牛早期卵母细胞和成熟前后的超微结构变化
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。
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.
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。
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.
引文
[1]洪涛.生物医学超微结构与电子显微镜技术(第一版).北京:科学出版社,1980,490～492
[2]秦鹏春.哺乳动物胚胎学(第二版).北京:科学出版社,2001:50～70
[3]金海霞.人类卵母细胞超微结构的研究进展.国外医学计划生育分册,2004,23(3):137～140
[4] Afzelius BA. The ultrastructure of the nuclear membrane of the sea urchin oocyte as studied with the electron microscope. Exp Cell Pes, 1995, 8(1): 147～158
[5]马泽芳,张林媛,郑丁团等.东北梅花鹿初级卵母细胞的超微结构.兽类学报,2006,26(1):59～63
[6]杨永杰,张燕君,张宝华等.体外培养的人卵母细胞的超微结构.解剖学报,2005,36(6):655～659
[7]雍艳红,余四九,巨向红等.牦牛卵泡细胞及其卵母细胞不同发育时期的结构变化.动物学报,2005, 51(6):1050～1057
[8]李健,胡建宏,韩增胜等.猪卵母细胞冷冻损伤的研究进展.中国农学通报,2005,21(10):21～24
[9]张玲,朱桂金,魏玉兰.丁内酯-Ⅰ对小鼠卵母细胞体外成熟、染色质构型和超微结构的影响.生殖医学杂志,2005,14(4):224～228
[10]高志花,周虚,王春清等.体外培养牛腔前卵泡卵母细胞的超微结构.中国兽医学报,2005,25(3):326～329
[11]P. Hyttel, T. Fair, H. Callesen and T. Greve. Oocyte growth, capacitation and final maturation in cattle. Theriogenology, 1997,47(1): 23～32
[12] T. Greising and L. Jonas. The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes. Theriogenology, 1999, 52(2): 303～312
[13] M. Schmidt, P. Hyttel, B. Avery and T. Greve. Ultrastructure of in vitro matured bovine oocytes after controlled freezing in 10% glycerol. Animal Reproduction Science, 1995, 37(3-4): 281～290
[14]M. Schmidt, P. Hyttel, T. Greve and B. Avery. Ultrastructure of frozen/thawed bovine in vitro matured oocytes. Theriogenology, 1993, 39(1): 304
[15] U. Süss, J. Kassner, M. Müller and G. Stranzinger. Ultrastructure of the intercellular coupling of cumulus cells before and after in vitro maturation of bovine oocytes.Theriogenology, 1988, 29(1):314
[16]M. H. Rajikin, M. Yusoff and R. B. Abdullah. Ultrastructural studies of developinggoat oocytes in vitro. Theriogenology, 1994, 42(6):1003～1016
[17]Vladimir Isachenko, Helmy Selman, Eugenia Isachenko. Effect of cryoprotectants on the ultrastructure of cooled human pronuclear oocytes. Fertility and Sterility, 2004, 81(3):720～722
