镉对小鼠卵母细胞成熟和早期胚胎发育的影响

发布时间：2018-06-19 10:30

本文选题：镉 + 小鼠　；参考：《扬州大学》2017年硕士论文

【摘要】：近年来,镉的毒性研究一直受到人们的广泛关注。机体摄入过量的镉可以引起多种器官和组织细胞的损伤,除主要危害肝脏、肾脏和骨骼外,对生殖系统也会造成损伤,例如危害精子的发生,改变卵巢的形态结构,抑制卵泡的生长与发育等。但是,环境镉暴露对卵母细胞以及后代的危害尚不完全清楚。饮用含镉量过高的水是机体摄入过量镉的一种主要方式。本研究以ICR小鼠连续35天自由饮用含镉水(64mg/L的CdCl2·2.5H2O)复制动物模型,研究镉对小鼠卵母细胞成熟以及早期胚胎发育的影响。1.镉对小鼠卵母细胞成熟的影响为了消除自来水中镉的影响,对照组雌鼠饮用Milli-Q超纯水,实验组雌鼠饮用Milli-Q水配置的含镉水。通过超数排卵实验,检测卵泡的发育和成熟,体内和体外实验检测卵母细胞的成熟情况,应用免疫荧光方法检测卵母细胞成熟过程中染色体、纺锤体、肌动蛋白帽、线粒体的形态和功能,并检测卵母细胞内ATP含量的变化;通过检测卵母细胞内组蛋白H3K9甲基化和H4K12乙酰化水平来反应镉对表观遗传修饰的影响。结果表明,镉处理导致雌性小鼠超排后卵母细胞数量的显著减少(p0.05);体内和体外实验均表明,镉处理后卵母细胞生发泡破裂(GVBD)无变化,但是大部分卵母细胞停滞在第一次减数分裂中期(MⅠ期),与对照组相比,成熟卵母细胞(MⅡ)所占比例显著下降,说明镉处理不会抑制卵母细胞减数分裂的恢复,但干扰了第一次减数分裂的正常完成。免疫荧光显示,镉处理导致MⅡ期卵母细胞染色体、纺锤体和细胞极性的异常,镉处理后MⅡ期卵母细胞的染色体并未整齐排列,纺锤体形态没有呈现典型的梭形,同时Actin cap出现丢失,说明镉处理导致MⅡ期卵母细胞的质量出现下降。进一步检测了线粒体的数量和功能,结果表明,镉处理导致MⅡ期卵母细胞中线粒体数目出现显著下降,虽然线粒体功能得到了增强,但却未能挽救ATP含量的下降。组蛋白修饰的检测结果显示,镉处理显著增强了 MⅡ期卵母细胞H3K9的甲基化水平和H4K12的乙酰化水平。通过孤雌激活实验来观察镉对卵母细胞第二次减数分裂的影响,结果显示,虽然镉处理延缓了第二极体的排出和原核的形成,但并未改变最终形成原核的数目,说明镉处理并未显著影响第二次减数分裂的最终完成。2.镉对小鼠早期胚胎发育的影响将镉处理的雌鼠与正常雄鼠交配后,采集受精卵进行体外培养,以观察镉对早期胚胎发育的影响;结果显示,镉处理显著降低了同一时期4细胞胚胎和囊胚的发育率。在进一步的实验中,采集镉处理雌鼠的卵母细胞进行体外受精,与对照组相比,囊胚发育率同样下降。这说明镉处理降低了成熟卵母细胞的质量,并进一步影响了其受精后早期胚胎的正常发育。综上所述,饮水型慢性镉中毒不仅会降低雌性小鼠卵母细胞排卵率,而且通过影响减数分裂进程、线粒体功能和组蛋白修饰来降低卵母细胞的质量,并最终导致胚胎发育能力的下降。
[Abstract]:In recent years, the toxicity of cadmium has been widely concerned. Excessive intake of cadmium can cause damage to various organs and tissue cells. In addition to the main harm to the liver, kidney and bone, it can also cause damage to the reproductive system, such as the occurrence of sperm, the morphologic changes of the ovaries, the inhibition of the growth and development of follicles. However, the harm of environmental cadmium exposure to oocytes and offspring is not completely clear. Drinking too high cadmium content water is a major way of excessive cadmium intake. In this study, ICR mice were free to drink cadmium water (64mg/L CdCl2 / 2.5H2O) for 35 days to copy animal models, and to study the maturation of oocytes and early embryos of mice by cadmium. The effect of.1. cadmium on the maturation of mouse oocyte in order to eliminate the effect of cadmium in the tap water. The female rats in the control group drank the ultra pure water of Milli-Q, and the female rats in the experimental group drank the cadmium water in the Milli-Q water. The development and maturation of the follicles were detected by the superovulation experiments. The maturation of oocyte in vivo and in vitro should be tested in vivo and in vitro. Detection of chromosomes, spindles, actin caps, morphologies and functions of mitochondria during oocyte maturation and detection of changes in ATP content in oocytes by immunofluorescence, and the effects of cadmium on epigenetic modification by detecting the levels of H3K9 methylation and H4K12 acetylation in oocytes. The number of oocytes in female mice decreased significantly (P0.05), and in vitro and in vitro experiments showed that there was no change in the germinal vesicle rupture (GVBD) of oocytes after cadmium treatment, but most oocytes were stagnated at the middle of the first meiosis (M I), and the proportion of mature oocytes (M II) decreased significantly compared with those in the group. Cadmium treatment does not inhibit the restoration of meiosis in oocyte, but interferes with the normal completion of the first meiosis. Immunofluorescence shows that cadmium treatment leads to the chromosomes of M II oocyte, the abnormal spindle and cell polarity, and the chromosomes of M II oocytes after cadmium treatment are not arranged neatly, and the spindle shape is not typical. At the same time, the loss of Actin cap showed that cadmium treatment resulted in a decrease in the quality of M II oocytes. The number and function of mitochondria were further detected. The results showed that the number of mitochondria in M II oocytes decreased significantly and the mitochondrial function was enhanced, but the content of ATP was not saved. Declines. The results of histone modification showed that cadmium treatment significantly enhanced the methylation level of H3K9 in M II oocyte and the level of acetylation of H4K12. The effects of cadmium on the second meiosis of oocytes were observed by parthenogenetic activation. The results showed that although cadmium treatment postpones the discharge of the second polar body and the formation of the prokaryotic cells, The effect of cadmium treatment did not change the number of prokaryotic cells, indicating that cadmium treatment did not significantly affect the second meiotic division. The effect of cadmium on the development of early embryo in mice was not significantly affected by the effect of cadmium on the development of early embryos in mice. After mating with normal male rats, the female mice were collected by the cadmium treated mouse and cultured in vitro to observe the effect of cadmium on the development of the early embryos. The results showed that cadmium treatment showed that cadmium treatment showed that cadmium treatment was significant. The development rate of 4 cell embryos and blastocysts in the same period was reduced. In further experiments, the oocytes of the female rats were fertilized in vitro. Compared with the control group, the development rate of blastocyst was also decreased. This indicates that cadmium treatment reduces the quality of mature oocytes and affects the normal hair of the early embryos after their fertilization. In summary, drinking water type chronic cadmium poisoning not only reduces the ovulation rate of oocyte in female mice, but also affects the meiosis process, mitochondrial function and histone modification to reduce the quality of oocyte, and eventually lead to the decline of embryonic development ability.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.8

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