Vitelline Coat Research Articles

Physiological functions of proteasomes in ascidian fertilization and embryonic cell cycle.

Fertilization is initiated by sperm binding to the vitelline coat, a glycoprotein coat, of the eggs. After the sperm binding, sperm undergoes the acrosome reaction, an exocytosis of the acrosome. By this acrosome reaction, sperm proteases or lytic agents in the acrosome which allows sperm to penetrate through the vitelline coat are exposed and released. Subsequently, the sperm-egg membrane fusion occurs, which triggers a transient increase in intracellular calcium ions, leading to the exocytosis and the resumption of the meiotic division cycle, i.e., egg activation.

Ascidian Larvae: The Role of Test Cells in Preventing Hydrophobicity

Abstract Ascidian test cells co‐differentiate on the surface of each ovarian oocyte beneath the vitelline coat. They become vacuolated and later occupy the perivitelline compartment of each egg and embryo. In some species, their vacuoles contain submicroscopic granules or filaments called ‘ornaments’ and acidic glycosaminoglycans. These test cells deposit their products on the surface of the larval tunic in late embryogenesis. In these species, the test cells are lost at hatching. In other species, the test cell vacuoles contain acidic glycosaminoglycans, but no ornaments. In these species, the test cells attach to the larval tunic and probably secrete acidic glycosaminoglycans. We deprived the embryos of seven species of ascidians of their test cells and vitelline coats during midembryogenesis. After completing their development, the larvae of both kinds of species were hydrophobic. They easily become trapped on the surface of sea water in cultures. Normal larvae (controls), bearing test cell secretions, are hydrophilic and never become trapped. We infer that negatively charged secretions of the test cells make normal larvae hydrophilic. Some molgulids with direct development have no test cells, no fins and no swimming larva. We reason that the test cells of these species may have been lost during evolution because they no longer had an important role in preventing hydrophobicity.

Existence of Three Mechanisms for Blocking Polyspermy in Oocytes of the Mussel Mytilus edulis.

We found the existence of a three-step mechanism to block polyspermy in the oocyte of the mussel Mytilus edulis. When the oocytes were inseminated within 30 min after spawning, they underwent monospermic fertilization over a wide range of sperm-oocyte ratios up to 5 x 103. A transient depolarization of the oocyte plasma membrane (fertilization potential) was observed immediately after insemination. Low-sodium seawater induced polyspermy and decreased the amplitude of the fertilization potential, suggesting the existence of a fast block to polyspermy that is dependent on depolarization of the plasma membrane. When the fertilized oocytes were inseminated again at a sperm-oocyte ratio that is great enough to give a high rate of polyspermy in initial insemination, many sperm could not undergo the acrosomal reaction and thus could not penetrate fertilized oocytes. The remaining sperm underwent an acrosomal reaction and the acrosomal process protruded through the vitelline coat, but it did not fuse with the oocyte plasma membrane. These findings suggest the existence of two strategies constituting a late polyspermy block: suppression of acrosomal reaction and block of contact or fusion between the plasma membranes of sperm and oocyte.

The 220-kDa Vitelline Coat Glycoprotein Mediates Sperm Binding in the Polarized Egg of Unio elongatulus through O-Linked Oligosaccharides

In previous studies we found that two glycoproteins of 220 and 180 kDa account for 80-90% of the material dissolved from the vitelline coat (VC) of Unio elongatulus egg (Focarelli and Rosati, 1993). The two glycoproteins were purified by electroelution and used to raise the corresponding polyclonal antibodies. Immunofluorescence experiments showed that the 180-kDa protein species is ubiquitous in the VC, whereas the 220-kDa protein is concentrated in a restricted region of the vegetal pole, the crater region, where the sperm-egg interaction occurs. Binding assays indicated that only the 220-kDa protein interacted with the sperm and that the protein bound in the apical region and triggered acrosomal changes in sperm. Competition binding assays showed that O- and not N-linked oligosaccharides derived from the 220-kDa protein competed with the binding of the protein to sperm and that fucose is involved in the ligand role of the 220-kDa protein.

