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During first gamete interaction, oligosaccharides on the glycoproteins present in oocyte envelopes of the rock shrimp, Rhynchocinetes typus, play an important role in spermatozoon recognition prior to the onset of penetration. These oligosaccharides have terminal monosaccharides that may be recognized by means of specific lectins. A variety of marine species have monosaccharides on their oocyte envelopes, allowing them to interact with the spermatozoa. The number and location of the monosaccharides varies during oocyte maturation. However, this phenomenon has not been studied in shrimp. The present study evaluates the presence and location of monosaccharides on oocyte envelopes during maturation using seven specific lectins conjugated to FITC (GNL, LCA, Con-A BS-1, LTA, GLS-II,DBA); these recognize mannose (α 1-3) mannose, α-mannose, α-glucose, glucose, galactose, fucose, N-acetyl-glucosamine, and N-acetyl-galactosamine, respectively. Participation of these carbohydrates in the fertilization process was determined through the insemination of fresh oocytes with spermatozoa previously incubated with the different monosaccharides that block the active sites of the spermatozoa, inhibiting fertilization. The results showed the presence of N-acetyl-glucosamine and mannose on the oocyte envelopes, indicating that these sugars could be the receptors during the first gamete interaction. The results also suggested that glucose could be a complementary receptor to N-acetyl-glucosamine and mannose since, despite the low concentration of glucose on the envelope, this sugar generated a high degree of fertilization inhibition.Durante la primera interacción gamética, los oligosacaridos unidos a las glicoproteínas de las cubiertas ovocitarias del camarón de roca Rhynchocinetes typus tienen un importante rol en el reconocimiento del espermatozoide antes de iniciar la penetración. Estos oligosacáridos presentan en su zona terminal, monocacáridos que pueden ser reconocidos por lectinas específicas. Diversas especies marinas tienen monosacáridos en la cubierta externa de sus ovocitos que le permiten interactuar con los espermatozoides y su número y localización presenta cambios durante la maduración, sin embargo este fenómeno no ha sido estudiado en camarones. El presente estudio evalúa la presencia y localización de monosacáridos sobre la cubierta de los ovocitos durante su maduración usando siete lectinas específicas conjugadas con FITC (GNL, LCA , Con-A BS-1, LTA, GLS-II y DBA) las cuales reconocen manosa (α 1-3) manosa, α-manosa, α-glucosa, glucosa, galactosa, fucosa, N-acetil-glucosamina y N-acetilgalactosamina respectivamente. La participación de estos carbohidratos en el proceso de fecundación fue determinada mediante la inseminación de ovocitos frescos con espermatozoides incubados previamente con los diferentes monosacáridos que bloquean los sitios activos de los espermatozoides inhibiéndo la fecundación. Los resultados mostraron la presencia de N-acetil-glucosamina y manosa en la cubierta externa de los ovocitos maduros los cuales podían ser los receptores espermáticos durante la primera interacción gamética. También se sugiere que la glucosa podría ser un receptor complementario a los anteriormente mencionados debido que a pesar que su concentración en las cubiertas es baja, genera una alta inhibición de la fecundación.

During first gamete interaction, oligosaccharides on the glycoproteins present in oocyte envelopes of the rock shrimp, Rhynchocinetes typus, play an important role in spermatozoon recognition prior to the onset of penetration. These oligosaccharides have terminal monosaccharides that may be recognized by means of specific lectins. A variety of marine species have monosaccharides on their oocyte envelopes, allowing them to interact with the spermatozoa. The number and location of the monosaccharides varies during oocyte maturation. However, this phenomenon has not been studied in shrimp. The present study evaluates the presence and location of monosaccharides on oocyte envelopes during maturation using seven specific lectins conjugated to FITC (GNL, LCA, Con-A BS-1, LTA, GLS-II,DBA); these recognize mannose (α 1-3) mannose, α-mannose, a-glucose, glucose, galactose, fucose, N-acetyl-glucosamine, and N-acetyl-galactosamine, respectively. Participation of these carbohydrates in the fertilization process was determined through the insemination of fresh oocytes with spermatozoa previously incubated with the different monosaccharides that block the active sites of the spermatozoa, inhibiting fertilization. The results showed the presence of N-acetyl-glucosamine and mannose on the oocyte envelopes, indicating that these sugars could be the receptors during the first gamete interaction. The results also suggested that glucose could be a complementary receptor to N-acetyl-glucosamine and mannose since, despite the low concentration of glucose on the envelope, this sugar generated a high degree of fertilization inhibition. [ABSTRACT FROM AUTHOR]

