调节环磷酸腺苷对绵羊卵母细胞减数分裂恢复的影响

Effects of regulating cyclic adenosine monophosphate on meiotic recovery in sheep oocytes

旨在探讨西洛酰胺(cilostamide)和雌二醇(E2)对绵羊卵母细胞和卵丘细胞中的环磷酸腺苷(cAMP)水平及卵母细胞减数分裂恢复的影响。首先在常规体外成熟体系中添加磷酸二酯酶3抑制剂西洛酰胺,探讨了其对绵羊卵母与卵丘细胞中cAMP水平调控的时间效应;随后在双相体外成熟系统中添加西洛酰胺或不同浓度的E2,利用竞争ELISA检测与DAPI染色技术,探讨了西洛酰胺和E2单独添加或联合添加对卵母和卵丘细胞内cAMP水平及卵母细胞核成熟的影响。结果显示:在常规体外成熟培养体系中,与对照组相比,培养30 min与2 h时,西洛酰胺组可显著提高卵母细胞内cAMP水平(P<0.05);在双相体外成熟系统中,与对照组相比,西洛酰胺组、0.5 ng/mL E2组和联合添加组(西洛酰胺+0.5 ng/mL E2)均可显著提升卵母细胞内cAMP水平,并且显著阻滞卵母细胞减数分裂恢复(P<0.05),同时联合添加组显示出最优的作用效果。综上,研究表明西洛酰胺与较低水平E2(0.5 ng/mL E2),具有改善绵羊卵母细胞体外成熟效率的作用,且联合添加具有明显叠加效应。

In order to further investigate the effects of cilostamide and estradiol(E2) on the regulation of cyclic adenylate monophosphate(cAMP) in ovine oocytes and cumulus cells and the recovery of meiosis in ovine oocytes. In this study, the time effect of phosphodiesterase 3 inhibitor cilostamideon the regulation of the cAMP level in sheep oocytes and cumulus cells was investigated by adding cilostamideto the routine in vitro maturation(IVM) system.Then, cilostamideor and E2 at different concentrations was added to the biphasic IVM system. ELISA and DAPI staining were used to determinethe effects of the addition of cilostamideor and E2 at different concentrations on the cAMP levels and oocyte maturation in oocytes and cumulus cells, respectively.The results showed that cilostamide significantly increased the level of intracellular cAMP(P<0.05). In biphasic IVM systems, cilostamide, 0.5 ng/mL E2, and combined addition(cilostamide+0.5 ng/mL E2) significantly increased the intracellular cAMP levels and significantly inhibited the meiosis recovery in the oocytes(P<0.05). At the same time, the combined addition group showed the optimal effect. In conclusion, the effects of cilostamide combined with a lower level of E2(0.5 ng/mL) rather than a higher level of E2(1 μg/mL) were found to have the potential to improve the IVM efficiency of sheep oocytes, and the combined additionhad a significant additive effect.