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.     

Polarity of sperm entry in the ascidian egg

We have investigated the point of sperm entry in denuded eggs of the ascidian Phallusia mammillata. In contrast to what is generally believed, the sperm show a strong tendency to enter the animal hemisphere rather than the vegetal hemisphere. After entry, the sperm nucleus is carried toward the vegetal pole of the egg during the cortical contraction which occurs within a few minutes after fertilization. This polarity of sperm entry is abolished and the entry point is randomized by pretreating the eggs with cytochalasin D. We suggest that cytochalasin may act by randomizing components needed for sperm attachment or fusion, or structures needed for sperm entry.     

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.     

Scanning electron-microscopical and other observations of sperm fertilization reactions in Limulus polyphemus L. (Merostomata: Xiphosura).

Sperm fertilization reactions of Limulus polyphemus were examined by scanning electron and/or light microscopy. The following were considered: sperm motility, attachment of sperm to egg, acrosome reaction, and penetration of the acrosomal filament. The spermatozoa after semination are non-motile and become active only in close proximity to a defined region surrounding the egg. Egg materials diffusing into this region induce sperm motility and stimulate large numbers of spermatozoa to move towards the egg surface. Each sperm initially attaches by the apical tip and undergoes the acrosome reaction which causes a more permanent secondary attachment by the adhesion of acrosomal contents to the egg surface. The acrosome reaction also initiates the penetration of the acrosomal filament through the egg envelope, an event occurring in 70-80% of the attached spermatozoa (about 10(6). Shortly after this penetration, a secondary reaction occurs which involves a spiralling of the flagellum and an incorporation into the sperm body of the flagellar fibrous components, which then become closely apposed to the sperm nucleus. These sperm fertilization reactions were performed or initiated with 0-34 M CaCl2 in whole eggs, egg sections, excised egg envelopes and/or the outer basement lamina of the egg envelope. The Limulus fertilization system is very valuable since sperm reactions can be examined biochemically, which may lead to a better understanding of the chemical mechanisms involved in sperm-egg interactions in all animal species.     

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。     

Light and Electron Microscopic Studies on Fertilization of Pelmatohydra Robusta I. Sperm Entry to a Specialized Region of the Egg

Fertilization of a fresh water polyp, Pelmatohydra robusta , was studied by light and electron microscopy. A small depression was observed in the animal pole of the unfertilized egg. The egg pronucleus was always situated in close contact with the bottom of the depression. Microvilli which were covered with an egg coat consisting of filamentous components were observed on the egg surface. Microvilli and the egg coat were not detected on the surface of the depression. Sperm were associated with the egg plasma membrane and entered the egg only at the bottom of the depression. Excess sperm aggregated around the depression of inseminated eggs. After fertilization, the egg made a protrusion in the region where the egg pronucleus and sperm were in close contact with each other. A new egg coat was formed on the entire surface of the fertilized egg. The restriction of sperm-egg interactions to a specialized region of the hydra egg is discussed in connection with the micropyle of Pisces eggs and the animal dimple of Discoglossus (Anura) eggs.     

A Fine Structural Study of the Fertilization Process of the Jellyfish Cladonema uchidai

This study described the fertilization process of the jellyfish Cladonema uchidai by means of transmission electron microscopy. Female pronucleus was situated in close vicinity to the animal pole of the spawned egg, where the surface of the egg was flat or slightly depressed. Microvilli were observed except on the surface at the animal pole. The egg was entirely covered with a coat composed of fibrous materials. The spermatozoon was of the primitive type, and the proacrosomal vesicles were found immediately beneath the plasma membrane of the antero-lateral region of the sperm head. Within 15 sec after insemination, spermatozoa were incorporated in the egg cytoplasm only at the microvilli-free surface at the animal pole. Neither opening of the proacosomal vesicles nor formation of the acrosomal process was observed. No appreciable changes of cortical cytoplasm could be detected, although the egg became sticky after fertilization. Decondensation of the incorporated sperm nucleus occurred without breakdown of the original nuclear envelope. Within 10 min after insemination, the sperm nucleus still under the process of its decondensation fused with the female pronucleus. These findings were discussed in comparison with the fertilization process of higher metazoans as well as of other cnidarians.     

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.     

