【作者】 邱志芳；

【导师】 章孝荣； 陶勇；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 本实验用透射电子显微镜观察体外成熟前后牛GV期卵母细胞的亚细胞结构,以及牛卵巢上不同发育时期卵母细胞过程中超微结构的变化,旨在为胚胎工程等相关研究提供依据。选择刚出生不久牛的卵巢和大量的性成熟前牛的卵巢作为研究对象进行电镜观察。电镜下观察发现,卵原细胞具有一个大的圆形细胞核,初级卵母细胞前细线期和细线期,细胞核基质中出现电子致密的线状结构,此时细胞核偏向于一侧,初级卵母细胞双线期出现了灯刷染色体。卵原细胞的细胞质中仅有少数的线粒体,线粒体的密度中等,初级卵母细胞前细线期和细线期,发现在Mi之间存在颗粒状或纤维状物质,双线期线粒体呈圆形,嵴很少,呈板状,有的甚至不见嵴;有腔卵母细胞是线粒体数量增多,呈圆形或椭圆形,基质电子密度较高,并进一步向皮质区迁移;直径2mm以上卵泡中卵母细胞的线粒体转变为带帽线粒体。卵原细胞与滤泡细胞之间的距离很小,滤泡细胞的核呈多形性;初级卵母细胞与相伴的卵泡细胞间靠得很紧密,二者间仅有窄小的间隙;有腔的卵母细胞与滤泡细胞之间的距离变大,滤泡细胞的核呈圆形。卵原细胞的细胞质中溶酶体含量较少;前细线期和细线期的初级卵母细胞溶酶体的数量增加,溶酶体和线粒体结构紧贴,卵丘细胞质中的溶酶体和线粒体密集分布;双线期的初级卵母细胞有大量体积小、电子密度较高的溶酶体,这些溶酶体常常联合在一起形成大的多泡体;有腔卵母细胞时,球状的溶酶体变成线状的溶酶体。卵原细胞的细胞质中囊泡数量较少,分布均匀;初级卵母细胞的前细线期和细线期数量显著增加且偏向于细胞的一侧;初级卵母细胞的双线期有的囊泡聚集在一起,大多有膜包围,电子密度极低,囊泡中似乎没有其他内容物;有腔卵母细胞时,有些囊泡中能看到一些絮状物,囊泡数量明显减少。以上关于牛初级卵母细胞不同时期超微结构变化的研究结果此前尚未见有相关报道。在牛的原始卵母细胞质中,未见有皮质颗粒;在初级卵母细胞前细线期和细线期皮质区出现比较少的明粒皮质颗粒;初级卵母细胞的双线期皮质区颗粒密集;有腔卵母细胞皮质颗粒比较丰富,有四、五层,大部分位于皮质区,这时皮质颗粒为暗粒。此外,牛卵巢上有些卵母细胞出现了细胞核皱缩、细胞器膨胀、自噬泡堆积,导致卵母细胞退化现象的发生;有些卵原细胞的细胞核与脂滴相伴存在;初级卵母细胞胞质中出现晕轮结构。直径为2～8㎜卵母细胞体外培养24h后的卵母细胞,透明带有增厚的趋势;但体外成熟的卵母细胞要比体外未成熟的卵母细胞和体外成熟后去级体卵母细胞增加的厚度要小。体外培养24h后的成熟卵母细胞,表面微绒毛又细又长,都出现了倒伏现象;体外未成熟的卵母细胞微绒毛却变得又短又粗,未见倒伏,体外成熟后去级体卵母细胞微绒毛变得稀少。体外培养后的卵母细胞,脂肪滴的直径有增大的趋势;而体外培养成熟的卵母细胞,脂肪滴有两种密度类型,并相伴存在,且其直径大小略有增加;体外培养未成熟的卵母细胞,脂肪滴显著变大,但密度不均一;体外培养成熟后又去极体的卵母细胞,与对照组比较,脂肪滴略有变大,但变化幅度不大,也有些脂肪滴的直径表现下降的趋势。三种处理的卵母细胞中,帽状线粒体、异常线粒体的含量都有下降的趋势;但体外成熟的卵母细胞中帽状线粒体含量的下降幅度最大,体外成熟的卵母细胞、体外成熟后去极体的卵母细胞中成熟线粒体的含量都显著提高,而体外培养未成熟的卵母细胞成熟线粒体有下降的趋势。体外培养成熟的卵母细胞中出现了残余成分,类似于溶酶体样的结构;体外培养成熟后又去极体的卵母细胞中出现基质固缩化。上述研究结果发现了牛卵母细胞发育过程中超微结构的某些变化规律,为探索牛卵母细胞的发育规律提供了初步的组织学和形态学依据。更多还原

【Abstract】 The aim of this study was to investigate the ultrastructureal change of bovine oocytes in development. The oocytes on ovaries at different stage and those before and after in vitro maturation were studied by transmission electromicrocopy. The results showed that the oogonium had a big and round nucleus. At early leptonema and late leptonema of elementary oocytes, a dense and linear structure in nucleus and ooplasm, was observed, and the nucleus yawed to one side. In contrast, at diplonema, lampbrush chromosome was found. Few mitochondrias were seen in the cytoplasm of oogonium and these mitochondrias’density was at middle level. At leptonema, the oocytes contained lots of granule and fibrin materials located between mitochondrias. In diplonema oocytes, the mitochondrias were round and had few cristas. In diplonema of elementary oocytes, the mitochondrias were round with few cristas .When the oocytes developed, the amount of mitochondrias became increased, and they were round and the density of plasm was high. In the follicles whose diameter was above 2mm, the mitochondrias formed hood. The distance between oogonium and cumulus oophorus was short. The nucleus of cumulus cells had many figures. There was only a narrow space between elementary oocytes and cumulus oophorus. The distance between antral follicle and cumulus oophorus