Downs S M
Department of Biology, Marquette University, Milwaukee, Wisconsin 53201-1881, USA.
Mol Reprod Dev. 1997 Feb;46(2):155-67. doi: 10.1002/(SICI)1098-2795(199702)46:2<155::AID-MRD6>3.0.CO;2-P.
This study was carried out to test the hypothesis that purine nucleotide-generating pathways are required for ligand-stimulated oocyte maturation in meiotically arrested cumulus cell-enclosed oocytes. Oocytes from hormonally primed, immature mice were cultured overnight in Eagle's minimum essential medium containing dibutyryl cyclic AMP (dbcAMP) (to maintain meiotic arrest), plus either mycophenolic acid or alanosine (inhibitors of guanyl and adenyl nucleotide production, respectively). Follicle-stimulating hormone (FSH) was added either at the outset of culture or after a 3-hr preincubation period. Under either of these conditions, the inhibitors suppressed FSH induction of germinal vesicle breakdown (GVB). In addition, the potency of FSH as an inducer of GVB was reduced following the 3-hr preincubation period, but this could be prevented if nucleotide precursors such as hypoxanthine, guanosine, or adenosine were included during the first 3 hr. Furthermore, preincubation had little effect on FSH induction of GVB when hypoxanthine was used to maintain meiotic arrest for the entire culture period. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, could not mimic this protective effect of hypoxanthine. Azaserine and aminopterin, inhibitors of purine de novo synthesis, blocked hormone-triggered maturation in dbcAMP-arrested oocytes, but had little effect on hypoxanthine-arrested oocytes. The effect of azaserine on dbcAMP-treated oocytes could be reversed by the inclusion of AICA riboside, a compound that can be taken up by cells and phosphorylated to form AICAR, which can enter the purine de novo pathway at a point distal to the sites of azaserine inhibition. FSH was stimulatory to purine de novo synthesis, while azaserine, aminopterin, hypoxanthine, and AICA riboside all suppressed de novo synthesis in the presence or absence of FSH, with dbcAMP having no effect. HPLC analysis of 14C-hypoxanthine metabolism in oocyte-cumulus cell complexes revealed that changes in the pattern of purine metabolism did not mediate the meiosis-inducing effect of FSH. These data support the conclusion that purine nucleotide-generating pathways are vital participants in the mechanism(s) regulating hormone-induced meiotic maturation, and that either the de novo or salvage pathway can fulfill this nucleotide requirement.
在减数分裂停滞的卵丘细胞包裹的卵母细胞中，配体刺激的卵母细胞成熟需要嘌呤核苷酸生成途径。将来自激素预处理的未成熟小鼠的卵母细胞在含有二丁酰环磷酸腺苷（dbcAMP）（以维持减数分裂停滞）的伊格尔最低必需培养基中培养过夜，再分别添加霉酚酸或阿拉诺新（分别为鸟苷酸和腺苷酸生成的抑制剂）。在培养开始时或3小时预孵育期后添加促卵泡激素（FSH）。在这两种条件下，抑制剂均抑制了FSH诱导的生发泡破裂（GVB）。此外，在3小时预孵育期后，FSH作为GVB诱导剂的效力降低，但如果在最初3小时内加入次黄嘌呤、鸟苷或腺苷等核苷酸前体，则可防止这种情况发生。此外，当使用次黄嘌呤在整个培养期维持减数分裂停滞时，预孵育对FSH诱导的GVB影响很小。磷酸二酯酶抑制剂3 - 异丁基 - 1 - 甲基黄嘌呤无法模拟次黄嘌呤的这种保护作用。嘌呤从头合成抑制剂重氮丝氨酸和氨甲蝶呤可阻断dbcAMP停滞的卵母细胞中激素触发的成熟，但对次黄嘌呤停滞的卵母细胞影响很小。重氮丝氨酸对dbcAMP处理的卵母细胞的作用可通过加入AICA核苷来逆转，AICA核苷是一种可被细胞摄取并磷酸化形成AICAR的化合物，AICAR可在重氮丝氨酸抑制位点远端的一点进入嘌呤从头合成途径。FSH对嘌呤从头合成有刺激作用，而重氮丝氨酸、氨甲蝶呤、次黄嘌呤和AICA核苷在有无FSH的情况下均抑制从头合成，dbcAMP则无影响。对卵母细胞 - 卵丘细胞复合物中14C - 次黄嘌呤代谢的HPLC分析表明，嘌呤代谢模式的变化并未介导FSH的减数分裂诱导作用。这些数据支持以下结论：嘌呤核苷酸生成途径是调节激素诱导的减数分裂成熟机制中的重要参与者，并且从头合成途径或补救途径均可满足这种核苷酸需求。