Brooding strategies in solitary ascidians: Corella species from north and south temperate waters

Corella inflata from the northwest Pacific coast of North America and Corella eumyota from southern New Zealand are both ovoviviparous solitary ascidians with very different methods of brood retention. Corella inflata eggs are covered with large follicle cells that are responsible for flotation. The floating eggs are trapped in the upwardly situated atrial chamber, where development ensues. Corella eumyota eggs have small follicle cells and do not float; the follicle cells produce a viscous glue that causes the eggs and embryos to stick to each other and to the right atrium. The follicle cells are not sticky when removed from the ovary but become so in about 15 min. In C. inflata, development to hatching requires 24–26 h at 13 °C and metamorphosis begins 17 h later. Corella eumyota tadpoles hatch at about 25 h at 15 °C and begin metamorphosis 11 h later. Both species retain the tadpoles well beyond hatching and release tadpoles fully competent to metamorphose. In C. inflata the tadpoles swim down and out of the atrium; C. eumyota tadpoles somehow break free of the glue-encrusted vitelline coat and swim out the atrial siphon. Tadpoles of both species swim vigorously when released and then rapidly metamorphose. Most tadpoles metamorphose in 15–20 min. Since both species spawn around dawn, tadpoles are released during the night.

Vitelline coat lysins from molluscan sperm and their use in microinjection

AbstractMicroinjection through the tough vitelline coats of oocytes from three molluscan species (Haliotis rufescens, Norrisia norrisi, and Astrea undosa) was facilitated by the local application of sperm vitelline coat lysin with a micropipet. Extracts containing lysin were the 12,000g supernates of frozen‐thawed sperm. In H. rufescens and N. norrisi, lysin extracts dissolved a small hole through the vitelline coat, whereas in A. undosa, the lysin created a pathway for microinjection by softening the vitelline coat without dissolving it. Similar effects were observed when oocytes were suspended in lysin extracts: Vitelline coats dissolved completely in H. rufescens (previously reported by Lewis et al. [1982], Dev. Biol. 92:227–239) and N. norrisi, but only softened and swelled in A. undosa (although they could then be removed by mechanical agitation). The lysin extracts were highly species specific and had no visible effects on heterologous vitelline coats even at concentrations higher than those required to dissolve/soften homologous coats. Evidence that N. norrisi lysin acts by a non‐enzymatic mechanism was provided by the observation that mobilities of radioactive bands in sodium dodecyl sulfate polyacrylamide gels were unchanged following dissolution of 125I‐labeled vitelline coats. Electron micrographs of the previously undescribed sperm from N. norrisi and A. undosa are also presented. © 1995 Wiley‐Liss, Inc.

N-Acetylglucosaminidase Inhibitor Isolated from the Vitelline Coat of Ascidian Eggs Is a Candidate Sperm Receptor

An inhibitor against the egg β-D- N-acetylglucosaminidase (GlcNAcase) was purified from the vitelline coat of eggs of the ascidian, Halocynthia roretzi. The purified inhibitor gave a single band with an apparent molecular mass of 70 kDa on SDS-PAGE under non-reducing conditions. The GlcNAcase inhibitor potently inhibited the ascidian egg GlcNAcase activity, and also substantially inhibited the GlcNAcase activity in the ascidian sperm extract, although to lesser extent. In contrast, it showed little inhibitory effect on the activity of GlcNAcase from Jack beans. This inhibitor was found to block the binding of sperm to the vitelline coat of the eggs in a concentration-dependent manner. These results strongly suggest that the GlcNAcase inhibitor isolated from the vitelline coat functions in the binding of sperm to the egg coat, as a possible sperm receptor.

Specific induction of self-discrimination by follicle cells in Ciona intestinalis oocytes.

