ucOCN Promotes Testosterone Synthesis via the PKA-MAPK/ERK-CREB Signaling Pathway in Porcine Leydig Cells

Bone health might be closely associated with male fertility, yet the molecular pathways remain poorly characterized. We demonstrate that undercarboxylated osteocalcin (ucOCN), a bone-derived hormone, initiates a signaling cascade that stimulates testosterone biosynthesis in porcine Leydig cells. Mechanistically, ucOCN binding to membrane receptor GPRC6A elevates intracellular cAMP levels and sequentially activates PKA, MEK, and ERK. ERK translocates to the nucleus and phosphorylates the transcription factor CREB. Activated CREB binds directly to promoter regions of the key steroidogenic genes and boosts testosterone production. The genetic or pharmacological inhibition of GPRC6A, PKA, MEK, or ERK signaling disrupts CREB activation and abolishes both steroidogenic gene expression and testosterone synthesis. Crucially, the phospho-switch S298 as a previously unrecognized phosphorylation site through which MEK regulates osteocalcin (OCN) signaling was identified. Collectively, our results indicate that ucOCN interacts with GPRC6A to promote testosterone synthesis in Leydig cells via the PKA-MAPK/ERK-CREB pathway. The above findings elucidate a fundamental endocrine axis between bone and the male reproductive system, offering novel mechanistic insights and potential therapeutic strategies for improving male fertility.