The changes in structural organization of actin in the sea urchin egg cortex in response to hydrostatic pressure

We have used hydrostatic pressure to study the structural organization of actin in the sea urchin egg cortex and the role of cortical actin in early development. Pressurization of Arbacia punctulata eggs to 6,000 psi at the first cleavage division caused the regression of the cleavage furrow and the disappearance of actin filament bundles from the microvilli. Within 30 s to 1 min of decompression these bundles reformed and furrowing resumed. Pressurization of dividing eggs to 7,500 psi caused both the regression of the cleavage furrow and the complete loss of microvilli from the egg surface. Following release from this higher pressure, the eggs underwent extensive, uncoordinated surface contractions, but failed to cleave. The eggs gradually regained their spherical shape and cleaved directly into four cells at the second cleavage division. Microvilli reformed on the egg surface over a period of time corresponding to that required for the recovery of normal egg shape and stability. During the initial stages of their regrowth the microvilli contained a network of actin filaments that began to transform into bundles when the microvilli had reached approximately 2/3 of their final length. These results demonstrate that moderate levels of hydrostatic pressure cause the reversible disruption of cortical actin organization, and suggest that this network of actin stabilizes the egg surface and participates in the formation of the contractile ring during cytokinesis. The results also demonstrate that actin filament bundles are not required for the regrowth of microvilli after their removal by pressurization. Preliminary experiments demonstrate that F-actin is not depolymerized in vitro by pressures up to 10,000 psi and suggest that pressure may act indirectly in vivo, either by changing the intracellular ionic environment or by altering the interaction of actin binding proteins with actin.     

Different cytoskeletal organization in two maturation stages of Discoglossus pictus (Anura) oocytes: thickness and stability of actin microfilaments and tropomyosin immunolocalization

In Discoglossus pictus oocytes, the germinative area (GA) contains long and irregular microvilli where actin microfilaments are located. In the egg, the funnel-shaped dimple that originates by invagination of the GA is present. In the dimple both microvilli and microfilament bundles have a very orderly appearance. This report extends previous observations (Campanella and Gabbiani, Gamete Res 3:99-114, 1980) and shows that GA microfilaments are thinner (36 A average) than dimple microfilaments (60 A average), as measured in ultrathin section. Moreover, the interfilament distance is smaller in GA bundles than in the dimple bundles. To get an insight into actin organization in oocytes and eggs, we used an actin-depolymerizing factor (ADF) in which cryostat sections were incubated prior to immunofluorescent staining with antiactin antibodies. The microfilaments of the GA microvilli and partially of the oocyte cortex are resistant to ADF when compared to those in the dimple and the rest of the egg cortex. We also investigated immunocytochemically the presence of tropomyosin and found that this protein is localized in the dimple and in the cortex of oocytes and eggs but is absent in the GA.     

An evolutionary interpretation of the “motivation to oviposit”

The ovipositional behavior of parasitoids and other insects is often described by phrases such as “motivation to oviposit” or “ovipositional drive”. This paper shows how an evolutionary (i.e. functional) interpretation can be given to such phrases. A detailed model for the parasitisation of Sycamore aphids by M. pseudoplatani is developed, using experiments by Collins and Dixon (1986). Two models are developed: i) one in which egg complement is the only state variable and ii) one in which egg complement and information concerning host densities are state variables. Comparisons of the behaviour of simulated parasitoids, using the decisions associated with the models, and the experiments suggest that both egg complement and information are important for the parasitoid's decision making. Accepting previously parasitized hosts may be optimal, and not simply an error in parasitoid perception. A number of other detailed predictions are made, such as the relative fitness of first and second eggs in superparasitized hosts and the nature of the memory of the parasitoid.     

Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs

We report on the internal ultrastructure of long, finger-like microvilli which cover the surface of the fertilized sea urchin egg. Eggs were attached to polylysine-coated surfaces; their upper portions were sheared away with a stream of buffer which left behind only their plasma membranes and adjacent cytoplasmic structures. Scanning electron microscopy (EM) of such fragments revealed intact thin protoplasmic projections radiating away from the body of the cortex. By transmission EM of cortices similarly prepared on grids, small bundles of microfilaments appear as cores within the thin cytoplasmic projections. These microfilaments are shown to be composed of actin by their ability to interact with muscle heavy meromyosin (HMM). HMM-decorated microfilaments possess repeating arrowheads which uniformly point toward the cell interior. Actin bundles in the microvilli of sea urchin eggs may mediate microvillus support and elongation.     

Cytoskeletal and contractile proteins in coelomic oocytes,unfertilized and fertilized eggs of discoglossus pictus (Anura)

The distribution of actin, myosin, and tubulin has been investigated in coelomic oocytes, unfertilized and fertilized eggs of Discoglossus pictus utilizing: (1) immunofluorescence; (2) electron microscopy; (3) incubation with heavy meromyosin (HMM), and (4) SDS-polyacrylamide gel electrophoresis (PAGE). In coelomic oocytes, the germinative area (GA) has long, irregular microvilli containing microfilaments. In the rest of the oocyte, the microvilli are shallow. During the transit of the oocyte in the oviduct, a dimple forms by the invagination of the GA. A palisade of microfialment bundles is present in the finger-shaped microvilli of the dimple and extends for about 10 μm in the cytoplasm. In the rest of the egg, microvilli are absent and only random filaments appear in the cortex. Following HMM incubation, the dimple microfilaments are decorated with arrowheads pointing toward the bulk of the cytoplasm. SDS-PAGE of egg extracts shows bands co-migrating with actin (43K), pyruvate kinase (57K), and phosphorylase (94K). As result fertilization, the pattern of microfilament bundles in the dimple disappers in parallel with the dimple invergination itself. Generally, the entire oocyte cortex is positive to immunofluorescent staining with anti-actin, antimyosin, and antitubulin antibodies. However, the pattern of distribution and intensity of immunofluorescent staining changes for each antiserum, during different stages. It is concluded that a contractile system is present in Discoglossus eggs, and it is particularly developed in the dimple. The dimple is probably a major compartment for the storage of unpolymerized tubulin.     

Cortical reaction in inseminated eggs of the horseshoe crab,Limulus polyphemus L

The egg cortical reaction in Limulus polyphemus is described in four events. Approximately 10 min after insemination, small “pits” are visible in the cortex of the inseminated Limulus egg. These pits progressively enlarge, eventually coalesce, and finally disappear, leaving a smooth-appearing surface approximately 60–90 min after insemination. Based on these visible changes, the following four events comprise the cortical reaction: (I) uninseminated egg—smooth surface, (II) inseminated egg—appearance and growth of pits, (III) inseminated egg—coalescence of pits, and (IV) inseminated egg—appearance of smooth surface. Preparation of these events for SEM studies demonstrated morphological characteristics and sequential development of the pits. Also numerous microvilli are found on the surface of uninseminated and inseminated eggs during the cortical reaction. An increase in their diameter during the reaction is particularly unique.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

pH regulates the polymerization of actin in the sea urchin egg cortex

The state of actin in the isolated cortex of the unfertilized sea urchin egg can be controlled by experimentally manipulating the pH of the isolation medium. Cortices isolated at the pH of the unfertilized egg (6.5--6.7) do not contain filamentous actin, while those isolated at the pH of the fertilized egg (7.3--7.5) develop large numbers of microvilli which contain bundles of actin filaments. Cortices that are isolated at pH 6.5 and then transferred to isolation medium buffered at pH 7.5 also develop actin filaments. However, the filaments are not arranged in bundles and microvilli do not form. Although the cortical granules in cortices isolated at pH 6.5 discharge at a free Ca++ concentration of approximately 10 micrometer, actin polymerization is not induced by increasing the Ca++ concentration of the isolation medium. These results suggest that the increase in cytoplasmic pH which occurs following fertilization induces the polymerization of actin in the egg cortex.     

