被刺戳与未被刺戳未受精蛙卵离心的差别

1.黑斑蛙和蟾蜍的受精卵与未受精卵在用×1500g 左右离心后,在形态上也像豹蛙的一样有区别。受精卵都形成绉褶脂类物质层而大部分的未受精卵则形成光滑的类脂物质层。2.用清洁玻璃针刺戳黑斑蛙末受精卵加×1500g 离心,所得的离心卵也像受精卵一样形成四层,并且具有绉褶的脂类物质层。3.我们根据已有的资料将蛙精子在受精时的作用分为三种,第一是使卵还原为双倍体,第二是刺激作用(Stimulus)和第三是调节作用(Regulation)。受精卵在离心后形成绉褶脂类物质层的现象是精子的刺激作用所造成的。用玻璃针刺戳也可引起这种现象。     

卵子皮层颗粒的功能

皮层颗粒是卵内一种特殊的、由膜包围的一种分泌颗粒,它位于质膜之下,许多无脊椎动物和脊椎动物单精受精卵都有此细胞器,生理多精受精卵则没有。当卵受精时发生胞吐作用,将其内含物释放,这过程被称为皮层反应。它的作用主要使卵的外膜发生变化,阻止多精受精以及保护胚胎发育,防止不利的环境条件。 1.卵子皮层卵子皮层是一位于质膜下呈凝胶的细胞质薄层,不同动物的厚度不同,1—5微米。皮层颗粒即位于此皮层中,若离心,卵内细胞质很易分层,而皮层颗粒不易被分离。因此皮层为一半固体的弹力层,其硬度主要取决于Ca~(2+)的存在。未受精卵的皮层含有单体的肌动蛋白,受精时随皮层反应肌动蛋白发生多聚作用而形成一凝胶的网状结构。从分离卵子表面的实验表明皮层颗粒牢固     

中华绒螯蟹成熟卵形态和超微结构的研究

中华绒螯蟹的成熟卵仅有初级卵膜，无次级和三级卵巢，质膜初期厚而多层，且具皱褶，卵核在卵的发育过程中变化很大，未发见中心粒，内质网和高尔基体均始见于蟹卵发育的初期，皮层颗粒先出现于蟹卵深部，随后移到卵的表层，无滋养细胞，蟹卵由卵泡细胞提供物质，形成卵黄，此外，还可直接从血淋巴内摄取卵黄前身物质。     

银鲫受精卵中卵黄粒和线粒体的超微结构变化

电镜观察到雌性银鲫肝脏中正在形成的卵黄物质具有晶形主体结构,银鲫卵的卵黄粒内无晶形主体,第一次卵裂前的受精卵卵黄粒内有两种形式的空泡。一种是一些中空的小空泡;另一种是一个大的空泡,泡内含有线粒体和颗粒状的核糖体,泡的边缘有片层状的类脂物。受精卵中的线粒体内部结构多样,有些含有颗粒状物,有些含有片层状的类脂物。       

唇受精卵的皮层反应及其引发机制

唇(鱼骨)皮层小泡1-4层,在光镜和透射电镜下均具5种形态,由外及内,小泡内颗粒直径逐渐减少.在H.E染色中,动物极低纬度区和精孔器附近的卵膜和质膜之间,具少量均匀的着色非常深的紫色斑点,与Ⅰ型皮层小泡内容物形态结构相似,在透射电镜下,这些斑点和卵膜、质膜有明显的界限,其外没有包被膜相结构,我们称之皮层反应引发斑,这是在鱼类受精卵中发现的一个新的结构.扫描电镜下,引发斑成絮状.引发斑对皮层反应的引发具有重要作用.皮层反应可分为潜伏期、始发期、高潮期、衰退期四个时期,潜伏期没有皮层反应发生,始发期只是位于受精卵外围的少量皮层小泡释放,高潮期为多个皮层小泡相互融合形成一个大的泡状体,泡状体再与质膜接触、融合后,随后破裂,释放内容物,可分为两个阶段,衰退期释放Ⅴ型皮层小泡和其他残存的皮层小泡以及未完全降解卵黄颗粒碎屑;皮层反应是由Ⅰ型皮层小泡和引发斑诱导的爆发性的链式反应,卵子外侧的皮层反应可以诱导内侧皮层反应.皮层反应有两个起始区域,在受精后35s开始于动物极低纬度区,稍后出现在精孔器前庭附近,随后在这两个始发区向四周扩散,并在前庭以外的区域愈合、打通.皮层小泡分批多次释放,质膜多次重组.精子入卵位点附近没有皮层小泡,不发生皮层反应,这提示皮层反应对鱼类多精受精的抑制效应有限.     

