| Cryopreservation of oocyte are particularly highlighted in vitro embryo production technology research. But in the process of its frozen, are susceptible to a series of injuries. With the rapid development of molecular biology technology, the researchers on oocyte freezing damage gradually turn to the molecular mechanism of the study, including the change of transmembrane protein may cause abnormal sperm-egg combination and fusion. CD9,which is identified to participate in sperm-egg fusion, is currently the only protein from egg, and the relevant reports show CD81 is also involved in the process. Interactions on CD81 and CD9 forms a complex which is likely to play a role in sperm-egg interaction. However, their roles in membrane fusion is unclear. Using immunofluorescence staining techniques, this study first detected the change of the cytoskeleton on different developmental stages of sheep oocytes before and after frozen-thawed; the distribution of CD81 in sheep ovarian tissue,testis and epididymis tissues,oocytes and sperm. By real-time fluorescent quantitative PCR technology,we tested the expression of CD81 mRNA in different developmental stages of sheep oocytes before and after frozen-thawed, and compared and analyzed the expression difference of CD81 mRNA. Using IVF, we alse examined Anti-CD81 monoclonal antibody and GV and MⅡoocytes of frozen-thawed effected on sheep sperm-egg fusion. Following results:1 The study on oocyte cytoskeleton after frozen-thawed.The results showed rate of normol morphology from frozen-thawed GV and MⅡstage oocyte is significantly lower,and microfilament, microtubule and nuclear had varying degrees of damage. Cryopreservation damaged its structure and cytoskeleton, and may affect the later development of oocytes.2 The detection on expression of CD81 in sheep ovarian tissue, oocyte, the organization of testis and epididymis and sperm.The results showed:(1) CD81 expressed on primary, secondary and tertiary follicles follicle cells, and expressed on the egg and mound, granulosa cells, basal membrane, endometrial cells and cells on the outer membrane from mature follicle;(2) CD81 expressed on GV and MⅡstage oocyte, but CD81 of GV stage oocytes mainly expressed on the cell membrane and that of MⅡ stage was on the egg cell membranes and transparent:(3) CD81 expressed on sheep testicular seminiferous tubule epithelial cells, and near the lumen base signal was stronger. In epididymis tissue,CD81 were detected on ciliated epithelium cells and the free edge of the cilia of pseudostratified ciliated columnar epithelium cells. CD81 expressed significantly positive signal on lumen of epididymidis corpus and cauda,basal cell layer expression;(4) CD81 expression was strong in the acrosome of sperm, no CD81 protein signal in the rest of the parts;3 The analysis of CD81 mRNA expression in different development stages of freezing oocytes. The result showed:CD81 mRNA expression quantity of fresh MⅡoocytes was significantly higher than the frozen MⅡ(P<0.01), and frozen MⅡ was significantly higher than fresh GV and frozen GV(P<0.01). But fresh GV had not significant difference with frozen GV;4 CD81 monoclonal antibody and Cryopreservation effected on sperm-egg combination and fusion. Oocytes with CD81 mAb incubation experiment results showed that:adhesion counts of single sperm and penetration rate of single sperm on oocyte(4.2±0.8,30%)was significantly lower than the control group (13±1.22,85%), and the differences were significant. Effect of GV and MⅡ stage oocytes after frozen-thawed on IVF results showed that adhesion counts of single sperm and penetration rate of single sperm on oocyte respectively (5.8±0.8,45%)was significantly lower than the control group (13±1.22,85%), Samely, that of MⅡ stage oocytes respectively (7.0±1.0,62%)was significantly lower than the control group, the differences were alse significant (P<0.01),and MⅡ and GV stage oocytes had significant difference (P<0.01). |
PDF Full Text Request