Effects Of BCL2L10 On In Vitro Maturation Of Buffalo Oocytes

BCL2-like protein-10(BCL2L10),a member of the BCL-2 family,it has been reported that it abundantly and specifically expressed in adult human and mouse oocytes and played a very important role in oocytes maturation and early embryonic development.This study aims to investigate the expression pattern of BCL2L10 in buffalo tissues and its effect on the in vitro maturation of buffalo oocytes,so as to realize the further mechanism of oocytes maturation and provide theoretical guidance for improving the in vitro maturation rate of buffalo oocytes.The thesis consists of three chapters:1.Cloning of buffalo BCL2L10 gene and its expression in various tissues.The results showed that the gene we cloned was 600 bp,and the coding sequence was 558 bp,coding 185 amino acids.Sequence analysis showed that the nucleotide sequence of BCL2L10 gene was high homologies while the amino acid sequence was low homologies with cattle,sheep,mice,wild boar and human,indicating that this protein had some evolution among different species.Using bioinformatics technology to predict the structure and function of BCL2L10 protein,it was found that BCL2L10 was membrane protein through the C-terminal transmembrane(TM)domain anchored in the mitochondrial membranes or other organelles membranes,which belong to BCL-2 family and may be involved in cell apoptosis.Furthermore,the qPCR results showed that BCL2L10 was expressed predominantly in ovary,smaller amounts in testis,and none in other tissues,while BAX was expressed in all buffalo tissues.2.The establishment of the expression patterns of BCL2L10 and BAX in buffalo oocytes and early embryos.The expression of BCL2L10 and BAX in the ovary,oocytes and early embryos of buffalo were detected by immunohistochemistry,immunofluorescence staining and real-time quantitative PCR.The results showed that BCL2L10 protein was mainly concentrated in the oocytes but none of staining reaction was found in granular cells and cumulus cells in ovary.The expression of BCL2L10 was up-regulated in the process of oocyte maturation in buffalo,while BAX gene was no significantly changed.Furthermore,during the buffalo early embryos development,the expression level of BCL2L10 presented earlier increase and later decrease trend,and highest in 8 cell stage,on the contrary,the expression level of BAX was low initial stage while rapid rise after 8 cell stage,and highest in blastocyst stage.3.Effect of inhibiting the expression of BCL2L10 gene by siRNA interference on in vitro maturation of buffalo oocytes.siRNA was transfected into GV buffalo oocytes using the method of cationic liposome transfection to explore the effect of down-regulating the expression of BCL2L10 gene on maturation rate and cleavage rate of parthenogenetic activation.The results showed that the expression of BCL2L10 gene can be effectively inhibited by siRNA interference,and the maturation rate and cleavage rate of buffalo oocytes were significantly decreased(48.18%?46.65%vs 33.5%;54.72%?61.32%vs 37.52%,p<0.01)after down-regulating the expression of BCL2L10 gene.At the same time,the expression of BAX,BCL-2,Caspase-3 and Caspase-9 genes were detected to verify the effect on apoptosis related genes after down-regulating the BCL2L10 gene.The results showed that the expression level of Caspase-9 gene was up-regulated through down-regulating BCL2L10 gene,and the apoptotic process was initiated.In conclusion:(1)The coding sequence of buffalo BCL2L10 gene is 558 bp and coding 185 amino acid.BCL2L10 protein have a certain evolution in different species,and mainly expressed in ovary,while BAX is expressed in all buffalo tissues.(2)The expression of BCL2L10 protein is mainly concentrate in oocytes in buffalo ovarian tissues,meanwhile,BCL2L10 and BAX completely express in the process of oocyte maturation and the early embryonic development.(3)The maturation rate of buffalo oocytes is significantly decrease through down-regulating BCL2L10 gene probably due to the up-regulation of Caspase-9,and then initiating the apoptotic process and disrupting the process of meiotic.