鳙鱼受精早期扫描电镜研究

镛鱼(Aristichthys nobilis)受精是精子通过卵膜孔附着于卵质膜表面精子穿入部的微绒毛,两者迅即发生融合,但未见到有明显的受精锥。授精一分钟,精子整个头部已与卵的质膜发生融合,并看到有精子整个尾部已被微绒毛包裹的情况。在受精精子附近有一尚未与卵完全分开的第一极体。本文还讨论了精子穿入部的功能。     

华鲮受精早期的电镜观察

采用扫描和透射电镜观察了华鲮(Sinilabeo rendahli)成熟卵子、精子的形态特征和精子入卵的过程。结果显示:华鲮成熟卵子直径1.2 mm左右,仅有一个受精孔,卵膜表面有大量的不规则褶皱,精孔器表面平整;成熟精子全长约30μm,头部圆形无顶体;授精1 s精子大量附着于精孔器周围,仅有一个精子进入卵中,授精30~60 s受精孔内形成“受精栓”,精子开始溶解,卵膜逐渐隆起,褶皱消失。     

黄颡鱼受精早期精子入卵扫描电镜观察

用扫描电镜对黄颡鱼(Pseudobagrus fulvidraco)成熟精、卵及授精早期精子入卵过程进行了观察。成熟精子为鞭毛型形态,全长为11·2～12·4μm,头部直径1·1～1·3μm,鞭毛长10·0～11·3μm。成熟的黄颡鱼卵呈圆形,具单一受精孔,卵膜上以受精孔为中心分布有无数辐射状沟嵴。授精前,受精孔暴露在外面;授精2s时,受精孔被纤维状物质覆盖,之后大量精子很快黏附在覆盖物上;至授精10s,漏斗状受精孔又暴露出来。黄颡鱼在授精10s～1min内完成精子入卵过程,可观察到几乎所有样品的精孔区出现一圈环状隆起。大量精子处于隆起外侧,只有少数越过隆起到达受精孔前庭。授精1·5min,精孔区的隆起变成两圈,精子鞭毛解体。授精3min,可见迟到的精子被挡在外面。授精5min,精孔区的精子头部解体,受精孔几乎被分泌物覆盖,受精塞清晰可见。至授精20min,精子几乎全部解体。讨论了精子入卵的动力作用、精卵识别和单精受精机制。     

小鼠和豚鼠精子在附睾成熟过程中Ca~(2 )分布变化规律的研究

小鼠附睾头精子,其头部Ca~(2 )在顶体前区顶体外膜内侧结合最多,Ca~(2 )沉淀反应颗粒于该处呈连续层状。附睾头豚鼠精子其头部结合Ca~(2 )含量很少,且主要结合于顶体前区腹面顶体外膜内侧。小鼠附睾体和附睾尾精子Ca~(2 )的分布特征基本上和附睾头精子相同。但豚鼠附睾尾精子顶体外膜内侧无Ca~(2 )结合。和附睾头、附睾尾的附睾液相比,附睾体附睾液基质内具有大量Ca~(2 )存在。附睾体柱状上皮细胞的微绒毛切面上也具有Ca~(2 )沉淀反应颗粒,微绒毛可能与附睾液Ca~(2 )含量的调节有关。精子尾部Ca~(2 )主要分布于线粒体内,在质膜内、外两侧和线粒体外膜外侧也结合有少量的Ca~(2 )。和小鼠精子相比,豚鼠精子尾部线粒体内具有大量的Ca~(2 )。     

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。     

尼罗罗非鱼[Tilapia nilotica （L）]卵子结构扫描电镜观察

本文对尼罗罗非鱼(亦称尼罗丽鲷)Ⅳ期末至Ⅴ期初的成熟卵子进行光镜与扫描电镜观察,发现其卵子的细胞核相,正处于第二次成熟裂中期。卵子皮质部的皮层颗粒小,排列不规则。 卵子质膜外有厚薄不同的两层膜,合为壳膜(chorion)。在卵子动物极的壳膜中央有一个卵膜孔(Micropyle)。受精后,可见精子从此孔迸入。这是精子入卵的唯一通道。受精时,壳膜上附有许多精子,但是由于精孔管径与精子头部横径大小相近,首先进入卵子的仅有一个精子。     

利用胞浆内单精子注射技术研究水牛与猪的种间受精:阶段性报告

胞浆内单精子注射(intracytoplasmic sperm injection,ICSI)是一种研究种间受精机制的有效工具。本文旨在采用ICSI方法来构建水牛-猪种间显微授精卵,探讨了水牛-猪种间显微授精卵早期发育并检验了几种提高其发育潜力的方法。研究表明:(1)经ICSI方法制备的水牛-猪种间显微授精卵可以形成双原核,并能够发育到囊胚期;(2)PZM-3相对于SOF而言要更适于水牛-猪种间ICSI胚胎的培养;(3)用甲基化酶抑制剂5-azacytidine(5-Aza C)对水牛精子进行处理可以在一定程度上提高水牛-猪种间ICSI卵的原核形成率,但并不能提高其发育潜力。综上,本研究可为进一步探索水牛-猪种间受精机制及精卵互作奠定基础。     

