De Felici M, Dolci S, Siracusa G
Department of Public Health and Cell Biology, 2nd University of Rome, Italy.
J Exp Zool. 1991 Dec;260(3):401-5. doi: 10.1002/jez.1402600314.
The involvement of calcium ions in the mechanism of meiotic resumption has been studied in mouse oocytes made resistant to the lethal effects of calcium-free medium (CFM) by zona pellucida removal (De Felici et al., '89). We show here that such oocytes undergo meiotic resumption in CFM (as evaluated by germinal vesicle breakdown, GVBD) at a rate comparable to that shown by oocytes cultured in medium containing 1.7 mM Ca2+. The addition to CFM of 50 u M Quin2/AM (a membrane permeable, high affinity Ca2+ chelator) totally prevents GVBD, while purported antagonists of Ca2+ release from intracellular stores, such as 150 uM 8-(N,N-diethylamino)octyl-3-4-5 trimethoxybenzoate (TMB-8) or 300 uM chlortetracycline, only cause a slight meiotic delay. On the other hand, if the oocytes are pre-incubated for 30 min in CFM supplemented with 100 uM TBM-8 plus 0.2 mM dibutyryl-cyclic AMP (dbcAMP, a reversible inhibitor of GVBD), and then cultured in the same medium, without dbcAMP, a sustained inhibition of meiotic maturation is obtained. Our observations suggest that an increase in intracellular free Ca2+ is essential for meiotic resumption by mouse oocytes; in the experimental absence of external Ca2+, release of the cation from internal stores is sufficient to allow meiotic resumption.
通过去除透明带使小鼠卵母细胞对无钙培养基（CFM）的致死效应产生抗性，在此基础上研究了钙离子在减数分裂恢复机制中的作用（De Felici等人，1989年）。我们在此表明，此类卵母细胞在CFM中经历减数分裂恢复（通过生发泡破裂，即GVBD来评估）的速率与在含有1.7 mM Ca2+的培养基中培养的卵母细胞所显示的速率相当。向CFM中添加50 μM Quin2/AM（一种膜通透性、高亲和力的Ca2+螯合剂）可完全阻止GVBD，而细胞内钙库Ca2+释放的所谓拮抗剂，如150 μM 8-（N，N-二乙氨基）辛基-3,4,5-三甲氧基苯甲酸酯（TMB-8）或300 μM氯四环素，仅导致轻微的减数分裂延迟。另一方面，如果将卵母细胞在补充有100 μM TBM-8加0.2 mM二丁酰环磷酸腺苷（dbcAMP，一种GVBD的可逆抑制剂）的CFM中预孵育30分钟，然后在不含dbcAMP的相同培养基中培养，则可获得对减数分裂成熟的持续抑制。我们的观察结果表明，细胞内游离Ca2+的增加对于小鼠卵母细胞的减数分裂恢复至关重要；在实验性缺乏外部Ca2+的情况下，阳离子从内部钙库的释放足以允许减数分裂恢复。
