Jablonka-Shariff A, Olson L M
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63167, USA.
Mol Reprod Dev. 2000 Apr;55(4):412-21. doi: 10.1002/(SICI)1098-2795(200004)55:4<412::AID-MRD9>3.0.CO;2-W.
This study was conducted to determine whether endothelial-derived nitric oxide synthase (eNOS) affects meiotic maturation of mouse oocytes in vitro. Cumulus-oocyte complexes (COC) were isolated from ovarian follicles of 27-day-old PMSG-primed wildtype (WT), and eNOS-knockout (eNOS-KO) females, and cultured in drops of medium under oil at 37 degrees C for 16-18 hr. Experiment 1 was carried out to determine effects of eNOS deficiency on the ability of COC to mature in vitro. To determine whether acute synthesis of nitric oxide (NO) was required for oocyte maturation, COC collected from WT mice were cultured in medium without (control) or with different doses of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS (exp. 2). To assess effects of NO deficiency on the kinetics of germinal vesicle breakdown (GVBD), COC from WT and eNOS-KO females were observed for 3.5 hr. COC from WT females were also incubated in medium without or with L-NAME (exp. 3 and 4). After the culture period, cumulus cells were removed, and oocytes were counted and classified as metaphase II (M II), metaphase I (M I) or showing atypical (degenerative) morphology. To determine viability and nuclear morphology of oocytes, they were stained with fluorescein diacetate or 4,6-diamidine-2'-phenylindole dihydrochloride, respectively. There were no differences in body weights but ovarian weights were lower in eNOS-KO mice compared with WT mice (P < 0.05). Ovaries from eNOS-KO mice contained fewer COC collected relative to WT mice (P < 0.01). Maturation of COC from eNOS-KO mice or WT oocytes treated with L-NAME resulted in a lower percentage of oocytes at M II stage (P < 0.01 and P < 0.05, respectively) and a higher percentage of oocytes at M I or atypical stages compared with those from WT (P < 0.01 and P < 0.05, respectively). Many oocytes that showed either an arrest in M I stage or abnormal morphology were not viable. Several oocytes in M II stage demonstrated abnormalities in distribution of maternal chromosomes. Our data demonstrate that eNOS-derived NO is a key modulator of oocyte meiotic maturation in vitro. These results support our previous observations in vivo and indicate that eNOS/NO has independent functions in both oocyte maturation and follicular/oocyte development.
本研究旨在确定内皮型一氧化氮合酶（eNOS）是否会影响小鼠卵母细胞的体外减数分裂成熟。从27日龄经孕马血清促性腺激素（PMSG）预处理的野生型（WT）和eNOS基因敲除（eNOS-KO）雌性小鼠的卵巢卵泡中分离出卵丘-卵母细胞复合体（COC），并在37℃的油滴培养基中培养16 - 18小时。实验1旨在确定eNOS缺乏对COC体外成熟能力的影响。为了确定卵母细胞成熟是否需要一氧化氮（NO）的急性合成，将从WT小鼠收集的COC在不含（对照）或含有不同剂量的NOS抑制剂N（ω）-硝基-L-精氨酸甲酯（L-NAME）的培养基中培养（实验2）。为了评估NO缺乏对生发泡破裂（GVBD）动力学的影响，观察来自WT和eNOS-KO雌性小鼠的COC 3.5小时。来自WT雌性小鼠的COC也在不含或含有L-NAME的培养基中孵育（实验3和4）。培养期结束后，去除卵丘细胞，对卵母细胞进行计数并分类为中期II（M II）、中期I（M I）或显示非典型（退化）形态。为了确定卵母细胞的活力和核形态，分别用二醋酸荧光素或4,6-二脒基-2'-苯基吲哚二盐酸盐对它们进行染色。eNOS-KO小鼠与WT小鼠的体重没有差异，但eNOS-KO小鼠的卵巢重量低于WT小鼠（P < 0.05）。相对于WT小鼠，eNOS-KO小鼠卵巢中收集到的COC较少（P < 0.01）。与WT小鼠相比，来自eNOS-KO小鼠的COC或用L-NAME处理的WT卵母细胞成熟后，处于M II期的卵母细胞百分比更低（分别为P < 0.01和P < 0.05），处于M I期或非典型期的卵母细胞百分比更高（分别为P < 0.01和P < 0.05）。许多停滞在M I期或形态异常的卵母细胞没有活力。一些处于M II期的卵母细胞显示出母源染色体分布异常。我们的数据表明，eNOS衍生的NO是体外卵母细胞减数分裂成熟的关键调节因子。这些结果支持了我们之前体内观察的结果，并表明eNOS/NO在卵母细胞成熟和卵泡/卵母细胞发育中具有独立的功能。
