in osteocalcin-treated groups were similar to control groups. While there was no significant difference in the number of Leydig cells, the presence of a significant increase in INSL3 and GPRC6A expressions, serum testosterone and ucOC levels were remarked. When all the findings are evaluated together; it was determined that the application of osteocalcin did not make a significant difference in the number of Leydig cells, but increases the level of testosterone by improving the function of existing adult Leydig cells during normal postnatal developmental process and post-EDS regeneration. This positive association between osteocalcin-testosterone and osteocalcin-INSL3 was concluded to be independent from LH in vivo conditions.