【作者】 隋宏书；

【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】 辅助生殖技术在治疗雌雄生殖不育方面是非常有效的。卵巢刺激并结合IVF或ICSI目前已经成为治疗卵巢周期异常、输卵管疾患、精子抗体症、少精或无精症方面的常用方法。但是,对于那些不能产生具有受孕能力配子的患者来说(如患有卵巢异常或卵子缺陷的妇女和患有精子发生失败的男子),目前还没有办法获得他们自己遗传基因的后代,只能使用捐赠的配子或胚胎来治疗。近年来,来源于克隆的一种独特技术——体细胞单倍体化可能为那些不能产生配子的不育患者提供治愈的机会。体细胞单倍体化是指体细胞(二倍体)被诱导进入减数分裂而使二倍体的染色体形成单倍体的过程。由于哺乳动物卵母细胞能够提供减数分裂的胞质,比较适合用来进行体细胞单倍体化。卵母细胞在体外很容易获得,而且近年来显微操作技术也不断地完善,因此体细胞单倍体化是可以发生的。但是,使用体细胞进行单倍体化形成人工配子以及使用它正常受精的报道甚少。到目前为止,还没有子代成功出生的报道。很多报道都显示,体细胞在卵母细胞内发生单倍体化过程中出现许多异常现象,如染色单体不能正确地分离、细胞骨架系统遭到破坏、MII期染色体凝集能力较差以及蛋白激酶活性偏低等。因此,有必要对体细胞单倍体化过程中所发生的减数分裂事件进行较为系统的研究。本实验采用体外培养、药物处理、激光共聚焦显微术以及蛋白激酶活性分析等方法系统地研究了初级精母细胞核替换生发泡(GV)后的重构卵在体外成熟以及激活期间所发生的减数分裂事件;同时,检测了重构卵中MAPK活性以探讨重构卵减数分裂调控的分子机理。主要实验结果如下:1. GV互换重组的卵母细胞成熟率与未经显微操作的裸卵无差异,而且都无大极体形成,染色体100%凝集,说明我们的显微操作系统对卵母细胞没有造成伤害。2.初级精母细胞(PS)植入GV期卵胞质后,能够完成第一次减数分裂,但很多重构卵都排出异常大的第一极体,染色体也不能完全凝集。初级精母细胞质的量对重构卵的成熟率、大极体形成以及染色体的凝集都没有影响。在重构卵成熟期间用OA处理显著减低大极体的形成比例,提高含有完全凝集染色体卵母细胞的比例;成熟期间使用U0126处理重构卵大极体比例显著升高,但不影响染色体凝集。3.重构卵第一极体排出的速度加快,发生在注核后4.5-6.5h,比对照组裸卵和GV互换卵早4 h。4.重构卵排出第一极体后老化40h,第一极体几乎不发生退化,而大多数对照组裸卵和GV互换卵的极体都已退化。在成熟期间添加OA,能够提高重构卵第一极体的退化率,但是在激活前使用U0126处理却不增加极体退化率。使用OA和U0126联合处理(即在成熟期间添加OA,在激活前再添加U0126),重构卵极体退化率与单独使用OA组差异不显著。说明调控极体退化的事件只发生在成熟期间,一旦极体排出,任何处理都无效。5.重构卵的激活率和原核形成率与GV互换组相比都显著降低。在成熟期间添加OA或激活前添加U0126处理后,重构卵激活率以及原核形成率显著升高,而且两种处理之间没有差异。使用二者联合处理(即先在成熟期间添加OA,然后在激活前添加U0126)后,重构卵的激活率以及原核形成率显著高于单独使用OA或U0126处理组。6.通过抗-lamina抗体染色后,核纤层蛋白在原核外周着色的强弱分为三种,即高度着色、中等着色和不着色。对照组裸卵和GV互换组中核纤层蛋白都发生高度着色。但是重构卵(GV+PS)中核纤层蛋白只发生中等着色。使用OA和U0126分别在重构卵成熟期间以及激活前处理,都能明显提高核纤层蛋白在原核中的表达,OA处理组要比U0126组效果好。使用OA和U0126组合共同处理效果更明显。7.在MII期,重构卵的MAPK和MPF活性都显著低于GV互换卵。在成熟期间使用OA或U0126处理后,分别提高或降低成熟重构卵中的MAPK活性,但对MPF活性没有影响。说明重构卵中大极体的产生、极体不发生退化及染色体完全凝集能力较差可能是由于成熟期间MAPK活性低造成的。8.从MII期到激活前(老化12h)和激活后(原核期),GV互换卵MPF活性逐渐下降,但重构卵中MPF活性下降幅度明显小于GV互换卵。在成熟期间添加OA或激活前使用U0126处理后,重构卵中MPF活性没有发生显著变化。9.从MII期到激活前和激活后,GV互换卵MAPK活性逐渐下降,但重构卵MAPK活性在此期间不发生显著下降。应当指出,MII期重构卵中MAPK活性本来就比GV互换卵低很多。成熟期间添加OA显著提高MII期重构卵的MAPK活性,因此在激活前重构卵中MAPK活性也明显下降。在激活前使用U0126处理后,重构卵中MAPK活性显著下降。在成熟期间添加OA,然后在激活前添加U0126处理,重构卵中MAPK活性下降的幅度显著高于单独使用OA或U0126处理组。10.总之,体细胞单倍体化的重构卵出现减数分裂异常(如大极体、染色体凝集不好等)、第一极体不退化以及激活后不能形成原核等异常现象。检测MAPK活性和用调节MAPK活性药物处理证明,这些异常可能与成熟卵母细胞的MAPK活性及其在老化和激活后的变化有关。更多还原

【Abstract】 Reproductive technologies are effective in overcoming male and female infertility. Ovarian stimulation combined with IVF or ICSI has become standard treatment in cases of ovarian cycle anomalies, tubal pathology, sperm antibodies, oligoasthenoteratozoospermia and even azoospermia. Only for patients without gametes are there currently no treatment options leading to their own genetic children. Women with ovarian failure or defective oocytes or men with spermatogenic failure are sterile and can only be treated by using donor gametes or embryos or by adoption. Recently, novel technology (somatic cell haploidization)derived from cloning may offer an alternative strategy for treatment of male and female sterility by creating gametes .Somatic cell haploidization is defined as a process whereby a somatic nucleus (2N) undergoes induced meiosis, during which the diploid chromosomes are reduced to haploid (1N). Mammalian oocytes are ideally suited for use as haploidization, because they are easily obtained and manipulation techniques are well established. Despite the success in using germ cells to show that, in principle, haploidization is possible, successful generation and fertilization of artificially hapoidized