Piezo介导的辅助孵化对小鼠胚胎着床和发育影响的研究

发布时间：2019-07-02 12:17

【摘要】：透明带(zona pellucida,ZP)是包裹在卵母细胞外的一层由糖蛋白组成的外被结构,在初级卵泡早期阶段由卵母细胞和颗粒细胞共同分泌形成。在受精过程和早期胚胎发育中起着重要作用。然而,透明带的厚化和硬化不利于胚胎的孵化。因此,我们尝试利用辅助孵化(assisted hatching,AH)技术提高囊胚的孵化率,从而提高胚胎的着床率以及子代的出生率。辅助孵化是通过人为的方法对透明带进行处理,使胚胎从透明带释放出的技术。目前,辅助孵化技术有许多种,我们组早期的实验发现,不同的辅助孵化技术对不同时期的胚胎进行操作会有不同的效果。Piezo介导的各种辅助孵化技术中,在桑椹胚期进行透明带打小孔处理后(打孔尺寸约为10 μm),可获得最高的囊胚孵化率。而该种方法是否能够提高胚胎的着床率以及子代的出生率仍有待考究。本实验利用该种辅助孵化方法处理胚胎,再进行胚胎移植(embryo transfer,ET),统计其胚胎着床率、母鼠妊娠率、子代出生率以及子代生长曲线等数据,以探明piezo介导的辅助孵化对小鼠胚胎着床和发育的影响。本研究对以下三种不同来源的胚胎进行分析,包括:(1)自然交配组(mating组),用作非辅助孵化、非胚胎移植操作的对照组;(2)体内受精获得的胚胎,仅进行胚胎移植,而不进行辅助孵化处理,用于观察胚胎移植对妊娠效率及后代发育的影响(embryo transfer,ET组);(3)体内受精获得的胚胎,进行辅助孵化处理后,再进行胚胎移植,用于观察辅助孵化和胚胎移植对妊娠效率及后代发育的影响(small hole,SH组)。我们的研究结果表明,与Mating组相比,ET组中的胚胎移植操作会降低胚胎着床数,着床后的胚胎发育迟缓,孕天延迟,子代出生数降低,并影响子代生长曲线;但对胚移后妊娠率、活胎率、性别都没有影响。胚移后得到的子代小鼠再进行自然交配,其孕天以及生育能力没有受到影响。与ET组相比,SH组虽然能够提高胚胎的体外孵化效率,但着床后胚胎发育迟缓,并影响子代生长曲线。打孔之后的胚胎在胚移之后,母鼠存活率、着床数、着床率、妊娠率、孕天、子代小鼠出生数、出生率、活胎率、性别、孕天以及子代小鼠生育能力等都没有影响。SH组与Mating组相比较,会降低胚胎着床数,着床后的胚胎发育更加迟缓,孕天延迟,子代出生数降低,并影响子代生长曲线;但对胚移后妊娠率、活胎率、性别都没有影响。胚移后得到的子代小鼠再进行自然交配,其孕天以及生育能力没有受到影响。综上所述,piezo介导的透明带打小孔的辅助孵化方式并不能够提高胚胎的着床率、母鼠的妊娠率和子代的出生率。相反,经过辅助孵化和胚胎移植操作后,会导致胚胎发育紊乱,对着床后胚胎的发育和子代生长曲线都有影响,但并不影响子代小鼠的性别和生殖能力。因此,我们认为,依赖于打孔方式的辅助孵化技术,在临床上应该被谨慎使用。
[Abstract]:Pellucida (zona pellucida,ZP) is a layer of glycoprotein wrapped in the outer layer of oocytes, which is co-secreted by oocytes and granulosa cells in the early stage of primary follicles. It plays an important role in the process of fertilization and early embryonic development. However, the thickening and hardening of pellucida is not conducive to embryo hatching. Therefore, we try to use auxiliary hatching (assisted hatching,AH) technology to improve the hatching rate of blastocysts, so as to improve the implantation rate of embryos and the birth rate of offspring. Assisted hatching is a technique in which the embryo is released from the pellucida by artificial treatment of the pellucida. At present, there are many kinds of auxiliary hatching techniques, and early experiments in our group have found that different auxiliary hatching techniques have different effects on embryos of different periods. Among the various auxiliary hatching techniques mediated by Piezo, the highest blastocyst hatching rate can be obtained after pellucida microporous treatment in morula stage (the perforation size is about 10 渭 m),). Whether this method can improve the implantation rate of embryos and the birth rate of offspring remains to be studied. In this experiment, embryos were treated with this auxiliary hatching method, and then embryo transfer (embryo transfer,ET) was carried out. The implantation rate of embryos, pregnancy rate of female mice, birth rate of offspring and growth curve of offspring were counted in order to find out the effect of piezo mediated assisted hatching on implantation and development of mouse embryos. In this study, the following three different sources of embryos were analyzed, including: (1) natural mating group (mating group), which was used as a control group for non-auxiliary hatching and non-embryo transfer; (2) embryos obtained from in vivo fertilisation were transferred only without auxiliary hatching, and the effects of embryo transfer on pregnancy efficiency and offspring development were observed (embryo transfer,ET group), and the results were as follows: (1) the natural mating group (embryo transfer,ET group) was used to observe the effect of embryo transfer on pregnancy efficiency and the development of offspring. (3) the embryos obtained by in vivo fertilisation were treated with auxiliary hatching, and then the embryos were transferred to observe the effects of assisted hatching and embryo transfer on pregnancy efficiency and the development of offspring (small hole,SH group). Our results showed that compared with Mating group, embryo transfer in ET group decreased the number of embryo implantation, delayed embryo development after implantation, delayed gestational days, decreased the number of offspring, and affected the growth curve of offspring, but had no effect on the pregnancy rate, live pregnancy rate and sex after embryo transfer. The offspring obtained after embryo transfer were mated naturally, and their gestational days and fertility were not affected. Compared with ET group, SH group could improve the hatching efficiency of embryos in vitro, but the embryo development after implantation was retarded, and the growth curve of offspring was affected. After embryo transfer, the survival rate, implantation number, implantation rate, pregnancy rate, gestational day, birth rate, live pregnancy rate, sex, gestational day and fertility of offspring in SH group were not affected. Compared with Mating group, SH group decreased the number of embryos implantation, delayed embryo development after implantation, decreased the number of offspring born, and affected the growth curve of offspring. However, it had no effect on the pregnancy rate, live pregnancy rate and sex after embryo transfer. The offspring obtained after embryo transfer were mated naturally, and their gestational days and fertility were not affected. In conclusion, the assisted hatching of pellucida mediated by piezo could not increase the implantation rate of embryos, the pregnancy rate of female mice and the birth rate of offspring. On the contrary, after assisted hatching and embryo transfer, it will lead to the disorder of embryo development, which has an effect on the development of postimplantation embryos and the growth curve of offspring, but does not affect the sex and reproductive ability of offspring. Therefore, we believe that auxiliary hatching technology, which depends on drilling, should be used with caution in clinic.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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