唇受精卵的皮层反应及其引发机制

唇皮层小泡1-4层,在光镜和透射电镜下均具5种形态,由外及内,小泡内颗粒直径逐渐减少。在H．E染 色中,动物极低纬度区和精孔器附近的卵膜和质膜之间,具少量均匀的着色非常深的紫色斑点,与Ⅰ型皮层小泡内 容物形态结构相似,在透射电镜下,这些斑点和卵膜、质膜有明显的界限,其外没有包被膜相结构,我们称之皮层反 应引发斑,这是在鱼类受精卵中发现的一个新的结构。扫描电镜下,引发斑成絮状。引发斑对皮层反应的引发具 有重要作用。皮层反应可分为潜伏期、始发期、高潮期、衰退期四个时期,潜伏期没有皮层反应发生,始发期只是位 于受精卵外围的少量皮层小泡释放,高潮期为多个皮层小泡相互融合形成一个大的泡状体,泡状体再与质膜接触、 融合后,随后破裂,释放内容物,可分为两个阶段,衰退期释放Ⅴ型皮层小泡和其他残存的皮层小泡以及未完全降 解卵黄颗粒碎屑;皮层反应是由Ⅰ型皮层小泡和引发斑诱导的爆发性的链式反应,卵子外侧的皮层反应可以诱导 内侧皮层反应。皮层反应有两个起始区域,在受精后35s开始于动物极低纬度区,稍后出现在精孔器前庭附近,随 后在这两个始发区向四周扩散,并在前庭以外的区域愈合、打通。皮层小泡分批多次释放,质膜多次重组。精子入 卵位点附近没有皮层小泡,不发生皮层反应,这提示皮层反应对鱼类多精受精的抑制效应有限。     

唇(鱼骨)受精卵的皮层反应及其引发机制

唇[鱼骨]皮层小泡1-4层,在光镜和透射电镜下均具5种形态,由外及内,小泡内颗粒直径逐渐减少。在H．E染色中,动物极低纬度区和精孔器附近的卵膜和质膜之间,具少量均匀的着色非常深的紫色斑点,与Ⅰ型皮层小泡内容物形态结构相似,在透射电镜下,这些斑点和卵膜、质膜有明显的界限,其外没有包被膜相结构,我们称之皮层反应引发斑,这是在鱼类受精卵中发现的一个新的结构。扫描电镜下,引发斑成絮状。引发斑对皮层反应的引发具有重要作用。皮层反应可分为潜伏期、始发期、高潮期、衰退期四个时期,潜伏期没有皮层反应发生,始发期只是位于受精卵外围的少量皮层小泡释放,高潮期为多个皮层小泡相互融合形成一个大的泡状体,泡状体再与质膜接触、融合后,随后破裂,释放内容物,可分为两个阶段,衰退期释放Ⅴ型皮层小泡和其他残存的皮层小泡以及未完全降解卵黄颗粒碎屑;皮层反应是由Ⅰ型皮层小泡和引发斑诱导的爆发性的链式反应,卵子外侧的皮层反应可以诱导内侧皮层反应。皮层反应有两个起始区域,在受精后35s开始于动物极低纬度区,稍后出现在精孔器前庭附近,随后在这两个始发区向四周扩散,并在前庭以外的区域愈合、打通。皮层小泡分批多次释放,质膜多次重组。精子人卵位点附近没有皮层小泡,不发生皮层反应,这提示皮层反应对鱼类多精受精的抑制效应有限。     