[18]Carolina M. Lucci, R. V. Silva, C. A. Carvalho, et al. Light microscopical and ultrastructural characterization of goat preantral follicles. Small Ruminant Research, 2001, 41(1):61～69
[19]谭景和,孙青原,杨增明.山羊卵母细胞发育的超微结构研究.解剖学报,1992,23(1):106～110
[20]孙青原,谭景和.山羊卵母细胞闭锁的超微结构研究.兽医大学学报,1993,13(2):129～132
[21]秦鹏春,吴光明.猪卵母细胞体外成熟与体外受精的超微结构研究.解剖学报,1994,25(3):281～285
[22] 钱菊汾,刘灵.山羊卵母细胞成熟的超微结构研究.西北农业大学学报,1995,23(3):16～20
[23]牧人,张锁链.羔山羊卵巢卵母细胞超微结构及核质成熟分析.内蒙古大学学报:自然科学版,1996,27(2):250～255
[24]孟庆刚,孙凤俊.海狸鼠卵巢卵母细胞体外成熟培养前后的超微结构研究.黑龙江动物繁殖,1997,5(2):3～6
[25]周琪,刘英举.家兔卵母细胞及早期胚胎发育的微细结构研究.黑龙江动物繁殖,1998,6(2):3～7
[26]芮荣,曹贵方,张涌.山羊腔前卵泡卵母细胞体外发育的超微结构.四川农业大学学报,1998,16(4):464～467
[27]陈振文, 叶华虎, 任文陟等.犬卵母细胞体成熟培养前后的超微结构变化.实验动物科学与管理,1998,15(4):5～9
[28]张育辉,刘全宏.中国大鲵卵母细胞发育的显微和超微结构.动物学报,1999,45(1):15～22
[29]刘哲,李荣凤.体外培养绵羊卵母细胞超微结构的变化.内蒙古大学学报(自然科学版),2000,31(2):223～227
[30]张志和,何光昕.大熊猫卵母细胞体成熟及受精后的超微结构观察.中国兽医学报,2000,20(5):501～504
[31]刘海军,张美佳.玻璃化冷冻对山羊卵母细胞超微结构的影响.发育与生殖生物学报:英文版,2001,10(B10):116～117
[32]刘海军,张美佳.玻璃化冷冻对山羊卵母细胞超微结构的影响.西北农林科技大学学报,2001,29(3):9～12
[33]孙凤俊,邴印忠,刘春龙等.银狐卵母细胞发育和老化过程的细胞学研究,黑龙江畜牧兽医,2001,(6):28～28
[34]苗竹林,王自能,唐海兰等.大鼠窦状卵泡卵母细胞超微结构变化.广州医学院学报,2004,32(4):17～20
[35]杨永杰,张燕君,张宝华等.体外培养的人卵母细胞的超微结构.解剖学报,2005,36(6):655～659
[36] 刘玉堂 秦鹏春.水貂卵母细胞发育过程中卵泡细胞的微细结构研究.兽类学报1995,15(4):273～278
[37] 谭景和,杨增明,秦鹏春.体外培养前黄牛卵母细胞的分类、形态结构及成熟能力的研究.中国农业科学,1989,22(1):83～88 [38 ]T. Greising and L. Jonas. The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes. Theriogenology, 1999, 52(2):303～312
[39]高志花,周虚,付永斌等.体外培养牛腔前卵泡卵母细胞微绒毛的超微结构观察.畜牧与兽医,2004,36(9):31～32
[40]雍艳红,余四九,巨向红等.牦牛卵泡细胞及其卵母细胞不同发育时期的结构变化.动物学报,2005,51(6):1050～1057
[41]陈子江,李梅,马金龙等.不同成熟期人卵母细胞慢速冷冻的初步研究.北京大学学报:医学版,2004,36(6):571～574
[42]高志花,周虚,高庆华.哺乳动物卵泡发育过程中的超微结构研究进展.黄牛杂志,2003,29(2):28～31
[43]李子义, 周琪, 邹啸环等.电活化前后小鼠卵母细胞的超微结构. 中国兽医学报, 1999, 19(2): 193~196.
[44] 千日成,秦鹏春.哺乳动物卵子的透明带.生物学杂志,1990,2:10-12,19
[45] M. H. Rajikin, M. Yusoff and R. B. Abdullah. Ultrastructural studies of developing goat oocytes in vitro Theriogenology, 1994, 42(6):1003～1016
[46] 刘玉堂,秦鹏春.水貂卵母细胞发育过程中脂滴的形态学及组织化学变化的研究.东北农学院学报,1993,24(4):382～387
[47]Rho GJ, Kim S, Yoo JG, Balasubramanian S, Lee HJ, Choe SY. Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes. Mol Reprod Dev. 2002, 63(4): 464～470.
[48]孙青原,高绍荣.小鼠卵母细胞体外受精中精核转化的机制研究.动物学报.1997,43(4): 382～389.
