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。     

中华鲟授精过程扫描电镜观察

在扫描电镜下观察到:中华鲟的成熟卵具有一层放射膜,两层卵黄膜;卵的动物极端有9—15个受精孔,受精孔直径约为12.7—13.9μm;精子由伞状顶体、棍棒状头部、漏斗状中段及扁平细长的尾部组成;各受精孔都能接纳多个精子;卵受精5秒后,精子才开始入卵,且精子入卵速度各不相同。受精时间持续较长,可由5秒延长至5分钟;卵受梢后,受精管道内除缠绕成团的精子尾部外,未发现其他堵塞物。     

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。     

金鱼精子入卵过程的扫描电镜观察

本文采用扫描电镜观察了金鱼(Carassius auratus)卵壳膜(chorion)表面结构和精子入卵过程。在壳膜的卵膜孔(micropyle)区有5—10条沟和嵴。位于精孔管下面,卵的质膜为一束较长的微绒毛组成的精子穿入部(sperm entry site)。授精5s,精子头的顶部已附着于精子穿入部,随即两者的质膜发生融合,而围于精子头部四周的微绒毛迅速伸长形成一受精锥,它不断将精子头部包裹。授精110s,精子的头部和颈部已完全进入卵内,受精锥本身也渐趋消失,但精子尾部仍平躺于卵的表面。皮层小泡是在授精30s后才开始破裂并释放其内含物,导致卵子表面呈蜂窝状,并在无膜内表面附着了大量球状物。     

三疣梭子蟹精卵相互作用的扫描电镜观察

应用扫描电镜技术观察了三疣梭子蟹的精卵相互作用。未受精成熟卵表面较光滑、无受精孔,但有许多微孔。成熟卵外被卵膜,内为卵母细胞。在卵自然产出后,精子迅速发生顶体反应使顶体囊外翻并压入卵膜,而核仍留于卵膜外,核辐射臂不收缩且仍附着于卵膜上。三疣梭子蟹为多精着卵和多精入卵膜。精子外翻顶体囊压入卵膜后,核辐射臂陆续回缩直至消失。作用于顶体丝上的卵母细胞主动拖精作用对入卵膜精子的进一步入卵、受精至关重要,环状卵膜突起的向心伸展也有一定的协助作用。探讨了着卵精子的顶体反应、精子入卵膜的机制及卵子在精子入卵过程中的作用     

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。     

大银鱼卵膜孔结构的电镜观察

扫描电镜下，大银鱼成熟卵卵膜孔区域呈现奇异的放射沟脊状结构。在授精开始的３０秒内，测得卵膜附近约８６％的精子沿着凹沟进入精孔管；还拍摄到了通过卵孔中心的纵切面映象，通过电子计算机模拟，验证了卵膜孔的这种结构，为精子的云集和进入精孔管所提供有利条件，从而，于泥鳅之后又发现了一种真骨鱼，其精子入卵除化学因素外，还存在着不容忽视的机械动力因素。     

中华鲟受精细胞学研究

中华鲟(Acipenser sinensis Gray)的成熟卵具有一层放射膜及二层卵黄膜。在动物极有9—15个受精孔。每一受精孔有一大的入口(12.7—13.9μm直径)及一细长的受精管道(1.2—1.3μm直径)。 进入受精孔的许多精子只能按序入卵。其中只有一个精子的头部膨大核化,最后形成雄性原核。同时活跃的卵子也形成雌性原核。雌、雄原核彼此接触,最后融合成合子核,随后分成两个子核。 中华鲟的受精方式为多精入卵,单精受精。     

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。     

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。     

日本鳗鲡精子形成过程中的形态结构特点

本文通过扫描电镜、透射电镜观察了日本鳗鲡（Anguilla japonica）精子形成的特殊过程及产生细胞器的特殊结构。由精细胞变成精子包括四个特殊阶段,即经过早期、中期、晚期和精子期．最后形成正常成熟的精子．（1）早期阶段：其特征是细胞核由椭圆形逐步变成长条形;在细胞核的一端．有一个大的圆的染色较浅的形状似球形的特殊结构,约占细胞核的三分之一,其内含有少量着色深的颗粒状和线条状物质,外面由质膜包被着与细胞核分开,该结构和细胞核的外层还有一层质膜包着形成一个整体：精子早期阶段没有形成独立的线粒体和中心粒。（2）中期阶段：其特征是细胞核呈长条形．有球形结构的一端成为细胞核的上端,无球形结构的一端成为细胞核的下端,在下端出现鞭毛的原基;球形结构伴随着精子的发生也发生变化,内部逐步分化出中心粒和线粒体等细胞器：在细胞核的中段有明显的溶酶体分布。（3）晚期阶段：其特征是细胞核呈“眉形”或“新月形”．中心粒从球形结构中释放出来形成独立结构．球形结构中只剩下还没有形成独立结构的线粒体：在细胞核的下端鞭毛的原基处长出较长的鞭毛,这时期的精子已具有运动能力。（4）精子期：其特征是细胞核呈圆形,中心粒位于植入窝内,线粒体分布在细胞核的下面．在线粒体的下面有袖套腔形成,此时形成的鞭毛为“9＋2”结构。日本鳗鲡精子经过四个时期的变态．才能形成真正成熟的精子。     

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.     

