Fertilization Potential of the Medaka Egg

Fertilization of the medaka egg in 10% Ringer's solution generates a depolarization of 4 mV just before the appearance of a characteristically longer hyperpolarization (25). The depolarization appears to be the result of a nonspecific leak triggered by sperm stimulation and the amplitude of the depolarization is thought to be independent of [Ca²⁺]_o (25). We have investigated the ionic dependence of this depolarization. An initial small depolarization (3–4 mV; duration, 5–8 sec) is followed by a rising phase of a spike-like depolarization in the range of 10–60 mV. The amplitude of this spike-like depolarization is propodional to log [Ca²⁺], ranging from 0.33–18 mM. Calcium antagonists, e.g. 10 mM cobalt or 10 μg/ml verapamil in 10% Ringer do not block the depolarization in the presence of 1.1 mM CaCl₂.     

Effects of Microinjected Cations on the Early Event of Fertilization in the Medaka Egg

Effects of microinjected cations on the early events of fertilization were examined using eggs of Oryzias latipes . Microinjection of either Ca²⁺, Ba²⁺ or Sr²⁺ into the thin cortical cytoplasm induced breakdown of cortical alveoli (vesicles) (CABD) under Ca-Mg-free conditions, but microinjection of Mg²⁺, Mn²⁺ or Co²⁺ prevented CABD at the injected region when the eggs were inseminated in regular saline. Under Ca-Mg-free conditions, CABD could also be induced by microinjection of various solutions (NaCl, choline chloride, sucrose, pH buffer) without any divalent cations or ionophore A23187. Ca²⁺ microinjected into the cortical cytoplasm did not play a role in sperm penetration. Upon microinjection with either Ca²⁺, Mg²⁺ or K⁺, the resting membrane potential leakage was transiently observed. However, depolarization of the membrane followed by slow hyperpolarization was observed only upon microinjection of Ca²⁺. From these experiments, it was inferred that microinjected divalent cations such as Ca²⁺, Ba²⁺ or Sr²⁺ do not act directly upon the cortical alveolus membrane, but trigger the induction of CABD via depolarization of the membrne and increase in intracellular Ca²⁺.     

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.     

Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg-free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca²⁺. The effect of extracellular Ca²⁺ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca²⁺. Verapamil significantly reduced the action of extracellular Ca²⁺ or Ba²⁺ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No ⁴⁵Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca²⁺ from the external medium is not required.     

Effects of Forskolin on Fine Structures of Medaka Follicles

Effects of forskolin (FK), which stimulates production of 17α, 20β-dihydroxy-4-pregnen-3-one and estradiol-17β, on the fine structure of preovulatory follicles of Oryzias latipes were examined. Granulosa cells incubated in culture medium containing FK exhibited dislocation of the nucleus from the chorion side to the basement membrane side, vesiculation of conspicuous dilated endoplasmic reticulum (ER) with electron-dense material and Golgi lamellae, and development of large oval mitochondria with an electron-dense matrix. Moreover, a thin vesicular layer adherent to the outermost layer of the chorion was found in all immature oocytes at the end of incubation in the presence of FK. Intercellular junctions between granulosa cells and the oocyte gradually decreased during incubation in the presence of FK, and were finally lost with closure of the radial canals in the chorion at the end of the incubation. On the other hand, intrafollicular oocytes that were first incubated with FK for 10 hr, matured normally when they were incubated an additional 8 hr in plain medium. In granulosa cells of these follicles, the dilated ER and vacuolated Golgi lamellae were no longer detectable. These observations suggest that the development of dilated ER and vacuolated Golgi lamellae is characteristic of granulosa cells induced by FK.     

The Program of and Mechanisms of Fertilization in the Echinoderm Egg

Current research on the mechanisms of sperm-egg fusion, theblock to polyspermy, and metabolic activation are described.A cinemicrographic analysis of fertilization reveals that fusionof sperm and egg occurs between non-motile gametes, indicatingthat the flagellar motion of sperm is not required. The blockto polyspermy is reviewed, emphasizing recent work on the roleof cortical granule protease in altering sperm receptors ofthe vitelline layer. Metabolic activation or derepression at fertilization is highlyregulated and occurs in a definite sequence. The primary eventappears to be release of intracellular Ca²⁺. The timing of metabolicderepression is different in starfish oocytes. Here, a partof the derepression occurs during maturation and another partat fertilization.     