东方扁虾卵子发生的超微结构

根据卵细胞的形态、内部结构特征及卵母细胞与滤泡细胞之间的关系,东方扁虾的卵子发生可划分为卵原细胞、卵黄发生前卵母细胞、卵黄发生卵母细胞和成熟卵母细胞等四个时期。卵原细胞胞质稀少,胞器以滑面内质网为主。卵黄发生前卵母细胞核明显膨大,特称为生发泡;在靠近核外膜的胞质中可观察到核仁外排物。卵黄发生卵母细胞逐渐为滤泡细胞所包围;卵黄合成旺盛,胞质中因而形成并积累了越来越多的卵黄粒。东方扁虾卵母细胞的卵黄发生是二源的。游离型核糖体率先参与内源性卵黄合成形成无膜卵黄粒。粗面内质网是内源性卵黄形成的主要胞器。滑面内质网、线粒体和溶酶体以多种方式活跃地参与卵黄粒形成。卵周隙内的外源性物质有两个来源:滤泡细胞的合成产物和血淋巴携带、转运的卵黄蛋白前体物。这些外源性物质主要通过质膜的微吞饮作用和微绒毛的吸收作用这两种方式进入卵母细胞,进而形成外源性卵黄。内源性和外源性的卵黄物质共同参与成熟卵母细胞中富含髓样小体的卵黄粒的形成。卵壳的形成和微绒毛的回缩被认为是东方扁虾卵母细胞成熟的形态学标志。       

文昌鱼受精机理研究——受精卵的超显微结构

青岛文昌鱼受精后受精膜即刻明显举起,皮层颗粒以完整形式胞吐到卵周腔中,分散后大部分皮层颗粒物质与卵黄膜结合一起组成三层结构的受精膜,随着受精膜的举起,它由厚变薄。雄性原核以核膜破裂,染色质去浓缩,扩大重建原核膜形成雄性原核,雌性原核形成是由分离的具有双层膜的染色体联合,膨大而成,雄性原核形成早于雌性原核。雄性原核的迁移受微丝控制。文昌鱼卵黄颗粒中存在线行和环形结构,这种亚显微结构电子密度很高,经实验证明,它既不是微管又不是微丝。     

凡纳滨对虾卵母细胞卵黄发生的超微结构

利用电镜研究凡纳滨对虾卵母细胞卵黄发生的全过程。结果表明 :凡纳滨对虾卵黄的发生是双源性的。卵黄发生早、中期是内源性卵黄大量合成的阶段 ,卵黄发生中、后期则以外源性卵黄的合成为主。内源性卵黄主要由内质网、线粒体、核糖体、溶酶体、高尔基器等多种胞器活跃参与形成。其中数量众多的囊泡状粗面内质网是形成内源性卵黄粒的最主要的细胞器 ;部分线粒体参与卵黄粒的合成并自身最终演变为卵黄粒 ;丰富的游离核糖体合成了大量致密的蛋白质颗粒并在卵质中直接聚集融合成无膜的卵黄粒 ;溶酶体通过吞噬、消化内含物来形成卵黄粒和脂滴 ,且方式多样 ;高尔基器不直接参与形成卵黄粒。外源性卵黄主要通过卵质膜的微吞饮活动从卵周隙或卵泡细胞中摄取外源物质来形成     

Studies on the Mitotic Apparatus of the Sea Urchin by Means of Antigen-Antibody Reactions in Agar

The primary purpose of the experiments reported in this paper was to gain information on the molecular origin of the mitotic apparatus. Antisera were prepared against unfertilized sea urchin (Strongylocentrotus purpuratus) egg antigens and mitotic apparatus antigens. These were permitted to react with various antigen solutions in Ouchterlony agar gel diffusion plates, and the resultant precipitation patterns analysed. The results revealed that the mitotic apparatus contains probably no more than two antigens (precursor-1 component and precursor-2 component) and that these are shared by the unfertilized egg. Absorption and fractionation techniques indicated that in the unfertilized egg the precursor-1 component is present both as a "soluble" protein and as an insoluble form tenaciously associated with intracellular structural elements. A survey of dividing and non-dividing tissues for the precursor-1 component revealed that it was restricted to tissues in which mitotic activity could be detected microscopically. No immunochemical relationship could be detected between the mitotic apparatus and proteins extracted, by various methods, from the lantern muscle.     

