Bu Shumin, Xia Guoliang, Tao Yong, Lei Lei, Zhou Bo
College of Biological Sciences, China Agricultural University, Beijing, 100094, PR China.
Mol Cell Endocrinol. 2003 Sep 30;207(1-2):21-30. doi: 10.1016/s0303-7207(03)00213-2.
The present experiment used cultured mouse cumulus cell-enclosed oocytes (CEOs) and denuded oocytes (DOs) to study the function of nitric oxide (NO) in mouse oocyte meiotic maturation. Either positive or negative actions of NO on meiotic maturation has been observed when CEOs or DOs were cultured for 24 h in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, or in maturation medium (without HX) supplemented with different doses of sodium nitroprusside (SNP, a NO donor), N-omega-nitro-L-arginine methyl ester (L-NAME) or N(w)-nitro-L-arginine (L-NNA) (two inhibitors of NO synthase, NOS), and L-arginine (the only substrate of NOS). Both NOS inhibitors suppressed the formation of first polar body (PB1) of the oocytes in CEOs in a dose dependent manner, but no effect on germinal vesicle break down (GVBD) was observed. An optimal inhibitory effect was observed with either 10(-3) M L-NAME (P<0.01) or 10(-3) M L-NNA (P<0.01) and the inhibition could be reversed by the addition of SNP (10(-5) M). The above mentioned optimal concentration of L-NAME or L-NNA on CEOs exhibited no effect on oocyte meiotic maturation of DOs. Treatments of low concentrations of SNP (10(-7), 10(-6), 10(-5) M) stimulated significantly the oocyte meiotic maturation of CEOs which were inhibited with HX, but had no effect on DOs in the same culture medium. While, the treatment with high concentrations of SNP (0.1-4 mM) during the CEOs cultured in maturation medium resulted in a lower percentage of oocytes at PB1 stage and a higher percentage of atypical oocytes in a dose dependent manner compared with control. A dose of SNP at 1 mM exhibited significant inhibitory effect on the formation of PB1, but without effect on the number of atypical oocytes compared with control, while, this SNP dosage not only inhibited the oocyte PB1 formation but also increased the percentage of dead oocytes in DOs. Although oocytes of all groups underwent GVBD at the end of the culture in the spontaneous maturation medium, the results of the kinetics showed that the treatment of the optimal concentration of SNP (1 mM) could significantly delay GVBD during the first 5 h culture period. The concomitant addition of L-NAME with SNP did not reverse the inhibitory effect of SNP on CEOs. Similarly, neither pre-incubation nor illumination by ultraviolet ray could balance the inhibitory effect of SNP. Finally, when added alone at a concentration of 4 mM, L-arg caused extensive death of both CEOs and DOs. While, administration of 4 mM L-arg and 1 mM L-NAME to both CEOs and DOs simultaneously resulted in markedly reduced CEOs death percentage as compared with L-arg treatment alone, but not in DOs. These data support the idea that NO could act with a dual action (stimulation or inhibition) in mouse meiotic maturation depending on its concentration.
本实验使用培养的小鼠卵丘细胞包裹的卵母细胞（CEOs）和裸卵（DOs）来研究一氧化氮（NO）在小鼠卵母细胞减数分裂成熟中的作用。当CEOs或DOs在含有4 mM次黄嘌呤（HX）的培养基中培养24小时以维持减数分裂停滞时，或者在补充有不同剂量硝普钠（SNP，一种NO供体）、N-ω-硝基-L-精氨酸甲酯（L-NAME）或N（ω）-硝基-L-精氨酸（L-NNA）（两种NO合酶抑制剂，NOS）以及L-精氨酸（NOS的唯一底物）的成熟培养基（不含HX）中培养时，已观察到NO对减数分裂成熟的正向或负向作用。两种NOS抑制剂均以剂量依赖性方式抑制CEOs中卵母细胞第一极体（PB1）的形成，但未观察到对生发泡破裂（GVBD）有影响。用10⁻³ M L-NAME（P<0.01）或10⁻³ M L-NNA（P<0.01）观察到最佳抑制效果，并且通过添加SNP（10⁻⁵ M）可以逆转这种抑制作用。上述L-NAME或L-NNA对CEOs的最佳浓度对DOs的卵母细胞减数分裂成熟没有影响。低浓度SNP（10⁻⁷、10⁻⁶、10⁻⁵ M）处理显著刺激了被HX抑制的CEOs的卵母细胞减数分裂成熟，但对相同培养基中的DOs没有影响。然而，在成熟培养基中培养CEOs期间用高浓度SNP（0.1 - 4 mM）处理，与对照相比，导致处于PB1期的卵母细胞百分比降低，非典型卵母细胞百分比以剂量依赖性方式升高。1 mM的SNP剂量对PB1的形成表现出显著抑制作用，但与对照相比，对非典型卵母细胞数量没有影响，而该SNP剂量不仅抑制DOs中卵母细胞PB1的形成，还增加了死亡卵母细胞的百分比。尽管所有组的卵母细胞在自发成熟培养基中培养结束时都经历了GVBD，但动力学结果表明，最佳浓度SNP（1 mM）处理在培养的前5小时内可显著延迟GVBD。L-NAME与SNP同时添加并未逆转SNP对CEOs的抑制作用。同样，预孵育或紫外线照射均不能平衡SNP的抑制作用。最后，当单独以4 mM的浓度添加时，L-精氨酸导致CEOs和DOs大量死亡。然而，同时向CEOs和DOs施用4 mM L-精氨酸和1 mM L-NAME，与单独L-精氨酸处理相比，CEOs的死亡百分比显著降低，但DOs没有。这些数据支持这样的观点，即NO在小鼠减数分裂成熟中可根据其浓度发挥双重作用（刺激或抑制）。