[49]孙青原,刘灵. 小鼠卵母细胞体外成熟受精过程中细胞核的时空变化规律.DEVELOPMENTAL AND REPRODUCTIVE BIOLOGY(发育与生殖生物学报). 1996, 5(1): 24-33, T001～T004.
[50] Stojkovic M, Machado SA, Stojkovie P, Zakhartvhenko V, Hutzler P, Goncalves PB, Wolf E. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol Reprod, 2001, 64: 904～910.
[51] Carmen Diez, Paloma Duque, Enrique Gómez, Carlos O. Hidalgo, Carolina Tamargo, Aida Rodríguez, Lina Fernández, Santiago de la Varga, Alba Fernández, Nieves Facal and Maite Carbajo. Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability. Theriogenology, 2005, 64(2):317～333
[52]孙青原, 谭景和.山羊卵母细胞闭锁的超微结构研究.兽医大学学报, 1993,13(2): 129～132.
[53]孙青原, 秦鹏春, 刘国艺等. 牛卵丘-卵母细胞复合体体外成熟前后的超微结构变化. 东北农业大学学报, 1996, 27(2): 158～164.
[54] T. Greising and L. Jonas.The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes Theriogenology, 1999,52(2): 303～312
[55] 孙青原, 秦鹏春. 牛卵母细胞发育的超微结构研究. 山东农业大学学报, 1989, 20(4): 9～16
[56] 孙青原, 秦鹏春. 牛卵母细胞发育过程的细胞核变化. 东北农学院学报, 1990, (21): 234～248
[57] Carolina M. Lucci, R. V. Silva, C. A. Carvalho, R. Figueiredo and N. Báo .Light microscopical and ultrastructural characterization of goat preantral follicles. Small Ruminant Research, 2001, 41(1):61～69
[58] M. Kafi, F. Mesbah, H. Nili and A. Khalili. Chronological and ultrastructural changes in camel (Camelus dromedarius) oocytes during in vitro maturation. Theriogenology, 2005, 63(9):2458～2470
[59] Vladimir Isachenko, Helmy Selman, Eugenia Isachenko, Markus Montag, Imam El-Danasouri and Frank Nawroth. Effect of cryoprotectants on the ultrastructure of cooled human pronuclear oocytes Fertility and Sterility, 2004, 81(3):720～722
[60] Lucinda L. Veeck and Robert N. Clark, El Shafie M., Sousa M., Windt M.-L., Kruger T. F. An Atlas of the Ultrastructure of Human Oocytes: A Guide for Assisted Reproduction, First Edition. New York: The Parthenon Publishing Group, 2000. Fertility and Sterility, 2001, 75(4):838～839
[61] P. Hyttel, T. Fair, H. Callesen and T. Greve. Oocyte growth, capacitation and final maturation in cattle. Theriogenology, 1997, 47(1):23～32
[62] M. A. Sirard and P. Blondin. Oocyte maturation and IVF in cattle .Animal Reproduction Science, 1996, 42(1-4):417～426
[63]陈大元编著.受精生物学.北京:科学出版社,2002:30～100
[64]杨增明,孙青原,夏国良主编.生殖生物学.北京:科学出版社,2005:400～500
[65] Plante L, King WA. Light and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization. J Assist Reprod Genet, 1994, 11: 515～529.
[66] Rho GJ, Kim S, Yoo JG, Balasubramanian S, Lee HJ, Choe SY. Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes. Mol Reprod Dev. 2002, 63(4): 464～470.
