【作者】 刘娜；

【导师】 谭景和；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 卵母细胞排卵后老化显著影响其胚胎发育。排卵后老化的卵母细胞受精后出生的小鼠表现出神经敏感和情绪的异常。与输卵管内滞留一样,体外培养成熟卵母细胞也会导致卵母细胞老化。然而,人类和一些动物通常可以在发情周期的任何一天从事性行为,这可能导致排卵后老化的卵母细胞受精。另外,许多研究和临床应用的实验设计都涉及成熟卵母细胞在显微操作和授精前的体外培养。因此,研究卵母细胞老化的机制并且调控卵母细胞老化无论对人类还是非人类动物的健康生殖都是非常重要的。老化卵母细胞最明显的特征包括对激活刺激敏感性的增加、MPF活性降低、后期II的启动、皮质颗粒部分胞吐、线粒体聚集、微管组装变化,并出现细胞凋亡的征兆,例如胞质和DNA断裂以及Bcl-2蛋白水平的降低。另外,老化的小鼠卵母细胞中组蛋白的某些赖氨酸位点乙酰化水平增加,而且由于老化过程中的氧化应激造成ROS的累积,并改变卵母细胞内的氧化还原状态,造成卵母细胞内总的GSH量减少以及GSH/GSSG比例的降低。我们之前的研究证明卵丘细胞能够促进小鼠卵母细胞老化。然而,卵丘细胞促进卵母细胞老化的确切机制还不清楚。据报道成熟卵母细胞能够代谢丙酮酸,并且在不含葡萄糖的培养基中卵丘卵母细胞复合体(COCs)比裸卵(DOs)消耗更多的丙酮酸。我们猜测卵母细胞在不含丙酮酸的条件下老化的更快,并且卵丘细胞可能通过消耗不含葡萄糖的CZB中的丙酮酸从而促进卵母细胞老化。为了验证这个猜测,我们将新排卵的小鼠卵母细胞培养在添加不同浓度丙酮酸的CZB中,然后进行激活处理或通过放射自显影的方法分析MPF活性。结果发现丙酮酸以剂量依赖性的方式降低COCs和DOs的激活率,而升高其MPF活性,卵丘细胞存在的情况下需要更多的丙酮酸。新鲜排卵的DOs培养在不另外添加丙酮酸的COCs条件化的CZB中,其激活率显著升高,MPF活性显著降低,然而,当在条件化培养基中添加10 mM丙酮酸后,DOs的激活率和MPF活性都与新鲜卵母细胞无差别。我们通过毛细管电泳试验证明,COCs和卵丘细胞单层制备的CZB条件化培养基中丙酮酸的含量显著低于新鲜制备的CZB、单独培养的CZB以及DOs条件化的CZB中丙酮酸的含量。这些结果有力地验证了我们的猜测:在不含葡萄糖的培养液中卵丘细胞通过消耗丙酮酸从而促进卵母细胞老化。为了检测丙酮酸对卵母细胞其他老化指标的影响,我们通过免疫荧光显微镜检查法观察了皮质颗粒的提前胞吐、线粒体分布、微管组装、抗凋亡蛋白Bcl-2水平、组蛋白乙酰化水平,通过Hoechst33342染色观察了后期II的启动,通过体外受精及其随后的胚胎发育能力检测了丙酮酸对老化卵母细胞发育潜力的影响。结果证明,丙酮酸能够显著抑制老化卵母细胞皮质颗粒的提前胞吐、线粒体聚集、微管组装的变化;并且显著抑制了Bcl-2、组蛋白乙酰化水平以及胞质碎裂比例的升高,说明丙酮酸能够显著抑制老化小鼠卵母细胞的凋亡。另外,我们的数据还证明老化过程中添加丙酮酸能够显著抑制老化对卵母细胞受精及其发育能力所造成的损伤。这些数据表明,丙酮酸能够显著抑制COCs和DOs的老化。为了验证丙酮酸是否通过其抗氧化作用从而抑制卵母细胞老化,我们通过2’,7’-dichlorodihydrofluorescein diacetate (DCHFDA)染色的方法检测了老化卵母细胞内ROS的水平,并通过DTNB-GSSG还原酶实验检测了老化卵母细胞内总GSH、还原型GSH水平以及GSH/GSSG比例。结果表明,丙酮酸能够显著抑制老化卵母细胞中ROS水平的增加,并且能够显著抑制总GSH和GSH水平以及GSH/GSSG比例的降低。这些数据表明,丙酮酸可能通过其抗氧化作用维持细胞内的氧化还原水平,显著削弱卵母细胞老化过程中的氧化应激对其造成的损伤。因此,我们得出结论:丙酮酸能够抑制小鼠COCs和DOs的老化,卵丘细胞通过消耗培养液中的丙酮酸从而促进卵母细胞老化。而且,从我们现在的数据可以看出,很显然在卵母细胞老化过程中添加丙酮酸能够显著提高排卵后老化卵母细胞的质量,并降低其凋亡指标。丙酮酸很可能通过其抗氧化作用从而维持卵母细胞GSH/GSSG比例,并阻止总GSH含量的降低以及胞质内ROS水平的升高,从而显著抑制凋亡的发生。更多还原

【Abstract】 Postovulatory aging of oocytes significantly affects embryonic development. Fertilization of postovulatory aged oocytes gives rise to mice suffering from nervous and emotional abnormalities and decreased reproductive fitness and longevity Like detention in the oviduct, in vitro culture of matured oocytes also led to oocyte aging. However, human beings and some animals potentially undertake sexual activity on any day of the estrous cycle, which may cause fertilization of aged ovulated oocytes. In addition, many experimental designs in both research and clinical applications involve culture of matured oocytes prior to micromanipulation or insemination. Therefore, studies on the mechanisms and control of oocyte aging are important for the healthy reproduction of both human beings and nonhuman mammals.The most prominent manifestations of aged oocytes include an increased susceptibility to activating stimuli, a decrease in MPF activity, the onset of anaphase II, a partial exocytosis of cortical granules, mitochondria aggregation, changes in microtubule assembly and signs of apoptotic cell death such as increased cytoplasmic and DNA fragmentation and decreased levels of Bcl-2 proteins. In addition, an increased acetylation on some lysines of histones and intracellular levels of ROS was increased for the reason of oxidative stress during aging process, subsequently decreased the level of total GSH and the ratio of GSH/GSSG. Our previous studies showed that cumulus cells accelerated aging of mouse oocytes.However, the exact mechanism by which cumulus cells promote oocyte aging is unknown. Since it was reported that matured oocytes were capable of metabolizing pyruvate and that cumulus-oocyte complexes (COCs) consumed much more pyruvate than cumulus-denuded oocytes (DOs) in the absence of glucose, we hypothesized that oocytes would age quickly in the absence of