Ultrastructure and Ultracytochemistry of Fertilization Envelope Formation in the Carp Egg

This paper describes ultrastructural and ultracytochemical events occurring during the process of transformation of the vitelline envelope (VE) into the fertilization envelope (FE) in the egg of Cyprinus carpio. The VE is composed of four layers, except the micropylar region. The outermost (first) layer can be subdivided into a double layer (upper and lower halves) by cytochemical differences. The upper half is more protein-rich and positive for acid phosphatase (AcPase) activity, while the lower one is more carbohydrate-rich and negative for AcPase.
The most striking differences between the VE and the FE appear in the first layer component and the thickness. The FE first layer, ultrastructurally and cytochemically consisting of a single layer, gradually grows to be about five-fold as thick as the VE first layer by 40 min after fertilization. This may be due to displacement of the former VE first layer by deposits of the cortical alveolar exudate.     

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.     

Ultrastructure of the Mature Egg and Fertilization in the Fern Ceratopteris thalictroides

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thalictroides (L.) Brongn. were observed by transmission electron microscopy. The results revealed that the mature egg possesses an obvious egg membrane at the periphery of the egg. Furthermore, a fertilization pore was identified in the upper egg membrane of the mature egg. The structure of the pore is described for the first time. The fertilization experiment indicated that spermatozoids crowd into the cavity above the egg through the neck canal of the archegonium; however, only one of these can penetrate into the egg through the fertilization pore. Immediately on penetration of the spermatozoid, the egg begins to shrink. The volume of the fertilized egg decreases to almost one-half that of the unfertilized egg. As a result, the protoplasm of the fertilized egg becomes dense and opaque, which may lead to a situation where the organelles of both the egg and the fertilizing spermatozoid become indistinguishable. Simultaneously, abundant vesicles containing concentric membranes or opaque materials appear near the fertilization pore in the cytoplasm of the fertilized egg. These vesicles are considered to act as a barrier that prevents polyspermy. The present study provides a new insight into the ultrastructure of the mature egg and the cytological mechanism of fertilization in ferns.     

Ultrastructure of the Mature Egg and Fertilization in the Fern Ceratopteris thafictroides

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thalictroides (L.) Brongn. were observed by transmission electron microscopy. The results revealed that the mature egg possesses an obvious egg membrane at the periphery of the egg. Furthermore, a fertilization pore was identified in the upper egg membrane of the mature egg. The structure of the pore is described for the first time. The fertilization experiment indicated that spermatozoids crowd into the cavity above the egg through the neck canal of the archegonium; however, only one of these can penetrate into the egg through the fertilization pore. Immediately on penetration of the spermatozoid, the egg begins to shrink. The volume of the fertilized egg decreases to almost one-half that of the unfertilized egg. As a result, the protoplasm of the fertilized egg becomes dense and opaque, which may lead to a situation where the organelles of both the egg and the fertilizing spermatozoid become indistinguishable. Simultaneously, abundant vesicles containing concentric membranes or opaque materials appear near the fertilization pore in the cytoplasm of the fertilized egg. These vesicles are considered to act as a barrier that prevents polyspermy. The present study provides a new insight into the ultrastructure of the mature egg and the cytological mechanism of fertilization in ferns.     

Ultrastructure of the Mature Egg and Fertilization in the Fern Ceratopteris thalictroides

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thafictroides （L.） Brongn. were observed by transmission electron microscopy. The results revealed that the mature egg possesses an obvious egg membrane at the periphery of the egg. Furthermore, a fertilization pore was identified in the upper egg membrane of the mature egg. The structure of the pore is described for the first time. The fertilization experiment indicated that spermatozoids crowd into the cavity above the egg through the neck canal of the archegonium; however, only one of these can penetrate into the egg through the fertilization pore. Immediately on penetration of the spermatozoid, the egg begins to shrink. The volume of the fertilized egg decreases to almost one-half that of the unfertilized egg. As a result, the protoplasm of the fertilized egg becomes dense and opaque, which may lead to a situation where the organelles of both the egg and the fertilizing spermatozoid become indistinguishable. Simultaneously, abundant vesicles containing concentric membranes or opaque materials appear near the fertilization pore in the cytoplasm of the fertilized egg. These vesicles are considered to act as a barrier that prevents polyspermy. The present study provides a new insight into the ultrastructure of the mature egg and the cytological mechanism of fertilization in ferns.     

