Formation and structure of egg envelopes in Russian sturgeon Acipenser gueldenstaedtii (Acipenseriformes: Acipenseridae)

The covering of the eggs in Russian sturgeon Acipenser gueldenstaedtii consists of three envelopes (the vitelline envelope, chorion and extrachorion) and is equipped with multiple micropyles. The most proximal to the oocyte is the vitelline envelope that consists of four layers of filamentous and trabecular material. The structural components of this envelope are synthesized by the oocyte (primary envelope). The chorion encloses the vitelline envelope. The extrachorion covers the external surface of the egg. Examination of the arrangement of layers that comprise the egg envelopes together with the ultrastructure of follicular cells revealed that the chorion and extrachorion are secondary envelopes. They are secreted by follicular cells and are built of homogeneous material. During formation of egg envelopes, the follicular cells gradually diversify into three morphologically different populations: 1) cells covering the animal oocyte region (cuboid), (2) main body cells (cylindrical) and (3) micropylar cells. The apical surfaces of follicular cells from the first two populations form processes that remain connected with the oocyte plasma membrane by means of gap junctions. Micropylar cells are located at the animal region of the oocyte. Their apical parts bear projections that form a barrier to the deposition of materials for egg envelopes, resulting in the formation of the micropylar canal.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

A unique adhesion apparatus on the eggs of the catfishClarias gariepinus (Teleostei,Clariidae)

Eggs of the catfishClarias gariepinus were investigated for the first time using light and electron microscopy (SEM and TEM). In shape, they differ strongly from those of other teleosts, their characteristic profile resembling a fur cap. This shape is unique among the eggs of teleostean fishes. The eggs ofC. gariepinus are attached to substrata at their animal pole, which shows an annular bulge consisting of numerous tiny attaching-filaments. These filaments seem to be a part of the zona radiata externa. The micropyle, located within the center of the annular bulge, is a straight opening in the zona radiata. Different ways of egg adhesion in teleosts were compared and discussed as well as the position of the micropyle with respect to fertilization.     

Ovulation and egg segregation in the tunic of a colonial ascidian,Diplosoma listerianum (Tunicata,Ascidiacea)

Summary The process of egg segregation in the tunic of the ovoviviparous ascidian Diplosoma listerianum was studied by light and electron microscopy. One egg at a time was seen to mature in each zooid. The eggs had large yolk and grew on the ovary wall enveloped in four layers: (1) outer follicle cells (OFC), long and rich in RER (rough endoplasmic reticulum) and with dense granules in the Golgi region; (2) flat inner follicle cells (IFC); (3) a loosely fibrillar vitelline coat (VC); (4) test cells encased on the egg surface. The growing egg protrudes from the ovary wall and presses on the contiguous epidermis. Granulocytes enter the space between the epidermis and the egg and insinuate cytoplasmic protrusions, disrupting the continuity of the OFC layer. At ovulation, OFC and IFC are discharged and form a post-ovulatory follicle (corpus luteum). The epidermis shrinks and closes, possibly by activation of microfilaments, causing the egg to be completely surrounded by the tunic. In the zooid, the wound caused by the passage of the egg is repaired both by contraction of the epidermis and by phagocytic activity. Altered spermatozoans are found in phagocytosing cells in the lumen of the ovary. These are presumably remnants of those which entered to fertilize the egg before segregation.     

Involvement of an acrosin-like enzyme in the acrosome reaction of sea urchin sperm

Extracts of the jelly coat of eggs of several marine invertebrates are known to induce in homologous sperm morphological changes known as the acrosome reaction. When sperm of the sea urchin Strongylocentrotus purpuratus are treated with low concentrations (0.2 μg fucose/ml) of egg jelly coat or 30 mM CaCl₂ in artificial seawater the acrosome reaction does not occur. However, either of these treatments causes the exposure of an acrosin-like enzyme to exogenous substrate and inhibitors. Subsequent addition of jelly coat to 3.7 μg fucose/ml to sperm in this “initial stage” induces the acrosome reaction (as judged by the appearance of an acrosomal filament). This concentration is also effective for untreated sperm. If inhibitors of the enzyme (diisopropylphosphofluoridate or phenylmethanesulfonyl fluoride) are added to sperm in the initial stage, no acrosomal filaments are observed when the high concentration of jelly coat is added. Whether other morphological changes occur in these sperm has not been examined. If phenylmethanesulfonyl fluoride is added 4 sec after the jelly coat, the acrosomal filaments are observed, but the sperm still fail to fertilize eggs. These results suggest a dual role for the acrosin-like enzyme(s), first in the mechanism of the acrosomal filament formation and then in a subsequent event in the fertilization process.     