Mitotic apparatus-surface interaction and cell division

Summary

Results of recent investigations concerning the mechanisms of animal cell division are reviewed. The mitotic apparatus was aspirated from a blastomere of a sand dollar (Echinarachnius parma) egg before second cleavage, and the time interval between removal and the appearance of the furrow in the control companion blastomere was measured. When the mitotic apparatus is removed 4 min or less before the furrows appear in the controls, furrows also develop in the operated cells. These results show that 4 min before furrowing begins, the surface changes which lead to formation of the division mechanism have become irreversible. When the mitotic apparatus of a cylindrical cell is shifted by pushing in one of the poles when the furrow appears, a new furrow develops in association with the new position of the mitotic apparatus. The same mitotic apparatus could elicit as many as 13 furrows over a 24.5 min period following the appearance of the first furrow. The results show that, in the proper geometrical circumstances, the mitotic apparatus and the surface can interact over a longer period than they do in normal cells.

By artificially constricting sand dollar eggs with a glass loop, the normal distance relations between the astral centers and the polar and equatorial surfaces can be reversed. Constricted cells cleave normally. The blocking effect of ethyl urethane can be reversed by moving the equatorial surface closer to the spindle portion of the mitotic apparatus. Relocation of other parts of the surface closer to the mitotic apparatus was ineffective. These results help elucidate the geometrical relations that are essential for furrow formation between the mitotic apparatus and the surface.

In cylindrical sand dollar eggs, single asters and the widely separated asters of a broken mitotic apparatus can cause furrow-like constrictions in the adjacent cylindrical surface. This reaction can be blocked by treating cells with ethyl urethane, which reduces astral size. The nature of the shape change that the aster causes depends upon the surface region affected. These results aid in understanding the nature of the change in surface physical activity caused by the mitotic apparatus.     

IMMUNOCHEMICAL STUDIES OF 22S PROTEIN FROM ISOLATED MITOTIC APPARATUS

Evidence is presented that the "22S protein" of mitotic apparatus isolated from sea urchin eggs is not microtubule protein. An antibody preparation active against 22S protein is described, and immunochemical studies of the distribution of 22S protein in various cellular fractions and among morphological features of mitotic apparatus are reported. The protein is ubiquitous in the metaphase egg fractions that were tested but is not found in sperm flagella. It is immunologically distinct from proposed microtubule protein isolated from mitotic apparatus by the method of Sakai, and from proposed microtubule protein obtained after extraction with mild acid. It exists in nontubule material of isolated mitotic apparatus but is not detectable in microtubules.     

细胞骨架与卵子减数分裂器的再启动和旋转关系的研究

本文采用体外授精(IVF)技术,通过小鼠受精卵早期发育过程中的形态变化,来研究受精过程中减数分裂器的重新启动与旋转。得到以下几个结果:①精子是在小鼠卵子的减数分裂器旋转之前进入卵子,随后精核发生去凝缩,从而使停滞的减数分裂器复苏启动。②雄性原核的形成过程,伴随着减数分裂器的旋转过程,雄性原核的形成要比雌性原核的为早。③在减数分裂器旋转过程中,微丝不仅参与,并且控制着旋转;解聚微丝,将阻止减数分裂器的旋转。④微管是减数分裂器的主要构成部分,它的稳定为减数分裂器形成提供了保证;解聚微管,将使减数分裂器解体。     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding to Sea Urchin Eggs and the Mitotic Apparatus

