Study On BMP15Signal Pathway To Regulate The Apoptosis Of Porcine Cumulus Cells

Somatic cells are closely related to the growth and development of oocyte inovarian mammalian. Granulosa cells are the main types of cells in ovary which aredivided into two types by the structure and function: the parietal cells which aredistribution in the follicle walls were called follicles mural granulosa cells (MGC),mainly secrete steroid hormones, such as follistatin, etc.; The cells around the oocytewere called cumulus cells (CC). Oocytes were tightly wrapped in CC around, whichwere highly specific cytoplasmic projections through the zona pellucida withproximity oocyte to form gap junctions to transfer nutrients for oocyte. Numerous studiesshow that oocytes are the strong irritants to the proliferation MGC and CC, but also caninhibit the death or apoptosis. Recent studies focused on the oocyte secreted factors, majorityare the study on GDF9and BMP15. BMP15attracted the majority of researchers’ attention asan oocyte-specific secreted factor. The effect of BMP15on granulosa cells apoptosis can beinhibited by the follistatin which is by the mural granulosa cells secretion. Thus, this studyused the BMP15and cumulus cells as the starting point to clarify the pathway factors inBMP15inhibition cumulus cell apoptosis.Cultured porcine cumulus cells to filter medium supplemented with the appropriateconcentration of BMP15, Fow cytometry was used to detect apoptosis in cumuluscells.The results showed that BMP15added with increasing concentrations lead to theapoptosis rate of cumulus cells decreased. Most of the cumulus cells were death when theconcentration of BMP15was200ng/ml; The shape and growth of cumulus cells in goodcondition when the concentration of BMP15was100ng/ml; compared to the concentration ofBMP15was0,25,50ng/ml, the apoptosis rate was significantly reduced.To clarify the associated pathways of the effect of BMP15on cumulus cells, weuse high-throughput sequencing to detect the different gene expression of cumuluscells with BMP15+and BMP15-, and use RT-qPCR to test and verify the sequencingresults. Cumulus cells with BMP15treatment generate195differentially expressedgenes compared with untreated cumulus cells, including123genes were up-regulated, 72genes were down-regulated, only14genes associated with apoptosis, the CCL2was up-regulated and the FBN1was down-regulated which change is obvious.Primary cultured cumulus cells in vitro then silencing CCL2showed that, thegene expression changes of CCL2can affect the effect of BMP15on cumulus cellsapoptosis. Flow cytometry results showed that the apoptosis rate of cumulus cells withBMP15treatment and silencing CCL2gene displayed significantly increasedapoptosis rate (p <0.05). Detect the expression of apoptosis-related genes found eithermRNA levels or protein levels, the ratios of Bcl2to Bax were significantly reduced,similarly shows that cumulus cells with BMP15treatment and silencing CCL2geneincreased the apoptosis rate. MTS method was applied to meature the proliferation ofcumulus cell, the results show that cumulus cells with BMP15treatment and silencingCCL2gene lead to the cell proliferation weakened or even decreased, indicating thatin the case of CCL2gene missing, the effect of BMP15promote cumulus cellproliferation were suppressed.FBN1gene silencing also affects the effect of BMP15protection cumulus cellsapoptosis. Apoptosis results showed that the apoptosis rate of cumulus cells withBMP15treatment and silencing FBN1gene was significantly lower (p <0.05). Detectthe expression of apoptosis-related genes found either mRNA levels or protein levels,the ratios of Bcl2to Bax were significantly increased, indicated that the cumulus cellswith BMP15treatment and silencing FBN1gene reduce cumulus cell apoptosis rate.The MTS results show that cumulus cells with BMP15treatment and silencing FBN1gene promote the cell proliferation,indicating in the case of FBN1gene missing,theeffect of BMP15promote cumulus cell proliferation were strengthened.Therefore, BMP15play an important role in cumulus cells apoptosis, the effectof BMP15protecting cumulus cells apoptotis were influenced by the expression ofCCL2and FBN1. BMP15may promote oocyte growth and development byprotecting cumulus cell apoptosis, which is providing a new insight involving in theregulatory mechanisms of the female germ cells development for BMP family.