[67] Isachenko V., Isachenko E., Michelmann HW, Alabart JL, Vazquez I., Bezugly N., Mawroth F.. Lipolysis and ultrastructual changes of intracellular lipid vesicles after cooling of bovine and porcine GV-oocytes. Embryol, 2001, 30: 333～338
[68] Shiloh H., Baratz SL, Koifman M, Ishai D, Bidder D, Weiner Z, Meganzi. M. Dirnfeld. The impact of cigarette smoking on zona pellucida thickness of oocytes and embryos prior to transfer into the uterine cavity. Hum Reprod. 2004, 19(1): 157～159
[69] Lee J, Miyano T, Moor RM. Spindle formation and dynamics of gamma-tubulin and nuclear mitochondrias apparatus protein distribution during meiosis in pig and mouse oocytes. Biol Reprod, 2000, 62: 1184～1192
[70] M. Schmidt, P. Hyttel, B. Avery and T. Greve. Ultrastructure of in vitro matured bovine oocytes after controlled freezing in 10% glycerol Animal Reproduction Science, 1995, 37(3-4):281～290
[71] Sabine K,Miodrag S,Sven R.,Horst-Dieter R.,Eckhard W,Fred S. Effects of growth hormone on the ultrastrure of bovine preimplantation embryos. Cell Tissue Res,2004,314:101～108
[72]翟中和, 王喜忠, 丁明孝主编. 细胞生物学. 北京高等教育出版社, 2001:200～400
[73] Q.Z. Yang, Q.Y. Sun, G.Y. Liu, P.C. Qin and H.L. Feng. Developmental competence and ultrastructure damage of cryopreserved GV-stage bovine oocytes Theriogenology, 1994, 41(1): 342
[74] M. Schmidt, P. Hyttel, T. Greve and B. Avery.Ultrastructure of frozen/thawed bovine in vitro matured oocytes. Theriogenology, 1993, 39(1):304
[75]马所峰,苗义良,李 威等.卵泡表面直径判断山羊卵母细胞的发育能力.畜牧兽医学报,2004,35(4): 399～403
[76] Pelletier C, Keefe DL, Trimarchi JR. Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos. Fertil Steril, 2004 Suppl 1, 81: 850～857.
[77] O’Brien JK, Dwarte D, Ryan JP, Maxwell WMC, Evans G. Comparison of in vitro maturatin, in vitro fertilization, metabolism and ultrastructure of oocytes from prepubertal and adults pigs. Reprod Domest Anim, 2000, 35: 101～107.
[78]韩毅冰,杨庆章,秦鹏春.牛卵母细胞体外成熟的研究.生物技术,1995,5(5):6-10
[79] Nottola SA, Makabe S, Stallone T, Familiari G, Macchiarelli G, Motta PM. In vitro fertilization human eggs. An electron microscopic study. Ital J Anat Embryol, 2001, 106(2 suppl 2): 75～83.
[80]张志和, 张美佳, 侯蓉等.大熊猫卵母细胞体外成熟及受精后的超微结构观察.中国兽医学报, 2000, 20(5): 501～504.
[81]Abe H, Matsuzaki S, Hoshi H. Ultrastructural differences in bovine morulae classified as high and low qualities by morphological evaluation. Theriogenology, 2002a, 57(4): 1273～1283.
[82] Memili E, Dominko T, First NL. Onset of transcription in bovine oocytes and preimplantation embryos. Mol Reprod Dev. 1998, 51(1): 36～41.
[83] Isachenko V, Selman H, Isachenko E, Montag M, Ei-Danasouri I, Nawroth F. Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. Reprod Biomed Online, 2003, 7(2): 211～216.
[84]Au HK, Yeh TS, Kao SH, Tzeng CR, Hsieh RH. Abnormal mitochondrial structure in human unfertilized oocytes and arrested embryos. Ann NY Acad Sci, 2005, 1042: 177-185.
[85]De Loos F, Van VC, Van MP, Kruip TA. Morphology of immature bovine oocytes. Gamete Research, 1989, 24: 197～204.
[86]Gjorret JO, Crichton EG, Loskutoff NM, Armstrong DL, Hyttel P. Ultrastructure of oocyte maturation, fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica). Mol Repord Dev, 2002, 63(1): 79～88.
[87]Greising T, Jonas L. The influences of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes. Theriogenology, 1999, 52(2); 303～312
[88]Hyttel P, Fair T, Avery B, Callesen H, Greve T. Transcriptional activity and ultrastructure in bovine oocytes. Reprod Domest Anim, 1999, 34: 247～254.