Colchicine forms a complex in vivo with a protein present in fertilized or unfertilized sea urchin eggs; similar binding was obtained in vitro with the soluble fraction from egg homogenates. Kinetic parameters and binding equilibrium constant were essentially the same in vivo and in vitro. The binding site protein was shown to have a sedimentation constant of 6S by zone centrifugation. The protein was present in extracts of the isolated mitotic apparatus at a concentration which was several times higher than in whole-egg homogenates. It was extracted from the mitotic apparatus at low ionic strength under conditions which lead to the disappearance of microtubules. No binding could be detected to the 27S protein, previously described by Kane, which is a major protein component of the isolated mitotic apparatus. The properties of the colchicine-bindinG protein, (binding constant, sedimentation constant, Sephadex elution volume) are similar to those obtained with the protein from mammalian cells, sea-urchin sperm tails, and brain tissue, and thus support the conclusion that the protein is a subunit of microtubules.     

THE MITOTIC APPARATUS ISOLATED IN GLYCEROL-CONTAINING MEDIUM

The mitotic apparatus of the sea urchin egg was isolated at 30°C in an isolation medium containing glycerol which is known to stabilize microtubules. After isolation in the 1 m glycerol-isolation medium, the mitotic apparatus was stabilized on addition of glycerol to a final concentration of 3 to 4 m. Without the addition, the chromosomes were disjoined from the spindle and the interzonal region between separating chromosomes was fragile resulting in separation of half spindles. Lowering the temperature of the isolation medium to 20°C or below, the isolation procedure allowed to isolate spindles. The isolated spindle behaved in a manner similar to the mitotic apparatus on the effect of glycerol concentration.
The glycerol-mitotic apparatus contained tubulin which was extractable with the isolation medium containing Ca ions or an organic mercurial. Tubulin was also extracted upon lowering the temperature to 0°C in the presence of GTP. Addition of KCl to a final concentration of 0.6 m immediately dispersed the mitotic apparatus. The extract revealed a colchicine binding of 0.001 mole per 105,000 × g of protein. The colchicine binding complex was found to have a molecular weight of 105,000. The DEAE Sephadex column chromatography of the KCl extract allowed to elute tubulin fraction which bound 0.1 mole colchicine per 105,000 × g of protein. The mitotic apparatus tubulin was shown to contain α and β subunits with mobilities quite identical with those of brain tubulin subunits. The molecular weights of the α and β subunits were 55,000 ± 1,000 and 51,000 ± 1,000, respectively.     

HURP is part of a Ran-dependent complex involved in spindle formation

BACKGROUND: GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. RESULTS: Here we identify a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. CONCLUSIONS: HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization.     

EMBRYO SAC DEVELOPMENT IN SOYBEAN: THE CENTRAL CELL AND ASPECTS OF FERTILIZATION

Development of the soybean central cell and events associated with fertilization are described. Central cell development involves the simultaneous formation of numerous multigrain amyloplasts, development of wall ingrowths, reduction of the cell's major vacuole, and nuclear migration. As these processes reach completion, a phase of lytic activity begins. This activity ultimately results in both the breakdown of most multigrain amyloplasts in the central cell and the reestablishment of the cell's large vacuole. While these processes occur, the egg and synergid cells continue development. Examination of the egg apparatus after plasmogamy but prior to karyogamy reveals several changes. Whereas the synergid containing the pollen tube is degenerate and the distribution of organelles in the persistent synergid has changed, the egg cell with a free sperm nucleus exhibits higher levels of metabolite storage and changes in its wall structure. The walls of the newly formed sporophyte are similar to those of egg apparatus cells (Folsom and Cass, Canadian Journal of Botany 68: 2135–2147 [1990]) in that they seem to lack a middle lamella. This layer becomes apparent only in the two-celled proembryo. While these changes occur in the egg, zygote, and proembryo, synergids undergo different degradative pathways.     

Microtubules in ascidian eggs during meiosis, fertilization, and mitosis

The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic crescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.