Downs S M
Biology Department, Marquette University, Milwaukee, Wisconsin 53233.
Dev Biol. 1995 Feb;167(2):502-12. doi: 10.1006/dbio.1995.1044.
The effects of glucose and cumulus cells on oocyte ATP levels and germinal vesicle breakdown (GVB) in isolated mouse oocytes have been examined. Oocyte-cumulus cell complexes or denuded oocytes (DO) from pregnant mare serum gonadotropin-primed immature mice were cultured in minimum essential medium containing 4 mM hypoxanthine and 1 mM pyruvate, in the absence or presence of 0.55 mM glucose. After 17-18 hr in the presence of glucose, ATP in both the oocyte-cumulus cell complexes and oocytes derived from such complexes (cumulus cell-enclosed oocytes; CEO) was elevated, and less than one-half of the oocytes had resumed maturation (48% GVB). Removal of glucose caused a decrease in ATP levels in complexes and CEO and reversed the meiotic arrest in CEO (98% GVB), and these effects were mimicked by iodoacetate treatment. Higher frequencies of GVB were observed in glucose-free medium after more than 6 hr of culture, while ATP levels were reduced within 3 hr. Glucose had no effect on ATP levels or the meiotic state of denuded oocytes. Interestingly, iodoacetate had a stimulatory effect on GVB in DO (86% GVB compared to 57% in controls), but did not effect ATP levels. Glycerrhetinic acid, a gap junction uncoupler, completely suppressed oocyte-cumulus cell coupling in cultured complexes (0.15% coupling compared to 16.6% in controls) and reversed the inhibitory effect of glucose on oocyte maturation (91 and 95% GVB at 10 and 25 microM compared to 42% GVB in controls). This agent also prevented follicle-stimulating hormone-induced meiotic maturation in dibutyryl cAMP-arrested CEO. These results thus implicate mediation by gap junctions of both inhibitory and stimulatory signals from the cumulus cells. Comparison of ATP levels in spontaneously maturing CEO in vitro with those from oocytes maturing in vivo in response to human chorionic gonadotropin revealed that a decrease in oocyte ATP preceded or accompanied GVB in spontaneously maturing oocytes but not in those maturing in vivo. The results of this study indicate that glucose-derived elevation of oocyte ATP contributes to meiotic arrest in cumulus cell-enclosed oocytes that is dependent on patient gap junctions. Furthermore, a model for reinitiation of oocyte maturation is supported in which spontaneous GVB results from cessation or interruption of inhibitory influences, while ligand-provoked GVB is brought about by the generation of stimulatory signals that override inhibitory input.
研究了葡萄糖和卵丘细胞对分离的小鼠卵母细胞ATP水平和生发泡破裂（GVB）的影响。从孕马血清促性腺激素预处理的未成熟小鼠获取卵母细胞-卵丘细胞复合体或裸卵（DO），在含有4 mM次黄嘌呤和1 mM丙酮酸的最低限度基本培养基中培养，培养基中添加或不添加0.55 mM葡萄糖。在葡萄糖存在的情况下培养17 - 18小时后，卵母细胞-卵丘细胞复合体以及由此类复合体衍生的卵母细胞（卵丘细胞包被的卵母细胞；CEO）中的ATP水平升高，且不到一半的卵母细胞恢复成熟（48%发生GVB）。去除葡萄糖导致复合体和CEO中的ATP水平下降，并逆转了CEO中的减数分裂阻滞（98%发生GVB），碘乙酸处理可模拟这些效应。培养超过6小时后，在无葡萄糖培养基中观察到更高频率的GVB，而ATP水平在3小时内降低。葡萄糖对裸卵的ATP水平或减数分裂状态没有影响。有趣的是，碘乙酸对DO中的GVB有刺激作用（86%发生GVB，而对照组为57%），但不影响ATP水平。甘草次酸是一种缝隙连接解偶联剂，它完全抑制培养复合体中的卵母细胞-卵丘细胞偶联（0.15%偶联，而对照组为16.6%），并逆转葡萄糖对卵母细胞成熟的抑制作用（在10和25 microM时分别为91%和95%发生GVB，而对照组为42%发生GVB）。该试剂还可防止促卵泡激素诱导的二丁酰环磷腺苷阻滞的CEO减数分裂成熟。因此，这些结果表明卵丘细胞的抑制和刺激信号通过缝隙连接介导。将体外自发成熟的CEO中的ATP水平与体内响应人绒毛膜促性腺激素而成熟的卵母细胞中的ATP水平进行比较，发现自发成熟的卵母细胞中，卵母细胞ATP水平的下降先于或伴随GVB发生，但在体内成熟的卵母细胞中并非如此。本研究结果表明，葡萄糖导致的卵母细胞ATP水平升高有助于依赖缝隙连接的卵丘细胞包被的卵母细胞中的减数分裂阻滞。此外，支持一种卵母细胞成熟重新启动的模型，即自发的GVB是由抑制性影响的停止或中断导致的，而配体引发的GVB是由产生的刺激信号超过抑制性输入引起的。
