不同形态卵母细胞与不同浓度精子组合对受精及早期胚胎发育的影响

发布时间：2018-10-31 15:13

【摘要】：体外受精(in vitro fertilization, IVF)是一项应用最为广泛的辅助生殖技术(assisted reproductive technology, ART)。卵母细胞质量和精子浓度在精卵结合过程中起到至关重要的作用,然而由于一些疾病或是自身排卵原因,亦或是取卵过程中人为操作的原因,临床上经常会获得无卵丘包裹的卵母细胞称为裸卵,(Denuded Oocyte, DO)甚至是无透明带的卵母细胞(Oocyte Zona pellucida Free, OZ-)。虽然围绕卵丘-卵母细胞复合体(cumulus-oocyte complex, COC)开展了许多研究,但对于DO或OZ-的卵子能否受精,以及受精的最佳精子浓度等问题,尚无系统报道。本研究以小鼠为实验对象,获得体内成熟的COC,用透明质酸酶去除卵丘获得DO,以及利用酸性M2操作液去除透明带获得OZ-,对于这三种在结构上逐步递减的卵母细胞进行了如下实验,以确定受精时的最佳精子浓度：1：将同一类型的卵母细胞与不同浓度的精子混合,确定每一类型卵母细胞的最佳受精精子浓度；2、将同一浓度的精子与不同类型的卵母细胞混合,以确定每一精子浓度梯度最适合的卵母细胞类型。我们的实验结果显示：对于体内成熟的COC,IVF最适精子浓度为106个/ml；对于DO,IVF最适精子浓度为107个/ml；而对于体内成熟的OZ-,IVF最适精子浓度为107个/m1。精子浓度为103个/ml或104个/ml时,体内成熟的DO获得最高的受精率和胚胎发育率；精子浓度为105个/ml或106个/ml时,体内成熟的COC获得最高的受精率和胚胎发育率；精子浓度为107个/ml时,OZ-获得最高的受精率。我们使用以上最适浓度的精子分别使COC、DO、OZ-体外受精后,获得的囊胚免疫荧光染色统计囊胚细胞数和直径,测量的结果表明COC组与DO组间没有显著差异,这两组显著高于OZ-组。综上所述,COC在106个/ml精子浓度下受精率和胚胎发育率最高,但是如果不能获得足够的COC,可以使用DO进行体外受精,DO的最适精子浓度为107个/m1。因为OZ-后期的胚胎发育率极低,所以不建议使用OZ-用于体外受精操作。
[Abstract]:In vitro fertilization (in vitro fertilization, IVF) is one of the most widely used assisted reproductive technology (assisted reproductive technology, ART). The quality of oocytes and the concentration of sperm play a vital role in the process of spermatozoa binding. However, due to some diseases or self-ovulatory reasons, or because of human manipulation in the process of ovulation, Oocytes without cumulus are often obtained clinically, called naked oocytes, (Denuded Oocyte, DO) or even pellucidal oocytes (Oocyte Zona pellucida Free, OZ-). Although many studies have been carried out around the cumulus oocyte complex (cumulus-oocyte complex, COC), there are no systematic reports on whether the eggs of DO or OZ- can be fertilized and the optimal sperm concentration of fertilization. In this study, mature COC, was obtained from mice to obtain DO, by removing cumulus with hyaluronidase, and OZ-, was obtained by removing pellucidal zone from acid M2 operation solution. The following experiments were carried out to determine the best sperm concentration during fertilization: 1. Mix the same type of oocytes with different concentrations of sperm. Determine the optimal fertilization sperm concentration for each type of oocyte; 2. The same concentration of spermatozoa was mixed with different types of oocytes to determine the most suitable type of oocytes for each sperm concentration gradient. Our results show that the optimal sperm concentration for mature COC,IVF is 106 / ml; for DO,IVF and 107 / ml; for mature OZ-,IVF. When the sperm concentration was 103 / ml or 104 / ml, the mature DO obtained the highest fertilization rate and embryo development rate, while the mature COC obtained the highest fertilization rate and embryo development rate when the sperm concentration was 105 / ml or 106 / ml. When sperm concentration was 107 / ml, OZ- obtained the highest fertilization rate. The number and diameter of blastocyst cells were counted by immunofluorescence staining of blastocysts after in vitro fertilization of COC,DO,OZ- with the optimal concentration of spermatozoa. The results showed that there was no significant difference between COC group and DO group. These two groups were significantly higher than OZ- group. In conclusion, the fertilization rate and embryo development rate of COC were the highest at 106 / ml sperm concentration, but if enough COC, could not be obtained, DO could be used for in vitro fertilization, and the optimal sperm concentration of DO was 107 / M1. OZ- is not recommended for in vitro fertilization because of the very low rate of late OZ- embryo development.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R714.8

【共引文献】