[89] Lou Z,Robinson J M. Co-localization of an endocytic marker and acid phosphatase in a tubular/reticular compartment in macrophage.J Histochem Cytochem,1992,40(1):93
[2]秦鹏春.哺乳动物胚胎学(第二版).北京:科学出版社,2001:50～70
[3]金海霞.人类卵母细胞超微结构的研究进展.国外医学计划生育分册,2004,23(3):137～140
[4] Afzelius BA. The ultrastructure of the nuclear membrane of the sea urchin oocyte as studied with the electron microscope. Exp Cell Pes, 1995, 8(1): 147～158
[5]马泽芳,张林媛,郑丁团等.东北梅花鹿初级卵母细胞的超微结构.兽类学报,2006,26(1):59～63
[6]杨永杰,张燕君,张宝华等.体外培养的人卵母细胞的超微结构.解剖学报,2005,36(6):655～659
[7]雍艳红,余四九,巨向红等.牦牛卵泡细胞及其卵母细胞不同发育时期的结构变化.动物学报,2005, 51(6):1050～1057
[8]李健,胡建宏,韩增胜等.猪卵母细胞冷冻损伤的研究进展.中国农学通报,2005,21(10):21～24
[9]张玲,朱桂金,魏玉兰.丁内酯-Ⅰ对小鼠卵母细胞体外成熟、染色质构型和超微结构的影响.生殖医学杂志,2005,14(4):224～228
[10]高志花,周虚,王春清等.体外培养牛腔前卵泡卵母细胞的超微结构.中国兽医学报,2005,25(3):326～329
[11]P. Hyttel, T. Fair, H. Callesen and T. Greve. Oocyte growth, capacitation and final maturation in cattle. Theriogenology, 1997,47(1): 23～32
[12] T. Greising and L. Jonas. The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes. Theriogenology, 1999, 52(2): 303～312
[13] M. Schmidt, P. Hyttel, B. Avery and T. Greve. Ultrastructure of in vitro matured bovine oocytes after controlled freezing in 10% glycerol. Animal Reproduction Science, 1995, 37(3-4): 281～290
[14]M. Schmidt, P. Hyttel, T. Greve and B. Avery. Ultrastructure of frozen/thawed bovine in vitro matured oocytes. Theriogenology, 1993, 39(1): 304
[15] U. Süss, J. Kassner, M. Müller and G. Stranzinger. Ultrastructure of the intercellular coupling of cumulus cells before and after in vitro maturation of bovine oocytes.Theriogenology, 1988, 29(1):314
[16]M. H. Rajikin, M. Yusoff and R. B. Abdullah. Ultrastructural studies of developinggoat oocytes in vitro. Theriogenology, 1994, 42(6):1003～1016
[17]Vladimir Isachenko, Helmy Selman, Eugenia Isachenko. Effect of cryoprotectants on the ultrastructure of cooled human pronuclear oocytes. Fertility and Sterility, 2004, 81(3):720～722
[18]Carolina M. Lucci, R. V. Silva, C. A. Carvalho, et al. Light microscopical and ultrastructural characterization of goat preantral follicles. Small Ruminant Research, 2001, 41(1):61～69
