Effects of Mouse Cumulus Cells on Bovine Oocytes Maturation In vitro and Development After Parthenogenesis | 小鼠卵丘细胞对牛卵母细胞体外成熟和孤雌发育的影响

Chino. 本实验通过牛生发泡(GV)期无卵丘细胞卵母细胞-裸卵(denuded oocytes, DOs)与小鼠卵丘细胞(mouse cumulus cells, mCCs)共培养，研究异种mCCs对牛DOs体外成熟和孤雌激活后发育的影响，进而探讨卵丘细胞对卵母细胞的作用机制，为牛卵母细胞体外成熟效率提高提供实验依据。牛卵母细胞分为COCs(cumulus-oocytes complexes)组、DOs+bCCs(bovine cumulus cells, bCCs)组、DOs+mCCs组和DOs组4个组合，体外成熟22~24 h后，检测各组卵母细胞第一极体排出率、MⅡ期卵母细胞中谷胱甘肽(glutathione，GSH)含量和孤雌激活后的发育能力，以及卵母细胞内骨形态发生蛋白15(BMP15)和生长分化因子9(GDF9)mRNA相对表达水平。结果表明：(1) DOs+mCCs组和DOs+bCCs组卵母细胞核成熟率[(68.48±5.71)%，(71.00±4.27)%]显著低于COCs组(84.70±3.41)%(P0.05)，但均显著高于DOs组(54.26±5.03)%(P0.05)；(2) DOs+mCCs组MⅡ卵母细胞GSH含量和孤雌激活囊胚率[(2.48±0.24) pmol/oocyte，(44.19±4.03)%]与DOs+bCCs组[(2.49±0.18)pmol/oocyte，(48.57±5.20)%]和DOs组[(2.40±0.17)pmol/oocyte，(43.47±7.34)%]之间没有差异，但分别显著低于COCs组[(9.67±0.18)pmol/oocyte，(59.93±6.13)%](P0.05)，各组孤雌激活卵裂率之间没有显著差异[(98.85±4.32)%，(92.11±2.00)%，(95.40±4.11)%，(93.18±3.19)%](P0.05)；(3)DOs+mCCs组、DOs+bCCs组和COCs组卵母细胞中BMP15和GDF9 mRNA相对表达水平均显著性高于DOs组(P0.05)，其中，DOs+mCCs组和COCs组GDF9 mRNA相对表达水平显著高于DOs+bCCs组(P0.05)。实验结果表明，mCCs能提高牛DOs的核成熟率及BMP15和GDF9 mRNA相对表达水平，但对牛DOs的细胞质成熟和MⅡ卵母细胞孤雌激活胚胎囊胚发育没有促进作用，说明卵丘细胞通过一些旁分泌因子促进卵母细胞核成熟，且这些因子的作用可能没有种属特异性。

Inglés. To research the effects of heterogeneous mouse cumulus cells (mCCs) on in vitro maturation of bovine denuded oocytes(DOs) and its development after parthenogenesis, bovine denuded germinal vesicle (GV) stage DOs were co-cultured in vitro with a mCCs monolayer in this study. The effect mechanism of cumulus cells on bovine DOs after in vitro maturation(IVM) and parthenogenesis were also discussed, and this could help to provide experimental bases for increasing maturation rate of bovine oocytes in vitro. Bovine oocytes were divided into four groups, namely COCs (cumulus-oocytes complexes) group, DOs+bCCs (bovine cumulus cells) group, DOs+mCCs group and DOs group. The extruded rate of oocytes in different groups of first polar body, glutathione (GSH) content and parthenogenetic development of MⅡ stage oocytes, relative expression levels of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) mRNA of oocytes were detected after in vitro mature for 22~24h. The results showed that (1) Nuclear maturation rate of DOs+mCCs group and DOs+bCCs group [(68.48±5.71)%, (71.00±4.27)%] were significantly lower than that of COCs group (84.70±3.41)% (P0.05), but significantly higher than that of DOs group (54.26±5.03)% (P0.05); After parthenogenetic activation the GSH content and blastocyst rate of MⅡ oocytes of DOs+mCCs group [(2.48±0.24)pmol/oocyte, (44.19±4.03)%] were similar to those of DOs+bCCs group [(2.49±0.18)pmol/oocyte, (48.57±5.20)%] and DOs group [(2.40±0.17)pmol/oocyte, (43.47±7.34)%], but were all significantly lower than those of COCs group [(9.67±0.18)pmol/oocyte, (59.93±6.13)%)](P0.05), and cleavage rate of different groups showed no significant difference [(98.85±4.32)%, (92.11±2.00)%, (95.40±4.11)%, (93.18±3.19)%] (P0.05); (3)Relative expression levels of BMP15 and GDF9 mRNA of DOs+mCCs, DOs+bCCs and COCs group were all significantly higher than that of DOs group (P0.05), but relative expression levels of GDF9 mRNA of DOs+mCCs and COCs group were significantly higher than that of DOs+bCCs group (P0.05). The result indicated that mCCs could improve nuclear maturation rate and BMP15, GDF9 mRNA relative expression levels of bovine DOs, but could not enhance cytoplasmic maturation of DOs and parthenogenetic embryo development of MⅡ oocytes. It's suggested that cumulus cells can promote bovine oocytes nuclear maturation in vitro through some paracrine factors, and the effects of these factors may have no species specificity .