Studies On Expression Pattern Of ZNF33B In Bovine Ovary And Its Regulating Mechanism In Oocyte Maturation

Zinc finger protein which wildly expressed in eukaryotes and prokaryotes, selective binds with specific target structure as a transcriptional factor. It plays an important role in the regulation of gene expression, cell differentiation, gamete function and embryonic development.Bovine ZNF33 B has a full-length of 35,440 bp, c DNA contains a full-length of 1358 bp, which encodes a 137 amino acid protein. ZNF33 B contains krüppel correlation Conservative C2H2 type zinc protein, also known as KRAB zinc protein. ZNF33 B m RNA expressed widely in other organs(tissue), So far, few researches are focused on the functions of ZNF33 B.Especially in the ovary, only a small amount of literature showed that the expression of ZNF33 B gene was significantly changed in the cumulus cells in superovulation ovary, but the expression pattern of ZNF33 B and its role in ovarian tissure, as well as its functions are still unclear.We found a mutation of g.-61G>T in 5’UTR in bovine ZNF33 B. The frequency of allele gene G and T are 0.3489 and 0.6511, respectively. And the frequency of genotype AA, AB and BB are 0.1017, 0.4964 and 0.4029 respectively. The statistical analysis showed that BB cattle had significantly more total oocytes, available embryos,degenerated embryos and unfertilized oocytes than AA ones(P<0.05 or P<0.01). AB type individuals’ superovulation character is between the former two. Meanwhile, we also found a 124-bp insertion in the first intron, and the ratio of the mutation is 12.95%. However, there is no significantly difference in the superovulation performance between the genotypes.(P>0.05).In order to investigate the expression of ZNF33 B gene in ovarian tissues, we study the expression pattern of ZNF33 B in bovine ovary by using the methord of QRT-PCR,Western Blot, immunohistochemistry and immunofluorescence. Results show that the expression of ZNF33 B in ovarian cortex in the follicular phaseand and medulla was much higher than ovarian cortex during corpus phase. The expression of ZNF33 B decreased gradually by the development of corpus luteum. ZNF33 B highly distributed in the cytoplasm of oocytes and granulosa lutein cells, this protein was also detected in the cytoplasm of cumulus cells and ovarian stroma. ZNF33 B distributed in nucleus of bovine early embryos which were generated by in-vitro fertilization or parthenogenetic activation.Based on the differential expression in bovine ovarian tissue and the potential role in oocyte maturation, of ZNF33 B gene in ovarian tissues and early embryo, we analyzed the role of this gene in the development of cumulus cells and oocyte maturation. First,QRT-PCR results showed that ZNF33 B m RNA significantly increased by the oocyte nucleus maturation in cumulus cells and oocytes(P<0.01), Silencing ZNF33 B gene by RNAi technique, the primary culture cumulus cells had much higher early apoptosis rate(p<0.05), late apoptosis rate(p<0.01) and more apoptosis cells(p<0.01), It maked that low expression of ZNF33 B promotes apoptosis of cumulus cells.Subsequently, in order to further explore the role and mechanism of ZNF33 B in oocyte maturation, in this study, we injected si RNA into GV cytoplasm of oocytes, and then they will go though in-vitro maturation, to clarify the effect of ZNF33 B on oocyte maturation by observing the first polar body.The oocytes which were tranfected by RNAi had lower maturation rate than the control(p<0.01);Several genes involves in oocyte maturation. We conducted further experiments to study the effect of ZNF33 B on oocyte maturation. We found several key genes which have important roles in oocyte maturation were significantly down-regulated in the ZNF33 B knockdown oocyte, included Cdc25 C, PDE3 A, CCNB1 and RBBP4(p<0.05 or p<0.01), but no change in the expression of EGFR or RBL2(P>0.01).In summary, ZNF33 B gene polymorphism effects greatly on the ovulation characteristics of cow, it can be used as a molecular marker of bovine superovulation and applied to early breeding. Expression of ZNF33 B has temporal and spatial characteristics, ZNF33 B could effect the growth and development of cumulus cells as well as influente oocyte maturation by regulating the expression of specific cytokines directly. In this study, the effect of ZNF33 B gene on oocyte maturation was identified from the level of gene and protein level.This study provides theoretical basis for analysising the molecular regulation pathway and effect of Zinc finger protein and mechanisms of oocyte maturation, but also provides a new evidence for improving the fertility of female animals and early selection.