[19]谭景和,孙青原,杨增明.山羊卵母细胞发育的超微结构研究.解剖学报,1992,23(1):106～110
[20]孙青原,谭景和.山羊卵母细胞闭锁的超微结构研究.兽医大学学报,1993,13(2):129～132
[21]秦鹏春,吴光明.猪卵母细胞体外成熟与体外受精的超微结构研究.解剖学报,1994,25(3):281～285
[22] 钱菊汾,刘灵.山羊卵母细胞成熟的超微结构研究.西北农业大学学报,1995,23(3):16～20
[23]牧人,张锁链.羔山羊卵巢卵母细胞超微结构及核质成熟分析.内蒙古大学学报:自然科学版,1996,27(2):250～255
[24]孟庆刚,孙凤俊.海狸鼠卵巢卵母细胞体外成熟培养前后的超微结构研究.黑龙江动物繁殖,1997,5(2):3～6
[25]周琪,刘英举.家兔卵母细胞及早期胚胎发育的微细结构研究.黑龙江动物繁殖,1998,6(2):3～7
[26]芮荣,曹贵方,张涌.山羊腔前卵泡卵母细胞体外发育的超微结构.四川农业大学学报,1998,16(4):464～467
[27]陈振文, 叶华虎, 任文陟等.犬卵母细胞体成熟培养前后的超微结构变化.实验动物科学与管理,1998,15(4):5～9
[28]张育辉,刘全宏.中国大鲵卵母细胞发育的显微和超微结构.动物学报,1999,45(1):15～22
[29]刘哲,李荣凤.体外培养绵羊卵母细胞超微结构的变化.内蒙古大学学报(自然科学版),2000,31(2):223～227
[30]张志和,何光昕.大熊猫卵母细胞体成熟及受精后的超微结构观察.中国兽医学报,2000,20(5):501～504
[31]刘海军,张美佳.玻璃化冷冻对山羊卵母细胞超微结构的影响.发育与生殖生物学报:英文版,2001,10(B10):116～117
[32]刘海军,张美佳.玻璃化冷冻对山羊卵母细胞超微结构的影响.西北农林科技大学学报,2001,29(3):9～12
[33]孙凤俊,邴印忠,刘春龙等.银狐卵母细胞发育和老化过程的细胞学研究,黑龙江畜牧兽医,2001,(6):28～28
[34]苗竹林,王自能,唐海兰等.大鼠窦状卵泡卵母细胞超微结构变化.广州医学院学报,2004,32(4):17～20
[35]杨永杰,张燕君,张宝华等.体外培养的人卵母细胞的超微结构.解剖学报,2005,36(6):655～659
[36] 刘玉堂 秦鹏春.水貂卵母细胞发育过程中卵泡细胞的微细结构研究.兽类学报1995,15(4):273～278
[37] 谭景和,杨增明,秦鹏春.体外培养前黄牛卵母细胞的分类、形态结构及成熟能力的研究.中国农业科学,1989,22(1):83～88 [38 ]T. Greising and L. Jonas. The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes. Theriogenology, 1999, 52(2):303～312
[39]高志花,周虚,付永斌等.体外培养牛腔前卵泡卵母细胞微绒毛的超微结构观察.畜牧与兽医,2004,36(9):31～32
[40]雍艳红,余四九,巨向红等.牦牛卵泡细胞及其卵母细胞不同发育时期的结构变化.动物学报,2005,51(6):1050～1057
[41]陈子江,李梅,马金龙等.不同成熟期人卵母细胞慢速冷冻的初步研究.北京大学学报:医学版,2004,36(6):571～574
[42]高志花,周虚,高庆华.哺乳动物卵泡发育过程中的超微结构研究进展.黄牛杂志,2003,29(2):28～31
[43]李子义, 周琪, 邹啸环等.电活化前后小鼠卵母细胞的超微结构. 中国兽医学报, 1999, 19(2): 193~196.
[44] 千日成,秦鹏春.哺乳动物卵子的透明带.生物学杂志,1990,2:10-12,19
[45] M. H. Rajikin, M. Yusoff and R. B. Abdullah. Ultrastructural studies of developing goat oocytes in vitro Theriogenology, 1994, 42(6):1003～1016
[46] 刘玉堂,秦鹏春.水貂卵母细胞发育过程中脂滴的形态学及组织化学变化的研究.东北农学院学报,1993,24(4):382～387
[47]Rho GJ, Kim S, Yoo JG, Balasubramanian S, Lee HJ, Choe SY. Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes. Mol Reprod Dev. 2002, 63(4): 464～470.
[48]孙青原,高绍荣.小鼠卵母细胞体外受精中精核转化的机制研究.动物学报.1997,43(4): 382～389.
[49]孙青原,刘灵. 小鼠卵母细胞体外成熟受精过程中细胞核的时空变化规律.DEVELOPMENTAL AND REPRODUCTIVE BIOLOGY(发育与生殖生物学报). 1996, 5(1): 24-33, T001～T004.