Analysis of fertilization and polyspermy in serotonin-spawned eggs of the zebra mussel,Dreissena polymorpha

Eggs isolated from animals spawned with 10⁻³ M serotonin were inseminated with sperm concentrations ranging from 10³–10⁶ sperm/ml. Multiple sperm attached to the surface of the egg and sperm incorporation occurred within 3 min postinsemination (PI). Sperm mitochondria, centrioles, and flagellum were also incorporated. Incorporation was essentially complete by 6 min PI. In the egg cortex, the sperm head rotated 180°, and a rapid translocation of the sperm through the cytoplasm towards the egg interior began by 5–6 min PI. In heavily polyspermic inseminations, translocations of the sperm were either minimal or nonexistent. In monospermic eggs, nuclear decondensation occurred after translocation was complete, beginning by 9–10 min PI. A male pronucleus began to develop in the cytoplasm by 21 min PI and enlarged to 20 μm before fusing with the female pronucleus. Oscillation of the egg cytoplasm and mitotic spindle apparatus was observed immediately prior to cleavage. Cleavage occurred at 60 min PI. Sperm incorporation and pronuclear formation were confirmed with fluorescent and confocal microscopy using the DNA-specific dyes Hoescht 33342 and 7-aminoactinomycin D. In sperm concentrations >10⁴ sperm/ml, 26–76% of the eggs exhibited polyspermy. The high incidence of polyspermy suggests that rapid, effective blocks to polyspermy were not present or were ineffective in a significant proportion of serotonin-spawned eggs. © 1996 Wiley-Liss, Inc.     

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。     

Time Sequence of Early Events in Fertilization in the Medaka Egg

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes . The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 10⁸/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.     

Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs

To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca²⁺ and exocytosis of cortical alveoli (CABD). The sperm‐penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although it decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at ≤40 min PI. Chromosome condensation in nonactivated eggs was not inhibited by actinomycin D, nor was the high activity of histone H1 kinase reduced. In the presence of cycloheximide or 6‐dimethylaminopurine (6‐DMAP), however, the compact sperm nucleus and the MII chromosomes transformed to interphase nuclei without CABD or extrusion of the polar body, although the activity of histone H1 kinase remained high. These results suggest that in the fish egg, transformation of MII chromosomes to an interphase nucleus may not be caused by loss of MPF activity, but rather than by the loss of activity of a short‐lived protein kinase(s), sensitive to 6‐DMAP that is independent of CABD in the cascade reactions triggered by increased cytoplasmic calcium. Dev. Genet. 25:137–145, 1999. © 1999 Wiley‐Liss, Inc.     

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca²⁺ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca²⁺ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca²⁺ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca²⁺ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca²⁺ into the cortex also induced a Ca²⁺ wave. When the egg was stimulated by microinjection of Ca²⁺ at the equatorial region, the Ca²⁺ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca²⁺ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca²⁺ wave preceded the wave of cortical change.
A Ca²⁺ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.     

Sperm entry induces polarity in fucoid zygotes

Fucoid zygotes establish a rhizoid-thallus growth axis in response to environmental signals; however, these extrinsic cues are not necessary for polarization, suggesting that zygotes may have inherent polarity. The hypothesis that sperm entry provides a default pathway for polarization of zygotes cultured in the absence of environmental signals was tested, and was supported by several lines of evidence. First, an F-actin patch, a cortical marker of the rhizoid pole, formed at the sperm entry site within minutes of fertilization. Second, the sperm entry site predicted the site of polar adhesive secretion (the first morphological manifestation of the rhizoid pole) and the position of rhizoid outgrowth. Third, when fertilization was restricted to one hemisphere of the egg, rhizoid outgrowth always occurred from that hemisphere. Fourth, delivery of sperm to one location within a population of eggs resulted in polarization of both adhesive secretion and rhizoid outgrowth toward the sperm source. Finally, induction of polyspermy using low sodium seawater increased the frequency of formation of two rhizoids. Sperm entry therefore provides an immediate default axis that can later be overridden by environmental cues.     

