Orexin-A Regulates Follicular Growth, Proliferation, Cell Cycle and Apoptosis in Mouse Primary Granulosa Cells via the AKT/ERK Signaling Pathway

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.

Integrated analysis of lncRNA and mRNA for the apoptosis of porcine ovarian granulosa cells after polyphenol resveratrol treatment

“…The ovary is one of the important reproductive organs of female animals, regulating follicle development and hormone secretion ( 23 ). Proliferation and apoptosis are natural physiological processes that occur naturally during the growth and development of ovarian follicles ( 24 ). In particular, the apoptosis of POGCs plays a vital role in the induction of atresia in porcine ovarian follicles ( 25 ).…”

Kurarinone attenuates hydrogen peroxide‐induced oxidative stress and apoptosis through activating the PI3K/Akt signaling by upregulating IGF1 expression in human ovarian granulosa cells

“…Furthermore, IGF1 silencing attenuated the protective effects of kurarinone on H 2 O 2 ‐induced granulosa cell damage, indicating that kurarinone regulated H 2 O 2 ‐induced granulosa cell damage through increasing IGF1 expression. The PI3K/Akt signaling is associated with granulosa cell survival and death 5,44 . Multiple evidences have confirmed that activated PI3K/Akt pathway attenuated H 2 O 2 ‐induced oxidative stress and apoptosis in granulosa cells 25,43,45 .…”

“…The ovarian disorders are the main reproductive pathologies contributing to infertility in female, including follicular atresia 3 . Granulosa cells are essential for follicular development, and dysfunctional granulosa cells are linked to follicular atresia progression 4,5 . The oxidative stress is one of the causal mediators of female reproductive diseases, 6 which leads to apoptosis of granulosa cells and subsequent follicular atresia 7 .…”

“…3 Granulosa cells are essential for follicular development, and dysfunctional granulosa cells are linked to follicular atresia progression. 4,5 The oxidative stress is one of the causal mediators of female reproductive diseases, 6 which leads to apoptosis of granulosa cells and subsequent follicular atresia. 7 Hence, exploring the potential strategy for protecting granulosa cells against oxidative stress and apoptosis is conducive to find new treatment for follicular atresia.…”

Avian Orexin: Feed Intake Regulator or Something Else?

“…This indicated how deficiency in orexin contributes to peripheral insulin resistance. The Akt pathway has also been implicated under orexin-A treatment in the granulosa cells of mice, where orexin-A and OX 1 R impacted the phosphorylation of this pathway and had impacts on downstream proliferation and apoptosis [ 82 ]. The Akt pathway, while it can be linked to ERK1/2 upstream, resulted in varying and different cellular responses than the ERK1/2 pathway [ 83 ].…”