绵羊卵母细胞体外核成熟抑制及其对胚胎发育潜力的影响

本研究旨在探讨次黄嘌呤 +dbcAMP或IBMX +dbcAMP对绵羊卵母细胞体外成熟的可逆性抑制作用 ,以及这种抑制作用对卵母细胞胚胎发育潜力的影响。自绵羊卵巢分离卵丘 -卵母细胞复合物进行体外培养 ,培养基中分别加入或不加入上述抑制剂。培养 6h后各组取部分卵母细胞固定染色检查卵母细胞核成熟情况 ;将其余的卵母细胞分别移入无抑制剂的成熟培养基中继续培养 18h后 ,再次检查各组卵母细胞核成熟情况 ,并进行体外受精和胚胎培养。结果表明 :次黄嘌呤 +dbcAMP或IBMX +dbcAM都分别能使 6 0 %以上的绵羊卵母细胞抑制在GV期。这种抑制是可逆性的 ,去除抑制剂后卵母细胞能恢复减数分裂 ,并加快由GVBD到MⅡ的成熟过程。各处理组受精率、卵裂率和囊胚发育率与对照组相比无显著性差异 ,表明卵母细胞的胚胎发育潜力没有受损。上述物质对卵母细胞成熟的可逆性抑制可用于研究卵母细胞成熟及其胚胎发育潜力的调节机制。

DEVELOPMENTAL COMPETENCE OF OVINE OOCYTES AFTER IN VITRO INHIBITION OF MEIOTIC RESUMPTION

The aims of the present study were to assess the effect of various inhibitors(hypoxanthine + dbcAMP or IBMX +dbcAMP)on meiotic resumption and subsequent development to the blastocyst stage of ovine oocytes. Ovine ovaries were acquired from a local abattoir and the immature cumulus oocyte complexes (COC) were dissected and cultured for 6 h in 1) Control: M199 supplemented with 10% fetal calf serum; 2) HX 6 h: dbcAMP 1 0 mM + hypoxanthine (HX) 3 mM; 3) IBMX 6 h: dbcAMP 1 0 mM+IBMX 0 1 mM. Subsequently, the oocytes from different treatment groups were washed and cultured for an additional 18 h in fresh M199+FCS. Then, the oocytes were inseminated with thawed spermatozoa and cultured. At different periods, a representative sample of the oocytes was fixed and stained with carmine to observe the nuclear status. The results showed: 1) both hypoxanthine + dbcAMP and IBMX + dbcAMP could prevent over 60% of oocytes from undergoing meiosis maturation after 6 h culture with an inhibitor; 2) there was no significant difference in the maturation rate of oocytes among the three groups (71 82 %, 73 72% and 74 13% respectively) after the additional 18 h culture, so the initial inhibitory effect was reversible and the timing of polar body extrusion was accelerated following the withdrawal of the inhibitors; 3) since the fertilization rate (64 71%, 61 74% and 58 41% respectively), cleavage rate (61 35%, 63 39% and 55 31% respectively) and blastocyst yields on day 8 of culture (27 78%, 27 50% and 22 05% respectively) were not significantly different among treatments we conclude that ovine oocyte development competence in vitro was not affected after meiotic resumption had been inhibited for 6 h. The results suggested that meiotic inhibition in vitro could be a tool in the study of the mechanisms involved in the acquisition of meiotic and developmental competence in ovine oocytes.