Sperm penetration and transformation of sperm entry site in eggs of the freshwater teleost Rhodeus ocellatus ocellatus

Eggs of bony fishes are enveloped by an egg envelope (chorion) in which a micropyle is present near the animal pole. Therefore, sperm penetration into the eggs is limited to the sperm entry site (SES), a region of plasma membrane just beneath the micropyle. In rose bitterling eggs, the SES transforms from a tuft of microvilli into a swollen mass (SM) that continues to plug the micropyle after sperm penetration. The present observations using the rose bitterling Rhodeus ocellatus ocellatus were conducted to examine: 1) whether or not sperm penetration is necessary for formation of the SM and 2) whether or not actin microfilaments are involved in the formation of the SM. Water activation without sperm transformed the SES from a tuft of microvilli into the SM, although it took a longer time for the transformation and the SMs were smaller than in the case of inseminated eggs. The SES presumably has the ability to transform into the SM upon activation of eggs in the present species. Cytochalasin B, which acts on actin microfilaments, did not prevent formation of the SM, irrespective of insemination or activation. The present observations suggest that sperm penetration is not necessary for SM formation and actin microfilaments do not participate in SM formation. © 1996 Wiley-Liss, Inc.     

Repeated inseminations required for natural fertility in a wild bird population

In most bird species, pairs copulate many times before egg laying. The exact function of repeated inseminations (i.e. successful copulations) is unknown, but several suggestions have been made. We tested the hypothesis that repeated inseminations are required to ensure fertilization of eggs, by using an experimental method where free-ranging male collared flycatchers (Ficedula albicollis) were prevented from inseminating their mates. We show that egg fertility was lower when females had not copulated during the studied part of their fertile period. By counting sperm on the inner perivitelline layer of eggs, we estimated that a minimum of 86 sperm must reach the site of fertilization to ensure average fertility. Using the timing of inseminations and the numbers of sperm on successive eggs, we show that repeated copulations are necessary to achieve an average rate of fertilization of a single clutch. Our results thus provide evidence that repeated inseminations function to ensure fertilization success. We discuss possible constraints on sperm production and utilization that may have contributed to this pattern.     

Temporal and spatial correlation of fertilization current, calcium waves and cytoplasmic contraction in eggs of Ciona intestinalis

Eggs of the ascidian Ciona intestinalis were loaded with the calcium indicator fura-2 via whole-cell clamp electrodes and changes in cytoplasmic calcium and cell currents were monitored during fertilization either in separate eggs or simultaneously in the same egg. The first indication of egg activation was the fertilization current; which reached peak values around 1 nA after 30 s. A wave of elevated calcium was detectable between 5 s and 30 s (mean = 21 s) after the start of the fertilization current. This wave spread across the egg increasing cytoplasmic calcium levels to at least 10 microM. When the fertilization current and calcium wave were complete and cytoplasmic calcium levels were decreasing to prefertilization levels, a cortical contraction wave spread across the egg surface. In eggs showing normal fertilization current, the calcium wave and the contraction wave were in the same direction. A region of elevated calcium persisted at the animal pole. Changing cytoplasmic calcium levels locally by local application of ionophore A23187 caused a contraction wave originating at the site of ionophore application. Increasing cytoplasmic calcium uniformly by facilitating calcium entry through voltage-regulated channels did not result in a contraction wave.     

Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization

Actin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM), or 5-iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10-20 sec. In the case of Clypeaster japonicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC-actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM- or IAF-actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC- or IAF-actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 microM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracellular free Ca2+ concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.     

Nucleoprotein changes during male pronuclear development as determined by the ammoniacal silver reaction

Sperm nucleoprotein changes during male pronuclear development in fertilized sea urchin (Arbacia punctulata) eggs have been examined utilizing the ammoniacal silver reaction (ASR) at the light and electronic microscopic levels of observation. Previous studies and control preparations indicated that the ASR has an affinity for basic proteins, staining intensely those rich in arginine residues. Differences in the affinity of the paternally derived chromatin to the ASR prior to, during, and following pronuclear development were observed. Relative to the female pronucleus the unincorporated sperm nucleus was densely stained. Upon its entry into the egg the sperm nucleus showed a two-fold increase in staining, indicating an augmentation in the availability of reactive sites already present in the paternally derived chromatin or an accumulation of “new” reactive sites from the egg cytoplasm. With the dispersion of the sperm nucleus there was a progressive decrease in staining intensity of the paternally derived chromatin. Subsequent to pronuclear fusion the paternally derived chromatin, recognized by its relatively dense staining, was seen at one pole of the zygote nucleus. With time there was a gradual regression in the size and staining intensity of the paternally derived chromatin within the zygote nucleus. Changes in reactivity of sperm-derived chromatin are discussed in reference to previous studies of chromatin transitions at fertilization.