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.     

MAGNESIUM ION-REQUIRING STEP IN FERTILIZATION OF SEA URCHINS*

The magnesium ion-requiring step in fertilization of sea urchins was investigated. When eggs were inseminated in Mg-free sea water, several spermatozoa were found to bind to each egg surface with their reacted acrosomes without elevation of fertilization membrane. The number of binding jelly-treated spermatozoa to an egg did not differ regardless of the presence or virtual absence of magnesium ions. Although fertilization did not occur in Ca, Mg-deficient sea water (CM-deficient SW) even when jelly-treated spermatozoa were employed, some eggs could be fertilized by the addition of magnesium to the CM-deficient SW 60 sec after insemination, when jelly-treated spermatozoa had completely lost their fertilizing capacity in the CM-deficient SW. The acrosomal process of jelly-treated spermatozoa appeared to penetrate the vitelline layer in the CM-deficient SW. DTT- or pancreatin-treated eggs could not be fertilized in the virtual absence of magnesium. Re-fertilization using the fertilized eggs deprived of fertilization membrane did not occur under conditions of magnesium deficiency. These results suggest that external magnesium ions are indispensable at least for the fertilization process following penetration of the vitelline layer by the spermatozoa, such as fusion of the plasma membrane between an egg and a reacted spermatozoon, or the subsequent step(s) such as sperm penetration into egg interior and egg activation which precedes the cortical reaction.     

Rat eggs normally exhibit a variety of surface phenomena during fertilization

The surface topography of the rat egg was examined during fertilization in vitro and in vivo. Using phase optics, 348 in vitro fertilized and 50 in vivo fertilized eggs were continuously monitored throughout the 7-hour period of sperm incorporation. A myriad of different surface configurations were seen, with each egg exhibiting one or more of the following changes. A small number of eggs (4–6%) formed surface elevations over the sperm head after its detachment from the flagellum, 15–30 min after sperm-egg fusion; 1 to 1.5 hr after fusion, 40–50% of the eggs produced the so-called incorporation cone, a prominent surface elevation over the decondensing sperm nucleus. The vast majority of eggs (74–82%) formed surface elevations over the proximal tip of the flagellum 2–3 hr after sperm-egg fusion. These had no association with the decondensing sperm nucleus. A few eggs (11–12%) exhibited multiple protrusions that were distributed randomly about the egg surface, whereas 14–20% did not manifest any surface elevations and remained spherical throughout the sperm incorporation period. Regardless of the type of surface change, all of the eggs resumed a spherical shape by the time sperm incorporation was complete. These observations are in contrast to the conclusions by previous authors that formation of the so-called incorporation cone over the decondensing sperm nucleus is a ubiquitous event.     

SPERM PENETRATION AND THE FORMATION OF A FERTILIZATION CONE IN THE COMMON CARP EGG*

Sperm penetration and the formation of a fertilization cone in the micropylar canal of the egg of the common carp were examined by electron microscopy. The overwhelming majority of inseminated eggs fixed without immersion in fresh water showed that the first spermatozoon had penetrated into the ooplasm before the cortical reaction had occurred, and in many cases had formed a fertilization cone to plug the micropylar canal. At this stage the sperm head was usually located at the base of the cone, and the tail part did not participate in the formation of the cone. Inseminated eggs fixed soon after immersion in fresh water showed that the elevation of the fertilization membrane and the simultaneous recession of the fertilization cone often permitted the penetration of a few supernumerary spermatozoa into the perivitelline space near the micropylar canal, but polyspermic fertilization was never observed. The mechanism of the block to polyspermy in the egg of the common carp is discussed in connection with the fertilization cone.     

ELECTRON MICROSCOPE STUDIES OF EARLY STAGES OF SPERM PENETRATION IN HYDROIDES HEXAGONUS (ANNELIDA) AND SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA)

