BMP15/GDF9 Regulates Proliferation And Apoptosis Of Bovine Cumulus Cells By BMPR2

During the development of mammalian follicles,granulosa cells being the character protect and provide nutrients to oocytes.Oocytes are not actually a passive recipient.Oocytes can synthesize factors that act on granular cells by paracrine manner,that regulate the physiological state of granulosa cells.The bidirectional signal exchange process between oocytes and granulosa cells promotes the normal and orderly development of follicles.Among the factors derived from oocytes,the TGFβ family is a very important class.Bone morphogenetic protein 15（BMP15）and Growth differentiation factor 9（GDF9）are two growth factors secreted by oocytes and belong to the TGFβ superfamily.BMP15 and GDF9 can bind to receptors on cumulus cells which would cause a cascade of downstream genes,affecting the proliferation and apoptosis of cumulus cells,therefore regulating follicular development and oocyte maturation.BMPR2 is a TGFβ type II receptor expressed on cumulus cells,and BMP15 and GDF9 bind to BMPR2 through different type I receptors,respectively,thereby regulating downstream metabolic reactions.Our previous research has proved that the addition of appropriate concentrations of BMP15 and GDF9 in bovine cumulus cells can promote the expression of BMPR2 and regulate cell proliferation and apoptosis.However,the mechanism of how the two affect BMPR2 is not yet clear.In order to further explore the mechanism of BMP15 and GDF9 mediated by BMPR2 in the regulation of bovine cumulus cells,the following work was carried out.First,we use high-throughput sequencing technology to construct mRNA differential expression profiles of BMP15 and GDF9-treated cumulus cells and control groups.The results showed that 246 genes were up-regulated and 183 genes were down-regulated by BMP15 alone;218 genes were up-regulated and 150 genes were down-regulated by GDF9 alone;228 genes were up-regulated and 202 genes were down-regulated by adding BMP15 and GDF9.We performed statistical and bioinformatic analysis of these genes,as well as screened four genes that may be involved in the synergistic effects of two cytokines regulating cumulus cell proliferation and apoptosis: USP42,YWHAH,SMAD6,and NOG,as genes for subsequent studies.Next,the selected genes were subjected to siRNA knockdown to detect changes in BMPR2 expression,apoptosis,and proliferation.The results showed that the addition of BMP15 and GDF9 inhibited cumulus cell apoptosis.As knocking down USP42 and YWHAH,BMPR2 was up-regulated;there was no significant change in apoptosis.CCND2,PCNA and CDC42 were significantly up-regulated.After knockdown of SMAD6 and NOG,BMPR2 was down-regulated significantly,apoptosis was increased,p53 was significantly up-regulated,and bcl-2 was significantly down-regulated.These results indicate that BMP15 and GDF9 are regulating downstream genes mediated by USP42,YWHAH,SMAD6 and NOG,thereby regulating cell proliferation and apoptosis.In order to initially explore the effect of non-coding RNA on the regulation of BMP15 and GDF9 on cumulus cells,we performed sequencing of the differential expression profiles of circRNAs in the four groups.1706 circRNAs were obtained by sequencing.We were carrying on bioinformatics analysis of these circRNAs and screening for differential circRNAs.Through functional annotation and enrichment analysis of the source genes,we found that the differential circRNA is related to exercise,reproduction,bio-adhesion,growth,and hormone secretion.The differential gene results showed that 3 circRNAs were up-regulated and 6 circRNAs were down-regulated in BMP15 group;12 up-regulated and 24 down-regulated in GDF9 group;15 circRNAs up-regulated and 13 down-regulated in BMP15 and GDF9 group.Circ15/a₇5,circ_ENSBTAG00000012691₁,and circ_n/a₃03 are different in the BMP15 and GDF9 groups in the joint addition combination group.It is speculated that they may be related to the additive effects of BMP15 and GDF9.Quantitative PCR analysis indicated that the expression levels of these three circRNAs were consistent with the sequencing results.We predicted the target miRNAs of these three circRNAs and verified by fluorescence quantitative PCR that the target miRNA expression was just opposite to the circRNA trend.These results indicate that BMP15 and GDF9 may regulate miRNA expression through circRNA as a miRNA sponge,thereby affecting downstream target genes,regulating the state of cumulus cells and affecting follicular development.To further investigate the role of circRNA in the regulation of BMP15/GDF9 on cumulus cells,this study screened miRNAs that specifically target BMPR2,and identified BMPR2 as a target gene for miR-187 by dual luciferase assay;bta_circRNA₁1396 specifically binds to miR-187.After inhibiting bta_circRNA₁1396 by siRNA,we detected the expression of miR-187 and the target gene BMPR2.The results showed that knockdown of bta_circRNA₁1396 up-regulated the expression of miR-187,down-regulated the expression of BMPR2,and increased apoptosis.Therefore,bta_circRNA₁1396 may act as the miRNA sponge,promote BMPR2 expression,inhibition of apoptosis by inhibiting the activity of miR-187.In summary,the results provide a molecular basis for further revealing the mechanism of action of BMP15 and GDF9 on bovine cumulus cells,which provides a theoretical foundation for further study of follicular development.