Fertilization Cone of Carp Eggs as Revealed by Scanning Electron Microscopy

The process of formation of the fertilization cone in carp eggs was examined by scanning electron microscopy. The fertilized eggs responded to penetration of one sperm by primary and secondary steps of formation of a fertilization cone of unique morphology. In the primary step, the earliest fertilization cone was seen at the superior or anterosuperior part of a fused sperm head in inseminated eggs fixed 20 sec after immersion in fresh water. The cone reached a maximum of more than 10 μm in length and 3–4 μm in thickness by 40 sec, resulting in a transient plugging of the micropylar canal. In the secondary step, usually seen at 105–120 sec, a conformation reminiscent of a very small caldera volcano was formed, with the shortened earlier cone and part of the sperm tail at its top. By 2.5 min, the fertilization cone had become conical, and the sperm tail still extended from its top. At 3 min, the sperm tail was often not detectable, but a cytoplasmic eminence was still seen as a trace of the fertilization cone. The role of the earlier fertilization cone in blocking polyspermy is discussed.     

家养鱼类受精生物学的研究 Ⅱ.几种淡水鱼类成熟卵球的精孔器与精子入卵通路的光镜与扫描电镜观察

作者对我国四种淡水养殖鱼类——团头鲂、草鱼、白鲢和花鲢卵球的精孔器作了光学显微镜和扫描电子显微镜的比较描述,在扫描电子显微镜下观察到这几种鱼类的精子均直接经精孔器前庭穿过精孔管进入卵内,并对精孔细胞、受精孔与精子入卵的关系以及精孔的位置进行了讨论。       

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。     

Surface ultrastructure of paddlefish eggs before and after fertilization

The surface ultrastructure of eggs of the paddlefish Polyodon spathula was investigated by scanning electron microscopy. Mature eggs of paddlefish possess four to 12 micropyles in the animal polar region. There are sperm entry sites in the egg surface under the micropyles which consist of tufts of microvilli. Five to nine sperm entry sites were observed on mature eggs. Probably, the number of sperm entry sites corresponds to the number of micropyles. In a few eggs, 1 min after fertilization the ball-like enlarged top of a cytoplasmic process (probably a full-grown fertilization cone) had reached the external aperture or the canal of several micropyles. In other micropyles of the same egg, a few smaller cytoplasmic processes or flocculent material were found in the micropylar canal. With one exception, no sperm tails were found there. The formation of the full-grown cytoplasmic process is possibly initiated before the cortical reaction has started in an area of the animal hemisphere. Three, 10 and 20 min after fertilization, the uneven surface of the cortical cytoplasm in the animal polar region rose gently where microvilli were much less than the in other area and together with a secondary polar body at the latter stage. Taken together, paddlefish eggs may have sperm entry sites corresponding to the number of micropyles and respond to the stimulus of fertilization by forming a few cytoplasmic processes–fertilization cones (larger and smaller). Sperm penetration into the egg may be achieved at an earlier stage of fertilization (sperm-egg contact), as inferred from the fact that a secondary polar body was formed at the 20-min stage irrespective of the exceptional finding of the sperm tail.     

Karyotypic study of some species of family Mochokidae (Pisces, Siluriformes): evidence of female heterogamety

The relationship between the specific anatomy of tilapia gametes and their function was studied in the sequence of events which follow artifical fertilization. Scanning electron microscopic observations showed that some events of the fertilization process, involving spermatozoon migration through the micropylar canal until reaching the villous plasma membrane of the eggs and its penetration into the egg cytoplasm, occur very rapidly (fractions of a second). However, the spermatozoon tail remains outside for about 1–2 min. Then, following the zygote formation and the elevation of the chorion after its separation from the plasma membrane, numerous sperm cells could be found in the vicinity of the micropyle. This cell mass, which seemed to be trapped by a network of microfilaments, was suggested to be the result of the evacuation of excess sperm cells throughout the micropylar canal. The significance of these results for sperm and egg plasma membrane interaction and for prevention of polyspermy are discussed.     

