Phospholipase C inhibits apoptosis of porcine primary granulosa cells cultured in vitro

Huali Chen,Youlin Wang,Xiaohan Jiang,Huanshan He,Yifei Pei,Jie Wang,Lin Wu,Xiaoya Li,Li Yang,Jiaxin Duan,Dejun Xu,Qingwang Li,Rongmao Hua,Jianyong Cheng,Yuan Li,Youfu Yang,Yamei He,Dingbang Liu

doi:10.1186/s13048-019-0567-4

Abstract

Phospholipase C (PLC) can participate in cell proliferation, differentiation and aging. However, whether it has a function in apoptosis in porcine primary granulosa cells is largely uncertain. The objective of this study was to examine the effects of PLC on apoptosis of porcine primary granulosa cells cultured in vitro. The mRNA expression of BAK, BAX and CASP3, were upregulated in the cells treated with U73122 (the PLC inhibitor). The abundance of BCL2 mRNA, was upregulated, while BAX and CASP3 mRNA expression was decreased after treatment with m-3M3FBS (the PLC activator). Both the early and late apoptosis rate were maximized with 0.5 μM U73122 for 4 h. The rate of early apoptosis was the highest at 4 h and the rate of late apoptosis was the highest at 12 h in the m-3M3FBS group. The protein abundance of PLCβ1, protein kinase C β (PKCβ), calmodulin-dependent protein kinaseII α (CAMKIIα) and calcineurinA (CalnA) were decreased by U73122, and CAMKIIα protein abundance was increased by m-3M3FBS. The mRNA expression of several downstream genes (CDC42, NFATc1, and NFκB) was upregulated by PLC. Our results demonstrated that apoptosis can be inhibited by altering PLC signaling in porcine primary granulosa cells cultured in vitro, and several calcium−sensitive targets and several downstream genes might take part in the processes.

Highlights

In mammalian ovaries, granulosa cells have been demonstrated to play a critical role in deciding the destiny of follicles, follicular growth and maintenance as well as their apoptotic process [1]
The results of immunofluorescence showed that the rate of Follicle-stimulating hormone receptor (FSHR)-positive cells was more than 98%, which indicated that the purity of the porcine granulosa cells isolated from porcine ovaries was high enough for them to be used subsequently
The influence of Phospholipase C (PLC) inhibitor or activator on PLC expression in porcine granulosa cells in vitro The PLCB1 mRNA abundance supplied with 0.5 μM U73122 was the lowest (p < 0.05) in porcine granulosa cells after treatment for 2 h, 4 h, 8 h, 12 h and 24 h in porcine granulosa cells, and did not change after treatment for 48 h (Additional file 1: Figure S1A-E)

Summary

Introduction

Granulosa cells have been demonstrated to play a critical role in deciding the destiny of follicles, follicular growth and maintenance as well as their apoptotic process [1]. PLCβ plays an important role in the Wnt/Ca2+ pathway, which promotes the release of intracellular Ca2+ and affects Ca2+ sensitive targets, containing protein kinase C (PKC), Ca2+-calmodulin-dependent protein kinaseII (CAMKII) and Ca2+-calmodulin-sensitive protein phosphatase calcineurin (Caln) [12, 13]. Both CAMKII and PKC activate NFκB, and Caln activates cytoplasmic protein nuclear factor associated with T cells (NFAT) via dephosphorylation [14, 15]

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