Fine structure of the chorionic projections of the egg of Rhithrogena kimminsi Thomas (Ephemeroptera : Heptageniidae) and their role in egg adhesion

Scanning (SEM) and transmission electron microscopical (TEM) observations of the eggs of Rhithrogena kimminsi (Ephemeroptera : Heptageniidae), a species of the alpestris group, revealed 2 kinds of chorionic projections, both characterized by knob-terminated coiled threads (KCTs). The former are concentrated at one egg pole, and arise directly from the shell surface. The latter are scattered on the egg chorion and are supported by a basal excrescence, giving them a peglike feature. At TEM level, KCTs, arising directly from the chorion, appear to be composed of fibers that are enveloped by filaments. The fibers are tightly twisted together and joined at their apicals, which end in a terminal knob. KCTs, supported by peglike projections, show a similar organization, but each thread derives from a single coiled fiber. The different numbers of fibers form wider threads at the egg polar region and thinner ones on the peglike projections. The involvement of both kinds of KCTs in egg adhesion is documented through the discharge of their threads.     

Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat

As the result of a combined biochemical and electron microscopic investigation, hitherto unrecognized structural features of the mouse egg extracellular coat, or zona pellucida, have been revealed. Specimens were prepared for electron microscopy by spraying individually isolated zonae pellucidae onto a substrate and were observed by both rotary shadowing and negative staining techniques. Results of these experiments suggest that the three zona pellucida glycoproteins, ZP1 (200,000 Mr), ZP2 (120,000 Mr) and ZP3 (83,000 Mr), are organized into long filaments. Negatively stained zona pellucida filaments resemble "beads-on-a-string", with each bead (9.5 nm in diameter) located every 17 nm or so (center-to-center distance) along the axis of the filament. The filaments, in turn, appear to be interconnected by one of the three zona pellucida glycoproteins, ZP1, giving rise to a three-dimensional matrix. Proteolysis of ZP1 by chymotrypsin or reduction of intermolecular disulfides of ZP1 by dithiothreitol results in both solubilization of zonae pellucidae and disruption of interconnections between individual zona pellucida filaments. These observations suggest that the zona pellucida, which plays important roles both during and after fertilization of mammalian eggs, is a highly organized extracellular coat in which glycoproteins are assembled into filaments possessing a recognizable structural repeat.     

Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.     

Seed coat development, anatomy and scanning electron microscopy of Harpagophytum procumbens (Devil's Claw), Pedaliaceae

Seed coat development of Harpagophytum procumbens (Devil's Claw) and the possible role of the mature seed coat in seed dormancy were studied by light microscopy (LM), transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM). Very young ovules of H. procumbens have a single thick integument consisting of densely packed thin-walled parenchyma cells that are uniform in shape and size. During later developmental stages the parenchyma cells differentiate into 4 different zones. Zone 1 is the multi-layered inner epidermis of the single integument that eventually develops into a tough impenetrable covering that tightly encloses the embryo. The inner epidermis is delineated on the inside by a few layers of collapsed remnant endosperm cell wall layers and on the outside by remnant cell wall layers of zone 2, also called the middle layer. Together with the inner epidermis these remnant cell wall layers from collapsed cells may contribute towards seed coat impermeability. Zone 2 underneath the inner epidermis consists of large thin-walled parenchyma cells. Zone 3 is the sub-epidermal layers underneath the outer epidermis referred to as a hypodermis and zone 4 is the single outer seed coat epidermal layer. Both zones 3 and 4 develop unusual secondary wall thickenings. The primary cell walls of the outer epidermis and hypodermis disintegrated during the final stages of seed maturation, leaving only a scaffold of these secondary cell wall thickenings. In the mature seed coat the outer fibrillar seed coat consists of the outer epidermis and hypodermis and separates easily to reveal the dense, smooth inner epidermis of the seed coat. Outer epidermal and hypodermal wall thickenings develop over primary pit fields and arise from the deposition of secondary cell wall material in the form of alternative electron dense and electron lucent layers. ESEM studies showed that the outer epidermal and hypodermal seed coat layers are exceptionally hygroscopic. At 100% relative humidity within the ESEM chamber, drops of water readily condense on the seed surface and react in various ways with the seed coat components, resulting in the swelling and expansion of the wall thickenings. The flexible fibrous outer seed coat epidermis and hypodermis may enhance soil seed contact and retention of water, while the inner seed coat epidermis maintains structural and perhaps chemical seed dormancy due to the possible presence of inhibitors.     

