In vitro effects of uncarboxylated osteocalcin on buffalo Leydig cell steroidogenesis

Uncarboxylated osteocalcin (UcOCN), a bone derived circulating protein, has been demonstrated to influence steroidogenesis in testicular Leydig cells of murine and human species. However, the role of UcOCN in testosterone biosynthesis remains unexplored in domestic animals. The present study aimed to investigate the impact of UcOCN on the expressions of steroidogenic genes (HSD3β1, HSD3β6, CYP17A1, CYP11A1), testosterone production and GPRC6A receptor localization in buffalo Leydig cells. Leydig cells from the testes of adult Murrah buffalo were isolated, with an average cell count and viability after digestion and Percoll enrichment of 1.43 × 10 6 cells/g of testes and 78.5%, respectively. Immunophenotyping of Percoll-enriched cell suspension by flow cytometry showed populations of Leydig cells ranging between 69 and 73.9%. Immunostaining confirmed the presence of GPRC6A receptors and CYP11A1 positive Leydig cells. When these cells were cultured and incubated with varying levels of UcOCN (6, 12, 24, and 48 ng/ml) and LH, there was a significant ( P