【作者】 苗德强；

【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】 第一章能量代谢在小鼠卵老化中的作用停滞在MII期的哺乳动物卵母细胞随着在输卵管或培养液中停留时间的延长而发生老化。老化的卵母细胞受精导致胚胎发育异常,而且卵母细胞老化也不利于体外受精和体细胞核移植等体外卵母细胞操作。在人上,有时一次体外受精失败后需要进行第二次授精,体外培养系统也可能影响到第二次授精的效果。因此有必要研究卵母细胞老化的机理。以前的报道证明卵母细胞老化过程中促成熟因子（maturation promoting factor,MPF）活性下降,从而导致对孤雌激活的敏感性增加,激活率升高。因此,激活率是评价卵母细胞老化程度的重要指标。我们通过向成分简单的CZB培养液中添加葡萄糖、乳酸钠和丙酮酸钠等能量物质,以及添加这些物质代谢途径的特异性抑制剂,研究了小鼠卵母细胞老化过程中能量代谢,为通过改变能量物质调节体外卵母细胞老化提供理论依据:对于优化卵母细胞体外培养条件也有重要意义。我们的实验结果如下:1.卵母细胞在M199+FCS/BSA与CZB+FCS/BSA/PVA中老化情况基本相同,其中去卵丘的卵母细胞（denuded oocyte,DO）在这些培养液中几乎不老化,而卵丘卵母细胞复合体（cumulus-oocytes complex,COC）一半以上发生老化,而且在M199中老化比率更高（74.5%和85%比56%和55%）;DO和COC在M199±PVA培养液中老化模式与以上几组显著不同,其中DO在这两种培养液中发生快速老化,而COC不发生老化。2.在不添加葡萄糖、丙酮酸钠和乳酸钠三种物质的培养液中,DO和COC都发生快速老化,激活率分别为93.3%和91.8%。而在单独添加丙酮酸或者乳酸的培养液中,DO和COC都不发生老化,激活率都低于5%。在单独添加葡萄糖的培养液中,DO发生快速老化,激活率为95.3%,而COC几乎不老化,激活率为0。在添加三种物质或者其中两种的培养液中,DO和COC激活率都很低。3.在含有葡萄糖的CZB培养液中分别添加100μM和200μM的脱氢表雄酮（dehydroepiandrosterone,DHEA）阻断葡萄糖磷酸戊糖通路（pentose phosphatepathway,PPP）发现,COC激活率均小于5%。4.在含有乳酸钠的CZB培养液中添加乳酸脱氢酶（lactate dehydrogenase,LDH）抑制剂草氨酸钠,随着浓度增加,卵母细胞孤雌激活率逐渐升高;添加10mM和20mM的草氨酸钠激活率分别为49.2%和59.6%。而在含有丙酮酸钠的培养液中添加草氨酸钠不影响卵母细胞激活率。5.在含有丙酮酸钠的CZB培养液中添加不同浓度的线粒体抑制剂鱼藤酮,随着浓度升高,卵母细胞激活率逐渐升高,0.05μM鱼藤酮就有30%卵母细胞能够孤雌激活,当浓度升高到0.1μM时,84.8%的卵母细胞能够孤雌激活。6.在不含有葡萄糖、丙酮酸钠和乳酸钠的CZB培养液中添加不同浓度的巯基乙醇,即使浓度升高到1mM卵母细胞激活率仍然维持在90%以上,显然巯基乙醇不能阻止卵母细胞老化。7.卵母细胞在不含葡萄糖、丙酮酸钠和乳酸钠的CZB培养液中添加不同浓度的二硫苏糖醇（dithiothreitol,DTT）,即使浓度升高到500^tM激活率仍然维持在90%以上,而且DO在添加500μM二硫苏糖醇的CZB培养液中大部分死亡,只有约20%存活下来,且存活下来的卵母细胞100%发生激活。COC在添加同样浓度的二硫苏糖醇的CZB培养液中不发生死亡。8.卵母细胞在三种CZB培养液中培养24h,其中不添加葡萄糖、乳酸钠和丙酮酸钠的CZB中,80%的卵母细胞发生死亡,存活的卵母细胞100%发生自发激活;而在单独添加乳酸钠的CZB中,卵母细胞老化24h没有发生死亡,但几乎所有卵母细胞（约97%）发生自发激活,而且卵母细胞形态极差,卵母细胞表现为严重畸形和碎裂;单独添加丙酮酸钠的一组卵母细胞保持正常形态,且没有发生激活。以上实验结果说明:1)培养液M199和CZB对卵母细胞老化影响不同。2)卵母细胞在单独添加乳酸钠或者丙酮酸钠的CZB中,DO和COC都不老化:而在单独添加葡萄糖的CZB中DO老化,COC不老化,这说明卵母细胞能够利用乳酸和丙酮酸,但只有通过卵丘细胞才能够利用葡萄糖。3)COC对葡萄糖的利用不是通过PPP途径,抑制PPP途径不会影响卵母细胞在添加葡萄糖的CZB培养液中的老化。4)乳酸必须通过LDH调节生成丙酮酸才能发挥作用,调节卵母细胞老化。5)抑制丙酮酸在线粒体内的代谢,减少ATP的产生会导致卵母细胞老化。6)单独添加巯基乙醇或者二硫苏糖醇不能阻止卵母细胞老化,说明丙酮酸的抗老化作用不是单独通过其抗氧化作用实现的。7)体外长时间（24h）培养卵母细胞,单独添加丙酮酸比单独添加乳酸有更好的抑制卵母细胞老化的作用。第二章小鼠胚胎发育的雄性效应研究虽然成功的胚胎发育依赖于雄性和雌性两方面的遗传和外遗传因素,但雄性对胚胎发育影响的研究却很少。还不清楚通过受精后胚胎发育对卵母细胞进行的评价是否会因不同雄性个体间精子的差异而受影响。本研究首次比较了不同成熟质量的卵母细胞与不同公鼠附睾精子体外受精和Sr2+孤雌激活发育能力之间的差异,并采用随机选择卵母细胞和分别来自附睾尾、输精管或者电射精精子体外受精及发育的方法检测雄性对受精和胚胎发育的影响。结果表明附睾精子和输精管精子受精率和囊胚率显著高于射精精子,但同一公鼠不同次射精精子间差异不显著。平均数和变异系数的标准误分析证明,只要涉及到多只公鼠受精率和受精后的囊胚发育率均显著高于Sr²⁺孤雌激活后的激活率和囊胚发育率,无论其精子是采自附睾尾、输精管还是射精精子,也不论卵母细胞质量如何。总结如下:（1）同等条件下,小鼠附睾尾精子受精率高于射精精子;（2）和大家畜类似,虽然小鼠被认为在遗传学上差异很小,但在体外生产胚胎上对其发育潜能仍存在显著的雄性效应;（3）孤雌激活用来评价卵母细胞质量更加合适,能够消除雄性效应。更多还原