Self-incompatibility, a mechanism that prevents self-fertilization in ascidians, is based on the ability of the oocyte vitelline coat to distinguish and accept only heterologous spermatozoa. In Ciona intestinalis self-discrimination is established during late oogenesis and is contributed or controlled by products of the overlying follicle cells. In this study we have further investigated the role of the follicle cells in the onset of self-discrimination by using in vitro maturation of ovarian oocytes deprived of the follicle cells and incubated with either autologous or heterologous follicle cells. Fertilization assays demonstrate that the action of the follicle cells is exerted even when they are detached from the vitelline coat and that only autologous follicle cells can promote the induction of self-sterility on the egg coat. Electron microscopy of the oocytes during maturation reveals that the switch from self-fertility to self-sterility is accompanied by the appearance of a thin electron-dense layer on the outer surface of the vitelline coat. We suggest that the formation of this layer is the result of the interaction between products of the follicle cells and the autologous vitelline coat.

Specific inhibition of sperm β-N-acetylglucosaminidase by the synthetic inhibitor N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime inhibits fertilization in the ascidian, Phallusia mammillata.

Increasing evidence has evolved from studies in ascidians and mammals that sperm β-N-acetylglucosaminidase (GlcNAc'ase) plays a crucial role in fertilization. In the ascidian Phallusia mammillata, GlcNAc'ase is the predominant sperm-bound glycosidase and N-acetylglucosamine (GlcNAc) is the prevailing glycoside residue on the vitelline coat. We report here that the GlcNAc'ase inhibitor O-(2-acetamido-2-deoxy-D-glucopyrano-sylidene)-amino-N-phenylcarbamate (PUGNAC) is a potent competitive inhibitor of sperm-bound GlcNAc'ase in P. mammillata. The inhibitor constant Ki for the isolated enzyme is 47 nmol/L. Fertilization of eggs is inhibited by PUGNAC in a dose dependent competitive manner with 50% inhibition at an inhibitor concentration of 85 μmol/L. Further experiments, in which intact eggs possessing an egg coat were mixed with eggs from which the coat had been removed, showed that only fertilization of intact eggs was inhibited by PUGNAC. This finding suggests that PUGNAC prevents the binding of the sperm-associated GlcNAc'ase to terminal GlcNAc residues on the vitelline coat, thus inhibiting sperm binding and subsequently fertilization. Furthermore and most importantly, it shows that treatment with PUGNAC does not affect the viability of sperm and that the process of sperm-egg fusion is not affected.

Origin and Differentiation of the Inner Follicular Cells during Oogenesis in Molgula pacifica (Urochordata), an Ascidian without Test Cells

AbstractIn Molgula pacifica small previtellogenic oocytes are found between cells of the ovarian epithelium. Each oocyte subsequently grows within a compartment of the epithelium known as a primary follicle. The wall of the primary follicle is composed of outer follicular epithelial cells. While growing from about 15–70 μm in diameter, each oocyte gradually recruits a set of about 950 non‐epithelial inner follicular cells. These cells co‐differentiate in sets with each oocyte, but test cells never appear. The first filamentous components of the vitelline coat appear on the surface of an oocyte in places where it is in contact with undifferentiated (stage 2) inner follicular cells. Each fully differentiated inner follicular cell stores adhesive precursors in a large compartment of the endoplasmic reticulum and probably secretes components of the vitelline coat. There is no evidence that the outer follicular epithelial cells transform into inner follicular cells by dedifferentiation as has often been assumed. Inner follicular cells, in stage 1, are nearly identical to hemoblasts. Hemoblasts may form the inner follicular cells, but to do this they would have to cross the outer follicular epithelium and this phenomenon has not yet been seen.

Evidence for the involvement of the Rho GTP-binding protein in egg activation of the ascidian Halocynthia roretzi.

Eggs of the ascidian Halocynthia roretzi are activated by insemination and by treatment with calcium ionophore, leading to elevation of the vitelline coat. Here we describe the effects on egg activation of microinjection of guanosine 5'-(γ thio) triphosphate (GTPγS, a non-hydrolyzable GTP analog), heparin (an antagonist of the inositol 1,4,5-trisphosphate receptor) and a monoclonal antibody to the Rho GTP-binding protein. Microinjected GTPγS induced elevation of the vitelline coat, but not when it was co-injected with EGTA or heparin. Pre-injected heparin or the anti-Rho monoclonal antibody blocked subsequent sperm-induced elevation of the vitelline coat, but not calcium ionophore-induced elevation. We also demonstrated that the amount of cytosolic inositol 1,4,5-trisphosphate was increased by insemination. These results strongly suggest that the Rho GTP-binding protein functions prior to the heparin-blocked inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release in the sperm induced activation process of H. roretzi eggs.

