Effects Of Cumulus Cells On Maturarion In Vitro And Subsequent Developmental Competence Of Porcine Oocytes

With the development of embryo biotechnology, the demands for oocyte quality derived from maturation in vitro are increasing. The research on maturarion in vitro and subsequent developmental competence of oocytes becomes the key step for the technology of in vitro fertilization (IVF), in vitro production of embryos (IVP) and somatic cell nuclear transfer (SCNT) etc. The quality of oocytes maturated in vitro is currently not as good as those maturated in vivo. It's still one of the most important tasks to understand deeply the rule of maturarion of oocytes, to improve in vitro culture technology and consummate in vitro maturation system of oocytes. Lots of results suggest that cumulus cells are very important during oocyte maturation. To research effectes of cumulus cells on maturion in vitro and subsequent developmental competence of oocytes has the important theory significance and the practical value. In this study, naked oocytes (NO), porcine cumulus cells (pCC) co-culture with naked oocytes, cumulus oocyte complex (COC) and single ovarian follicle were cultured in vitro in order to research effectes of cumulus cells on maturation of oocytes in vitro and development of IVF embryos. Real-time PCR was used for the quantitation of Fas/FasL mRNA to analysis expression of Fas/FasL mRNA during oocytes IVM, IVF and embryos development, provide a reference for further understanding of molecular mechanism of effectes of cumulus cells on oocytes IVM, IVF and subsequent developmental competence. The results are listed as follows:1. The naked oocytes, oocytes co-culture with pCC and the COC were cultured for 48h, all of their zona pellucida diameter of oocytes significantly grew bigger than GV stage oocytes (158.41±5.82,160.02±4.05,160.38±5.98 vsl48.96±5.30; P>0.05), but the zona pellucida diameter of oocyte among all three groups were not significantly different (P>0.05). 2. The pbl rate of COC group was highest (71.25%). It was significantly higher than pCC co-culture group NO group and follicular group (49.32%,31.46% and 27.14%; P<0.05). The pbl rate of pCC co-culture group was higher than NO group and follicular group. The follicular group's pbl rate was the lowest. The rate of Class A oocyte of COC group was significantly higher than the other groups The rate of Class A oocyte of co-culture group is significantly higher than NO group and follicular group. The rate of Class C oocyte of COC group was the lowest (8.33%). The rate of Class C oocyte of follicular group was 18.57% and pCC co-cultrue group was 24.43%. The rate of Class C oocyte of NO group was the highest (44.60%). The Survival rate of COC group (90.83%) is significantly higher than the others. NO group was the lowest (53.33%). All the results suggest that oocyte maturation is regulating by cumulus cells through both gap junction and paracrine.3. Although the rate of pbI of follicular group was lower; the survival rate of follicular was higher than NO group and the rate of Class B oocyte of follicular group is significantly higher than the others. The rate of pbl have some connection with oocyte nuclear maturation, maybe there are some factors to depressing oocyte nuclear maturation in whole follicular culture, which cause the rate of pbI keep low. But the high rate of class B oocyte at least suggests that whole follicular culture is a good way to portect oocytes.4. The developmental and survival rate of COC group embroys were significantly higher than pCC co-cultrued group and NO group. in cleavage rate,4 cell rate,and blastocyst rate; pCC co-cultrued group was higher than no group,but there were no significantly difference between them.5. Expression of Fas/FasL mRNA of oocyte did not grow significantly from GV stage to MⅡstage. the level of Fas/FasL mRNA were low at GV oocyte and MⅡstage in all groups. Expression of Fas/FasL mRNA of MⅡoocyte in COC group pCC co-cultrue group and NO group were higher than GV oocyte, but no significantly difference. During oocyte maturation, Fas/FasL signal path may be not the main regular way, it suggest there are another signal path to regular oocyte apoptosis in IVM. Further research is required.6. Expression of Fas/FasL mRNA was detected in all stage of early embryos. At 2 cell stage, Fas/FasL mRNA expression in NO group sharply increased, being significantly higher than that of pCC co-cultrue group, and pCC co-cultrue group was significantly higher than that from COC group. At 4 cell stage, Fas/FasL mRNA expression in pCC co-cultrue group were higher than that of COC group significantly (P>0.05). It suggests that the cumulus cells behave evidently inhibitive effects on the expression of Fas/FasL mRNA of IVF embryos during IVM of oocytes.