The micropyle: a sperm guidance system in teleost fertilization

The micropylar region of the Rosy barb, Barbus conchonius, egg consists of 7-10 grooves and ridges, which drain directly into a funnel-shaped vestibule, the only point on the chorion through which sperm-egg contact is achieved during fertilization. Results of time-lapse video microscope study and computer-aided analysis of sperm motility pattern in the micropylar region showed that the fertilizing sperm, usually the first to enter the micropylar region, always travelled preferentially along the grooves into the micropylar pit. Subsequently, 86% of sperm arriving the micropylar region within 30 s travelled preferentially along the grooves into the immediate vicinity of the micropylar pit. The sperm guidance role of the micropylar region was calculated to enhance chances of egg penetration/fertilization by as much as 99.7% once sperm were within the micropylar region, possibly in response to some form of chemo-attractant(s) from the egg. Sperm agglutination post-fertilization was also found to occur preferentially along the grooves. Results of our in vitro fertilization experiments showed association between point of sperm entry and blastodisc formation: the blastodisc formed directly beneath the micropyle in all undisturbed eggs.     

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。     

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.     

泥鳅精子入卵的动力作用

在扫描电镜下,泥鳅成熟卵卵膜孔外围呈现完整的左涡旋状结构,受精时精子是顺着涡旋的流线进入卵膜孔。涡旋纹理接近对数螺线。本文分析了真骨鱼类的受精因素,除已知的化学因素外,还存在物理因素。也讨论了泥鳅成熟卵卵膜孔形态形成的必然性。     

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.     

Closure of the micropyle during embryonic development of some pelagic fish eggs

The fine structure of the egg envelope and micropyle was studied in unfertilized and developing eggs of the flounder Paralichthys olivaceus (Temminck & Schlegel), the Alaska pollack Theragra chalcogramma (Pallas), the Japanese tilefish Branchiostegus japonicus (Houttuyn) and the porgy Pagrus major (Temminck & Schlegel). The outer envelope surface of the unfertilized egg was wrinkled, while the inner surface was folded. The micropyle of the unfertilized egg consisted of a shallow vestibule and a distinct canal. The micropylar region of the inner surface of the envelope had a conical- or bowl-shaped protrusion. In developing eggs, the thickness of the envelope decreased and showed smooth outer and inner surfaces which indicated that it had been stretched tangentially at the time of the perivitelline space formation. The lumen of the micropylar canal was invariably occupied with envelope material. We postulate that the blockage of the micropylar canal is a result of the stretching of the envelope. The closure of the micropyle inhibits sperm and external pathogens from penetrating into the perivitelline space and seems to be involved in both the permanent prevention of polyspermy and the protection of the developing embryo from bacterial infection.     

Factors inducing closure of the micropylar canal in the chum salmon egg

The micropylar canal of the chum salmon egg was almost completely closed following egg activation caused by incubation in a hypotonic salt solution (HSS) for I h. The closure occurred in both inseminated and parthenogenetically activated eggs. Incubation of isolated envelopes from non-activated eggs in HSS or perivitelline fluid (PVF) did not induce any modification in micropylar structure, indicating that normal organization of the egg is essential for inducing closure. To reduce the volume of the perivitelline fluid, the eggs were activated in PVF or HSS containing 8 mM Dextran, Although the envelope showed hardening, closure of the micropyle was not observed in these eggs. The wall of the micropylar canal, however, possessed a slightly rough surface. Following activation in a Ca-free hypotonic salt solution with 10 mM EDTA, hardening of the egg envelope was completely inhibited. Although such eggs possessed an apparent perivitelline space, neither closure of the micropylar canal nor roughening of the canal surface were detected. We conclude that the synergistic action of perivitelline turgor pressure and perivitelline material is responsible for the closure of the micropyle.     

Response to Sperm Penetration of the Cortex of Eggs of the Fish, Plecoglossus altivelis

The response of the egg to sperm penetration was examined in eggs of the fish, Plecoglossus altivelis , by scanning electron microscopy. Eggs responded to sperm penetration by forming a fertilization cone at a "sperm entry site", which is a specialized structure in the egg surface under the micropyle. Within one minute, the fertilization cone showed dramatic morphological changes from its earliest appearance, through full two-storied growth to its marked recession. The sperm entry site in the egg surface is discussed as a morphologically specialized organ responsible for the entrance of a fertilizing spermatozoon. The morphological characteristics of the egg and sperm are also described.     

