无卵丘的水牛卵母细胞体外成熟方法的研究

卵母细胞生长发育和成熟调控机理的研究,是生殖生物学领域研究的热点和难点。研究发现,卵丘细胞的存在与否严重影响卵母细胞的体外成熟质量,且小鼠无卵丘的卵母细胞经体外成熟并常规体外受精后未能得到“试管后代”,表明常规体外培养方法明显降低无卵丘的卵母细胞发育潜能。可能的原因是:(1)卵母细胞与颗粒细胞间的间隙连接丢失,这种连接对卵母细胞的胞质成熟是至关重要的。(2)皮层颗粒的减少或继续生成路径的障碍。(3)卵母细胞易于贴底,造成变形,从而可能引起卵母细胞内细胞器机械性移位,造成损伤,且贴底后可能影响卵母细胞与培养基的物质交换。因此,探明卵丘细胞在卵母细胞体外成熟的作用及机制,探索培养无卵丘的水牛卵母细胞的新方法,对于今后进一步提高卵母细胞成熟质量具有重大的指导意义。本研究针对去卵丘细胞的卵母细胞体外培养存在的问题,以卵巢组织、卵丘细胞为介质来模拟卵母细胞在体内的状态,试验分为5组:M1,直接培养法(对照组);M2,与单层卵丘细胞共培养法;M3,未扩展卵丘细胞包围法;M4,扩展卵丘细胞团支撑法;M5,卵巢组织薄片包围法。培养24 h后计算第一极体(PB1)排出率,随后对这些卵母细胞进行孤雌激活。结果发现:成熟24 h时,M4组的第一极体排出率明显高于M1组和M5组,其他各组间没有显著差异(P>0.05);孤雌激活结果显示M5组的卵裂率显著低于M1组(P<0.05),而M2组与M1组间没有差异(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)。结论:(1)扩展卵丘细胞团支撑法支持无卵丘的水牛卵母细胞的体外核质成熟,而未扩展卵丘细胞包围法促进卵母细胞胞质的成熟,这可能是通过重新构建卵丘-卵母细胞间的间隙连接实现的;(2)与单层卵丘细胞共培养法并不能促进无卵丘的水牛卵母细胞的核成熟及其发育潜力;(3)用卵巢组织包围不能模拟体内卵巢的环境,且卵巢组织可能分泌抑制卵母细胞胞质成熟的因子。目前对无卵丘卵母细胞的培养方法的研究甚少,而我们建立的卵丘细胞团包围/支撑法及卵巢组织包围培养法未见类似的报道。本研究初步建立了水牛无卵丘卵母细胞的有效培养方法,将为研究卵母细胞生长发育和成熟调控机理提供新的模型,为利用无卵丘的水牛卵母细胞提供理论和技术支持。

Primary study on the mature methods of the in vitro buffalo denuded oocytes

The culture methods of denuded buffalo oocytes were explored using buffalo ovarian tissues and cumulus cells as medium to simulate the ovarian status in vivo in order to supply with the models to investigate the mechanisms of oocyte maturation.The culture methods were:control group(cultured in medium directly,M1),co-cultured with monolayer cumulus cells sticking the dish(M2),denuded oocytes surrounded by no expanding cumulus cells piece(M3),denuded oocytes supported by expanding cumulus cells clump(M4) and denuded oocytes surrounded by ovarian tissue pieces(M5).After matured in vitro for 24 h,the first polar body(PB1) was assessed,then the oocytes were activated by 6-dimethylaminopurine combined with Ionomycin,and then the cleavage rate and blastocyst rate were checked.The results showed that the PB1 rate in M4 group was significantly higher than M1 group and M5 group(P<0.05),no significant different was checked between other groups(P>0.05);The results of oocyte parthenogenetic development showed that cleavage rate of M5 group was significantly lower than M1 group(P<0.05),no significant different in cleavage rate and blastocyst rate was checked between M1 and M2 group(P>0.05);but the blastocyst development rates of M3 and M4 groups were significantly higher than those of M1 and M5 groups(P<0.05).These results indicated that:(1) M3 and M4 group can improve the maturation of the nuclear and cytoplasm of buffalo denuded oocytes effectively;(2) Co-culture with monolayer cumulus cells sticking the dish can not improve the maturation of nuclear and developmental ability of buffalo denuded oocytes;(3)The ovarian tissues may secrete some factors that inhibit the maturation of the nuclear and cytoplasm.