Racowsky C
J Exp Zool. 1986 Aug;239(2):263-75. doi: 10.1002/jez.1402390214.
The effect of increasing cytoplasmic calcium on cyclic adenosine monophosphate (cAMP)-dependent meiotic arrest (%GV where GV is germinal vesicle) in hamster oocytes was investigated. The hypotheses tested were that calcium is required for the spontaneous maturation of hamster oocytes, elevation of calcium in the oocyte-cumulus complex can antagonize cAMP-dependent meiotic arrest, and the intraoocyte level of cAMP remains unchanged, but heterologous metabolic coupling decreases, concomitant with calcium-stimulation of germinal vesicle breakdown (GVBD). Levels of cAMP were elevated by culturing cells in the presence of dibutyryl cAMP (dbcAMP), isobutylmethylxanthine (IBMX) or forskolin and intracellular levels of calcium were manipulated by altering the CaCl2 concentration in the medium and/or by utilizing EGTA or A23187. Intracellular cAMP was determined by RIA, functional metabolic coupling was assessed by determination of the fraction of radiolabeled uridine marker transferred from the cumulus mass to the oocyte, and meiotic stage was determined cytogenetically. Compared with the proportion of oocytes that underwent meiotic maturation in control medium containing 1.53 mM CaCl2, that of cumulus-free (denuded) oocytes was unaffected by culture in the absence of added CaCl2, while that of cumulus-enclosed (intact) oocytes was significantly decreased (%GV = 59.5 +/- 4.8 and 4.2 +/- 0.9 in 0 and 1.53 mM CaCl2, respectively, P less than 0.001, where GV is germinal vesicle). EGTA prevented, in a dose-dependent manner, the spontaneous maturation of denuded oocytes that occurred in 0 mM CaCl2 (ID50 = 0.05 mM, where ID50 is the dose of EGTA that inhibited GVBD in 50% cultured oocytes). In contrast, compared with the control, less than 1 mM EGTA failed to increase the %GV of intact oocytes, although 5 mM EGTA significantly increased meiotic arrest. The %GVBD of oocytes cultured in medium containing 0 mM CaCl2 was dose-dependent on A23187 for both intact oocytes (ID50 = 3.0 microM) and for denuded oocytes cultured in the presence of 0.5 mM EGTA (ID50 = 2.7 microM). Elevated extracellular calcium significantly antagonized dbcAMP-maintained meiotic arrest in both types of oocyte and the %GV was significantly correlated with the pH of the medium [(r) = -0.78 and -0.60 for intact and denuded oocytes, respectively, P less than 0.001 in both cases]. Both CaCl2 and A23187 induced dose-dependent antagonistic effects on forskolin-maintained meiotic arrest in intact oocytes but neither antagonism was accompanied by significant dose-dependent decreases in either the intraoocyte content of cAMP or the extent of heterologous metabolic coupling.(ABSTRACT TRUNCATED AT 400 WORDS)
研究了增加细胞质钙对仓鼠卵母细胞中环磷酸腺苷（cAMP）依赖性减数分裂阻滞（以生发泡期（GV）占比表示，其中GV为生发泡）的影响。所检验的假说是，钙是仓鼠卵母细胞自发成熟所必需的，卵丘 - 卵母细胞复合体中钙的升高可拮抗cAMP依赖性减数分裂阻滞，并且卵母细胞内cAMP水平保持不变，但异源代谢偶联减少，同时伴随着生发泡破裂（GVBD）的钙刺激。通过在二丁酰cAMP（dbcAMP）、异丁基甲基黄嘌呤（IBMX）或福斯可林存在下培养细胞来提高cAMP水平，并通过改变培养基中CaCl2浓度和/或利用乙二醇双（2 - 氨基乙基醚）四乙酸（EGTA）或A23187来调控细胞内钙水平。通过放射免疫分析（RIA）测定细胞内cAMP，通过测定从卵丘团转移到卵母细胞的放射性标记尿苷标记物的比例来评估功能性代谢偶联，并通过细胞遗传学方法确定减数分裂阶段。与在含有1.53 mM CaCl2的对照培养基中经历减数分裂成熟的卵母细胞比例相比，无卵丘（裸）卵母细胞在未添加CaCl2的培养基中培养时不受影响，而有卵丘（完整）卵母细胞的比例则显著降低（在0和1.53 mM CaCl2中，GV占比分别为 = 59.5 +/- 4.8和4.2 +/- 0.9，P < 0.001，其中GV为生发泡）。EGTA以剂量依赖性方式阻止了在0 mM CaCl2中发生的裸卵母细胞的自发成熟（半数抑制剂量（ID50） = 0.05 mM，其中ID50是抑制50%培养卵母细胞中GVBD的EGTA剂量）。相比之下，与对照相比，低于1 mM的EGTA未能增加完整卵母细胞的GV占比，尽管5 mM EGTA显著增加了减数分裂阻滞。对于完整卵母细胞（ID50 = 3.0 microM）以及在0.5 mM EGTA存在下培养的裸卵母细胞（ID50 = 2.7 microM），在含有0 mM CaCl2的培养基中培养的卵母细胞的GVBD占比均呈剂量依赖性依赖于A23187。细胞外钙升高在两种类型的卵母细胞中均显著拮抗dbcAMP维持的减数分裂阻滞，并且GV占比与培养基的pH显著相关（完整和裸卵母细胞的相关系数（r）分别为 - 0.78和 - 0.60，两种情况下P均 < 0.001）。CaCl2和A23187在完整卵母细胞中均对福斯可林维持的减数分裂阻滞产生剂量依赖性拮抗作用，但两种拮抗作用均未伴随着卵母细胞内cAMP含量或异源代谢偶联程度的显著剂量依赖性降低。（摘要截断于400字）