became big, and the nucleus of cumulus oophorus was round. There were only a few lysosomes in the oogonium; when the oocytes reached early leptonema and leptonema, the number of lysosomes increased. Moreover, the lysosomes clang to mitochondrias. The lysosomes and mitochondrias densely distributed in the cumulus oophorus. In diplonema of elementary oocytes, there were lots of dense lysosomes. These lysosomes formed a large vesicle. In antral follicles, the globular lysosomes turned to linear lysosomes. Few vesicles distributed uniformity in the cytoplasm of the oogonium. The amount of vesicles significantly increased and yawed to one side in the early leptonema and leptonema’s oocytes. Many vesicles congregated most of which was besieged by membrane in the diplonema oocytes. There were materials in the vesicles of the antral follicle oocytes, and the vesicles diminished. CGs were not found in the cytoplasm of bovine oogonium. However, there were light CGs in the oolemna of early leptonema and leptonema oocytes. CGs congregated in the diplonema oocytes. In antral follicle oocytes, there were lots of dark CGs, most of which distributed in the oolemna. Some oocytes in the ovary degenerated; Some nucleus of antral follicle oocytes accompanied lipid droplets; Moreover, the halos appeared in the cytoplasm of cumulus oophorus.Zona pellucida (ZP) of oocytes matured in vitro had a tendency to become thicker. But ZP of oocytes with first pobar body was thinner than that of oocytes without first polar body (PB1) after in vitro maturation. Microvilli of oocytes cultured in vitro appeared falling. However, the microvilli of oocytes matured in vitro was thinner and longer, and the microvilli of oocytes without PB1 become shorter and thicker; The oocytes whose nucleus was removed had few microvilli. Lipid droplets’diameter of oocytes cultured in vitro was big. Lipid droplet of ooctyes matured in vitro had two types, moreover these lipid droplets showed up accompanying and become slightly bigger. Lipid droplet of oocytes without PB1 appeared heterogeneous and become markedly bigger. Lipid droplets’density of oocytes without nucleus after maturation appeared dark, and the diameter of these had a descendent tendency. The hooded mitochondrias showed up in three types of oocytes; the proportion of abnormal mitochondrias all descended. The decadency arrangement was largest in oocytes matured in vitro; the amount of mature mitochondrias in oocytes matured in vitro and oocytes after enucleation improved markedly; these mitochondrias in oocytes unmatured in vitro decreased. Oocytes matured in vitro emerged lysosomes-like residual materials. Cytoplasm of oocytes which is deleted after matured in vitro appeared partly solid state. These results show the characteristic of ultrastructural changes during bovine oocyte development, and provide histology and morphologic gist for bovine oocytes development.更多还原