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 cortical contraction related to the ooplasmic segregation inCiona intestinalis eggs

Summary The egg cytoplasm of ascidian,Ciona intestinalis, segregates towards both the animal and vegetal poles within a few minutes of fertilization or parthenogetic activation with ionophore A23187. A constriction appears first on the egg surface near the animal pole and then moves to the vegetal pole. Carmine granules and spermatozoa attached to the egg surface move towards the vegetal pole with the movement of the constriction. Microvilli, which are distributed uniformly in unfertilized egg, disappear on the animal side of the constriction and became more dense on the vegetal side of the constriction. Transmission electron microscopy revealed that sub-cortical cytoplasm, containing numerous mitochondria and sub-cortical granules, moves towards the vegetal pole with the movement of the constriction and then concentrates into a cytoplasmic cap at the vegetal pole. An electron-dense layer appears in the cortex of the cap. The ooplasmic segregation and the cortical contraction were inhibited by cytochalasin B and induced by ionophore A23187. These observations suggest that ooplasmic segregation is caused by the cortical contraction which is characterised by a surface constriction and by the formation of an electron-dense layer.     

Animal-vegetal polarity in the plasma membrane of a molluscan egg: a quantitative freeze-fracture study

Using freeze-fracture electron microscopy, the numerical particle distribution in the fertilized Nassarius egg plasma membrane has been analyzed in four areas at different positions along the animal-vegetal axis of the egg. These areas can be distinguished by distinct microvilli patterns and differences in microvilli densities. In all areas, more IMPs (intramembrane particles) are present on the P face than on the corresponding E face. The ratio of the number of IMPs present on E and P face is similar in all areas (0.48-0.55) except for the most animal part of the vegetal hemisphere, where relatively more IMPs remain attached to the exterior half of the fractured membrane (E/P ratio = 0.88). The IMP density at the vegetal pole of the egg is considerably higher than in the animal hemisphere and in the animal part of the vegetal hemisphere. This difference is due to an increased number of IMPs in all size classes (4-18 nm). In the area adjacent to the vegetal pole the density of particles is also higher than in the two more animal areas, but here the difference is exclusively due to the smaller IMP size classes (4-8 nm). Statistical analysis of our data reveals that the area adjacent to the vegetal pole patch is significantly different from the other areas with respect to the distribution of the IMPs over the different IMP size classes. These results demonstrate the polar organization of the Nassarius egg plasma membrane. The possible role of this surface heterogeneity in the spatial organization of the egg cell and the later embryo is discussed.     

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca²⁺] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal–vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.     

The effect of local cortical microfilament disorganization on ooplasmic segregation in the loach (Misgurnus fossilis) egg

Injections of cytochalasin D (CD) or DNase I under the surface of fertilized loach egg result in local disorganization of microfilamentous cortex (MC) as revealed by transmission electron microscopy. This effect correlates with the loss of the cortex ability to contract in vitro. The disorganization of MC in the vegetal hemisphere of the egg does not affect the ooplasm segregation or blastodisk cleavage. Injection under the animal pole suppresses blastodisk formation and results in the autonomous separation of ooplasm in the central part of the egg. The experiments suggest that (1) autonomous separation of ooplasm from the yolk granules can proceed in the central part of the egg without the participation of MC; (2) normal segregation of ooplasm at the animal pole requires that the structures of microfilaments in the animal hemisphere (but not in the vegetal one) be preserved.     

The plasma-membrance IMP pattern as related to animal/vegetal polarity in the amphibian egg

Freeze-fracture electron microscopy of the plasma membrane of the fertilized, uncleaved Xenopus egg shows that intramembranous particles (IMPs) range in size from ca. 50 to 200 Å and that more IMPs are attached to the E-face than to the P-face. The overall IMP densities of the animal and the vegetal hemisphere do not differ significantly. IMP-free regions (?, ca. 0.1 μm) on the tips of surface protrusions were irregularly distributed in the animal and the vegetal half (E-face) occupying ca. 8.5 and 2%, respectively of the free area. The relative densities for 16 different IMP sizes have been compared, on the basis of seven animal and seven vegetal halves, counting (E-faces only) ca. 10,000 IMPs in each hemisphere. For IMP sizes of ≤81 Å, a significant difference (P < 0.0005) was found, more small IMPs being present in the animal half. Some evidence for IMP-associated thin elements was found. These findings are discussed in relation to plasma membrane anisotropy and the morphogenetic role of the egg cortex.     