Eggs and clutches of the Spheroolithidae from the Cretaceous Tiantai basin,Zhejiang Province,China

Numerous discoveries in the Tiantai basin of Zhejiang Province, China, enrich our understanding of the parataxonomy, paleobiology and taphonomic histories of fossil eggs from a diverse array of Cretaceous oofamilies. We describe the most abundant of these egg types catalogued in the Zhejiang Museum of Natural History, Spheroolithus cf. zhangtoucaoensis (oofamily Spheroolithidae). Scanning electron microscopy, here utilised for the first time on Spheroolithus eggs from Tiantai, and petrographic microscopy reveal 0.81–1.37-mm thick eggshell composed of a single structural layer of calcite with slightly flaring shell units, irregular pores, horizontal accretion lines and a sweeping extinction pattern under crossed polars. This contradicts previous reports of the presence of two structural layers in Tiantai Spheroolithus. Clutches consist of 2–13 eggs arranged in an irregular, single-layered pattern. As these eggs are among the oldest Asian examples of Spheroolithus currently known, they may shed light on the early evolution of this oogenus. This study establishes the definitive presence of the Spheroolithidae in the Tiantai basin, contra recent reports, and provides the framework for ongoing examination of egg diversity and taphonomy in the Tiantai basin.     

Scanning electron microscopy of the eggs of Palingenia longicauda (Olivier) (Ephemeroptera : Palingeniidae)

The eggs of Palingenia longicauda (Ephemeroptera : Palingeniidae) were studied by scanning electron microscopy (SEM), and showed a biconvex shape with the 2 aspects joined along a thick peripheral border. The eggs were completely wrapped by an exochorion that differs in thickness and organization according to the region of the chorionic surface. The thickest part of the exochorion formed a plaque, which also covered part of the peripheral border, and was constituted of a network of filaments. The remaining part of the exochorion was composed of a thin wrinkled sheet. The micropyle, hitherto unknown in Palingeniidae, appeared as a round opening penetrating into the plaque. The fibrillar network surrounding the micropyle dove-tailed with the egg chorion, forming a differentiated raised process. This peculiar interconnection facilitates egg anchoring to the substratum, and is an adaptation of the fibrous coat to the aquatic environment.     

Surface morphology of eggs of Euproctis chrysorrhoea (Linnaeus, 1758)

Candan, S., Suludere, Z. and Bayrakdar, F. 2007. Surface morphology of eggs of Euproctis chrysorrhoea (Linnaeus, 1758). —Acta Zoologica (Stockholm) 88 : 000–000. Filaments covering the egg batches and chorion structure were studied both by light and scanning electron microscopy in the brown‐tailed moth Euproctis chrysorrhoea (Linnaeus, 1758). Females lay eggs in masses on the underside of apple leaves. The egg batches are covered with brown hairs derived from the bodies of the female. Each female lays about 200–400 eggs. The spherical eggs are about 0.84 mm long and 0.47 mm wide. Newly deposited eggs are golden‐yellow and darken after the onset of embryonic development. The micropylar area appears somewhat depressed and has a circular outline. The region is surrounded by a rosette of 10–12 petal‐shaped primary cells, which are completely surrounded by a series of secondary and tertiary cells. The remainder of the egg is largely smooth, but shows aeropyles. These are located in the corners of ill‐defined polygons.     

Filamentous structure of the external surface of abalone eggs revealed by quick-freeze deep-etch technique

The external surface of abalone eggs was examined by thin section and quick-freeze, deep-etch electron microscopy. In thin sections, networks of fine filaments were found interconnecting the adjacent microvilli on the surface of unfertilized eggs. Quick-freeze, deep-etch electron microscopy revealed the three-dimensional structure of these networks of filaments on the external surface of the egg. Mainly two networks of filaments were identified; one was composed of thicker (14–19 nm) filaments interconnecting with the neighboring microvilli nearly horizontally, and the other was composed of thinner (8–14 nm) branched filaments closely surrounding the microvilli surface as well as highly interconnecting neighboring microvilli in a polygonal pattern. The overall structure of the filamentous network on the egg surface showed no distinct alteration after fertilization. These networks of filaments observed on the egg surface may play a key role in sperm–egg interaction.     

A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response

Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.     

Egg envelope synthesis and chorion modification after oviposition in the dragonfly Libellula depressa (Odonata, Libellulidae).

Libellula depressa (Odonata, Libellulidae) is an exophytic dragonfly ovidepositing eggs in clutches on the surface of floating plants and algae. The present work investigates, at ultrastructural level, the gradual differentiation of the egg envelopes and the chorionic changes after egg deposition in water. The ovary of the mature female of L. depressa is composed of numerous strings of panoistic ovarioles, where the eggshell formation takes place gradually throughout the activity of the follicle cells. The present data show that the egg envelopes are constituted of a very thick electrondense vitelline envelope, a thin endochorion and an extremely thick exochorion composed of a fibrillar matrix resting on a thin electrondense layer. After deposition in water, L. depressa eggs, initially white and almost transparent, gradually become brown spots in a semitransparent jelly coat, rich of incorporated debris. The jelly coat enveloping the eggs of L. depressa derives exclusively from the exochorion, constituted of a fibrillar matrix, which swell at contact with water. The jelly-like coat performs an adhesive function and presumably a protective role during egg segmentation and ensuing larval hatching.     