【Abstract】 ChapterⅠ: Effects of energy metabolism during mouse oocyte agingmetaphase II （MII）-arrested oocyte undergone oocyte aging afer delay in oviduct or culture media. Fertilized aged oocytes usually result in developmental anomaly. In addition,oocyte aging impact in vitro oocyte operation ,such as in vitro fertilization and nuclear transplantation and so on. In human,another insemination is needed if the first insemination failed. In vitro culture system may influence the second insemination, so it is necessary to investigate the underlying mechanism of oocyte aging. The previous reports demonstrated MPF activation decreased during oocyte aging, which lead to increasing of the sensitiveness to parthenogenetic activation and rate of activation. The rate of activation can reflect the degree of oocyte aging, so we detected oocyte aging by observing the rate of oocyte activation. We studied energy metabolism during oocyte aging by supplying glucose, sodium lactate , sodium pyruvate and its specific inhibitors of metabolism pathway. By this way, we can provide proof for regulating oocyte aging by altering energy substance, which is very important to optimize in vitro culture condition of oocytes. The results are summarized as follows:1. The oocytes cultured in CZB+FCS/BSA/PVA undergone similar oocyte aging. DO in these medias didn’t undergo aging, but more than half COC did and the activation rate is significant higher in M199 than in CZB. COC in M199±PVA were obviously different from in above groups. DO undergone rapid oocyte aging, however COC didn’t.2. Both DO and COC undergone rapid oocyte aging afer being cultured in CZB without glucose, sodium pyruvate and sodium lactate, and the activation rates are 93.3% and 91.8% respectively, but neither DO nor COC undergone oocyte aging in meida supplied with either sodium pyruvate or sodium lactate alone, the activation rates were all lower than 5%; DO undergone rapid oocyte aging,with 95.3% activation rate, however COC didn’t ,with 0 activation rate, in CZB supplied with glucose alone; Few oocytes undergone aging in CZB supplied with three or two substance.3. The activation rate of COC cultured in CZB containing glucose supplied with 100μM or 200μM DHEA, inhibitor of PPP path, were all lower than 5%.4. The activation rate gradually increased when increasing the concentration ofoxamate, inhibitor of LDH, in CZB containing sodium lactate. Activation rates were49.2% and 59.6% respectively. But activation rate of oocytes cultured in CZBcontaining sodium pyruvate supplied with oxamate, remained unchanged.5.Activation rate gradually increased when the oocytes were cultured in CZBcontaining sodium pyruvate with increasing concentration of rotenone. When theconcentration of Rotenone were 0.05μM,30% oocytes were activated,while thecongcentration