人卵母细胞体外成熟前后线粒体分布的变化

发布时间：2018-06-28 04:21

本文选题：卵母细胞 + 体外成熟　；参考：《山东大学》2007年硕士论文

【摘要】： 研究目的：本研究旨在通过对人未成熟和体外培养成熟后卵母细胞中线粒体的分布的比较，阐明卵母细胞成熟和线粒体分布的关系。研究方法：(1)未成熟卵母细胞的来源：未成熟卵母细胞来源于自2006年3月至10月就诊于山东大学山东省立医院生殖医学中心，仅单纯男方因素或同时合并女方输卵管因素行卵母细胞内单精子显微注射的不孕症患者。经透明质酸酶消化后，所获未成熟卵母细胞经患者同意，并签署相关知情同意书后捐献用于医学科学研究。 (2)体内成熟的卵母细胞的来源：体内成熟的卵母细胞来源于同期在山东大学山东省立医院生殖医学中心就诊，行促排卵后同房或人工授精的不孕症患者，若注射HCG10 000IU 42 h后仍未排卵，则行人工破卵治疗。即将大卵泡通过经阴道穿刺的方法使之破裂，卵泡液流出，同时卵子也进入腹腔，患者将有妊娠的可能。如果卵泡较多，为避免卵巢过度刺激综合征和多胎妊娠，会对部分小卵泡进行穿刺，穿刺获得的成熟卵母细胞经患者同意，并签署相关知情同意书后捐献用于医学科学研究。将未成熟卵母细胞随机分为两组，采用荧光染色法，一组直接染色，另一组体外培养成熟后再进行染色。未成熟卵母细胞和成熟卵母细胞均用Mito Tracker Green FM染色，经多聚甲醛固定后，，在激光扫描共聚焦显微镜下观察线粒体的分布。结果：284个未成熟卵母细胞中有144个进行体外成熟培养，其中GV期卵母细胞88个，MI期卵母细胞56个。IVM 48h后，56个GV期卵母细胞达到MⅡ期，成熟率为63.64％；50个MⅠ期卵母细胞达到MⅡ期，成熟率为89.29％。MⅠ期卵母细胞的成熟率明显高于GV期卵母细胞，两者之间的差别有统计学意义(P=0.016)。线粒体在细胞质中的分布方式可分为以下三种：周边分布、半周边分布和均匀分布。在64.10％(50／78)的GV期卵母细胞中，线粒体呈周边分布。在MⅠ期卵母细胞中，线粒体呈均匀分布的占38.71％(24／62)，45.16％(28／62)的MⅠ期卵母细胞的线粒体仍维持周边分布。体外培养成熟以后，线粒体在75.47％(80／106)的卵母细胞中呈现均匀分布。GV期卵母细胞中线粒体以周边分布为主，与MⅠ期和MⅡ期卵母细胞相比，其差异有统计学意义。成熟卵母细胞中线粒体大多呈均匀分布，与未成熟卵母细胞相比，也存在显著差异。由此可以看出，人卵母细胞成熟前后，线粒体发生了明显的分布变化，由未成熟卵母细胞中以周边分布为主变为成熟卵母细胞中以均匀分布为主。体内成熟的11个卵母细胞中，线粒体呈均匀分布的有8个(72.73％)，另有3个呈周边分布(27.27％)。与体外培养成熟的卵母细胞相比，体内成熟的卵母细胞中线粒体分布的特点是胞质中央区域线粒体的浓集，荧光强度高于周边区域。结论：(1)人卵母细胞成熟前后，线粒体出现了明显的分布变化，由未成熟卵母细胞中以周边分布为主变为成熟卵母细胞中以均匀分布为主。(2)体内成熟的卵母细胞中线粒体的分布与体外成熟的卵母细胞略有不同。
[Abstract]:Objective: This study aims to elucidate the relationship between the maturation of oocyte and the distribution of mitochondria by comparing the distribution of mitochondria in the immature and in vitro cultured oocytes.
Research methods: (1) the source of immature oocyte: immature oocyte originates from March 2006 to October in the reproductive medical center of Shangdong Province-owned Hospital of Shandong University. Only male factor or single sperm microinjection in oocyte with female oviduct factors combined with female fallopian tube factors and hyaluronidase elimination. After that, the obtained oocytes were donated to medical research by consent of the patients and signing the relevant informed consent.
(2) the source of mature oocytes in the body: mature oocytes from the body of the body of the body from the same period at the reproductive medical center of Shangdong Province-owned Hospital of Shandong University, and the infertile patients with ovulatory or artificial insemination after ovulation induction. If HCG10 000IU 42 h were not ovulation, artificial ovulation was performed. The large follicles were punctured through the transvaginal puncture. The method makes it broken, the follicular fluid outflows and the egg enters the abdominal cavity. The patient will have the possibility of pregnancy. If there are more follicles, in order to avoid ovarian hyperstimulation syndrome and multiple pregnancy, some small follicles will be punctured, the mature oocyte obtained by the puncture is approved by the patient, and the related informed consent is signed and donated to medicine. Scientific research.
The immature oocytes were randomly divided into two groups, which were stained by fluorescent staining, a group of direct staining, and the other group was stained after maturation in vitro. The immature oocyte and mature oocyte were stained with Mito Tracker Green FM, and the distribution of mitochondria was observed under laser scanning confocal microscope after immobilization of polyformaldehyde.
Results: 144 of the 284 immature oocytes were cultured in vitro, including 88 GV oocytes and 56.IVM 48h for MI oocytes, 56 GV oocytes reached stage M II, the maturation rate was 63.64%, 50 M I oocytes reached M II stage, and maturity rate of 89.29%.M I oocytes was significantly higher than GV. The difference between the two oocytes was statistically significant (P=0.016).
The distribution of mitochondria in the cytoplasm can be divided into three types: peripheral distribution, semi circumjacent distribution and uniform distribution. In 64.10% (50 / 78) GV oocytes, mitochondria are distributed around the mitochondria. In M I oocytes, mitochondria are evenly distributed in 38.71% (24 / 62), 45.16% (28 / 62) M I oocyte lines The mitochondria were distributed in 75.47% (80 / 106) oocytes in 75.47% (80 / 106) oocytes, and the mitochondria in the oocyte were distributed mainly in the peripheral distribution. The difference was statistically significant compared with that of stage M I and M II oocytes, and the mitochondria in mature oocytes were mostly uniform and unmature. There are also significant differences in oocyte, and it can be seen that there are obvious changes in the mitochondria before and after the maturation of human oocytes, and the distribution of the oocyte from the immature oocyte to the mature oocyte mainly from the peripheral distribution to the mature oocyte.
In the 11 oocytes mature in vivo, there are 8 (72.73%) mitochondria in the mitochondria and 3 peripheral distribution (27.27%). Compared with the mature oocyte in vitro, the distribution of mitochondria in the mature oocyte is the concentration of mitochondria in the central region of the cytoplasm, and the fluorescence intensity is higher than that of the surrounding area.
Conclusions: (1) the distribution of mitochondria in the human oocyte before and after maturation is obviously distributed, and the distribution of the mitochondria from the immature oocyte to the mature oocyte mainly is mainly in the mature oocyte. (2) the distribution of mitochondria in the mature oocytes is slightly different from that in the mature oocytes.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321.1

【参考文献】