gametes from somatic cells has not been reported. A careful study of published data revealed similar meiotic abnormalities between the reconstructed oocytes with GV replaced with somatic cells. For examle, reconstructed oocytes frequently produced failure of chromosome segregation, abnormally large first polar bodies, diffused spindles, loosely condensed chromosomes and low activity of protein kinases. Therefore, it is necessary to study some meiotic events of haploization of somatic cells.The meiotic events of mouse oocytes reconstructed with germinal vesicles replaced with primary spermatocytes nuclei were studied by using in vitro culture, drug treatment, confocal microscopy, protein kinase assay in maturation and activation. Besides, we exmined MAP kinase activity in reconstructed oocytes to elucidate the molecular mechanisms regulating meiosis. The results are as follows:1. No difference was found in maturation rates, large polar body formation and chromosome condensation between intact denuded oocytes and germinal vesicle transferred oocytes at the end of the culture period. The results indicatied that the microsurgical procedure had no effect on these oocyts.2. Meiotic maturation could be achieved in reconstructed oocytes matured after primary spermatocyte was transferred into GV ooplasm, but the first polar body was very large and the proportion of oocytes with adequately condensed chromosome was very low. The cytoplasm amount of primary spermatocytes had no influence on the proportion of maturation rates, large polar body formation and adequately condensed chromosome in reconstructed oocytes. OA treatment during maturation reduced the proportion of reconstructed oocytes with large polar body and improved the proportion of reconstructed oocytes with adequately condensed chromosome. However, the proportion of reconstructed oocytes with large polar body was significantly improved by U0126 treatment during maturation, but this treatment had no effected on chromosome condensation.3. After PS was transferred into GV ooplasm, extrusion of the first polar body occurred at 4.5-6.5hr which was approximately 4hr earlier than DO or GV transferred oocytes.4. First polar body did not degenerate after aging for 40 hours in reconstructed oocytes and this was different from DO and GV transferred oocytes. It was OA treatment during maturation not U0126 treatment before activation that significantly improved the degeneration of first polar bodies in reconstructed oocytes with primary spermatocytes and GV cytoplasm. Moreover, when treated with OA during maturation followed by U0126 treatment before activation, the degeneration rates of first polar bodies in reconstructed oocytes was the same as reconstructed oocytes treated only with OA during maturation. The results mentioned above suggested that the events regulating the degeneration of first poalr body in renconstructed oocyte occurred during maturation. Once the first polar body was extrused, no treatment was effective.5. The percentages of activation and pronucleus formation in reconstructed oocytes were significantly lower than that in GV transferred oocytes. Either OA treatment during maturation or U0126 treatment before activation improved the activation and pronucleus formation in reconstructed oocytes and there was no difference between the two treatments. If treated with OA during maturation followed