Effects Of BMP15 And GDF9 Through MiRNA-375-BMPR2 Pathway On Bovine Cumulus Cells Proliferation And Apoptosis

Follicles are the basic developmental units in the mammalian ovaries.They are mainly composed of the oocyte and the surrounding cell layers,the follicular membrane and the follicular fluid.Granulosa cells in the cell layer can be divided into mural granulosa cells(MGC)and cumulus cells(CC),Both of which play a decisive role in follicular development.As a type of granulosa cell,cumulus cell proliferation and apoptosis play vital roles in follicular development and oocyte maturation.Bone morphogenetic protein 15(BMP15,or GDF9B)and growth differentiation factor 9(GDF9),which are secreted by oocytes,are important regulators of follicular growth and development and ovarian function.BMPR2 is the common and only type II receptor of BMP15 and GDF9.At present,it is known that multiple mirco RNAs can regulate the expression of the BMPR2 protein,but most previous studies have focused on stem cells or mesenchymal stem cells.The previous results from our group that BMPR2 is a direct target gene of mi R-375.Inhibition of BMPR2 expression has the same inhibitory effect on cell differentiation as does overexpression of mi R-375.Therefore,mi R-375 may have a potential role in regulating BMP15-and GDF9-related pathways.However,the specific routes of action and mechanisms are not clear.In this study,bovine cumulus cells were first separately incubated in vitro,using Cell Counting Kit-8(CCK-8)analyses and flow cytometry during the in vitro culture process.The concentrations of BMP15 and GDF9 alone or in combination are selected in the screening medium,were used to determine the effects of BMP15 and GDF9 on bovine cumulus cells proliferation and apoptosis.The results showed that the addition of 100 ng/m L BMP15 or 200 ng/m L GDF9 or the combined addition of 50 ng/m L BMP15 and 100 ng/m L GDF9 effectively inhibited bovine cumulus cell apoptosis and promoted cell proliferation.To investigate the regulatory effect of mi R-375 on BMPR2 in the proliferation and apoptosis,cumulus cells were treated with BMP15 and GDF9 alone or in combination.After addition of appropriate concentrations of BMP15,GDF9 and BMP15+GDF9,the expression of endogenous mi R-375 in cumulus cells was detected by Stem-loop RT-q PCR.The changes of BMPR2 m RNA and protein were then detected by quantitative PCR and Western Blot.The results can be seen that the addition of BMP15 or GDF9 caused mi R-375 down-regulation and BMPR2 up-regulation.BMP15/GDF9 negatively regulated mi R-375 expression and positively regulated BMPR2 expression.In addition to type II receptors,BMP15 and GDF9 can bind to their respective type I receptors ALK4,ALK5 and ALK6 to regulate follicular growth and granulosa cell growth and differentiation through the Smad signaling pathway.Therefore,mi R-375 mimics,mi R-375 inhibitor and BMPR2 si RNA were synthesized and used ribo for transfection experiments.Then,RT-q PCR and Western Blot analysis were used to detect changes after transfection in the expression levels of the BMP15/GDF9 type I receptors ALK4,ALK5 and ALK6;Western Blot analysis was used to detect changes the phosphorylation levels of Smad2/3 and Smad1/5/8(p-Smad1/5/8 and p-Smad2/3),which are key signaling pathway proteins downstream of BMP15/GDF9.The results showed that mi R-375 can indirectly regulate the expression of ALK4 via its target gene BMPR2 but has no effect on ALK5/6.ALK4 itself is a pro-apoptotic factor,the addition of mi R-375 also upregulates ALK4,which is consistent with the function of mi RNA-375 itself in promoting apoptosis.In addition,BMP15,GDF9,BMP15/GDF9,mi R-375 and BMPR2 had no significant effect on the level of Smad1/5/8 or Smad2/3 but had varying degrees of influence on p-Smad1/5/8 and p-Smad2/3.These results indicated that the BMP15 and GDF9 activation of Smad1/5/8 and Smad2/3 may involve multiple pathways,but mi R-375/BMPR2 is one of the key pathways.In terms of the added concentrations,BMP15/GDF9 has a pronounced synergistic effect on the p-Smad1/5/8 and p-Smad2/3 pathways.Cumulus cell expansion had important effects on oocyte maturation and quality,whereas PTX3,HAS2 and PTGS2 are important genes that affected cumulus cell proliferation in vitro COCs culture.Therefore,in this study,the regulation of mi RNA-375 and BMPR2 on these key factors was clarified by RT-q PCR and Flow cytometry.The results showed that the Bcl2: Bax ratio was significantly reduced and the proportion of cumulus cell apoptosis was increased after the over-expression of mi R-375 or inhibition of BMPR2.Overexpression of mi RNA-375 and knockdown of BMPR2 both inhibited the expression of PTX3,HAS2 and PTGS2,whereas the addition of inhibitor led to the opposite result.The addition of BMP15 and GDF9 did not prevent apoptosis caused by mi R-375 mimics but had a mitigating effect on apoptosis caused by inhibition of BMPR2.This study showed that mi R-375 could regulate the expression levels of PTX3,HAS2 and PTGS2 through its target genes.Under normal physiological conditions,although mi R-375/BMPR2 has a regulatory effect on PTX3,HAS2 and PTGS2,this effect is dependent on BMP15 and GDF9 or is dominated by BMP15 and GDF9,BMP15 and GDF9 have a more effective pathway for the regulation of cumulus cell proliferation.In summary,this study focused on mi R-375 and explored how BMP15/GDF9 affects the expression of mi R-375,how it acting on BMPR2 and regulating cumulus cell proliferation and apoptosis through the Smad2/3 and Smad1/5/8 pathways.The results of the study clarify the regulation of mi R-375 and BMPR2 by BMP15 and GDF9,found that BMP15 and GDF9 have significant synergistic effects in activating the Smad signaling pathway and cumulus cell proliferation.Our study provides a theoretical basis for gaining an in-depth understanding of the molecular regulation mechanism of follicular development and oocyte maturation and provides theoretical support for the establishment of a more comprehensive oocyte in vitro maturation system and for the full evaluation of animal egg resources.