Laforest Martin F, Pouliot Eric, Guéguen Laëtitia, Richard François J
Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Faculté des Sciences de l'Agriculture et d'Alimentation, Université Laval, Ste-Foy, Québec, Canada G1K 7P4.
Mol Reprod Dev. 2005 Mar;70(3):361-72. doi: 10.1002/mrd.20203.
The meiosis of mammalian oocytes begins during the fetal life and stops at the dictyate stage. This study has assessed the role of specific phosphodiesterase (PDE) inhibitors on the control of meiotic resumption in porcine oocytes investigating the influence of PMSG-hCG and cAMP stimulation. Cumulus-oocytes complexes (COCs) and denuded oocytes (DOs) were collected from gilt ovaries obtained at a local slaughterhouse. Oocytes were cultured in NCSU23 with different PDE inhibitors. The EC(50) for oocytes maintained in germinal vesicle (GV) stage was evaluated using different doses of both cilostamide (CIL), PDE3 inhibitor and 3-isobutyl-1-methylxanthine (IBMX), a nonspecific PDE inhibitor. In presence of PMSG-hCG, meiotic resumption is observed after 24 hr of culture. Both CIL and IBMX reversibly blocked meiotic resumption. In absence of PMSG-hCG, meiotic resumption is reduced after 24 hr of culture. After 48 hr of culture, only CIL significantly blocked meiotic resumption. Still in absence of PMSG-hCG, significant effect of treatment was only observed in COCs using the combination of CIL and rolipram (PDE3 and PDE4 inhibitor, respectively) compared to the use of IBMX. To assess the contribution of cAMP synthesis, a low dose of an adenylyl cyclase (AC) stimulator, forskolin, has been used in combination with CIL showing a significant effect of this combination. In CIL-treated COCs and DOs, significant higher percentages of oocytes were maintained in GV stage when cultured in combination with forskolin instead of PMSG-hCG. In conclusion, these results demonstrate that the control of meiotic resumption in porcine oocytes is highly regulated by cAMP. Both the degradation by specific PDE3 enzyme and the synthesis by an active AC are highly involved.
哺乳动物卵母细胞的减数分裂始于胎儿期，并在双线期阶段停止。本研究评估了特定磷酸二酯酶（PDE）抑制剂在控制猪卵母细胞减数分裂恢复中的作用，研究了孕马血清促性腺激素 - 人绒毛膜促性腺激素（PMSG - hCG）和环磷酸腺苷（cAMP）刺激的影响。从当地屠宰场获得的后备母猪卵巢中收集卵丘 - 卵母细胞复合体（COCs）和裸卵（DOs）。卵母细胞在含有不同PDE抑制剂的NCSU23中培养。使用不同剂量的西洛他唑（CIL，PDE3抑制剂）和3 - 异丁基 - 1 - 甲基黄嘌呤（IBMX，一种非特异性PDE抑制剂）评估维持在生发泡（GV）期的卵母细胞的半数有效浓度（EC50）。在PMSG - hCG存在的情况下，培养24小时后观察到减数分裂恢复。CIL和IBMX均可逆转性地阻断减数分裂恢复。在没有PMSG - hCG的情况下，培养24小时后减数分裂恢复减少。培养48小时后，只有CIL能显著阻断减数分裂恢复。同样在没有PMSG - hCG的情况下，与使用IBMX相比，仅在使用CIL和咯利普兰（分别为PDE3和PDE4抑制剂）组合的COCs中观察到显著的处理效果。为了评估cAMP合成的作用，低剂量的腺苷酸环化酶（AC）刺激剂福斯高林与CIL联合使用，显示出该组合的显著效果。在CIL处理的COCs和DOs中，与PMSG - hCG相比，与福斯高林联合培养时，维持在GV期的卵母细胞百分比显著更高。总之，这些结果表明猪卵母细胞减数分裂恢复的控制受cAMP高度调节。特定的PDE3酶降解和活性AC合成均高度参与其中。
