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

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.

Keywords: Apoptosis; Granulosa cells; Phospholipase C; Porcine.

Fig. 1

The identification of porcine granulosa cells by immunofluorescence. Porcine granulosa cells were stained with FSHR; The cell nuclei in the entire visual field were stained by DAPI; Most of the DAPI could be merged with FSHR. Magnification, × 600; Scale bar = 50 μM

Fig. 2

The influence of PLC inhibitor and activator on PLC mRNA and protein expression in porcine granulosa cells in vitro. Data are mean ± S.E.M.of three independent replicates. (A) The PLCB1 mRNA expression treated with 0.5 μM U73122 for the times given. (B) One representative western blot band chart for PLCB1 treated with 0.5 μM U73122 for 4 h. (C) The PLCB1 protein abundance treated with 0.5 μM U73122 for 4 h. (D) The PLCB1 mRNA expression treated with 0.5 μM m3M3FBS for the times given. (E) One representative western blot band chart for PLCB1 treated with 0.5 μM m3M3FBS for 4 h. (F) The PLCB1 protein abundance treated with 0.5 μM m3M3FBS for 4 h. One-way ANOVA was used for genes and independent sample t-test was used for proteins. For each treatment, means without common letters are significantly different (p<0.05) (A and D); means with ** indicate a difference at p<0.01 compared with control (C); means without asterisk are not significantly different (F)

Fig. 3

The influence of PLC inhibitor and activator on apoptosis of porcine granulosa cells in vitro. The cells were challenged with 0.5 μM U73122 or m-3M3FBS for 4 h (genes) or for the times given (percentage of apoptotic cells), which were processed for annexin V/PI staining and measured by flow cytometry assay. (A) The effect of U73122 on the abundance of the mRNAs that encode the transcription factors involved in the regulation of apoptosis of porcine granulosa cells. (B) The percentage of early apoptosis and late apoptosis treated with 0.5 μM U73122 at different times. (C) The effect of m3M3FBS on the abundance of the mRNAs that encode the transcription factors involved in the regulation of apoptosis of porcine granulosa cells. (D) The percentage of early apoptosis and late apoptosis treated with 0.5 μM m-3M3FBS at different times. Data are mean ± S.E.M. of three independent replicates. Independent sample t-test was used for (A) and (C), and one-way ANOVA was used for (B) and (D). For each treatment, means without asterisk are not significantly different, means with * indicate a difference at p <0.05 compared with control, means with ** indicate a difference at p <0.01 compared with control (A and C); or means without common letters are significantly different(p<0.05) (B and D)

Fig. 4

The effect of U73122 and m3M3FBS on intracellular Ca²⁺ in porcine granulose cells.The cells were challenged with 0.5 μM U73122 or m3M3FBS for 4 h. a Median fluorescence intensity of intracellular Ca²⁺ treated with U73122. b Median fluorescence intensity of intracellular Ca²⁺ treated with m-3M3FBS. Data are mean ± S.E.M. of three independent replicates. Independent sample t-test was used. For each treatment, means with ** indicate a difference at p<0.01 compared with control (b), and means with *** indicate a difference at p<0.001 compared with control (a)

Fig. 5

The effect of U73122 and m-3M3FBS on Ca²⁺ sensitive proteins (PKCβ, CAMKIIα and CalcineurinA protein) and mRNA expression of downstream genes and CTNNB in porcine granulosa cells. The cells were challenged with 0.5 μM U73122 or m-3M3FBS for 4 h. a One representative western blot band chart for PKCβ, CAMKIIα and calcineurinA treated with U73122. bThe protein abundance of PKCβ, CAMKIIα and calcineurinA treated with U73122. c The effect of U73122 on mRNA expression of downstream genes and CTNNB. d One representative western blot band chart for PKCβ, CAMKIIα and calcineurinA treated by m3M3FBS. e The protein abundance of PKCβ, CAMKIIα and calcineurinA added by m-3M3FBS. f The effect of m3M3FBS on mRNA expression of downstream genes and CTNNB. Data are mean ± S.E.M. of three independent replicates. Independent sample t-test was used. For each treatment, means without asterisk are not significantly different, means with * indicate a difference at p<0.05 compared with control, means with ** indicate a difference at p<0.01 compared with control, and means with *** indicate a difference at p<0.001 compared with control