Effect Of CD9 On The Developmental Competence Of Freezing And Thawing Oocytes In Sheep

Cryopreservation of mammalian oocytes have important value to biology and human medicine research and development, it can provide effective technical measure for biotechnology experimental material, like mammalian germplasm resource preservation, human assisted fertilization technology. The main reason, about oocytes are difficult to combine and fuse with sperm even fertilization, may oocytes irreversible changes in membrane protein structure during the ultra low temperature freezing preservation process, which changes in transmembrane protein maybe cause adhesion failure or sperm cannot penetrate the plasma membrane of oocytes.CD9 belongs to transmembrane 4 super family, is widely expressed in various tissues and cells, involved in sperm egg binding, fusion and other important life activities. In the study, as a model with sheep, using by immunofluorescence staining, quantitative real-time PCR and western blot technology to detect the differental expression of CD9 in fresh and freezing GV and MⅡ oocytes; through in vitro fertilization (IVF) technology, investigate effect of CD9 expression on sperm egg binding and fusion ability before and after cryopreservation. The following results were abtained by above research methods:1 In the study, the GV stage oocytes were collected, and GV stage oocytes in vitro maturation to MⅡ stage oocytes. In further, randomly divided them into fresh goup and frozen group. Investigate the effect of freezing on the oocytes developmental ability by using freezing solution with EG,DMSO and PROH. The results showed that the rate of frozen-thawed GV stage oocytes maturation (31%) was significantly lower than fresh group (75%) (P<0.05). The results of cell morphology observation showed that the normal rate of frozen-thawed GV and MⅡ stages oocytes respectively was 55% and 70%, the rate of fresh GV and MⅡ stages oocytes’ respectively was 92% and 90%, there was a significant difference between two groups of data (P<0.05).2 Immunohistochemical technique was used to locate CD9 protein in sheep ovary. Foud that the CD9 expressed in follicular granulosa cells and cumulus cells. And the CD9 fluorescence signal can be detected in the primordial follicle, the fluorescence signal was gradually increased with the follicle development, and it reached the strongest in the mature follicle.3 Quantitative real-time PCR detect CD9 mRNA expression level. The results showed that the expression of CD9 mRNA in fresh MII oocytes was significantly increased(P<0.05), then in frozen MII oocytes(P<0.05), and in frozen GV oocytes was the lowest(P<0.05). The results of western blot and the expression of CD9 fluorescence on the oocytes befor and after cryopreservation had a same trend with the results of expression of mRNA, it was means, CD9 expression in the fresh MII oocytes was the highest, then in the frozen MII oocytes, and frozen GV oocytes was the lowest.4 The effect of CD9 on the average number of sperm binding to oocytes and sperm penetration rate were detected by in vitro fertilization technology. The results showed that frozen GV oocytes after IVM-IVF, the average number of sperm binding to oocytes and the penetration rate respectively was 2.9±0.6,34%; frozen MII oocytes after IVF, the values respectively was 3.5±0.1,41%. Above two groups of experimental results were significantly lower than that of those binding to control(6.0±0.5,78%)(P<0.05).