Vitelline coat of Unio elongatulus: II. Biochemical properties of the 220‐ and 180‐kD components

In a previous study we found that two glycoproteins with apparent molecular weights of 220 kD and 180 kD account for 80-90% of the material dissolved from the vitelline coat of the egg of the bivalve mollusk, Unio elongatulus (Focarelli and Rosati, 1993: Mol Reprod Dev 35:44-51). In this study we isolated and purified these glycoproteins by electroelution. The two proteins differ in many respects: the 180-kD molecule is acidic in nature and highly heterogeneous, whereas the 220-kD protein is neutral and less heterogeneous. Both seem to have O- and N-linked oligosaccharide chains. The 180-kD protein contains 13-16% carbohydrate, whereas the 220-kD molecular contains only 7-8%. Amino acid analysis and peptide mapping also show that each protein represents a unique polypeptide chain.

Isolation, Characterization, and Primary Structure of Three Major Proteins Obtained from Mytilus edulis Sperm1

Acrosomal proteins from Mytilus edulis sperms were separated into 11 fractions by reverse phase HPLC. The three major proteins, named M3, M6, and M7, showed strong egg vitelline coat lysin and first polar body releasing activities. The amino acid sequences of these proteins were determined. M6 and M7 were composed of 180 amino acid residues and showed high sequence homology (76%), while M3 was composed of 149 residues and showed 26% homology with M6 and M7. The disulfide linkage motif of the three proteins was similar and resembled the carbohydrate recognition domain (CRD) of C-type lectin. The C-terminal half of these proteins showed sequence homology with CRD of C-type lectins, but no homology with vitelline coat lysins of other mollusks. The proteins bound to asialofetuin-Sepharose in the presence of Ca2+ and were eluted with EDTA, indicating that they are Ca(2+)-dependent carbohydrate-binding proteins.

Glycosidases, proteases and ascidian fertilization

Spermatozoa should bind to and then penetrate the vitelline coat for fertilization in ascidians and many other animals. There is substantial evidence that the binding of ascidian sperm is mediated by a sperm glycosidase and complementary saccharide chains of glycoproteins in the vitelline coat. Involvement of a sperm proteasome in the binding is also suggested. For the penetration, sperm proteases such as chymotrypsin-like enzyme, acrosin, spermosin and proteasome are suggested to play essential roles. Sperm glycosidase, that is translocated from the tip of sperm head to the surface overlying the mitochondrion, anchors the mitochondrion at the outer surface of vitelline coat. Therefore it assists sperm to penetrate the vitelline coat and traverse the perivitelline space. For fusion with egg plasma membrane, sperm metalloendoprotease seems to be involved. Egg glycosidases and proteases serve for some steps after fertilization, such as the prevention of polyspermy, expansion of perivitelline space and regulation of cell cycle.

Ovulation and embryo-parent relationships inBotrylloides leachi(Ascidiacea, Tunicata)

Summary The ultrastructural events of ovulation and differentiation of the brood pouch are studied in the colonial ascidian Botrylloides leachi. Each zooid matures one or two yolked oocytes enveloped by test cells, vitelline coat, and inner and outer follicle cells. A small vesicular oviduct lies between the outer follicle cells and the brood pouch, a flask-shaped structure differentiated from the wall of the atrial chamber. The oviduct epithelium fuses with the outer follicle and brood pouch cells and breaks, creating a pathway which drives the oocyte to the brood pouch. Embryos develop in isolation in the latter whose epithelium undergoes differentiation: the cell surface increases enormously, forming deeper and deeper baso-lateral foldings, cell interdigitations and apical polymorphic protrusions. Extended tight junctions separate the different domains of the plasmalemma and create a barrier between the pouch lumen and the blood. Large quantities of glycogen, mainly in the form of rosettes, are formed ...

