Downs Stephen M, Gilles Ryan, Vanderhoef Christopher, Humpherson Peter G, Leese Henry J
Biology Department, Marquette University, Milwaukee, Wisconsin, USA.
Mol Reprod Dev. 2006 Mar;73(3):379-89. doi: 10.1002/mrd.20416.
In this study we have examined the effects of denuded oocyte coculture with dissociated cumulus cells (CC) or intact oocyte-CC complexes on meiotic resumption. When denuded oocytes (DO) or cumulus cell-enclosed oocytes (CEO) were cultured in 40-microl drops of medium under oil, and held in meiotic arrest with 4 mM hypoxanthine plus 25 microM dbcAMP, they underwent germinal vesicle breakdown (GVB) at similar frequencies (34%-35%). Coculture of DO with complexes or dissociated CCs stimulated maturation (50% and 61% GVB, respectively), with no effect of DO on maturation of cocultured CEO (32% GVB). This coculture effect was increased with the number of CCs added to the culture drop. When either glucose or glutamine was eliminated from the medium, no meiotic induction resulted from cocultured CCs. When CEO were cultured alone in microdrops, increasing their number from 10 to 50 significantly lowered the percentage resuming maturation, an effect also reduced by removing glucose and/or glutamine from the medium. This effect was not observed with DO. When inhibitory medium was conditioned overnight with complexes, subsequent culture with DO led to higher maturation percentages than culture in unconditioned medium; however, when CEO were cultured in conditioned medium, there was either no effect or increased inhibition of maturation. Assay of glucose and pyruvate in spent medium showed that DO cultured alone consumed glucose and pyruvate, but under CC coculture conditions more glucose was consumed and significant amounts of pyruvate accumulated in the medium, changes that led to an increase in the maturation of DO. Further experiments showed that DO were more sensitive than CEO to the meiosis-inducing effect of pyruvate. These results demonstrate different responsiveness of DO and CEO to coculture conditions and question the physiological relevance of denuded oocyte/CC coculture to study meiotic induction.
在本研究中，我们检测了裸卵与解离的卵丘细胞（CC）或完整的卵母细胞-卵丘细胞复合体共培养对减数分裂恢复的影响。当裸卵（DO）或卵丘细胞包裹的卵母细胞（CEO）在油下40微升培养基滴中培养，并用4 mM次黄嘌呤加25 μM二丁酰环磷腺苷（dbcAMP）维持减数分裂阻滞时，它们发生生发泡破裂（GVB）的频率相似（34%-35%）。DO与复合体或解离的CC共培养可刺激成熟（分别为50%和61% GVB），而DO对共培养的CEO成熟无影响（32% GVB）。这种共培养效应随着添加到培养滴中的CC数量增加而增强。当培养基中去除葡萄糖或谷氨酰胺时，共培养的CC不会诱导减数分裂。当CEO单独在微滴中培养时，将其数量从10增加到50会显著降低恢复成熟的百分比，从培养基中去除葡萄糖和/或谷氨酰胺也会降低这种效应。DO未观察到这种效应。当抑制性培养基与复合体过夜孵育后，随后与DO共培养导致的成熟百分比高于在未处理培养基中培养；然而，当CEO在处理过的培养基中培养时，要么对成熟无影响，要么成熟抑制增加。对用过的培养基中的葡萄糖和丙酮酸进行检测表明，单独培养的DO消耗葡萄糖和丙酮酸，但在CC共培养条件下消耗更多葡萄糖，且大量丙酮酸在培养基中积累，这些变化导致DO成熟增加。进一步实验表明，DO比CEO对丙酮酸的减数分裂诱导作用更敏感。这些结果表明DO和CEO对共培养条件的反应不同，并对裸卵/CC共培养用于研究减数分裂诱导的生理相关性提出质疑。