[50] Stojkovic M, Machado SA, Stojkovie P, Zakhartvhenko V, Hutzler P, Goncalves PB, Wolf E. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol Reprod, 2001, 64: 904～910.
[51] Carmen Diez, Paloma Duque, Enrique Gómez, Carlos O. Hidalgo, Carolina Tamargo, Aida Rodríguez, Lina Fernández, Santiago de la Varga, Alba Fernández, Nieves Facal and Maite Carbajo. Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability. Theriogenology, 2005, 64(2):317～333
[52]孙青原, 谭景和.山羊卵母细胞闭锁的超微结构研究.兽医大学学报, 1993,13(2): 129～132.
[53]孙青原, 秦鹏春, 刘国艺等. 牛卵丘-卵母细胞复合体体外成熟前后的超微结构变化. 东北农业大学学报, 1996, 27(2): 158～164.
[54] T. Greising and L. Jonas.The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes Theriogenology, 1999,52(2): 303～312
[55] 孙青原, 秦鹏春. 牛卵母细胞发育的超微结构研究. 山东农业大学学报, 1989, 20(4): 9～16
[56] 孙青原, 秦鹏春. 牛卵母细胞发育过程的细胞核变化. 东北农学院学报, 1990, (21): 234～248
[57] Carolina M. Lucci, R. V. Silva, C. A. Carvalho, R. Figueiredo and N. Báo .Light microscopical and ultrastructural characterization of goat preantral follicles. Small Ruminant Research, 2001, 41(1):61～69
[58] M. Kafi, F. Mesbah, H. Nili and A. Khalili. Chronological and ultrastructural changes in camel (Camelus dromedarius) oocytes during in vitro maturation. Theriogenology, 2005, 63(9):2458～2470
[59] Vladimir Isachenko, Helmy Selman, Eugenia Isachenko, Markus Montag, Imam El-Danasouri and Frank Nawroth. Effect of cryoprotectants on the ultrastructure of cooled human pronuclear oocytes Fertility and Sterility, 2004, 81(3):720～722
[60] Lucinda L. Veeck and Robert N. Clark, El Shafie M., Sousa M., Windt M.-L., Kruger T. F. An Atlas of the Ultrastructure of Human Oocytes: A Guide for Assisted Reproduction, First Edition. New York: The Parthenon Publishing Group, 2000. Fertility and Sterility, 2001, 75(4):838～839
[61] P. Hyttel, T. Fair, H. Callesen and T. Greve. Oocyte growth, capacitation and final maturation in cattle. Theriogenology, 1997, 47(1):23～32
[62] M. A. Sirard and P. Blondin. Oocyte maturation and IVF in cattle .Animal Reproduction Science, 1996, 42(1-4):417～426
[63]陈大元编著.受精生物学.北京:科学出版社,2002:30～100
[64]杨增明,孙青原,夏国良主编.生殖生物学.北京:科学出版社,2005:400～500
[65] Plante L, King WA. Light and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization. J Assist Reprod Genet, 1994, 11: 515～529.
[66] Rho GJ, Kim S, Yoo JG, Balasubramanian S, Lee HJ, Choe SY. Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes. Mol Reprod Dev. 2002, 63(4): 464～470.