"两步法"体外培养山羊卵母细胞的超微结构观察

有假说认为,卵母细胞在体外培养过程中,如果延长GV期,可促进卵母细胞进一步成熟,因而提高发育潜能。采用山羊半卵泡和卵母细胞共培养,抑制卵母细胞GVBD发生,从而延长GV期。比较了共培养前后和恢复成熟培养后卵母细胞的超微结构变化,其目的从亚细胞水平寻找卵母细胞进一步成熟的证据。研究发现,常规成熟培养:有卵周隙存在,但不贯通,局部区域卵膜与透明带结合紧密;部分皮质区尚有细胞器存在;微绒毛大部分从透明带中撤出,倒伏于质膜表面,数量较多,形态较为粗大;皮层颗粒质膜下部分单层分布,部分散布于皮质区;线粒体均匀散布于卵质中央区。共培养前:卵母细胞的卵周隙尚未形成,微绒毛没有从透明带中撤出;线粒体等细胞器分布于皮质区,皮层颗粒成簇状分布,皮质区富含细胞器。共培养后:局部形成卵周隙,微绒毛已自透明带中撤出,数量较多,垂直或倒伏于卵膜表面;线粒体以簇状分批开始内移,皮层颗粒已部分单层分布于质膜下,部分皮质区缺乏细胞器。恢复成熟培养后:卵周隙进一步扩大并且贯通,微绒毛数量减少并且绝大多数垂直于卵膜;线粒体在卵质中央区均匀分布,皮层颗粒卵膜下单层分布,大部分皮质区无细胞器存在。利用“两步法”培养得到的卵母细胞与体外常规成熟培养的卵母细胞相比,更有利于皮层颗粒的质膜下单层分布,卵母细胞卵周隙的形成与贯通,微绒毛数量减少和垂直于卵膜表面,无细胞器皮层区的进一步形成。因此,更有利于卵母细胞胞质的进一步成熟。     

唇精子早期入卵观察

用扫描电镜对唇成熟卵子及早期精子入卵过程进行观察.结果 显示,唇成熟卵子在动物极中央有一深凹陷的表面光滑的精孔器,其外径2.512 μm,内径2.330 μm,精子直径1.567 μm.混匀的精卵刚遇水时,没有精子进入精孔器.受精后1 s,精孔器内出现精子.受精后5 s,组织切片显示,精子已经进入卵子内,并形成具有强烈抑制多精入卵作用的受精锥.受精后10 s,精子在精孔器前庭集结,尚未形成受精塞.受精后20 s,在精孔器内形成受精塞.受精塞没有阻塞精孔管,经分析它不是来源于皮层反应产物.受精塞形成后,可以吸附入卵的精子,这对多精入卵有积极的抑制作用;精子尾部在入卵过程中相互缠绕,这也是减少多精入卵的重要机制.受精后30 s, 受精塞和吸附的精子向精孔器外移动.受精后50 s, 受精塞和吸附的精子堵塞精孔器.受精后60 s, 受精塞吸附的精子开始解体,但是由于精孔管未封闭,还有精子通过精孔管进入到质膜.在人工受精过程中,卵子的单精受精屏障会因其周围精子密度大、精子与卵子距离短、精子运动速度快而被打破,从而导致这些卵子出现多精入卵的现象.受精后80 s, 精孔管仍然没有封闭,精孔器附近的精子明显出现活动能力的差异:精孔器外面的精子活动能力最强,精孔管旁边的精子活动能力较弱;精孔管外堆积的精子活性消失,受精塞吸附的精子已开始解体,经初步分析,这可能是进入其内的精子耗能有所差异的结果.受精后100 s,受精塞吸附的精子解体.     

受精过程中的顶体反应

各类动物精子的顶体结构大体相似,位于核的前端,为一顶体膜包围的囊状结构,这就是顶体囊.在它和核之间有一顶体下腔,内有未聚合的肌动蛋白.当精子遇到卵膜时,顶体膜和其外的质膜发生融合,释放内含的顶体酶.与此同时,顶体下腔内的肌动蛋白发生聚合,形成顶体突起.由此突起附着于卵膜,借精子的运动和顶体酶的作用,使精子穿过卵膜而与卵的质腹相遇融合而受精.     

Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg

Morphological studies on the gametes and entry of the spermatozoan into the egg of the zebra danio, Brachydanio rerio, were conducted primarily with scanning electron microscopy. The spermatozoan showed a spherical head, which lacked an acrosome, a midpiece containing several mitochondria, and a flagellum. Observations of the unfertilized egg confirmed and extended prior studies showing a distinct cluster of microvilli on the plasma membrane, identified as the sperm entry site, beneath the inner micropylar aperture (Hart and Donovan, '83). The fertilizing spermatozoan attached to the sperm entry site within 5 seconds of the mixing of a gamete suspension. Binding to the egg microvilli appeared restricted to the equatorial surface of the spermatozoan. Fusion between the plasma membranes of the interacting gametes was followed by the formation of a distinct, nipple-shaped fertilization cone. The sperm head was partially incorporated into the fertilization cone cytoplasm by 60 seconds postinsemination. The incorporation of the entire sperm head, midpiece, and a portion of the flagellum occurred between 1 and 2 minutes. During this time, the fertilization cone shortened and was transformed into a massive, blister-like cytoplasmic swelling. Concurrently, upward movements of the ooplasm resulted in the gradual disappearance of the original depression in the egg surface containing the sperm entry site. The second polar body, fully developed by 10 minutes postinsemination, formed approximately 10-15 microns from the site of sperm penetration. Development of the fertilization cone, formation of the second polar body and exocytosis of cortical granules at the sperm entry site readily occurred in parthenogenetically activated eggs, indicating that these surface rearrangements do not require sperm binding and/or fusion.     

Sperm Incorporation Independent of Fertilization Cone Formation in the Danio Egg

The responses of the egg to insemination in a modified Fish Ringer's solution (FRS) were examined in eggs of the zebrafish ( Brachydanio rerio ) primarily by scanning electron microscopy. FRS is a physiological saline which temporarily inhibits parthenogenetic activation of the egg for 5–8 min. Spermatozoa were collected in a small volume of water and pipetted over eggs in FRS. Eggs inseminated in FRS typically incorporated the fertilizing sperm within 3–4 min. Inseminated cells showed an absence of a fertilization cone and no cortical granule exocytosis. The deep conical depression in the egg surface beneath the micropyle remained unaltered. Control eggs inseminated in tank water developed a large fertilization cone during sperm incorporation. Occasionally, eggs inseminated in water were observed to incorporate the entire sperm head prior to egg activation. Our results corroborate earlier findings showing that in the zebrafish, cortical granule exocytosis, fertilization cone formation and elevation of the sperm entry site are not triggered by the fertilizing sperm in experimental conditions (18, 19). Furthermore, sperm incorporation requires neither egg activation nor formation of a fertilization cone in this fish.     

Ultrastructural and experimental investigations of sperm-egg interactions in fertilization ofHydra carnea

Summary Fertilization in the freshwater hydrozoanHydra carnea has been examined by light, scanning and transmission electron microscopy. Sperm penetrate the jelly coat which covers the entire egg surface only at the site of the emission of the polar bodies. The egg surface exhibits a small depression, the so called fertilization pit at this site. Sperm-egg fusion takes place only at the bottom of the fertilization pit.Hydra sperm lack a structurally distinct acrosome and in most of the observed cases, fusion was initiated by contact between the membrane of the lateral part of the sperm head and the egg surfacce. Neither microvilli nor a fertilization cone are formed at the site of gamete fusion. The process of membrane fusion takes only a few seconds and within 1 to 2 min sperm head and midpiece are incorporated in the egg.Electron dense material is released by the egg upon insemination but cortical granule exocytosis does not occur and a fertilization envelope is not formed. The possible polyspermy-preventing mechanisms in hydrozoans are discussed. Hydra eggs can be cut into halves whereupon the egg membranes reseal at the cut edges and the fragments assume a spherical shape. Fragments containing the female pronucleus can be inseminated and exhibit normal cleavage and development. The observation that in such isolated parts the jelly coat will not fuse along the cut edges was used to determine its role in site-specific gamete fusion. These experiments indicate that site-specificity of gamete fusion can be attributed to special membrane properties at the fertilization pit.     

Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin B sensitivity: Scanning electron microscopy and time-lapse video microscopy during fertilization of the sea urchin Lytechinus variegatus

Sperm incorporation and the formation of the fertilization cone with its associated microvilli were investigated by scanning electron microscopy of eggs denuded of their vitelline layers with dithiothreitol or stripped of their elevating fertilization coats by physical methods. The activity of the elongating microvilli which appear to engulf the entering spermatozoon was recorded in living untreated eggs with time-lapse video microscopy. Following the acrosome reaction, the elongated acrosomal process connects the sperm head to the egg surface. About 15 microvilli adjacent to the attached sperm elongate at a rate of 2.6 μm/min and appear to engulf the sperm head, midpiece, and sperm tail. These elongate microvilli swell to form the fertilization cone (average height, 6.7 ± 2.0 μm) and are resorbed as the sperm tail enters the egg cytoplasm 10 min after insemination. Cytochalasin B, an inhibitor of microfilament motility, completely inhibits the observed egg plasma membrane surface activity in both control and denuded eggs. These results argue for a role of the microfilaments found in the egg cortex and microvilli as necessary for the engulfment of the sperm during incorporation and indicate that cytochalasin interferes with the fertilization process at this site.     

