【作者】 苏静艳；

【导师】 严云勤；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2005， 硕士

【摘要】 哺乳动物卵母细胞减数分裂成熟和受精是一个重要和复杂的过程,研究卵母细胞的孤雌激活及其激活过程中细胞骨架组织和结构的动态变化将有助于加深对这一机制的认识。近年来,有些关于孤雌激活胚胎中细胞骨架分布和作用的报道,但对于不同刺激激活的孤雌胚中细胞骨架的动态性变化仍有待于更为系统的研究。目前,国内相关研究的报道很少。本实验主要利用卵母细胞的乙醇激活、胚胎体外培养和免疫荧光细胞化学方法对小鼠卵母细胞乙醇激活效率、影响因素、孤雌胚类型、发育、原核形成、迁移及早期胚胎卵裂过程中细胞骨架的组装、作用等进行了系统的研究和分析,结果显示: (1) 小鼠卵母细胞的乙醇激活结果表明,随着乙醇浓度的升高和作用时间的延长,卵母细胞激活率和发育率都显著提高,但乙醇浓度过高和作用时间过长会导致卵母细胞碎裂率的显著增加,7%乙醇作用卵母细胞7min 为最佳激活条件。随着卵龄增加和温度的升高,卵母细胞激活率、发育率提高,碎裂率增加显著。卵丘细胞对激活效果影响不大,性成熟小鼠卵母细胞激活效果较好。(2) 随着hCG 注射后卵龄增加,卵母细胞第一极体退化比例逐渐上升,无第一极体卵母细胞数量增加,但有无第一极体对小鼠卵母细胞孤雌激活率、发育率和碎裂率影响不显著,且有无第一极体卵母细胞具有相同的细胞骨架动态分布规律。(3) 免疫荧光细胞化学结果显示,MII 期卵母细胞微管主要集中在减数分裂纺锤体上,随着第二极体排出,纺锤体开始瓦解消失,形成富含微管的中体结构,连接发生分裂的细胞,中体在细胞分裂完成后最终消失。原核形成后逐渐由胞质边缘迁移到胞质中心。(4) 不同发育时期的卵细胞中,细胞皮层和极体中始终有微丝聚集。MII 期卵母细胞微丝主要集中在染色体所在的皮层区域,原核形成后微丝绕核重新聚集。极体排出和第一次有丝分裂时微丝在卵裂沟处富集。(5) 紫杉醇处理MII 期卵母细胞时,纺锤体组装异常,胞质中出现大量胞质微管星体,染色体排列异常,不能分离。洗脱药物后,激活的卵母细胞能够重新恢复减数分裂,纺锤体重新组装,卵母细胞排出第二极体、形成原核。实验表明,卵母细胞的孤雌激活受多种因素的影响,选择最佳的激活条件对孤雌激活效果尤为重要。卵母细胞减数分裂恢复和孤雌激活过程中,细胞骨架的组织和结构发生了一系列明显的变化,微丝和微管在减数分裂恢复、染色体运动、纺锤体形成、定位及旋转、极体排放、原核的形成和迁移及早期胚胎卵裂等事件中均具有重要的调控作用。更多还原

【Abstract】 Meiotic maturation and fertilization of mammalian oocytes is an important and complex process, the studies of oocytes parthenogenetic activation and the dynamic changes of cytoskeleton during oocytes activation will make us get a deeper understanding of the mechanism of this process. Recent years, there are some reports about the cytoskeletal distribution and roles of parthenotes, but the dynamic changes of cytoskeleton in the parthenotes activated by various stimuli are still a problem lacking systematic studies. To date, there are only a few internal reports about this field. This experiment aimed to systematically investigate and analyze the ethanol activated rates of mouse oocytes, affected factors of oocytes activation, types and development of parthenotes, and the organization and roles of the cytoskeleton during pronuclear formation and migration and early embryo cleavage by ethanol activation of oocytes, embryo culture in vitro and immunofluorescence cyto-chemistry. The results: (1) The ethanol activation of mouse oocytes showed that the activated and developmental rates of oocytes increased significantly with the increase of ethanol concentration and extension of exposure time, but over concentration and exposure time would result in increased fragment rates significantly. 7% ethanol treated oocytes for 7min was the optimum activated condition. With the increases of oocyte ages and temperature, the oocytes activation and development rates increased, while fragment rates increased significantly. Ovarian cumulus had no obvious effect on the activation of oocytes in vitro. Oocytes of sexual maturity had better activated effect than under sexual maturity mouse. (2) Degraded proportion of oocytes first polar body (PB1) and numbers of oocytes without PB1 both increased with the increases of oocyte ages after hCG injection. With or without PB1 had no significant effects on the activation, development and fragment rates. We also observed that oocytes with or without PB1 had the same distribution patterns of cytoskeleton. (3) Our fluorescence observation results indicated that microtubules were restricted to the region of the metaphase II (MII) meiotic spindle. With the extrusion of the second polar body (PB2), MII spindle began to disintegrate and disappear slowly, and the compact microtubule-containing structure known as the midbody was formed. The midbody located in the contact surfaces of cells that divided, and finally disappeared after cell division completed. Pronuclei migrated to the center of cytoplasm after their formation at the edge. (4) Microfilaments concentrated to the oocyte cortex and polar body at the different developmental stages of eggs. Microfilaments were mainly distributed in the cortex area which chromosomes located at mataphase of second meiosis (MII)and concentrated around pronuclei after pronuclear formation. After extrusion of polar body and the completion of the first cell cycle, microfilaments concentrated to the cleavage furrows. (5) The MII spindle showed abnormal assembly, and multiple microtubule asters formed in the cytoplasm, chromosomes were not being normal aligned and couldn’t separated each other after treating the oocytes with taxol. While washed taxol completely, the activated oocytes could resume meiosis II, the MII spindle reorganized, the oocytes extruded PB2 and formed pronuclei. Our results showed that the parthenogenetic activation of mouse oocytes was affected by many factors, so the selection of optimum activated conditions was very important for the effect of activation. The organization and structure of cytoskeleton changed obviously during the resumption of meiotic and parthenogenetic activation of mammalian oocytes. Microtubules and microfilaments played important regulated roles in many events such as the resumption of meiotic, chromosomal movement, the formation, location and rotation of meiotic spindle, polar body extrusion, pronuclear formation and migration and also early embryo cleavage.更多还原