Su Y Q, Xia G L, Byskov A G, Fu G D, Yang C R
College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.
Mol Reprod Dev. 1999 May;53(1):51-8. doi: 10.1002/(SICI)1098-2795(199905)53:1<51::AID-MRD6>3.0.CO;2-4.
The present experiments were conducted to examine the hypothesis that follicle-stimulating hormone (FSH) can stimulate the hydrolysis of phosphoinositide, generating the intracellular second messengers to activate protein kinase C and mobilizing intracellular calcium, thus inducing oocyte meiotic resumption. Pig cumulus cell-enclosed oocytes (CEO) were cultured for 24 hr in 4 mM hypoxanthine (HX)-supplemented medium and treated with different agents in the following designs: (1) CEO were treated with neomycin (an inhibitor of phosphoinositide hydrolysis) in the presence of FSH or only treated with 7,12-dimethylbenzin(a) anthracene (DMBA, a tumor promoter which can cause phosphorylation of phospholipase C (PLC), formation of inositol triphophate, and mobilization of intracellular calcium) to mimic the direct activation of PLC; (2) CEO were challenged by FSH, together with sphingosine or staurosporine (two kinds of PKC inhibitors); or treated with phorbol myristate acetate (PMA, an activator of PKC) separately; (3) CEO were primed with BAPTA/AM (an intracellular calcium chelator) or BAPTA/AM +FSH for 60 min, and then transferred into a new culture medium supplemented with FSH but without BAPTA/AM; total culture time was 24 hr. At the end of the culture, the incidence of germinal vesicle breakdown (GVBD) was calculated. The results showed that: (1) FSH (100 U/liter) could stimulate pig CEO to override the arrest of HX and resume meiosis; DMBA (10(-8)-10(-5) M) itself also had such a kind of effect; whereas neomycin, at the level of 10-20 mM, could dramatically inhibit the stimulatory effect of FSH. (2) Staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) could also inhibit the effect of FSH in a dose-dependent manner on stimulating CEO to resume meiosis. However, PMA (10(-8)-10(-5) M) alone had a dual effect on the meiotic resumption of pig CEO. PMA, at the level of 10(-8)-10(-6) M, could stimulate CEO to resume meiosis, and at high concentration of 10(-5) M , it could even enhance the inhibitory effect of HX. (3) Priming CEO with BAPTA/AM only or BAPTA/AM +FSH for 60 min could significantly inhibit the effect of FSH in a dose-dependent manner. These results indicate that in the process of ligand-mediated meiotic resumption of pig CEO, FSH can stimulate the hydrolysis of phosphoinositide leading to the activation of PKC and mobilization of intracellular calcium; and suggest that multiple signaling pathways and signal interaction are involved in this process.
促卵泡激素（FSH）可刺激磷酸肌醇水解，产生细胞内第二信使以激活蛋白激酶C并动员细胞内钙，从而诱导卵母细胞减数分裂恢复。将猪卵丘细胞包裹的卵母细胞（CEO）在添加4 mM次黄嘌呤（HX）的培养基中培养24小时，并按以下设计用不同试剂处理：（1）CEO在FSH存在下用新霉素（磷酸肌醇水解抑制剂）处理，或仅用7,12 - 二甲基苯并(a)蒽（DMBA，一种可导致磷脂酶C（PLC）磷酸化、肌醇三磷酸形成和细胞内钙动员的肿瘤促进剂）处理以模拟PLC的直接激活；（2）CEO用FSH刺激，同时加入鞘氨醇或星形孢菌素（两种蛋白激酶C抑制剂）；或分别用佛波酯（PMA，蛋白激酶C激活剂）处理；（3）CEO用BAPTA/AM（一种细胞内钙螯合剂）或BAPTA/AM + FSH预处理60分钟，然后转移到添加FSH但不含BAPTA/AM的新培养基中；总培养时间为24小时。培养结束时，计算生发泡破裂（GVBD）的发生率。结果表明：（1）FSH（100 U/升）可刺激猪CEO克服HX的阻滞并恢复减数分裂；DMBA（10(-8)-10(-5) M）本身也有这种作用；而10 - 20 mM水平的新霉素可显著抑制FSH的刺激作用。（2）星形孢菌素（10(-9)-10(-6) M）或鞘氨醇（(10(-8)-10(-5) M）也可剂量依赖性地抑制FSH刺激CEO恢复减数分裂的作用。然而，单独的PMA（10(-8)-10(-5) M）对猪CEO的减数分裂恢复有双重作用。10(-8)-10(-6) M水平的PMA可刺激CEO恢复减数分裂，而在10(-5) M的高浓度下，它甚至可增强HX的抑制作用。（3）仅用BAPTA/AM或BAPTA/AM + FSH预处理CEO 60分钟可剂量依赖性地显著抑制FSH的作用。这些结果表明，在猪CEO配体介导的减数分裂恢复过程中，FSH可刺激磷酸肌醇水解，导致PKC激活和细胞内钙动员；并提示该过程涉及多种信号通路和信号相互作用。