Cortical activity in vertebrate eggs. I: The activation waves

We present a physical model for the propagation of chemical and mechanical waves on the surface of vertebrate eggs. As a first step we analyzed the propagation of the calcium wave observed to sweep over the surface of the Medaka egg (Gilkey et al., 1978). It has been assumed that this wave is driven by a mechanism of calcium-stimulated-calcium-release. By formulating this hypothesis mathematically we can use the observed wavefront data to obtain a map of cortical reactivity. This map indicates a gradient of reactivity along the egg: highest in the animal hemisphere and tapering off towards the vegetal hemisphere. The cortex of Xenopus eggs is also capable of propagating a calcium wave (Busa & Nuccitelli, 1985). At about the same time a wave of expansion followed by a wave of contraction sweeps across the egg surface (Takeichi et al., 1984). We have proposed a mechanism for this wave pair based on the physical chemistry of actomyosin gels. The calcium wave activates solation factors which sever some of the actin chains which leads to an osmotic swelling of the gel. Calcium also activates the contractile machinery of the actomyosin system which causes the gel to contract. The contraction lags the swelling because of the nature of the kinetics: solation and swelling is a more rapid process than contraction. By writing the equations for gel expansion and contraction we can mimic the mechanical and chemical wave propagation by a computer simulation. If the model is correct this provides a method for using the waves as a diagnostic of the mechanochemical properties of the egg cortex.     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.     

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.     

'METACHRONOUS' CLEAVAGE AND INITIATION OF GASTRULATION IN AMPHIBIAN EMBRYOS

The cleavage pattern in the egg of Xenopus laevis has been investigated with the aid of time-lapse cinematography. From the 5th cleavage onward, divisions of the surface blastomeres are not synchronous but metachronous. A few blastomeres in a very restricted region which is situated in most cases in the dorsal side of the animal hemisphere, slightly distant from the median line and near the equatorial junction of the animal and vegetal hemispheres, divide before the other blastomeres, and a wave-like propagation of the divisions travels along the surface from that region toward the animal and vegetal poles. The wave-like propagation ends in the vegetal pole region. In the animal hemisphere, this pattern of cleavage is continued until the 13th cleavage and thereafter the divisions of surface blastomeres become asynchronous. In the vegetal pole region, however, the 14th metachronous division of blastomeres is clearly observed in the film. Gastrulation begins after 14 cleavages.     

Differential Emergence of Cortical Granule Breakdown and Electrophysiological Responses during Meiotic Maturation of Toad Oocytes

Oocytes of the toad, Bufo bufo japonicus , at various stages of progesterone-induced maturation were stimulated by pricking or treatment with Ca-ionophore A23187. Upon pricking, oocytes 14 hr after hormone treatment (PHT) underwent sequential activation responses, such as development of an activation potential, cortical granule breakdown (CGBD), and formation of a perivitelline space (PVS), like those of mature oocytes (18 hr PHT). When oocytes were pricked at 14 hr PHT, it took about 10 min for the wave of CGBD to spread over all the oocyte surface, in contrast to the case with mature oocytes in which it took about 150 sec. The rate of spread of CGBD was significantly less in the vegetal hemisphere than in the animal hemisphere in both mature and immature oocytes. Treatment with A23187 (1 μM) for 5 min induced an activation potential, and PVS formation by the oocytes from 10 hr PHT, which was 3–4 hr earlier than the time when these responses could be induced by pricking. Oocytes at 8–9 hr PHT also showed CGBD in response to A23187, but without formation of an activation potential. Several patches of local PVS caused by the non-propagating CGBD were observed in oocytes treated with the ionophore 5–7 hr PHT. When a high concentration (10 μM) of A23187 was employed, CGBD without PVS formation was induced even in oocytes at 0 hr PHT. These results indicate that the responsiveness to a Ca²⁺ surge that is a prerequisite for both CGBD and genesis of an activation potential is acquired for the repective responses at different stages of oocyte maturation.     

