CULTURE CONDITIONS AFFECT MEIOTIC REGULATION IN CUMULUS CELL-ENCLOSEDMOUSE OOCYTES

To test the hypothesis that culture conditions influence meiotic regul ation in mouse oocytes, we have examined the effects of six culture me dia, four organic buffers, and pH on spontaneous maturation, the maint enance of meiotic arrest and ligand-induced maturation in cumulus cell -enclosed oocytes from hormonally primed immature mice. The media test ed were Eagle's minimum essential medium (MEM), Ham's F-10 (F-10), M19 9, M16, Waymouth's MB 752/1 (MB 752/1), and Leibovitz's L-15 (L-15). A ll six media supported greater than or equal to 94% spontaneous germin al vesicle breakdown (GVB) during a 17-18 hr incubation period, but po lar body formation was lower in M199 and MB 752/1 than in the other me dia. The incidence of polar bodies could be increased in these two med ia by the addition of pyruvate. With the exception of M16 and MB 752/1 , 4 mM hypoxanthine maintained a significant number of cumulus cell-en closed oocytes in meiotic arrest. Inhibition could be restored by the addition of glutamine to M16 and pyruvate to MB 752/1. Follicle-stimul ating hormone (FSH) and epidermal growth factor (EGF) stimulated GVB i n those media in which hypoxanthine was inhibitory. dbcAMP was able to maintain meiotic arrest in all of the media, but was least effective in M16. FSH stimulated GVB in all dbcAMP-arrested groups except L-15, and FSH became stimulatory in L-15 when the pyruvate level was reduced to 0.23 mM and galactose was replaced with 5.5 mM glucose. When MEM w as buffered principally with the organic buffers MOPS, HEPES, DIPSO, o r PIPES (at 20 mM), high frequencies of GVB and polar body formation w ere observed in inhibitor-free medium. dbcAMP suppressed GVB in all gr oups; hypoxanthine also maintained meiotic arrest in all buffering con ditions, although this effect was nominal in PIPES-buffered medium. FS H and EGF stimulated GVB in all dbcAMP- and hypoxanthine-treated group s. When the concentration of HEPES was increased from 20 mM to 25 mM, a more pronounced suppressive effect on maturation in both dbcAMP- and hypoxanthine-supplemented groups was observed in the absence of FSH. But whereas HEPES reduced the induction of maturation by FSH in dbcAMP -arrested oocytes, this buffer had no effect on FSH action in hypoxant hine-treated oocytes. When MEM was buffered with HEPES and the pH was adjusted to 6.8, 7.0, 7.2, or 7.4, a dramatic effect of pH on meiotic maturation was observed. pH had no significant effect on hypoxanthine salvage by oocyte-cumulus cell complexes, but FSH-induced de novo puri ne synthesis was significantly augmented by increased pH, in parallel with increased induction of GVB. The results of this study demonstrate that the use of different culture media, or minor changes in culture conditions, can lead to significant variation in (1) the spontaneous m aturation of oocytes, (2) the ability of meiotic inhibitors to suppres s GVB, or (3) the efficacy of meiosis-inducing ligands. Furthermore, s uch observations provide a unique opportunity to examine specific mole cules and metabolic pathways that can account for this variation and t hereby gain valuable insights into the mechanisms involved in meiotic regulation. (C) 1997 Wiley-Liss, Inc.