Microvillar elongation following parthenogenetic activation of sea urchin eggs

Parthenogenetic activation of unfertilized sea urchin eggs with ammonium chloride at pH 8.0 resulted in a slow, but dramatic, reorganization of surface microvilli in four species of sea urchin eggs. Following NH4Cl treatment, elongation of microvilli on the egg surface was observed concomitant with the formation of microfilament bundles within the microvillar cores. A minimum of 2 h of treatment was required for elongation and microfilament bundle formation to occur. The maintenance of elongated microvilli was pH-sensitive; removal of the activating agent resulted in the retraction of extended microvilli while readdition of NH4Cl caused microvilli to elongate again. Accompanying microvillar elongation in activated eggs, there was an increased calcium uptake as measured by 45Ca uptake. Blocking calcium uptake by incubation in lanthanum chloride or zero-calcium seawater containing 2 mM EGTA prevented microvillar elongation. These results suggested that elongation of microvilli following parthenogenetic activation by NH4Cl is pH- and calcium-dependent and is similar to that observed during normal fertilization.     

Ground crickets singing in volcanic warm “islets” in snowy winter: Their seasonal life cycles,photoperiodic responses and origin

The ground cricket Dianemobius nigrofasciatus overwinters as an egg in Japan, being univoltine in Hokkaido and northern Honshu and bivoltine farther south. In Hokkaido, however, this cricket is heard singing in winter in several fumarolic fields covered with moss and grasses locally known as “bokke”. In such warm “islets” the adult density was high in early summer and again in autumn, indicating that the cricket is bivoltine in contrast to the univoltine life cycle outside the bokke habitats in Hokkaido. Eggs laid by females collected at regular intervals from a bokke habitat showed a clear seasonal cycle of diapause incidence. At 26°C, the bokke strains produced non‐diapause eggs under long days and diapause eggs under short days as in the southern bivoltine populations, although the critical day‐length was longer than in the south. Several strains derived from non‐bokke habitats in Hokkaido and northern Honshu produced high percentages of diapause eggs under long days as well as short days as expected for the univoltine life cycle. Winter adults singing in bokke habitats could be either survivors of the autumn generation or individuals derived from eggs laid in autumn and then matured in response to the high soil temperature. In the laboratory, the proportion of egg diapause in short days was decreased by selection only for several generations. Phylogenetic trees of bokke and non‐bokke populations, based on both the nucleotide sequence of the mitochondrial COI gene and four allozyme loci, suggest that bokke populations have not been isolated from non‐bokke populations for an evolutionarily significant time.     

Actin, microvilli, and the fertilization cone of sea urchin eggs

Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges.     

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.     

Protein kinase C activity in mouse eggs regulates gamete membrane interaction

Gamete membrane interaction is critical to initiate the development of a new organism. The signaling pathways governing this event, however, are poorly understood. In this report, we provide the first evidence that protein kinase C activity in mouse eggs plays a crucial role in the regulation of this process. Stimulating PKC activity in mouse eggs by phorbol 12-myristate 13-acetate (PMA) drastically inhibited the egg's membrane ability to bind and fuse with sperm. Surprisingly, this significant reduction of gamete membrane interaction was also observed in eggs treated with the PKC inhibitors staurosporine and calphostin c. In further analysis, we found that while no change of egg actin cytoskeleton was detected after either PMA or calphostin c treatment, the structural morphology of egg surface microvilli was severely altered in the PMA-treated eggs, but not in the calphostin c-treated eggs. Moreover, sperm, which bound but did not fuse with the eggs treated with the anti-CD9 antibody KMC8, were liberated from the egg membrane after PMA, but not calphostin c, treatment. Taken together, these results suggest that egg PKC may be precisely balanced to regulate gamete membrane interaction in a biphasic mode, and this biphasic regulation is executed through two different mechanisms.     

