【摘要】：透明帶(zona pellucida,ZP)是包裹在卵母細(xì)胞外的一層由糖蛋白組成的外被結(jié)構(gòu),在初級(jí)卵泡早期階段由卵母細(xì)胞和顆粒細(xì)胞共同分泌形成。在受精過(guò)程和早期胚胎發(fā)育中起著重要作用。然而,透明帶的厚化和硬化不利于胚胎的孵化。因此,我們嘗試?yán)幂o助孵化(assisted hatching,AH)技術(shù)提高囊胚的孵化率,從而提高胚胎的著床率以及子代的出生率。輔助孵化是通過(guò)人為的方法對(duì)透明帶進(jìn)行處理,使胚胎從透明帶釋放出的技術(shù)。目前,輔助孵化技術(shù)有許多種,我們組早期的實(shí)驗(yàn)發(fā)現(xiàn),不同的輔助孵化技術(shù)對(duì)不同時(shí)期的胚胎進(jìn)行操作會(huì)有不同的效果。Piezo介導(dǎo)的各種輔助孵化技術(shù)中,在桑椹胚期進(jìn)行透明帶打小孔處理后(打孔尺寸約為10 μm),可獲得最高的囊胚孵化率。而該種方法是否能夠提高胚胎的著床率以及子代的出生率仍有待考究。本實(shí)驗(yàn)利用該種輔助孵化方法處理胚胎,再進(jìn)行胚胎移植(embryo transfer,ET),統(tǒng)計(jì)其胚胎著床率、母鼠妊娠率、子代出生率以及子代生長(zhǎng)曲線等數(shù)據(jù),以探明piezo介導(dǎo)的輔助孵化對(duì)小鼠胚胎著床和發(fā)育的影響。本研究對(duì)以下三種不同來(lái)源的胚胎進(jìn)行分析,包括:(1)自然交配組(mating組),用作非輔助孵化、非胚胎移植操作的對(duì)照組;(2)體內(nèi)受精獲得的胚胎,僅進(jìn)行胚胎移植,而不進(jìn)行輔助孵化處理,用于觀察胚胎移植對(duì)妊娠效率及后代發(fā)育的影響(embryo transfer,ET組);(3)體內(nèi)受精獲得的胚胎,進(jìn)行輔助孵化處理后,再進(jìn)行胚胎移植,用于觀察輔助孵化和胚胎移植對(duì)妊娠效率及后代發(fā)育的影響(small hole,SH組)。我們的研究結(jié)果表明,與Mating組相比,ET組中的胚胎移植操作會(huì)降低胚胎著床數(shù),著床后的胚胎發(fā)育遲緩,孕天延遲,子代出生數(shù)降低,并影響子代生長(zhǎng)曲線;但對(duì)胚移后妊娠率、活胎率、性別都沒(méi)有影響。胚移后得到的子代小鼠再進(jìn)行自然交配,其孕天以及生育能力沒(méi)有受到影響。與ET組相比,SH組雖然能夠提高胚胎的體外孵化效率,但著床后胚胎發(fā)育遲緩,并影響子代生長(zhǎng)曲線。打孔之后的胚胎在胚移之后,母鼠存活率、著床數(shù)、著床率、妊娠率、孕天、子代小鼠出生數(shù)、出生率、活胎率、性別、孕天以及子代小鼠生育能力等都沒(méi)有影響。SH組與Mating組相比較,會(huì)降低胚胎著床數(shù),著床后的胚胎發(fā)育更加遲緩,孕天延遲,子代出生數(shù)降低,并影響子代生長(zhǎng)曲線;但對(duì)胚移后妊娠率、活胎率、性別都沒(méi)有影響。胚移后得到的子代小鼠再進(jìn)行自然交配,其孕天以及生育能力沒(méi)有受到影響。綜上所述,piezo介導(dǎo)的透明帶打小孔的輔助孵化方式并不能夠提高胚胎的著床率、母鼠的妊娠率和子代的出生率。相反,經(jīng)過(guò)輔助孵化和胚胎移植操作后,會(huì)導(dǎo)致胚胎發(fā)育紊亂,對(duì)著床后胚胎的發(fā)育和子代生長(zhǎng)曲線都有影響,但并不影響子代小鼠的性別和生殖能力。因此,我們認(rèn)為,依賴于打孔方式的輔助孵化技術(shù),在臨床上應(yīng)該被謹(jǐn)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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714.8

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