Influence of the calcium ionophore A23187 on rat egg behavior and cortical F-actin

Rat eggs treated with the calcium ionophore A23187 and subjected to long-term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time-lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore-treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD-phallacidin, the distribution of F-actin in ionophore-activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5-h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD-phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore-induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation of this animal egg.     

Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

Fertilization alters the spatial distribution and the density of voltage-dependent sodium current in the egg of the ascidian Boltenia villosa

The spatial distribution of voltage-dependent ionic currents was characterized in Boltenia villosa eggs before and after fertilization using two-microelectrode voltage clamp of paired animal-vegetal halves of eggs (merogones) made surgically. Major voltage-dependent conductances in the Boltenia egg are a transient inward Na current, a transient inward Ca current, and an inwardly rectifying K current. These currents were randomly distributed along the animal-vegetal axis in the unfertilized egg. When paired merogones (surgically prepared egg fragments) were made at the vegetal cap stage, 15-30 min after fertilization, Ca and K currents remained randomly distributed along the animal-vegetal axis. In contrast, the relative Na current density was found to be twofold lower in the vegetal vs the animal merogones made at the vegetal cap stage. By making pairs of merogones from unfertilized eggs and subsequently fertilizing one merogone of a pair, we showed that this change in current density ratio was due to a loss of absolute Na current density in the vegetal hemisphere shortly after fertilization. These results also show that this loss was intrinsic to the vegetal hemisphere, rather than being determined solely by the point of sperm entry. A second decrease in Na current was observed during the hour before first cleavage, 60-120 min after fertilization (M.L. Block and W.J. Moody, 1987, J. Physiol. 393, 619-634), both in fertilized eggs and in animal merogones fertilized after isolation. This second loss of Na current was not observed in vegetal merogones fertilized after isolation or in either animal or vegetal merogones made from fertilized eggs at the vegetal cap stage. Possible mechanisms for te rapid (complete by 40 min after fertilization) and the late (occurring from ca. 60 to 120 minutes after fertilization) Na current losses are discussed.     

The wave of activation current in the Xenopus egg

A ring-shaped wave of inward current, the activation current, propagates across the Xenopus egg from the site of activation during the positive phase of the activation or fertilization potential. This activation current wave is due to an increased chloride conductance and reflects the propagated of the ionic channels responsible for the fertilization potential. These channels are present in the animal and vegetal hemispheres; however, the magnitude of the activation current is 6-7 times greater in the animal hemisphere. Outward current of a smaller magnitude and spread out over a larger area precedes and follows the inward current except at the point of activation where the current is first inward. The inward current wave is detected in all eggs activated by sperm and in eggs activated by pricking with a sharp needle, by application of the Ca2+ ionophore, A23187, and by intracellular iontophoresis of Ca2+ or inositol 1,4,5-trisphosphate. Reduction of the inward current by TMB-8, which blocks intracellular calcium release in some cells, suggests that the activation current channels are calcium sensitive and that the current wave is concomitant with a wave of increased intracellular calcium initiated by sperm-egg interaction. The wave of cortical granule exocytosis and two or more contraction waves follow the current wave.     

Independence of two microtubule systems in fertilized frog eggs: the sperm aster and the vegetal parallel array

Two microtubule-containing structures are implicated in dorsoventral polarization of the frog egg, and we examined the relationship between them. The sperm aster provides a directional cue for a cortical rotation specifying polarity, and a vegetal cortical array of parallel microtubules is likely part of the rotational machinery. The growing aster has an accumulation of microtubules marking the path of the sperm pronucleus, and its microtubules extend into the egg cortex as well as the cytoplasm. To test whether the vegetal parallel array was an extension of astral cortical growth, fertilized or activated eggs were bisected into animal and vegetal fragments. The vegetal fragments formed parallel arrays, even when isolated within a few minutes of egg activation. Neither the sperm centrosome nor another microtubule organizing center in the animal half of the egg is required for formation of the parallel array, but some animal half activity is involved in its disappearance. Correspondence to: R.P. Elinson     

The Periodic Changes in Microvilli Density in Activated Xenopus Eggs That Correspond to the Cleavage Cycle