Sneaker “jack” males outcompete dominant “hooknose” males under sperm competition in Chinook salmon (Oncorhynchus tshawytscha)

In a variety of taxa, males deploy alternative reproductive tactics to secure fertilizations. In many species, small “sneaker” males attempt to steal fertilizations while avoiding encounters with larger, more aggressive, dominant males. Sneaker males usually face a number of disadvantages, including reduced access to females and the higher likelihood that upon ejaculation, their sperm face competition from other males. Nevertheless, sneaker males represent an evolutionarily stable strategy under a wide range of conditions. Game theory suggests that sneaker males compensate for these disadvantages by investing disproportionately in spermatogenesis, by producing more sperm per unit body mass (the “fair raffle”) and/or by producing higher quality sperm (the “loaded raffle”). Here, we test these models by competing sperm from sneaker “jack” males against sperm from dominant “hooknose” males in Chinook salmon. Using two complementary approaches, we reject the fair raffle in favor of the loaded raffle and estimate that jack males were ~1.35 times as likely as hooknose males to fertilize eggs under controlled competitive conditions. Interestingly, the direction and magnitude of this skew in paternity shifted according to individual female egg donors, suggesting cryptic female choice could moderate the outcomes of sperm competition in this externally fertilizing species.     

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.     

Microvilli on sea urchin eggs: a second burst of elongation

A scanning EM study reveals about 300,000 microvilli on each egg of the sea urchin Strongylocentrotus droebachiensis. The microvilli are about 0.2 μm long before fertilization, elongate to about 0.5 μm soon after fertilization (the “first burst” of microvillus elongation), and subsequently elongate again about midway between fertilization and first cell division (the “second burst” of elongation). The second burst occurs during a discrete 30-min period and results in some microvilli being as long as 10 μm, although the average length is about 1.8 μm. The surface area of the egg following the second burst is about 2.7 times the area of the unfertilized egg.     

MEGAGAMETOPHYTE ORGANIZATION IN A POLYEMBRYONIC LINE OF LINUM USITATISSIMUM

A haploid-diploid twin-producing polyembryonic line “RA91” of Linum usitatissimum L., flax, known to produce twin embryos with a frequency in excess of 32% was employed to determine the cytological factors relating to the production of haploid embryos using light and transmission electron microscopy. Potentially polyembryonic flax megagametophytes contain at maturity either a conventional 3-celled egg apparatus (36% of megagametophytes), or contain supernumerary eggs forming either a 4- (50%) or 5-celled (14%) egg apparatus. In megagametophytes with supernumerary eggs, one egg cell appears to occupy the conventional position, whereas additional supernumerary eggs surmount the synergids and are smaller cells presumably derived from division of the true egg. Although supernumerary eggs appear similar in ultrastructure, they differ in location, size, and unlike typical egg cells, appear to have a complete, contiguous cell wall adjacent to the synergid. Such supernumerary cells are postulated as the origin of such haploid embryos, with the diploid member formed by conventional fertilization events with the micropylar egg.     

Ultrastructural evidence for the presence of actin filaments in mouse eggs at fertilization

Summary Mouse eggs at fertilization were permeated with glycerol solutions and then reacted with heavy meromyosin to show actin filaments by electron microscopy. The meiotic area of the egg surface is devoid of microvilli and is supported by a thick layer (0.6–0.8 m in width) of submembranous filaments. A much thinner layer (less than 0.3 m) is present in the remaining non-meiotic microvillous area and underlying its membrane is a very thick layer of cross-filaments and filament bundles.     

Ultrastructure of the Respiratory Epithelium in the Lungs of the Newt Triturus cristatus

The respiratory epithelium in the lungs of the newt Triturus cristatus has been studied by electron microscopy. The entire pulmonary gas-exchange area is covered by a continuous epithelium, the cells of which are all of the same type and are termed “pneumonocytes”. Typically each pneumonocyte is squamous and has attenuated sheets of cytoplasm which cover the pulmonary capillaries. Its free surface bears microvilli while mitochondria, multivesticular bodies and small inclusions are prominent in its cytoplasm. Many pneuomonocytes send cytoplasmic processes deep into the substance of the lung wall. It is postulated that these processes may help to anchor the epithelium.     

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.