Cytochalasin B does not block sperm penetration into denuded starfish oocytes.

During fertilisation in starfish oocytes, the fertilisation cone develops temporarily beneath the penetrating sperm. The role of the fertilisation cone in sperm incorporation in the starfish Asterias amurensis was examined using cytochalasin B (CB). CB (2 microM) allowed sperm acrosomal process-egg plasma membrane fusion and egg activation, but inhibited the development of the fertilisation cone containing actin microfilaments. When sperm were added to intact oocytes (with the jelly coat and vitelline coat) in seawater containing CB, the sperm head did not penetrate the fertilisation membrane. Although the acrosomal process fused with egg plasma membrane, the sperm head remained outside the fertilisation membrane. On the other hand, denuded oocytes without the jelly coat and vitelline coat allowed sperm penetration even in the presence of 2 microM CB. Electron microscopy revealed that sperm organelles, including the acrosomal process, nucleus, mitochondrion and tail, were incorporated into the slightly electron-dense cytoplasm, which was similar to the cytoplasm of the fertilisation cone. These results show that the development of the fertilisation cone/actin filament complex is not essential for incorporation of the sperm, since incorporation can occur in denuded oocytes. However, the cone is required for fertilisation of intact oocytes, suggesting that this actin-filament-containing structure is necessary for getting the sperm through the outer egg coats.

Sperm-Surface Chymotrypsin-like Protease Activity Required for Fertilization in Ascidians

During fertilization, species-specific gamete binding must be followed by sperm penetration of egg vestments before gamete fusion can occur. Sperm proteases, called lysins, aid this process. Sperm from Ascidia ceratodes, Ascidia callosa, and Ascidia paratropa were found to have a surface-mounted chymotrypsin-like protease when studied by enzymology, biotinylation, immunolabeling, and histochemistry. Chymotrypsin substrates and inhibitors blocked fertilization in a concentration-dependent manner in A. ceratodes and decreased the number of sperm heads which penetrated the egg's vitelline coat, but had no effect on sperm binding to follicle cells. Sperm bound to agarose beads coated with the chymotrypsin inhibitor α2-macroglobulin. Chymotrypsin-like enzyme activity, assayed fluorimetrically using N -succinyl-leucinyl-leucinyl-valinyl-tyrosinyl-7-amido-4-methyl-coumarin as the substrate, was associated with head fractions prepared by differential centrifugation. Biotinylation of live sperm followed by detergent extraction showed that chymotrypsin-like activity could be removed from the detergent extract using avidinagarose beads. Indirect immunofluorescence of unreacted and reacted sperm heavily labeled membrane domains overlying the mitochondrion and at the base of the head with occasional labeling of the sperm tip. Histochemical studies, which used N -succinyl-alanyl-alanyl-prolyl-phenylalanyl-β-napthylamide as the substrate, colocalized enzyme activity in head regions of unreacted and near the mitochondrion of reacted sperm. Thus, we conclude that in ascidian sperm a chymotrypsin-like protease is exposed on the external surface of the plasma membrane of the head, is required for fertilization, and plays a role in sperm penetration but not binding.

Sexual reproduction in the compound ascidian Diplosoma listerianum (Tunicata). II. Sperm penetration through ovary wall and evidence of internal fertilization

Diplosoma listerianum differs from most ascidians in that, at ovulation, eggs are emitted at the bottom of the ovary and segregated into the tunic, so that fertilization occurs far from seawater. A fertilization canal, a hollow extension of the ovary, conducts sperm towards the egg. In the present paper, ultrastructural evidence is reported on the morphological relationship between the ovary, egg envelopes and oocyte and on the mechanism by which sperm-egg interaction is established. In the ovary, the very complex sperm, equipped with a spiral “dense groove”, undergo metamorphosis as the first step in a sperm reaction and then pass through the ovary epithelium insinuating themselves between the intercellular junctions which appear to be mouldable, although able to maintain the egg-ovary barrier. Sperm then reach the vitelline coat, where a further step in the sperm reaction occurs. Before the egg abandons the ovary, the sperm head is incorporated into the oocyte by a process recalling phagocytosis, with the formation of an engulfing pocket. Sperm-egg contact and incorporation in D. listerianum occur in a way, never previously reported for other ascidians, in which fusion of plasma membranes takes place immediately after sperm-egg contact. Unlike other cytoplasmic components, the dense groove persists until the sperm enters the egg. It gives a corkscrew-like configuration to the sperm head and allows close adhesion to cell membranes, facilitating sperm movement. Expulsion of numerous cortical granules and features of a cortical reaction were observed in the egg penetrated by the sperm. The mode of internal fertilization of this species in comparison with that of other tunicates and phylogenetic aspects are discussed. Ripe colonies of D. listerianum collected in the Lagoon of Venice, Italy in 1986 and 1991 and colonies reared on glass in aquaria were used for our investigations.