by U0126 treatment before activation, the percentage of activation and pronucleus formation in reconstructed oocytes was significantly higher than that obtained with either treatment only.6. Three different types of immunostaining were established : type I, high staining; type II, intermediate staining; type III, negative staining by using the confocal settings and anti-lamina antibody. DO and GV transferred oocytes showed only type I staining. But reconstructed oocytes with primary spermatocytes showed only type II staining. Either OA treatment during maturation or U0126 treatment before activation significantly improved the proportion of type I staining and the effect of OA treatment during maturation was better than U0126 treatment before activation. If treated with OA during maturation followed by U0126 treatment before activation, the percentage of type I staining in reconstructed oocytes was significantly higher than that obtained with either treatment only.7. MAP kinase and MPF activity were significantly lower in fresh MII reconstructed oocytes than that in fresh GV transferred oocytes at MII. OA treatment during maturation in reconstructed oocytes with primary spermatcytes and GV cytoplasm improved MAP kinase activity significantly and U0126 treatment during maturation had the opposite effect. Howerver, the two treatments during maturation had no influence on MPF activity. From the results mentioned above, we can conclude that large first poalr body formation, long-term persistence of first polar body, inability of bipolar spindle formation and adequately condensed chromosome may be due to the low MAP kinase activity during maturation in reconstructed oocytes.8. MPF activity in GV transferred oocytes from MII stage to the stage before and after activation decreased gradually. But the degree of MPF activity decline in reconstructed oocytes was smaller than that in GV transferred oocytes. OA treatment during maturation and U0126 treatment before activatioh did not affect MPF activity in MII reconstructed oocytes. 9. MAP kinase activity in GV transferred oocytes from MII stage to the stage before and after activation decreased gradually. Howerver, MAP kinase activity in reconstructed oocytes did not change significantly. MAP kinase activity was lower in reconstructed oocytes at MII than that in GV transferred oocytes and OA treatment during maturation improved the MAP kinase activity significantly in reconstructed oocytes at MII, thus MAPK activity in reconstructed oocytes also declined significantly. U0126 treatment before activation significnatly declined MAPK activity in reconstructed oocytes. If treated with OA during maturation followed by U0126 treatment before activation, the degree of MAP kinase activity decline in reconstructed oocytes was higher than that in reconstructed oocytes treated with OA or U0126 only.10. In conclusion, data mentioned above revealed many abnormalities in the reconstructed oocytes. For example, reconstructed oocytes frequently produced abnormally large first polar bodies, loosely condensed chromosomes, first polar body long-term persistence, and failure of pronucleus formation. These meiotic abnormal events were relative to the changes of MAP kianse activity in matured oocytes, oocytes before activation and after activation throughout detection of MAPK activity and drug treatment regulating MAPK activity.更多还原