银鲫精子在两性融合生殖鱼类卵质中的受精特征

红鲤(♀)×银鲫(♂)的杂种胚胎均不能成活。受精细胞学研究结果表明,雌核发育银鲫的精核在两性融合生殖的鲤鱼卵质中能转化为雄性原核,并和雌性原核融合成合子核。但在卵裂开始后,可观察到某些异常现象,如染色体丢失,多极纺锤体形成,以及第二次卵裂时受精卵的两分裂球的非同步性分裂等。我们初步认为,雌核发育银鲫的染色体和两性融合生殖的鱼类染色体之间存在某种不相容性,表现为彼此间的互相排斥。出现这种现象可能和银鲫染色体组的雌核发育遗传背景有关。     

THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.     

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。     

Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization

Unfertilized eggs of the rose bitterling (Rhodeus ocellatus ocellatus) were squeezed out of females that had an elongated ovipositor and were dechorionated mechanically with fine forceps in physiological saline. The dechorionated eggs were transferred into fresh water then inseminated at once by spermatozoa of the same species. A large number of spermatozoa was found on the surface of eggs that had not yet had cortical reaction following insemination. The surface of the naked eggs responded by formation of many small cytoplasmic protrusions (viz., fertilization cones) at sperm attachment sites. The formed fertilization cones were rosettelike structures formed by the aggregation of some bleblike swellings devoid of microvilli and microplicae. About 10 min after insemination, the fertilization cones retracted, but marks of their presence characterized by less microvilli and microplicae remained in the eggs 15 min after insemination. Many spermatozoa penetrated into the cytoplasm of each naked egg. The sperm nuclear envelope disappeared by means of vesiculation resulting from fusion of the inner and outer membranes. The sperm nucleus decondensed and developed into a larger male pronucleus. Smooth-surfaced vesicles surrounded the decondensing sperm nucleus and formed the new male pronuclear envelope. Sperm mitochondria and flagella were found in the egg 15 min after insemination. The response of the egg surface to sperm entry and pronucleus formation are discussed.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

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.     

The hagfish oocyte at late stages of oogenesis: structural and metabolic events at the micropylar region

The hagfishes (Class Agnatha), primitive vertebrates and massive secretors of mucus, occupy a unique niche in a marine benthic environment and exists in abundance despite an infrequent ovulation of a low number (12-45) of notably large, prolate ellipsoidal (~8 mm x 28 mm. width x length, respectively) eggs. To establish factors that might contribute to survivability of one species (Eptatretus stouti), we examined three groups of mature females and document structural events characteristic of oocytes at late developmental stages: attached to the gonad, after ovulation into the body cavity, and after deposition on aquaria substrata. Quantitative autoradiographic analyses following administration in vivo of [(3)H]-leucine was useful to confirm post-vitellogenic stages and late metabolic events at the micropylar region located at one end of each egg. After egg disassembly, a combination of light microscopy and scanning electron microscopy was used to delineate structural features and 3-D relationships in juxtaposition to the micropylar cup and canal. A thick (~200mum) complex tripartite chorion surrounds a large micropylar cup (~300mum, width at base) consisting of fused polygonal (mainly hexagonal) substructural units (3-4 mum, width) and collectively serves to stabilize the micropylar canal (of size to prevent polyspermy) that penetrates the ooplasm near the germinal vesicle. A tuft composed of numerous chorionic appendages ('anchor filaments', ~2-3 mm length) is enmeshed in a gel matrix and surrounds the cup. When the eggs are deposited, intermediate filament aggregates (IFA) from slime gland mucus become interspersed in the tuft/gel complex. A similar IFA/tuft/gel region found on the vegetal pole-end facilitates firm egg-end attachments and possibly assists localization of egg clutches to a favorable substratum. Although the mode of fertilization is as yet unknown, the collective structural characteristies of the hagfish egg arc indicative of physical strength and are markedly different from eggs of the other group of cyclostomes (lampreys) and from tcleosts. The hagfish produces fewer but larger eggs which are prolate ellipsoidal rather than spherical and have a thicker chorion, a greater chorion to egg width ratio, a much larger micropylar cup with a unique substructure, larger and more localized chorionic appendages, and appendage tufts interspersed with adhesive substances derived not only from the follicular epithelium but also from the slime glands.