Downs Stephen M, Verhoeven Amelia
Biology Department, Marquette University, Milwaukee, Wisconsin 53233, USA.
Mol Reprod Dev. 2003 Sep;66(1):90-7. doi: 10.1002/mrd.10326.
The selection of culture media and supplements therein has a tremendous impact on the regulation of oocyte maturation in vitro. In the present study, we have evaluated how altering the levels of glutamine in the presence or absence of glucose affects meiotic arrest in cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) when cultured in either the simple medium M16 or the more complex Eagle's minimum essential medium (MEM). We have also tested the effectiveness of follicle-stimulating hormone (FSH) in triggering germinal vesicle breakdown (GVB) and purine de novo synthesis in differing MEM culture conditions. When DO were cultured 17-18 hr in hypoxanthine (HX)- or dbcAMP-supplemented M16 medium, neither glucose nor glutamine had any effect on oocyte maturation, with dbcAMP the more effective inhibitor. In the absence of glutamine, cumulus cells promoted meiotic resumption, since significantly lower levels of meiotic arrest were maintained in CEO than in DO by either HX or dbcAMP, but addition of the amino acid dose-dependently decreased the maturation percentage in CEO below that observed in DO. In MEM, glutamine and glucose again had little effect on the maturation of DO, although the percentage of maturing DO in HX-supplemented medium was about 20% lower than that in M16 medium. In the absence of glucose, high levels of maturation were observed in CEO in glutamine-free medium that were dose-dependently lowered by the amino acid. However, when glucose was present, CEO were as effectively arrested as DO when glutamine was absent, with no further effect of the amino acid. This inhibitory action of glucose was dependent on the essential amino acids present in MEM. The effects of glutamine were not due to changes in metabolic coupling between the oocyte and cumulus cells. Measurement of purine de novo synthesis indicated that the maintenance of meiotic arrest as well as FSH induction of meiotic resumption were associated with increases in purine synthesis. We conclude that glucose and glutamine act cooperatively to promote the synthesis of new purine compounds within the somatic compartment and that the timing and duration of such synthesis determines whether meiotic resumption will be suppressed or promoted.
培养基及其添加成分的选择对体外卵母细胞成熟的调控具有巨大影响。在本研究中，我们评估了在有或无葡萄糖存在的情况下改变谷氨酰胺水平，对卵丘细胞包裹的卵母细胞（CEO）和裸卵（DO）在简单培养基M16或更复杂的伊格尔最低必需培养基（MEM）中培养时减数分裂阻滞的影响。我们还测试了促卵泡激素（FSH）在不同MEM培养条件下触发生发泡破裂（GVB）和嘌呤从头合成的有效性。当DO在添加次黄嘌呤（HX）或二丁酰环磷腺苷（dbcAMP）的M16培养基中培养17 - 18小时时，葡萄糖和谷氨酰胺对卵母细胞成熟均无任何影响，其中dbcAMP是更有效的抑制剂。在缺乏谷氨酰胺的情况下，卵丘细胞促进减数分裂恢复，因为与HX或dbcAMP相比，CEO中减数分裂阻滞水平显著低于DO，但添加该氨基酸会使CEO中的成熟率剂量依赖性降低至低于DO中的观察值。在MEM中，谷氨酰胺和葡萄糖对DO的成熟同样影响较小，尽管添加HX的培养基中成熟DO的百分比比M16培养基中低约20%。在无葡萄糖的情况下，在无谷氨酰胺培养基中观察到CEO有高水平的成熟，该氨基酸可使其剂量依赖性降低。然而，当存在葡萄糖时，在缺乏谷氨酰胺的情况下，CEO与DO一样有效地被阻滞，该氨基酸无进一步影响。葡萄糖的这种抑制作用取决于MEM中存在的必需氨基酸。谷氨酰胺的作用并非由于卵母细胞与卵丘细胞之间代谢偶联的变化。嘌呤从头合成的测量表明，减数分裂阻滞的维持以及FSH诱导的减数分裂恢复与嘌呤合成增加有关。我们得出结论，葡萄糖和谷氨酰胺协同作用促进体细胞区室中新嘌呤化合物的合成，并且这种合成的时间和持续时间决定减数分裂恢复是被抑制还是被促进。
