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

Muhammad Safdar; Aixin Liang; Shahid Ali Rajput; Nasir Abbas; Muhammad Zubair; Aftab Shaukat; Aziz ur Rehman; Huma Jamil; Yan Guo; Farman Ullah; Liguo Yang

doi:10.3390/molecules26185635

Molecules (Sep 2021)

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

Muhammad Safdar,
Aixin Liang,
Shahid Ali Rajput,
Nasir Abbas,
Muhammad Zubair,
Aftab Shaukat,
Aziz ur Rehman,
Huma Jamil,
Yan Guo,
Farman Ullah,
Liguo Yang

Affiliations

Muhammad Safdar: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China
Aixin Liang: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China
Shahid Ali Rajput: Faculty of Veterinary and Animal Sciences, Muhammad Nawaz Shareef University of Agriculture, Multan 66000, Pakistan
Nasir Abbas: Department of Animal Breeding & Genetics, PMAS-Arid Agriculture University, Rawalpindi 46300, Pakistan
Muhammad Zubair: Key Laboratory of Agricultural Microbiology, Education Ministry of China, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Aftab Shaukat: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China
Aziz ur Rehman: Department of Pathobiology, University of Veterinary and Animal Sciences, Jhang 35200, Pakistan
Huma Jamil: Department of Theriogenology, Faculty of Veterinary Science, University of Agriculture, Faisalabad 38000, Pakistan
Yan Guo: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China
Farman Ullah: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China
Liguo Yang: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China

DOI: https://doi.org/10.3390/molecules26185635
Journal volume & issue: Vol. 26, no. 18
p. 5635

Abstract

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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.

Published in Molecules

ISSN: 1420-3049 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Chemistry: Organic chemistry
Website: http://www.mdpi.com/journal/molecules

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