RFRP-3, the mammalian ortholog of GnIH, induces cell cycle arrest at G2/M in porcine ovarian granulosa cells

Xiaoye Wang; Xun Li; Chuanhuo Hu

doi:10.1016/j.peptides.2018.01.006

RFRP-3, the mammalian ortholog of GnIH, induces cell cycle arrest at G2/M in porcine ovarian granulosa cells

Peptides. 2018 Mar:101:106-111. doi: 10.1016/j.peptides.2018.01.006. Epub 2018 Jan 11.

Authors

Xiaoye Wang¹, Xun Li², Chuanhuo Hu³

Affiliations

¹ College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China.
² College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China. Electronic address: lixun198@163.com.
³ College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China. Electronic address: hch64815@gxu.edu.cn.

PMID: 29337271
DOI: 10.1016/j.peptides.2018.01.006

Abstract

RFamide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), has been proposed as a key inhibitory regulator of mammal reproduction. Our previous studies have demonstrated that RFRP-3 inhibited the expression of proliferation-related proteins in porcine granulose cells (GCs), but the inhibitory mechanism causing this has not been discovered. Here, we aim to elucidate the underlying mechanism and determine the cell cycle regulatory sites of action of RFRP-3 on porcine GC proliferation. To this end, the viability of porcine GCs was initially estimated by cell counting kit-8 (CCK-8). We confirmed that different doses of RFRP-3 decreased the cellular viability, suggesting that RFRP-3 could inhibit the proliferation of GCs. Subsequently, we evaluated the direct effects of RFRP-3 on the expression of cell cycle regulators. Compared to the control treated cells, 10^-6 and 10^-8 M of RFRP-3 effectively reduced the transcription of Cyclin B1 and CDK1 mRNAs. However, treatment with RFRP-3 did not alter Cyclin A2, Cyclin D1, CDK2, or CDK4 mRNA levels. These results suggest that RFRP-3 might be inducing G2/M-phase arrest in porcine GCs. Finally, to further determine the molecular mechanism underlying RFRP-3-mediated G2/M cell cycle arrest, we observed the levels of G2/M cell cycle regulatory factors in RFRP-3-treated porcine GCs. The results showed that RFRP-3 treatment significantly increased the expression of Myt1, p-Wee1 and p-Cdc2, whereas the level of Cyclin B1 significantly decreased in porcine GCs treated with 10^-6 M of RFRP-3. Taken together, our data suggest that RFRP-3 regulates the phosphorylation or expression of G2/M cell cycle regulatory factors to induce G2/M-phase arrest via inhibition Cyclin B-CDK1 complex activation in porcine GCs, which might provide an unfavorable condition for porcine GC proliferation.

Keywords: Cell cycle arrest; Gonadotropin-inhibitory hormone (GnIH); Porcine ovarian granulosa cells (GCs); RFamide-related peptide-3 (RFRP-3).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Female
G2 Phase Cell Cycle Checkpoints / drug effects*
Gene Expression Regulation / drug effects*
Glycoproteins / chemistry
Glycoproteins / pharmacology*
Granulosa Cells / classification
Granulosa Cells / metabolism*
M Phase Cell Cycle Checkpoints / drug effects*
Neuropeptides / chemistry
Neuropeptides / pharmacology*
Swine

Substances

Glycoproteins
Neuropeptides
RFamide peptide
gonadotropin inhibitor