The Effect Of Follistatin On The In Vitro Maturation Of Buffalo Oocytes

Follistatin,a member of the transforming growth factor-β(TGF-β)superfamily,has been identified as an inhibitor of follicle-stimulating hormone(FSH).With the exploration of the function of FST,it was found that it may play an important role in animal reproduction.Therefore,this study first analyzes the structural characteristics of buffalo FST and its expression in various tissues of buffalo,and then through the interference and overexpression of FST in the cumulus-oocyte complex(COCs),explored the effect of FST on buffalo oocytes IVM and its mechanism of action,meanwhile,provided certain theoretical guidance for further improving the efficiency of IVM in buffalo oocytes.The results of this study are as follows:1.The cloning and expression of buffalo FST gene encoding region were studied.Using the total RNA of buffalo ovary as a template,the cloned FST gene coding region has a total length of 1035 bp,which encodes a total of 344 amino acids.Comparing the cloned buffalo FST amino acid sequence with the homology of cattle,chicken,sheep,human,wild boar and other species,it is found that the homology between buffalo and cattle is the highest,and the homology with chicken is the lowest.Using bioinformatics technology to predict the structure and function of the FST protein,it is found that the buffalo FST protein is a classic secreted protein.The real-time fluorescence quantitative test results showed that the expression of Buffalo FST was the highest in the lung,followed by the ovary,and almost no expression in the testis.2 Analyzed the expression of buffalo FST during oocyte maturation and early embryonic development.The results showed that buffalo FST was expressed in granulosa cells,oocytes and basement membrane cells of buffalo follicles at different stages,and expressed in GV and MII buffalo oocytes,and FST expression was significantly up-regulated after maturation(P<0.05).During the early embryonic development of buffalo,the expression level of FST was significantly different in each developmental stage(P<0.05),showing a maternal gene expression pattern.3.The effect of interference or overexpression on the in vitro maturation of buffalo oocytes.During the maturation of buffalo oocytes,cationic liposome transfection was used to interfere with the expression of FST gene in COCs.It was found that si RNA basically inhibited the expression level of FST gene in buffalo cumulus cells.The expression of FST has no significant effect;while interference with the expression of FST gene in COCs has no significant effect on the elimination rate of the first polar body of buffalo oocytes(P>0.05),but the cleavage rate and blastocyst rate of embryos after parthenogenetic activation Significantly reduced(P<0.05);in addition,after interference with the FST gene expression in COCs,the location of mitochondria in buffalo oocytes was abnormal,the distribution of cortical granules did not change significantly,and the cumulus expansion area was significantly reduced(P<0.05).The apoptotic rate of cumulus cells was significantly increased(P<0.05),and the expression levels of TGF-β/SMAD pathway related genes TGF-β,SMAD2,and SMAD3 were significantly reduced(P<0.05),but the expression level of SMAD4 was significantly increased(P<0.05).When the FST gene was overexpressed in COCs,it had no significant effect on the discharge rate of the first polar body of the buffalo(P>0.05),but the cleavage rate and blastocyst rate of the embryo after parthenogenetic activation increased significantly(P>0.05);And after overexpression of FST,the cumulus expansion area increased significantly(P<0.05),and the expression levels of anti-apoptotic gene BCL2 and TGF-β/SMAD pathway related genes TGF-β,SMAD2,SMAD3 increased significantly(P<0.05),The expression levels of SMAD4 and the pro-apoptotic gene BAX decreased significantly(P<0.05)The above results indicate that FST gene is highly expressed in buffalo ovarian tissues,and interference with FST gene is not conducive to the maturation of buffalo oocyte cytoplasm,and overexpression of FST gene can increase the developmental potential of buffalo oocytes.This effect of FST may be related to the activation of the TGF-β/SMAD signaling pathway,the up-regulation of the cumulus expansion genes HAS2,PTX3,TNFAIP6 and the anti-apoptotic gene BCL2,and the down-regulation of the pro-apoptotic gene BAX.