孤雌活化诱导恢复的小鼠减数分裂期间细胞骨架的动态与作用

减数分裂的顺利完成是胞质分裂和核分裂在时间和空间上的协调结果,细胞骨架系统在减数分裂的一系列事件中具有重要的调节作用.实验通过孤雌活化诱导小鼠MⅡ期卵减数分裂恢复,采用激光共聚焦显微术检测了减数分裂期间的微管、微丝和核的动态变化,并通过细胞骨架药物处理,以分析微管和微丝在减数分裂事件中的不同作用.结果显示：纺锤体微管为核的定位、分离和运动所必需;纺锤体从与质膜平行旋转至与质膜垂直是极体排放的前提;微丝是控制纺锤体旋转的关键因素;纺锤体旋转完成后微丝随即解聚,不参与极体的最后排出,形成原核后再重新组装.     

草鱼人工雌核发育的细胞学观察

运用组织学方法研究了用紫外线辐射处理后的鲤鱼精子诱导草鱼卵子雌核发育的细胞学过程。结果表明精核在卵子内主要有２种变种（１）始终保持固缩状态,不形成雄性原核;（２）形成雄性原核。冷休克处理可破坏卵子第二次成熟分裂的纺锤体。冷休克处理后,室温孵化期卵子内变化有２种（１）卵子内重新形成了纺锤体和纺锤丝,其中部分卵子可排出第二极体;（２）卵子内纺锤体或纺锤丝不恢复。     

唇鱼骨受精的细胞学研究

唇精孔器属深凹陷、短孔径型。精子在受精后2s到达精孔管、5s进入卵子。受精后8—15min,卵子进入第二次减数分裂后期。受精后10min,开始形成雄性原核。受精后20min,进入第二次减数分裂末期。受精后25min,雌性原核形成。受精后30—35min,雌性原核向雄性原核移动。受精后40min,雌雄原核接近。受精后50min,雌雄原核结合。受精后70min,受精卵进入第一次有丝分裂中期,受精后80min,进入第一次有丝分裂后期,受精后120min,进入末期。卵黄降解与其内部或外周小泡的泡状缺口紧密相关。雌雄原核结合是精子星光扩张、牵引和细胞质流动的共同结果。有多精入卵的现象。     

微丝在卵子极性产生、分裂沟形成和极体排放中的作用

小鼠生发泡（ＧＶ）期卵母细胞在１μｇ／ｍｌ细胞松弛素Ｂ（ＣＢ）中培养,部分微丝解聚,卵母细胞不 能产生极性而在细胞中部形成分裂沟（假分裂）;极泡期印在１μｇ／ｍｌ ＣＢ中,分裂沟继续收缩,排放第 一极体。假分裂的分裂沟和极泡期的极区分裂沟形成均与分裂器中体位置相关。部分微丝解聚并不影响 假分裂和第一极体排放;全部微丝解聚（１０μｇ／ｍｌ ＣＢ）将中断假分裂和胞质分裂,分裂沟消失,卵恢 复球形。由此可见,成熟过程中卵母细胞极性的产生及分裂沟的形成都依赖于微丝的聚合。胞质分裂和 第一极体排放同样需一定量的微丝存在。     

中国对虾受精过程中精卵核的细胞学变化

中国对虾精子以其棘部顶端随机附着在卵上,精子在凝胶膜形成后,第一极体排出前入卵,精子入卵后,絮状的精核经过重建形成雄原核,中国对虾卵子排放时处于第一次成熟分裂的中期,卵子入海水时,纺锤体的长轴与质膜平行,卵子激活后,纺锤体的长轴开始旋转,旋转至纺鲑体长轴与质膜垂直时,由纺锤丝牵引着染色体向两极移动,外侧的染色体由质膜包裹形成第一极体,受膜举起后,由次级卵母细胞排放出第二极体,此后,单倍雌核重建形成雌原核,雄原核形成早于雌原核,雌雄原核于卵子中央联会形成联合核,受精后的50分钟纺锤丝牵关染色体称向两极,质膜内缢断裂形成两个细胞的胚胎。     

微丝在卵子极性产生,分裂沟形成和极体排放中的作用

小鼠生发泡期卵母细胞在１μｇ／ｍｌ细胞松弛素Ｂ中培养，部分微丝解聚，卵母细胞不能产生极性而在细胞中部形成分裂沟（假分裂）；极泡期卵在１μｇ／ｍｌ ＣＢ 中，分裂沟继续收缩，排和放第一极体，假分裂的分裂沟和极区分裂沟形成均与分裂器中体位置相关，部分微丝解聚并不影响假分裂和第一极体的排放，全部微丝解释（１０μｇ／ｍｌ ＣＢ）将中断假分裂和胞质分裂，分裂沟消失，卵恢复球形，由此可见，成熟过程中卵母细胞极     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

肌动蛋白解聚因子在动物生殖和胚胎发育中的作用

肌动蛋白解聚因子家族Cofilin/ADF(AC蛋白家族)属于肌动蛋白结合蛋白,是微丝骨架的一个重要调节者。AC蛋白家族能够截断微丝,促进肌动蛋白单体的解离和循环以及微丝解聚,调控微丝骨架的重建,进而影响与微丝骨架相关的一些生理功能如细胞增殖、迁移、凋亡及胚胎发育等。本文将着重介绍AC蛋白家族在动物生殖诸如精子发生、卵巢发育、卵子发生、卵裂,以及胚胎发育等过程中的调节与功能。     