Maternal energy investment in eggs and jelly coats surrounding eggs of the echinoid Arbacia punctulata

In free-spawning marine invertebrates, the amount of maternal energy that is invested in each egg has profound implications for all life-history stages of the offspring. The eggs of echinoids are freely spawned into the water and are surrounded by several structurally complex extracellular layers. These extracellular layers, or jelly coats, do not contribute energy to embryonic development but must impose an energy cost on the production of each egg. The investment of maternal energy reserves in the jelly coats of echinoid eggs may have important implications for the number of eggs that can be produced (i.e., fecundity) and the amount of energy that can be invested in each egg. We estimated the degree to which maternal energy is invested in the jelly coats surrounding eggs of the echinoid Arbacia punctulata. Estimates were derived from measurements of the amount of energy contained in the combined eggs and jelly coats, and in the eggs alone. The amount of energy contained in A. punctulata eggs ranged from 2.70 to 5.53 x 10(-4) J egg(-1). The amount of energy contained in the jelly coats ranged from 0.13 to 0.48 x 10(-4) J jelly coat(-1). The mean concentration of energy in the eggs was 2.15 mm(-3) and 0.29 J mm(-3) in the jelly coats. These results indicate that between 3% and 11% (mean = 7%) of the total energy invested in each A. punctulata egg is partitioned to the jelly coat alone. A significant positive relationship was found between the volumes of the jelly coats and the amount of energy they contained. Based on this relationship and an analysis of differences in the size of jelly coats between echinoid species, we suggest that the degree to which energy is invested in jelly coats may vary among echinoid species and is therefore likely to be an important life-history characteristic of these organisms.     

Fine structural investigation of the insemination response in Urechis caupo.

Electron microscopy of Urechis eggs revealed no changes in the egg cortex or investing layers until 4 min after insemination at 172C. From 4 min to about 30 min after insemination the surface coat gradually elevates, widening the perivitelline space. During this period, microvilli separate from the tightly woven layer of the surface coat, fibrogranular aggregates resembling surface coat material appear in the perivitelline space, and some cortical granules are extruded from the egg cortex into cytoplasmic processes. There is no statistically significant decrease in the number of cortical granules remaining in the egg surface during the first 95 min after insemination; many cortical granules persist in postgastrulae. Most of the cortical granules remain in fertilized eggs after removal of the surface coat with glucose-EGTA. We found no morphological correlates of the polyspermy block which is established within 1 min of insemination (Paul, 1975).     

A new extracellular matrix protein of the sea urchin embryo with properties of a substrate adhesion molecule

Summary A new embryonic extracellular matrix protein has been purified from eggs of the sea urchin Paracentrotus lividus. The molecule is a 210 kD dimer consisting of two 105 kD subunits that are held together by S-S bridges. In the unfertilized egg, the protein is found within granules uniformly distributed throughout the cytoplasm. After the egg is fertilized, the antigen is polarized to the apical surface of ectodermal and endodermal cells during all of the developmental stages examined, until the pluteus larva is formed. The protein promotes the adhesion of blastula cells to the substrate and is antigenically distinct from echinonectin, a well characterized substrate adhesion molecule. This report adds a new candidate to the list of known extracellular matrix molecules for the regulation of differentiation and morphogenesis in the sea urchin embryo. Offprint requests to: V. Matranga     

Multiple Lines of Egg Defense in a Neotropical Arachnid with Temporary Brood Desertion

Egg predation is the one of the main costs of brood desertion in many ectothermic animals. When stressful environmental conditions constrain parental activities to only some periods of the day, the combination of physical or chemical defenses may attenuate the costs related to egg loss during periods of temporary parental absence. Females of the harvestman Neosadocus maximus periodically abandon their clutches to shelter or forage. They also cover their eggs with a hygroscopic mucus coat and seem to lose fewer eggs to predation than other syntopic harvestmen whose eggs lack the mucus coat. Using two species of N. maximus egg predators, we demonstrate that eggs whose mucus coat was experimentally removed suffered higher predation rate than eggs whose mucus coat was left intact. We argue that this mucus provides physical protection against egg predators, especially small arthropods. A similar mucus coat has independently evolved in other two clades of Neotropical harvestman in which males care for the eggs and typically leave their clutches unattended for several hours a day. We propose that the presence of multiple lines of egg defense may have evolved as a way of lowering the costs imposed by intra‐ and interspecific egg predation during periods of temporary brood desertion.     

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat

In the initial stage of ascidian fertilization sequential sperm–egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66kDa, 120kDa and 140kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata .