【作者】 刘俊；

【导师】 孙少琛；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2016， 硕士

【摘要】 哺乳动物生命起源的一个重要基础的过程就是卵母细胞的成熟,这也是多年来科学研究的一个热点。卵母细胞的成熟涉及多个复杂的细胞内过程,其中,细胞骨架,包括微管和微丝在内,它们的组装以及动态变化对于卵母细胞的成熟极为重要。细胞骨架任意一部分受到任何的破坏都有可能导致卵母细胞成熟的失败或是异常,甚至后续的受精卵以及胚胎发育都会受到影响,进而导致婴儿的缺陷及畸形。然而,涉及到微丝以及微管的调控机制,目前的研究还不能完全解释清楚。因此,进一步研究卵母细胞成熟过程中的分子调控机制,对于完善人类对于哺乳动物生殖机理的理解,促进家畜繁殖的调控甚至人类的生殖健康都有着深远的意义。本试验以小鼠为研究对象,采用体外培养、抑制剂处理、吗啉代(morpholino,MO)注射、活细胞观察、免疫荧光染色和激光共聚焦及免疫蛋白印迹等方法,研究相关分子在小鼠卵母细胞成熟过程中的调控作用。先用免疫荧光染色法检测MKlp2和profilin1两种蛋白在小鼠卵母细胞成熟过程中的表达与定位,再分别通过蛋白特异性抑制剂处理或吗啉代(morpholino,MO)注射技术,破坏蛋白的活性或者功能,进而研究这两种蛋白对于卵母细胞成熟过程中相关过程包括纺锤体装配、染色体排列以及微丝的组装的影响,最终了解它们对卵母细胞极体排出的调控作用。此外,用蛋白免疫印迹(westernblot)技术,检测敲低profilin1后对微丝以及相关调控蛋白表达的影响,进一步揭示profilin1调控小鼠卵母细胞减数分裂成熟可能的分子机制,为完善哺乳动物卵母细胞调控机制提供重要证据。本研究共分2个部分,主要研究结果如下:试验一、MKlp2蛋白抑制剂 paprotrain影响小鼠卵母细胞成熟过程中的极体排出哺乳动物卵母细胞的减数分裂成熟过程中涉及一系列的重要过程,包括纺锤体的组装和迁移,皮质重组,以及极体的排出。这些过程由很多蛋白调控,但是,MKlp2(有丝分裂类驱动蛋白2;又称KIF20A),一个调控有丝分裂胞质分裂的微管结合蛋白,是否参与到卵母细胞成熟过程中我们尚不清楚。本实验就MKlp2在小鼠卵母细胞中的功能展开了试验研究,免疫荧光染色结果显示,MKlp2定位在卵母细胞中的微管上;活细胞观察发现用MKlp2的抑制剂破坏MKlp2的活性,导致第一极体排出的失败,并且能够在新鲜的培养液中恢复;细胞周期的分析结果显示大多数卵母细胞停滞在中期或者末期;但是,经过抑制剂的处理,卵母细胞的纺锤体结构和染色体的排列并没有遭到破坏。因此,本试验证明了 MKlp2通过调控第一极体排出来作用于小鼠卵母细胞的成熟。试验二、Profilin1在小鼠卵母细胞中微丝介导的极体排出过程中的反馈调控作用作为一个小的微丝绑定蛋白,profilin1在微丝组装过程中起着很重要的作用。但是,profilin1在哺乳动物卵母细胞中的功能尚不清楚,为了研究profilin1在卵母细胞中的功能,我们首先使用了免疫荧光染色来检测profilin1的定位。结果显示,profilin1定位在减数分裂纺锤体的周围,并且和胞质中的微丝共定位。通过注射特异性的吗啉代来敲低profilin1的表达之后,第一极体排出失败,这个失败是由于卵母细胞胞质中以及胞膜上的微丝表达量升高导致。进一步研究,免疫印迹分析显示在敲低profilin1之后,Rho相关激酶(ROCK)的表达水平以及肌球蛋白轻链(MLC)的磷酸化水平显著下降。因此,我们的结果表明了 profilin1在小鼠卵母细胞成熟过程中的调控作用是通过一个profilin-actin和ROCK-MLC2之间的反馈机制来实现的。更多还原

【Abstract】 One of the most important processes during the origin of mammalians’ lives is the oocyte maturation,which is a heated topic of many years scientific research.Oocyte maturation involves several complex cellular processes.The dynamics of cytoskeleton,including microtubule and actin,are essential for the maturation.Any defects during the processes may result in failure or abnormality of oocyte maturation,which can further affect the development of the zygote and early embryo and lead to the defect and malformation in infants.However,current research cannot full explain the underline mechanism of the regulation of actin and microtubule.Although many regulators and signal pathways have been demonstrated,there are still plenty of uncovered aspects in this field.Therefore,further research in molecular regulation mechanism of oocyte maturation has profound significance in consummating people’s understand of mammalian reproductive mechanism and improving livestock’s reproductive capacity and human’s reproductive health.In these experiments,mice were employed as the experimental animals.We used in vitro culture,inhibitor treatment,morpholino injection,time-lapse microscopy,immunofluorescence staining and western blot to investigate the roles of related molecules during oocyte maturation.First,immunofluorescence staining was employed to examine the localization of MKlp2 and profilin 1 during oocyte maturation.Then,we used inhibitor treatment and morpholino injection to disrupt protein activity or function and further explored their effects on spindle formation,chromatin alignment and actin assembly,which helped us learn how they regulate polar body extrusion during oocyte maturation.Besides,with the employment of western bolt,we examined the impact of profilin 1 knockdown on actin as well as the expression of several related molecules,which further indicated the possible mechanism of profilin 1 regulation on mouse oocyte meiotic maturation.These results provide vital evidences for completing the regulation mechanism of mammalian oocyte maturation.This study consists of the following two experiments.Project 1.MKlp2 Inhibition Results in Polar Body Extrusion Failure during Mouse Oocyte Maturation Mammalian oocyte meiotic maturation involves a number of important processes,including spindle assembly and migration,cortical reorganization and polar body extrusion.Numerous proteins contribute to these processes,but it is unknown whether MKlp2(mitotic kinesin-like protein 2;also called KIF20A),a microtubule-associated protein that regulates cytokinesis during mitosis,is involved in oocyte maturation.Here we examined the role of MKlp2 in mouse oocyte.Immunostaining results showed that MKlp2 localized to oocyte microtubules.Time-lapse microscopy showed that disrupting MKlp2 expression with paprotrain,a specific inhibitor of MKlp2,resulted in polar body extrusion failure.This could be rescued after rescuing oocytes from paprotrain in fresh medium.Cell cycle analysis showed that most oocytes were arrested at metaphase I or telophase I.However,oocyte spindle structure and chromosome alignment were not disrupted after the inhibition of MKlp2 by paprotrain.Taken together,our results demonstrated that MKlp2 is crucial for oocyte maturation by regulating polar body extrusion.Project 2.Profilin 1 plays feedback role on actin-mediated polar body extrusion in mouse oocytesAs a small actin-binding protein,profilin plays a central role in the regulation of actin assembly.However,the functions of profilin in mammalian oocytes are uncertain.To investigate the function of profilin in oocytes,immunofluorescent staining was first used to examine the localization of profilin 1.The results showed that profilin 1 was localized around the meiotic spindles and was eo-localized with cytoplasmic actin.Knockdown(KD)of profilin 1 with specific morpholino(MO)microinjection resulted in polar body extrusion failure.This failure was resulted from an increase of actin expression both at membranes and in the cytoplasm.Furthermore,western blot analysis revealed that the expression of ROCK and the phosphorylation level of myosin light chain(MLC)were significantly decreased after profilin 1 KD.Thus,our results indicated that a feedback mechanism between profilin-actin(PA)and ROCK-MLC2 regulates the actin assembly during mouse oocyte maturation.更多还原