In order to obtain the cytological basis for the periodic flattening and rounding-up of activated amphibian eggs, the surface ultrastructure and the cortical microfilament organization were studied in Xenopus laevis . Scanning electron microscopy (SEM) of the egg surface revealed that the density of microvilli at the animal pole region decreased significantly when the periodic flattening started, but increased again concomitantly with the commencement of the rounding-up. Isolated pieces of the cortices stained with rhodamine-phalloidin exhibited the periodic disorganization and reorganization of a meshwork with bright dots probably corresponding to microvilli, in good synchrony with the decrease and increase of the microvilli density. Study of appropriate batches of eggs in which the moving front of surface contraction waves (SCWs; 1) can be localized revealed that the decrease and increase of the microvilli density correspond to SCW-1 and -2, respectively. SEM and the cytochemical examination of the eggs from which the germinal vesicle (GV) had been removed revealed that none of these changes occurred in the enucleated eggs. These observations suggest that the GV-dependent regulation of the microfilament organization in an egg cortex constitutes the cytological basis for the SCWs and for the periodic flattening and rounding-up of denuded eggs.     

Ooplasmic segregation in theTubifex egg: Mode of pole plasm accumulation and possible involvement of microfilaments

Summary Ooplasmic segregation, i.e. the accumulation of pole plasm in theTubifex egg, consists of two steps: (1) Cytoplasm devoid of yolk granules and lipid droplets migrates toward the egg periphery and forms a continuous subcortical layer around the whole egg; (2) the subcortical cytoplasm moves along the surface toward the animal pole in the animal hemisphere and toward the vegetal pole in the vegetal hemisphere, and finally accumulates at both poles of the egg to form the animal and vegetal pole plasms. Whereas the subcortical layer increases in volume during the first step, it decreases during the second step. This is ascribed to the compact rearrangement in the subcortical layer of membraneous organelles such as endoplasmic reticulum and mitochondria. The number of membraneous organelles associated with the cortical layer increases during the second step. Electron microscopy reveals the presence of microfilaments not only in the cortical layer but also in the subcortical layer. Subcortical microfilaments link membraneous organelles to form networks; some are associated with bundles of cortical microfilaments. The thickness of the cortical layer differs regionally. The pattern of this difference does not change during the second step. On the other hand, the subcortical cytoplasm moves ahead of the stationary cortical layer. The accumulation of pole plasm is blocked by cytochalasin B but not by colchicine. The first step of this process is less sensitive to cytochalasin B than the second step, suggesting that these two steps are controlled by differnt mechanisms. The mechanical aspects of ooplasmic segregation in theTubifex egg are discussed in the light of the present observations.     

The ultrastructural organization of the isolated cortex in eggs ofNassarius reticulatus (Mollusca)

Summary We have studied the organization of the cortex in fertilized eggs ofNassarius reticulatus by examining rotary-shadowed whole mounts of isolated cortices in the transmission electron microscope. The following components were distinguished: (a) the plasma membrane, with clathrin-coated areas and coated pits, (b) microfilaments and microtubules, and (c) a tubulovesicular network of endoplasmic reticulum. Microfilaments were identified by labeling with heavy meromyosin, and microtubules with a monoclonal anti-tubulin antibody, using both immunofluorescence microscopy and immunogold labeling for transmission electron microscopy. The microfilaments are organized in a network parallel to and closely associated with the plasma membrane, with typical Y- and X-shaped intersections. The endoplasmic reticulum is associated with this microfilamentous lattice. The microtubules also run parallel to the plasma membrane, but they are located at a greater distance, as can be inferred from stereo images. In the uncleaved egg, numerous microtubules are present in the egg cortex. Shortly before polar lobe formation, at the onset of mitosis, the microtubules disappear almost entirely. They reappear again at the end of first cleavage, as the polar lobe is being resorbed. The synthesis of cortical microtubules at this stage appears to depend on the presence of microtubule-organizing centers in the animal hemisphere of the egg, since microtubules do not reappear in isolated polar lobes. Clathrin-coated areas are present in both the animal and vegetal hemisphere before polar lobe formation. During mitosis, the clathrin-coated plaques and pits are found almost exclusively in the animal hemisphere. After resorption of the polar lobe, at the two-cell stage, no clathrin-coated areas were found at all.