Effects of cytochalasins B and D on the fertilization of zebrafish (Brachydanio) eggs

The effects of selected concentrations of cytochalasins B (1-10 micrograms/ml; CB) and D (10, 50 micrograms/ml; CD) on the morphology and fertilization of zebra danio (Brachydanio) eggs were studied primarily with light and scanning electron microscopy. Eggs pretreated with either CB (10 micrograms/ml) or CD (10, 50 micrograms/ml) prepared in Fish Ringer's solution-0.5% DMSO showed a flattened shape, alterations in the form of surface microplicae and microvilli, and occasional spontaneous exocytosis of cortical granules. All eggs preincubated in either CB or CD were activated upon transfer to tap water, showing cortical granule exocytosis, elevation of the chorion, and formation of a fertilization cone. When eggs were pretreated for 5 minutes with 1-5 micrograms/ml CB or 10 micrograms/ml CD and inseminated, they incorporated the fertilizing sperm and typically developed to the two-cell stage. A single sperm cell attached to and fused with the sperm entry site microvilli but failed to enter the cytoplasm in eggs preincubated with 10 micrograms/ml CB. Eggs that were immersed continuously in either CB (10 micrograms/ml) or CD (50 micrograms/ml) 15 seconds after insemination also failed to incorporate the fertilizing sperm. Treatment of eggs after insemination with CD (10 micrograms/ml), however, did not prevent sperm cell incorporation or fertilization cone formation. Our drug data suggest the presence of actin-containing filaments in the danio egg before and following fertilization. These filaments appear to play a role in maintaining the shape of the egg cell and its surface specializations and in the incorporation of the fertilizing sperm. The fertilization cone appears to form independently of actin polymerization.     

革胡子鲶受精过程的扫描电镜观察

应用扫描电镜观察和描述了革胡子鲶成熟卵和精子的形态、卵壳膜的表面结构和形态、受精孔的位置和结构、精子入卵过程的程序和变化。讨论了精子入卵过程及精孔细胞在解体之后可能转变为一种能够吸引精子在精孔区聚集的“受精素”物质等问题。     

Early Events in Anuran Amphibian Fertilization: An Ultrastructural Study of Changes Occurring in the Course of Monospermic Fertilization and Artificial Activation

In unfertilized frog eggs, the plasma membrane displays an animal vegetal polarity characterized by the presence of short microvilli in the vegetal hemisphere and long microvilli or ridge-like protrusions in the animal hemisphere. The densities of microvilli are similar in the two hemispheres.
The fertilizing sperm always fuses with the animal hemisphere of the egg and induces a wave of exocytosis of cortical granules from its site of penetration. Similar spreading of the cortical reaction is seen on activation by pricking the egg cortex. The integration of the cortical granule membrane with the plasma membrane is rapidly followed by elongation of microvilli, which is progressively realized all over the egg surface from the site of sperm entry or the site of pricking. At this time, the length and shape of the microvilli in the animal and vegetal hemispheres are similar and their densities are the same as in unfertilized eggs.
A "smoothing" wave can be seen on the living egg, 40–60 seconds after pricking, starting around the site of pricking. This wave of microvillar elongation is accompanied by changes in intensity of diffracted light spots observed at the surface of the egg. This pattern might result from rapid and progressive thickening of the cortex that would drive pigment granules into the cytoplasm. The Brownian movement of these granules is thought to be responsible for the observed diffracted light spots.
Electrical stimulus or the ionophore A23187 induced activation reactions similar to those triggered by the sperm or by pricking, except that the cortical reaction began simultaneously in several distinct sites of the cortex.     

The sperm entry site during fertilization of the zebrafish egg: localization of actin.

The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.     

尼罗罗非鱼成熟卵结构及精子入卵早期的电镜观察

用扫描电镜观察尼罗罗非鱼(Tilopia nilotica)成熟卵卵膜孔结构和精子入卵的早期情况,用透射电镜观察成熟卵皮质,可见卵膜孔包括前庭和精孔管两部分,前庭壁及壳膜外表面上有许多小孔洞,精孔管壁呈阶梯状。卵膜孔下的卵皮质是一凹陷区,这一区域存在着皮质小泡。本实验见到5种形态的皮质小泡,其中大的皮质小泡靠近质膜。