pyruvate, and cumulus cells would accelerate oocyte aging by depleting pyruvate in the CZB medium that contains no glucose. The objective of this study was to test this hypothesis. Newly ovulated mouse oocytes were cultured in the CZB medium supplemented with different concentrations of pyruvate before treated for activation or assayed for MPF activity. While pyruvate decreased the susceptibility to activation stimulus while increased the MPF activity of both COCs and DOs in a dose-dependent manner, more pyruvate was needed in the presence than in absence of cumulus cells. When newly ovulated DOs were cultured in COCs-conditioned CZB without pyruvate supplementation, activation rates increased while MPF activity decreased significantly; however, when cultured in conditioned CZB supplemented with 10 mM pyruvate, both activation rates and MPF activity remained close to those of newly ovulated control oocytes. Capillary electrophoresis showed that CZB conditioned with COCs or cumulus cells contained significantly less pyruvate than freshly prepared CZB and CZB conditioned with DOs or without cells. These results have fully born out our hypothesis that cumulus cells accelerate oocyte aging by depleting pyruvate in the culture medium that contains no glucose.In order to detect the effects of pyruvate on the other indicators of oocytes aging, we observed the premature exocytosis of cortical granules, mitochondria distribution, microtuble assembly, levels of Bcl-2 and histone acetylation through immunofluorescence microscopy; Observed the onset of anaphase II by Hoechst 33342 staining. We also detected the effects of pyruvate on the development potential of aging oocytes through in vitro fertilizaion and subsequent embryonic development. This results suggested that pyrvate can significantly inhibit premature exocytosis of cortical granules, mitochondria aggregation and changes in microtuble assembly; and significantly prevented the decreases of levels of Bcl-2, histone acetylation and the rates of cytoplasmic fragmentation, indicated that pyruvate prevented apoptosis of aging mouse oocytes. In addation, the present results also suggested that pyruvate can significantly weaken the detrimental effects of postovulatory aging on the developmental potential of oocytes. This data confirmed that pyruvate inhibited aging of both COCs and DOs.In order to verify whether the pyruvate inhibited the aging of oocytes through its antioxidative ability we evaluated the intracellular level of ROS through 2 ’, 7’-dichlorodihydrofluorescein diacetate (DCHFDA) staining method; and assaied the levels of total GSH, reduced GSH and the ratio of GSH/GSSG through DTNB-GSSG Reductase experiment. The results showed that pyruvate could significantly prevent the increase in the level of ROS, and the decrease of levels of total GSH and reduced GSH and the ratio of GSH/GSSG. It is concluded that pyruvate could maintain the redox state of the aging oocytes and significantly weaken the injury of oocytes resulted from oxidative stress during postovulatory aging.Therefore, we conclude that pyruvate could inhibit the aging of both COCs and DOs, cumulus cells accelerate oocyte aging by depleting pyruvate in the culture medium that contains no glucose. And from the data presented, it is apparent that the supplementation of pyruvate during oocytes aging had several beneficial effects on the qualities of postovulated oocytes and reduced multiple measures of apoptosis. One beneficial effect of pyruvate in preventing the apoptosis is the favorable maintenance of the oocytes GSH-to GSSG ratio and prevention of a decrease in total glutathione content and the increase in ROS maybe through its antioxidative activites.更多还原