were 0.1μM, 84% oocytes were activated.6.Oocytes were cultured in CZB without glucose, sodium pyruvate and sodium lactatesupplied with different concentration of mercaptoethanol. With the concentration increasinguntil 1mM the activation rate remained more than 90%. It is obvious that mercaptoethanol can notprevent oocyte aging.7. Oocytes were cultured in CZB without glucose, sodium pyruvate and sodium lactate supplied with different concentration of dithiothreitol. With the concentration increasing until 500μM, the activation rate remained more than 90%. Furthermore, when 500μM dithiothreitol were supplied, more DOs died, and only about 20% survived, 100% of which undergone activation, hower COC did not die.8. 80% Oocytes died when they were cultured in CZB without glucose, sodium lactate, and sodium pyruvate for 24h, and all of the survived oocytes undergone spontaneous activation; When the oocytes were cultured in CZB supplied with sodium lactate 24h, all the oocytes survived and almost all of them （97%） undergone spontaneous activation, furthermore, they appeared severe abnormal and fragmental; All the oocytes cultured in CZB with sodium pyruvate remained normal and did not undergo activation.Our above results demonstrated that:1. Oocytes cultured in M199 and CZB undergo aging differently.2.Both DO and COC cultured in CZB supplied with sodium lactate or sodiumpyruvate did not undergo aging; But DO undergone aging ,however, COC not whencultured in CZB with glucose alone. This indicated that oocyte can use sodium lactate or sodium pyruvate, but utilize glucose only by cumulus cells.3. COC utilized glucose not by PPP, because of inhibiting PPP did not influence oocyte aging in CZB supplied with glucose.4. Lactate regulated oocyte aging depending on LDH ,which converted lactate to pyruvate.5.To Inhibit metablism of pyruvate in mitochondria can reduce ATP production, so it lead to oocyte aging.6. Oocyte aging can not be prevented when mercaptoethanol or DTT were supplied to CZB without glucose, lactate or pyruvate. This result indicated that pyruvate prevented oocyte aging not only by its antioxidation.7. Pyruvate can prevent oocyte aging more than lactate when they were cultured for 24h.ChapterⅡ: the study on the effects of male on the development of mouse embryoAlthough successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr²⁺ parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr²⁺ parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that （1） epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; （2） like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; （3） parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male.更多还原