无卵丘的水牛卵母细胞体外成熟方法的初步探讨

A Preliminary Study on the Maturation of Buffalo Denuded Oocytes in vitro

主要探讨无卵丘水牛卵母细胞体外成熟的可行性,以便为研究卵母细胞成熟机理提供模型。无卵丘的水牛卵母细胞随机分为5组,然后分别进行直接成熟培养(M1),与卵丘细胞单层共培养(M2),用未扩展的卵丘细胞块包围培养(M3),与扩展的卵丘细胞团共培养(M4)和用卵巢组织包围培养(M5)。无卵丘的水牛卵母细胞体外成熟培养24 h后检查第一极体(PB1)排出率,随后对这些卵母细胞进行孤雌激活,评定其成熟质量。结果发现,M4组的第一极体排出率明显高于M1组和M5组,其它各组间没有显著差异(P>0.05);M5组的孤雌激活卵裂率显著低于M1组和M4组(P<0.05),而与M2组和M3组没有显著差异(P>0.05),但M3和M4两组的囊胚发育率显著高于M1组和M5组(P<0.05)。这些研究结果表明:(1)未扩展卵丘细胞包围法和扩展卵丘细胞团支撑法可促进无卵丘水牛卵母细胞的体外成熟,但与卵丘细胞单层共培养没有作用;(2)卵巢组织包围培养不利于水牛卵母细胞的体外成熟。

The feasibility of maturing denuded buffalo oocytes in vitro was explored in this study, so as to provide a model to investigate the mechanisms of oocytes maturation. Denuded buffalo oocytes were allocated randomly to culture in medium directly （M1）, co-culture on cumulus cell monolayers （M2）, co-culture with surrounding cumulus cell pieces （M3）, co-culture on expanding cumulus cell clumps （M4） and co-culture with surrounding ovarian tissue pieces （MS）. At 24 h after maturation in vitro, rate of the first polar body （PB1） extruded was checkd, and the developmental competence of oocytes was evaluated by parthenogenetic activation using ionomycin and 6-dimethylaminopurine. The PB1 rate of oocytes matured using M4 was significantly higher than that of oocytes matured using M1 and M5 （P〈0.05）. Significantly more oocytes developed to blastocysts when they were matured using either M3 or M4 in comparison with them matured using M1 and M5 （P〈0. 05）. In addition, the cleavage rate of oocytes matured using M4 and M1 was also significantly higher than that of oocytes matured using M5 （P〈0.05）. In conclusion, co-culture with either cumulus cell pieces or expanding cumulus cell clumps can promote the maturation of buffalo denuded oocytes in vitro, but co-culture with cumulus cell monolayers does not work, and co-culture with ovarian tissues may be unfavorable to the maturation of buffalo denuded oocytes in vitro.