The Detrimental Effect Of Cumulus Denudation On Rabbit Oocytes

Maturation of oocytes after cumulus denudation is necessary in many experiments, such as germinal vesicle (GV) transfer and the investigation on the effect of cumulus cells on oocytes. Studies on bovine and mouse suggested that the denuded oocytes (DO) had the ability to mature and develope to blastocyst, although the rate of development is low when compared with cumulus-oocyte complexes (COC). Furthermore, the maturation and development of DOs in these animals can be partly improved when cocultured with cumulus cells. However, we found that rabbit oocytes could not mature when denuded. Therefore, we presumed that the process of denuding may have more detrimental effect on rabbit oocytes than on other animals. Because the oocyte communicates with corona cells through gap junction composed of connexons, denuding mechanically may have adverse effect on the oocyte membrane. In addition, it has been demonstrated that denuding by pipette may generate a shear stress that will induce apoptosis in oocytes. In order to solve the above problems, we compared the influence of denuding on the maturation and development of rabbit and mouse oocytes, two typical models. The results were summarized as follows:1. Experiments in which heptanol as a blocker suggested that cumulus cells promote maturation and cleavage in rabbit oocytes and development in mouse oocytes, but they had little effect on rabbit oocyte development and mouse oocyte maturtion.2. Experiments by denuding and coculture indicated that the maturation and development of oocyte was influenced by cumulus cells in a cumulus cell-oocyte gap junction dependent manner. 3. About 50% of rabbit oocytes matured to MII when the gap junctions were completely blocked by heptanol. However, when denuded, rabbit oocytes did not mature. The results suggested that the process of denuding injured rabbit oocytes severely. It wasn't this case in mouse oocyte and the process had little influence on mouse oocyte.4. The oocyte membrane integrity was assessed by supravital staining using Hoechst33258. Using this stain membrane injury was found in rabbit oocytes but not in mouse oocytes. The rabbit oocyte injured degenerated in culture.5. The apoptosis of oocyte was detected by Bcl-2 antibody. The results indicated that the process of denuding induced apoptosis in rabbit oocytes but not in mouse oocytes.