大葱卵器及受精后助细胞的超微结构

章丘大葱（Ａllium fistulosum L.cv.Zhangqiu)的卵器由１个卵细胞及２个助细胞组成,观察到不少卵器没有卵细胞,只有２个助细胞。卵细胞的核及大部分细胞质位于细胞的合点端,１个大液泡占据了细胞其他部位。卵细胞含有很多的核糖体及多聚核糖体、嵴明显的线粒体、粗面内质网、高尔基体具小泡,卵细胞似是一个活跃的细胞。细胞外被细胞壁,其合点端及侧方与助细胞共同壁不连续,助细胞有一较大的核,位于细胞膨大的部位,众多的小液泡遍布细胞中。核糖体及聚合核糖体、线粒体,粗面内质网及风心圆环状粗面内质丰富,高尔基体及小泡常见,反映了其活跃的代谢作用。助细胞合点端及侧方与卵细胞、中央细胞的共同壁不连续,与卵细胞共同壁含胞间连丝,壁不连续处,有不状多层膜结构伸入卵细胞质,显示助细胞可能对卵细胞提供营养,伟粉后,一个助细胞退化,宿存助细胞至随胚胚期尚存在,它经历了一个缓慢的退化过程,出现质壁分离,细胞质变稀,液泡扩大,细胞器逐渐减少,在椭形胚期,宿存助细胞核内的染色质及核仁消失,有细胞质侵入核内,因宿存助细胞壁变厚,细胞质出现现脂滴,宿存助细胞可能仍有合成功能,宿存助细胞壁出现若干无壁部位,细胞内的营养物质可能通过无壁部位向胚乳转运,供游离核胚乳及胚乳细胞化初期的发育。     

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 Penetration and in vitro Fertilization of the Egg of the House Cricket Acheta domesticus

Summary

A study of sperm penetration of the egg of the house cricket, Acheta domesticus, using a fluorescent microscope technique, showed that sperm penetration of the chorion, prior to fertilization, is not restricted to a specialised area of the egg surface, such as the micropyle. An acrosomal filament is seen on penetrating sperm. Polyspermy (multiple sperm attachment) is seen under normal conditions.

Eggs fertilized in vitro developed to the 4 day (pre-katatrepsis) stage, but did not undergo katatrepsis. Development was confirmed by cytogenetic studies. The percentage of eggs showing cleavage nuclei (i.e. initial development) was 59% after in vitro fertilization and 5% in ovarian eggs incubated in a hypotonic medium.     

怀头鲇胚胎发育过程卵膜形态结构变化及对孵化影响

采用扫描电镜和光学解剖镜,对黑龙江水域怀头鲇(Silirus soldatovi)成熟卵膜层次构造和受精卵胚胎发育过程中卵膜形态结构变化进行观察,并比较未脱黏和人工脱黏卵受精卵膜的表面超微结构变化.结果显示,受精卵膜的胶膜表面由一层薄而致密的物质组成,上有微孔构造.未脱黏受精卵膜表面胶膜光滑致密,多孔隙,内有小梁相连,随胚胎发育逐渐膨胀、展开、变薄,破膜期自然脱落.人工脱黏几乎全部脱去鱼卵的胶膜层,从而使卵失去黏性.脱去胶膜层的受精卵膜表面由不规则的颗粒状结构紧密嵌合而成,表面粗糙,胚胎发育过程中颗粒形状变化不大,但颗粒层逐渐变薄而且疏松,直至胚胎破膜而出:胚胎发育后期颗粒层有过早脱落和破洞出现.同时对活体鱼卵进行连续比较观察,讨论了卵膜结构及动态变化与孵化效果的关系.     

Microbial Community Response during the Iron Fertilization Experiment LOHAFEX

Iron fertilization experiments in high-nutrient, low-chlorophyll areas are known to induce phytoplankton blooms. However, little is known about the response of the microbial community upon iron fertilization. As part of the LOHAFEX experiment in the southern Atlantic Ocean, Bacteria and Archaea were monitored within and outside an induced bloom, dominated by Phaeocystis-like nanoplankton, during the 38 days of the experiment. The microbial production increased 1.6-fold (thymidine uptake) and 2.1-fold (leucine uptake), while total cell numbers increased only slightly over the course of the experiment. 454 tag pyrosequencing of partial 16S rRNA genes and catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) showed that the composition and abundance of the bacterial and archaeal community in the iron-fertilized water body were remarkably constant without development of typical bloom-related succession patterns. Members of groups usually found in phytoplankton blooms, such as Roseobacter and Gammaproteobacteria, showed no response or only a minor response to the bloom. However, sequence numbers and total cell numbers of the SAR11 and SAR86 clades increased slightly but significantly toward the end of the experiment. It seems that although microbial productivity was enhanced within the fertilized area, a succession-like response of the microbial community upon the algal bloom was averted by highly effective grazing. Only small-celled members like the SAR11 and SAR86 clades could possibly escape the grazing pressure, explaining a net increase of those clades in numbers.