Purification and properties of N‐acetylglucosaminidase from eggs of the ascidian, Halocynthia roretzi

In several ascidians, beta-D-N-acetylglucosaminidase (GlcNAcase) released from eggs following fertilization is proposed to play a key role in polyspermy block through its binding to the sperm receptor on the vitelline coat [C. C. Lambert (1989) Development 105, 415-420]. In the ascidian Halocynthia roretzi, GlcNAc-specific lectins inhibited the fertilization most strongly among various lectins. Furthermore, GlcNAcase activity was released from the eggs in response to the egg activation. The GlcNAcase was purified from the eggs to apparent homogeneity by chromatographies on DEAE-Toyopearl, SP-Toyopearl, Toyopearl HW-65, and Mono S. The purified enzyme gave a single band on isoelectric focusing with an isoelectric point of 7.0. It gave two bands on SDS/PAGE: the molecular masses of the bands were estimated to be 65 kDa/66 kDa, and 84 kDa/85 kDa under reducing/non-reducing conditions, respectively. The two bands were found to converge to a single band of 56 kDa after deglycosylation, which suggests microheterogeneity in the sugar moiety. The enzyme showed an oligomeric structure with an apparent molecular mass of 520 kDa, estimated by gel filtration. The optimum pH of the activity was around 4.5. The enzyme hydrolyzed both 4-methylumbelliferyl-GlcNAc and 4-methylumbelliferyl-GalNAc, suggesting that it should be characterized as a beta-D-N-acetylhexosaminidase, with Km values of 1.2 mM and 0.52 mM, respectively. The purified enzyme was found to be capable of binding to the vitelline coat in a GlcNAc-specific manner. Immunoblot analysis using antibody raised against the purified GlcNAcase revealed that the enzyme itself is released from the eggs upon fertilization. Thus, the GlcNAcase purified in this study is released from eggs following fertilization and bound to the vitelline coat in order to function in the polyspermy blocking mechanism.

Effects of protease inhibitors on binding of sperm to the vitelline coat of ascidian eggs: implications for participation of a proteasome (multicatalytic proteinase complex).

Among various protease inhibitors, chymostatin (an inhibitor of sperm chymotrypsin-like protease) strongly inhibited the binding of sperm to the vitelline coat of glycerinated eggs of the ascidian Halocynthia roretzi, whereas leupeptin (an inhibitor for sperm acrosin), antipain, and soybean trypsin inhibitor had no significant inhibitory effects. Dansyl-Val-Pro-argininal (an inhibitor of the sperm trypsin-like protease, spermosin) had an inhibitory effect on the binding of sperm that was much smaller than its effects on fertilization. Since the sperm chymotrypsin-like protease that is involved in ascidian fertilization has been identified as a proteasome (multicatalytic proteinase complex), we tested the effects of several peptidyl argininals, inhibitors of the activities of proteasomes, on this binding process. The ranking of the inhibitory effects of these compounds on the binding of sperm was the same as that of their effects on the chymotrypsin-like activity of the proteasome, reported previously. The potent inhibitors of binding used in these studies had no or minimal effects on sperm motility. These results suggest that a sperm chymotrypsin-like protease (most probably the chymotrypsin-like protease in the proteasome) plays a key role in binding of sperm to the vitelline coat of the ascidian egg.