[67] Isachenko V., Isachenko E., Michelmann HW, Alabart JL, Vazquez I., Bezugly N., Mawroth F.. Lipolysis and ultrastructual changes of intracellular lipid vesicles after cooling of bovine and porcine GV-oocytes. Embryol, 2001, 30: 333～338
[68] Shiloh H., Baratz SL, Koifman M, Ishai D, Bidder D, Weiner Z, Meganzi. M. Dirnfeld. The impact of cigarette smoking on zona pellucida thickness of oocytes and embryos prior to transfer into the uterine cavity. Hum Reprod. 2004, 19(1): 157～159
[69] Lee J, Miyano T, Moor RM. Spindle formation and dynamics of gamma-tubulin and nuclear mitochondrias apparatus protein distribution during meiosis in pig and mouse oocytes. Biol Reprod, 2000, 62: 1184～1192
[70] M. Schmidt, P. Hyttel, B. Avery and T. Greve. Ultrastructure of in vitro matured bovine oocytes after controlled freezing in 10% glycerol Animal Reproduction Science, 1995, 37(3-4):281～290
[71] Sabine K,Miodrag S,Sven R.,Horst-Dieter R.,Eckhard W,Fred S. Effects of growth hormone on the ultrastrure of bovine preimplantation embryos. Cell Tissue Res,2004,314:101～108
[72]翟中和, 王喜忠, 丁明孝主编. 细胞生物学. 北京高等教育出版社, 2001:200～400
[73] Q.Z. Yang, Q.Y. Sun, G.Y. Liu, P.C. Qin and H.L. Feng. Developmental competence and ultrastructure damage of cryopreserved GV-stage bovine oocytes Theriogenology, 1994, 41(1): 342
[74] M. Schmidt, P. Hyttel, T. Greve and B. Avery.Ultrastructure of frozen/thawed bovine in vitro matured oocytes. Theriogenology, 1993, 39(1):304
[75]马所峰,苗义良,李 威等.卵泡表面直径判断山羊卵母细胞的发育能力.畜牧兽医学报,2004,35(4): 399～403
[76] Pelletier C, Keefe DL, Trimarchi JR. Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos. Fertil Steril, 2004 Suppl 1, 81: 850～857.
[77] O’Brien JK, Dwarte D, Ryan JP, Maxwell WMC, Evans G. Comparison of in vitro maturatin, in vitro fertilization, metabolism and ultrastructure of oocytes from prepubertal and adults pigs. Reprod Domest Anim, 2000, 35: 101～107.
[78]韩毅冰,杨庆章,秦鹏春.牛卵母细胞体外成熟的研究.生物技术,1995,5(5):6-10
[79] Nottola SA, Makabe S, Stallone T, Familiari G, Macchiarelli G, Motta PM. In vitro fertilization human eggs. An electron microscopic study. Ital J Anat Embryol, 2001, 106(2 suppl 2): 75～83.
[80]张志和, 张美佳, 侯蓉等.大熊猫卵母细胞体外成熟及受精后的超微结构观察.中国兽医学报, 2000, 20(5): 501～504.
[81]Abe H, Matsuzaki S, Hoshi H. Ultrastructural differences in bovine morulae classified as high and low qualities by morphological evaluation. Theriogenology, 2002a, 57(4): 1273～1283.
[82] Memili E, Dominko T, First NL. Onset of transcription in bovine oocytes and preimplantation embryos. Mol Reprod Dev. 1998, 51(1): 36～41.
[83] Isachenko V, Selman H, Isachenko E, Montag M, Ei-Danasouri I, Nawroth F. Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. Reprod Biomed Online, 2003, 7(2): 211～216.
[84]Au HK, Yeh TS, Kao SH, Tzeng CR, Hsieh RH. Abnormal mitochondrial structure in human unfertilized oocytes and arrested embryos. Ann NY Acad Sci, 2005, 1042: 177-185.
[85]De Loos F, Van VC, Van MP, Kruip TA. Morphology of immature bovine oocytes. Gamete Research, 1989, 24: 197～204.
[86]Gjorret JO, Crichton EG, Loskutoff NM, Armstrong DL, Hyttel P. Ultrastructure of oocyte maturation, fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica). Mol Repord Dev, 2002, 63(1): 79～88.
[87]Greising T, Jonas L. The influences of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes. Theriogenology, 1999, 52(2); 303～312
[88]Hyttel P, Fair T, Avery B, Callesen H, Greve T. Transcriptional activity and ultrastructure in bovine oocytes. Reprod Domest Anim, 1999, 34: 247～254.
[89] Lou Z,Robinson J M. Co-localization of an endocytic marker and acid phosphatase in a tubular/reticular compartment in macrophage.J Histochem Cytochem,1992,40(1):93