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。     

THE MECHANISM OF SPERM HEAD TRANSFORMATION DURING MOUSE OOCYTE FERTILIZATION IN VITRO

用Ｈｏｅｃｈｓｔ３３３４２标记及氨银反应的方法在光镜和电镜水平上研究不同发育阶段的小鼠卵母细胞转化精核的能力。生发泡期卵母细胞质不能诱发精子组蛋白替代鱼精蛋白及精核解聚。生发泡破裂后,卵母细胞获得使精核解聚的能力,但直到卵母细胞完成成熟前,雄原核均不能形成。进一步研究表明,卵母细胞成熟过程中持续的蛋白质合成是雄原核形成所必需的,鱼精蛋白质磷酸化是精核解聚中的关键步骤。     

大熊猫与金黄地鼠体外异种受精的研究

在大熊猫精子与地鼠卵的体外异种受精中,发现大熊猫精子穿入地鼠卵后可以激活受精卵产生极区,释放第二极体,受精卵内雌性原核形成。与此同时,地鼠卵的胞质也能促使大熊猫精子头发育成雄性原核,异种精卵间的相互作用与同种受精的相似。 细胞松弛素B能阻抑大熊猫雄性原核从地鼠卵皮层迁移到卵的中央,实验表明大熊猫雄性原核的迁移也受异种卵的微丝的控制。     

Independence of two microtubule systems in fertilized frog eggs: the sperm aster and the vegetal parallel array

Two microtubule-containing structures are implicated in dorsoventral polarization of the frog egg, and we examined the relationship between them. The sperm aster provides a directional cue for a cortical rotation specifying polarity, and a vegetal cortical array of parallel microtubules is likely part of the rotational machinery. The growing aster has an accumulation of microtubules marking the path of the sperm pronucleus, and its microtubules extend into the egg cortex as well as the cytoplasm. To test whether the vegetal parallel array was an extension of astral cortical growth, fertilized or activated eggs were bisected into animal and vegetal fragments. The vegetal fragments formed parallel arrays, even when isolated within a few minutes of egg activation. Neither the sperm centrosome nor another microtubule organizing center in the animal half of the egg is required for formation of the parallel array, but some animal half activity is involved in its disappearance. Correspondence to: R.P. Elinson     

The Drosophila maternal gene sésame is required for sperm chromatin remodeling at fertilization

The spermatozoon features an extremely condensed and inactive nucleus. The unique sperm chromatin organization is acquired during the late stages of spermatid differentiation by the replacement of somatic histones with sperm-specific chromosomal proteins. At fertilization, the inactive sperm nucleus must be rapidly transformed into a DNA replication competent male pronucleus before the formation of the zygote. The sequential events of this crucial process are well conserved among animals and are controlled by molecules present in the egg. We have previously identified a Drosophila maternal effect mutation called sésame, which specifically arrests male pronucleus formation at a late stage of chromatin decondensation. In this study, we show that sésame affects maternal histone incorporation in the male pronucleus, a situation that is expected to prevent nucleosomal organization of the paternal chromatin. As an apparent consequence, the male pronucleus is arrested before the first S-phase and does not condense mitotic chromosomes. However, centromeric heterochromatin is present on paternal centromeres, which occasionally interact with microtubules. The abnormal chromatin organization of the male pronucleus does not prevent the formation of a male pronuclear envelope, which breaks down and reassembles in synchrony with maternally derived nuclei present in the same cytoplasm.     

FERTILIZATION PROCESS IN THE HEART-URCHIN,CLYPEASTER JAPONICUS OBSERVED WITH A DIFFERENTIAL INTERFERENCE MICROSCOPE*

The observations of the fertilization process in the heart-urchin, Clypeaster japonicus with a differential interference microscope indicate that the sperm pronucleus is carried to the center of the egg by the growth of the sperm aster as stated by Chambers (5), and that the egg pronucleus is carried to the center of the aster by a filamentous structure formed between them. The curved path of egg pronucleus in the fertilized egg is interpreted as the combination of the movement of the center of the aster and the movement of the egg pronucleus toward the center of the aster. The movement and the rotation of the sperm head result from pushing by the tail being engulfed in the egg.     

Cargo Transport by Cytoplasmic Dynein Can Center Embryonic Centrosomes

To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell’s center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.     

Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs.

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.     

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.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

PREMATURE CHROMOSOME CONDENSATION OF THE KARYOGAMY-BLOCKED SPERM PRONUCLEUS IN THE FERTILIZATION OF FUCUS DISTICHUS (FUCALES,PHAEOPHYCEAE)1

Mitosis of egg and sperm pronuclei of Fucus distichus subsp. evanescens (C. Agardh)Powell was examined by fluorescence and electron microscopy when migration of the sperm pronucleus and, as a result, karyogamy were blocked by colchicine treatment after plasmogamy. Chromosome condensation was obsewed in both pronuclei Microspectrophotometric studies after staining the nuclei with mithramycin A clearly showed that DNA synthesis ocurred in the egg pronucleus but not in the sperm pronucleus. This means that chromosomes condensed prematurely in the sperm pronucleus (premature chromosome condensation). In some cases, the egg chromosomes became arranged on a metaphase plate, whereas the sperm chromosomes lay scattered near the egg pronucleus. Immuno fluorescence microscopy using anti-β-tubulin antibody confirmed that a normal spindle was formed at the egg pronucleus. A pair of centrioles existed at the two poles of this spindle. The sperm nuclear membrane disappeared, and microtubules radiated to the sperm chromosomes from one pole of the egg spindle.