Aktas H, Wheeler M B, Rosenkrans C F, First N L, Leibfried-Rutledge M L
Department of Meat and Animal Science, University of Wisconsin, Madison 53706, USA.
J Reprod Fertil. 1995 Nov;105(2):227-35. doi: 10.1530/jrf.0.1050227.
The effects of high intracellular cAMP concentrations ([cAMP]i) on germinal vesicle maintenance of bovine cumulus-oocyte complexes were investigated, using 8-bromo-3',5'-cAMP (8-Br-cAMP) or an invasive adenylate cyclase from Bordetella pertussis to increase the [cAMP]i. The effects of interactions of these agents with macromolecular supplements in culture medium (fetal calf serum, FCS; polyvinylpyrrolidone, PVP; BSA), and different methods of processing complexes before culture, on subsequent germinal vesicle maintenance by invasive adenylate cyclase were studied. While 8-Br-cAMP was unable to maintain germinal vesicle arrest in the majority of oocytes for 20 h (36% with FCS, 24% with BSA, 18% with PVP), it maintained germinal vesicle arrest in a high proportion of cumulus-enclosed oocytes when BSA or PVP was used (37% with FCS, 52% with BSA, 53% with PVP). The difference in frequency of germinal vesicle maintenance between macromolecular supplements was not related to [cAMP]i when assayed after culture for 2 h with invasive adenylate cyclase. Complexes processed in whole follicular fluid were not maintained in meiotic arrest (26%) when cultured with invasive adenylate cyclase and PVP. Complexes processed in follicular fluid with 3-isobutyl 1-methylxanthine (IBMX) plus invasive adenylate cyclase were arrested at the germinal vesicle stage at high frequencies (65%), while those processed in IBMX or IBMX plus 8-Br-cAMP-supplemented follicular fluid had intermediate (43% and 49%, respectively) frequencies of intact germinal vesicles. Oocyte complexes processed in follicular fluid supplemented with IBMX and invasive adenylate cyclase formed morulae and blastocysts (27.2%), as did oocytes processed in follicular fluid alone (26%). Phosphoprotein profiles showed that control oocytes and 8-Br-cAMP-treated oocytes share a profile that is different from that of oocytes treated with invasive adenylate cyclase. These results show that increased [cAMP]i reversibly maintains bovine oocytes in meiotic arrest for an extended period without the occurrence of the post-translational protein modifications observed during meiotic resumption or transient arrest.
利用8-溴-3',5'-环磷酸腺苷（8-Br-cAMP）或百日咳博德特氏菌的侵袭性腺苷酸环化酶来提高细胞内cAMP浓度（[cAMP]i），研究了高细胞内cAMP浓度对牛卵丘-卵母细胞复合体生发泡维持的影响。研究了这些试剂与培养基中的大分子补充剂（胎牛血清，FCS；聚乙烯吡咯烷酮，PVP；牛血清白蛋白，BSA）之间的相互作用，以及培养前处理复合体的不同方法，对侵袭性腺苷酸环化酶随后维持生发泡的影响。虽然8-Br-cAMP在大多数卵母细胞中无法维持生发泡停滞20小时（FCS组为36%，BSA组为24%，PVP组为18%），但当使用BSA或PVP时，它能在高比例的卵丘包裹的卵母细胞中维持生发泡停滞（FCS组为37%，BSA组为52%，PVP组为53%）。用侵袭性腺苷酸环化酶培养2小时后测定，大分子补充剂之间生发泡维持频率的差异与[cAMP]i无关。用侵袭性腺苷酸环化酶和PVP培养时，在全卵泡液中处理的复合体不能维持减数分裂停滞（26%）。用3-异丁基-1-甲基黄嘌呤（IBMX）加侵袭性腺苷酸环化酶在卵泡液中处理的复合体在生发泡阶段高频停滞（65%），而在IBMX或补充了IBMX加8-Br-cAMP的卵泡液中处理的复合体完整生发泡的频率处于中间水平（分别为43%和49%）。在补充了IBMX和侵袭性腺苷酸环化酶的卵泡液中处理的卵母细胞复合体形成桑葚胚和囊胚（27.2%），单独在卵泡液中处理的卵母细胞也是如此（26%）。磷酸化蛋白谱显示，对照卵母细胞和8-Br-cAMP处理的卵母细胞具有与侵袭性腺苷酸环化酶处理的卵母细胞不同的谱。这些结果表明，升高的[cAMP]i可使牛卵母细胞在减数分裂停滞状态下可逆地维持较长时间，而不会出现减数分裂恢复或短暂停滞期间观察到的翻译后蛋白质修饰。