1. The early events of sperm entry in Saccoglossus and Hydroides are described and examined in relation to present knowledge of the acrosome reaction and of egg membrane lysins. In Saccoglossus and several other species these events occur in two phases. First. The acrosome filament of the spermatozoön spans the egg membrane barriers, reaches the reactive egg protoplasm, and causes the egg to begin its fertilization reaction. Second. The filament and its connected sperm head move through the egg membrane barriers and enter the egg proper. The first phase is completed in a matter of seconds but the second phase usually requires several minutes. 2. The peripheral areas of the eggs of the two species differ as seen in sections. In Hydroides, but not in Saccoglossus, the vitelline membrane is bounded by a distinct outer border layer of small concentrically differentiated bodies and penetrated by microvilli from the egg. 3. The acrosome filament, seen in the living condition as a delicate thread in Hydroides and as an exceedingly tenuous thread in Saccoglossus, appears to be tubular in both species when seen in electron micrographs of thin sections. 4. The acrosomal region of Hydroides appears to consist of two components—a peripheral one, which may collapse during the acrosome reaction, and a central one related to the acrosome filament. 5. Deliberately induced polyspermic material was used to increase the probability of finding examples of sperm penetration in thin sections. 6. As seen in sections, areas of low electron density, interpreted as spaces or pits from which the material of the membrane is absent, surround the attached or penetrating spermatozoa. (a) In Hydroides the spaces vary greatly in many characteristics including shape, position in the membrane, and size with relation to the enclosed sperm head. In one specimen a portion of the membrane is missing from border to border; no spermatozoön is seen but immediately beneath the space is the apex of a fertilization cone. (b) In every case in which a determination could be made, the spermatozoön in the membrane has undergone its acrosome reaction. (c) In Saccoglossus some pits are found with which several spermatozoa are associated. Generally, where the spermatozoa are more numerous the pit is larger. (d) Pits similar to those seen in Saccoglossus sections are observed in living eggs. They remain in Membrane I after sperm entry. (e) From the above and other considerations it is suggested that the pits and spaces are formed by local action of a lysin or lysins emanating from the individual spermatozoön at the site of sperm entry. 7. It is considered that the suggested lysin would participate in sperm entry by eroding the membrane barrier in the vicinity of the sperm head, thus permitting the sperm head to pass through the membrane. Since the acrosome filament much earlier stimulates the egg's initial fertilization response, this lysin would facilitate the second phase of the early events of sperm entry.     

Penetration of the zona-free mouse egg by capacitated epididymal sperm: Cinemicrographic observations

Conditions were established for routine cinemicrographic examination of sperm incorporation by living zona-free mouse eggs employing oil immersion objectives and Nomarski optics. Initial sperm attachment to the egg plasma membrane, which was reversible and appeared to require flagellar activity, involved localized areas of the head corresponding approximately to the position of the equatorial segment. Penetrating sperm lay flat on the egg and, during incorporation, appeared to sink into the egg cytoplasm, accompanied by short bursts of flagellar activity and subsequent rotation of the flagellum around its insertion point. Ensuing sperm head decondensation involved dissociation of individual particulate structures and a dramatic localized clearing in the egg cytoplasm. The normalcy of the penetration process and the potential applicability of this approach was attested to by the observations that polar body extrusion, male and female pronuclear formation, and migration through the egg cytoplasm in preparation for syngamy occurred in several sequences followed for extended time periods.     

雌核发育和两性融合发育鱼卵调控精核受精发育的生化特性研究

两性融合生殖的鱼卵受精后,精核能疏松、解凝,形成雄性原核：雌核发育银鲫卵子受精后,精核发育受到抑制,无法形成原核。采用显微注射去膜精核以及细胞学和电镜观察的方法,本文对两类鱼卵受精后精核早期发育的生化性质进行了初步探讨,并着重研究了雌核发育银鲫卵子控制精核发育的生化特征。实验结果显示,两性融合生殖鱼类卵质中,一定量的Ｃａ２＋的存在,二硫键的还原作用对于精核的发育显然是必要的;而在雌核发育银鲫卵中,Ｃａ２＋的功能和二硫键的还原作用与精核发育受到抑制之间并无直接联系。银鲫卵质中似乎显示出异常的磷酸酶脂解活性,导致磷酸化过程无法进行,使精核解凝受到阻碍。另外,两性融合生殖的鱼卵重质层中具有大量诱导精核原核化的有关因子,而银鲫卵质中则缺少该因子（或活性极低）。银鲫卵质中还可能缺乏某些与雄性原核的核膜重组装有关的大分子物质。     

Flagellar gyration and midpiece rotation during extension of the acrosomal process of Thyone sperm: how and why this occurs

The midpiece of Thyone sperm contains a large mitochondrion and a centriolar pair. Associated with one of the pair, i.e., the basal body of the flagellum, are satellite structures which apparently anchor the flagellar axoneme to the mitochondrion and to the plasma membrane covering the midpiece. Immediately before and as the acrosomal process elongates, the flagellum and the midpiece begin to rotate at 1-2 rotations per second even though the head of the sperm, by being firmly attached on its lateral surfaces to the coverslip, does not rotate at all. This rotation is not observed in the absence of flagellar beating whose frequency is much greater than that of its gyration. To understand how the midpiece rotates relative to the sperm head, it is first necessary to realize that in Thyone the flagellar axoneme projects at an acute angle to the principal axis of the sperm and is bent towards one side of this axis. Thus movement of the flagellum induces the sperm to tumble or yaw in solution. If the head is stuck, the midpiece will rotate because all that connects the sperm head to the midpiece is the plasma membrane, a liquid-like layer. A finger-like projection extends from the proximal centriole into an indentation in the basal end of the nucleus. In contrast to the asymmetry of the flagellum, this indentation is situated exactly on the principal axis of the sperm and, along with the finger-like projection, acts as a biological bearing to maintain the orderly rotation of the midpiece. The biological purpose of flagellar gyration during fertilization is discussed.