Abstract
Granulosa cells (GCs) are essential for follicular growth, development, and atresia. The orexin-A (OXA) neuropeptide is widely involved in the regulation of various biological functions. OXA selectively binds to orexin receptor type 1 (OX1R) and mediates all its biological actions via OX1R. This study aimed to explore the expression of OXA and OX1R and their regulatory role in GCs proliferation, cell cycle progression, apoptosis, oocyte maturation, and underlying molecular mechanisms of these processes and elucidate its novel signaling pathway. Western blotting and RT-qPCR showed that OXA and OX1R were expressed during different developmental stages of GCs, and siRNA transfection successfully inhibited the expression of OX1R at the translational and transcriptional levels. Flow cytometry revealed that OX1R knockdown upregulated GCs apoptosis and triggered S-phase arrest in cell cycle progression. RT-qPCR and Western blotting showed significantly reduced expression of Bcl-2 and elevated expression of Bax, caspase-3, TNF-α, and P21 in OX1R-silenced GCs. Furthermore, the CCK-8 assay showed that knockdown of OX1R suppressed GCs proliferation by downregulating the expression of PCNA, a proliferation marker gene, at the translational and transcriptional levels. Western blotting revealed that knockdown of OX1R resulted in a considerable decrease of the phosphorylation level of the AKT and ERK1/2 proteins, indicating that the AKT/ERK1/2 pathway is involved in regulating GCs proliferation and apoptosis. In addition, OX1R silencing enhanced the mRNA expression of GDF9 and suppressed the mRNA expression of BMP15 in mouse GCs. Collectively, these results reveal a novel regulatory role of OXA in the development of GCs and folliculogenesis by regulating proliferation, apoptosis, and cell cycle progression. Therefore, OXA can be a promising therapeutic agent for female infertility.