Abstract: Sperm trypsin-like proteases are known to play important roles in fertilization, but their detailed functions are still unknown. We previously explored the binding partners of sperm trypsin-like proteases, HrProacrosin and HrSpermosin, in the ascidian Halocynthia roretzi, and we isolated several candidate proteins on the vitelline coat. We found that some of these proteins are identical to the C-terminal coding region (CT) and von Willebrand factor type D (vWF-D) domain of vitellogenin. We also found that CT on the vitelline coat disappears after fertilization. Vitellogenin is a large lipid transfer protein that is enzymatically processed during vitellogenesis. Although the processed domains including phosvitin and lipovitellin are known to function as yolk nutrient proteins, the roles of the CT and vWF-D domain remain elusive. Our results showed that the CT and vWF-D domain of vitellogenin are processed and attached to the vitelline coat, which in turn participate in fertilization as the binding partners of sperm proteases. [Copyright &y& Elsevier]

We have previously reported that two trypsin-like enzymes, acrosin and spermosin, play key roles in sperm penetration through the vitelline coat of the ascidian (Urochordata) Halocynthia roretzi [Sawada et al . (1984), J. Biol. Chem. 259 , 2900–2904; Sawada et al . (1984), Dev. Biol. 105 , 246–249]. Here, we show the amino-acid sequence of the ascidian preprospermosin, which is deduced from the nucleotide sequence of the isolated cDNA clone. The isolated ascidian preprospermosin cDNA consisted of 1740 nucleotides, and an open reading frame encoding 388 amino acids, which corresponds to a molecular mass of 41 896 Da. By sequence alignment, it was suggested that His178, Asp230 and Ser324 make up a catalytic triad and that ascidian spermosin be classified as a novel trypsin family member. The mRNA of preprospermosin is specifically expressed in ascidian gonads but not in other tissues. Purified spermosin consists of 33- and 40-kDa bands as determined by SDS/PAGE under nonreducing conditions. The 40-kDa spermosin consists of a heavy chain (residues 130–388) and a long light chain designated L1 (residues 23–129), whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain designated L2 (residues 97–129). The L1 chain contains a proline-rich region, designated L1(ΔL2) which is lacking in L2. Investigation with the glutathione-S -transferase (GST)–spermosin-light-chain fusion proteins, including GST–L1, GST–L2, and GST–L1(ΔL2), revealed that the proline-rich region in the L1 chain binds to the vitelline coat of ascidian eggs. Thus, we propose that sperm spermosin is a novel trypsin-like protease that binds to the vitelline coat and also plays a part in penetration of sperm through the vitelline coat during ascidian fertilization. [ABSTRACT FROM AUTHOR]

When unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, are released naturally they are strictly self-sterile, whereas almost all ovarian eggs isolated after spawning are self-fertile. Self-sterile eggs are prepared within a relatively short period of several hours before the spawning. The morphological changes in ovarian eggs during late oogenesis were studied with special reference to the establishment of self-sterility. Four types of eggs at serial developmental stages were classified according to the morphology of their external envelopes. Self-sterility was established in the last stage, from the ovarian egg type 3 (OVE3) to UFE stages. Ovarian eggs which had become committed to UFE were denoted as full-grown ovarian eggs (FOE). FOE were able to differentiate into self-sterile UFE in vitro, whereas OVE3 could not. Several morphological differences between OVE3 and UFE were found. OVE3 had a germinal vesicle (GV), a type of vitelline coat (VC-OVE3) and no expanded perivitelline space, whereas UFE had completed germinal vesicle break down (GVBD), had another type of coat (VC-UFE) and showed an expanded perivitelline space. There were also some differences in the mode of fertilization between OVE3 and UFE. In UFE, sperm became bound firmly to the vitelline coat and passed through the coat with the help of follicle cells, whereas in OVE3, sperm did not bind so strongly and entered the perivitelline space without the aid of follicle cells. The relationships between the establishment of self-sterility and these morphological and functional changes in ovarian eggs are discussed. [ABSTRACT FROM AUTHOR]

The egg coats of a starfish ( Patiria miniata) are examined before, during, and after the cortical reaction by scanning and transmission electron microscopy. The unfertilized egg is closely invested by a vitelline coat about 300 μm thick, and cortical granules are scattered in the peripheral cytoplasm. After insemination, as the cortical granules undergo exocytosis, the cortical reaction sweeps over the egg surface. Much of the material ejected from the cortical granules adheres to the inner surface of the vitelline coat as a dense layer about 40 μm thick and as scattered spheres and hemispheres, each about 1 μm in diameter. Together, the vitelline coat and the adherent cortical granule material form the fertilization envelope, which becomes separated from the plasma membrane of the egg by a perivitelline space. The perivitelline space contains some flocculent material, which is too diffuse and discontinuous to be considered a hyaline layer. Possible functions of the starfish egg coats are discussed. [ABSTRACT FROM AUTHOR]

We have studied the differentiation of the vitelline coat (VC) of the ascidian Ciona intestinalis. In the young previtellogenic oocyte the vitelline coat precursor material (VCPM) makes its first appearance as patches of fibrous material in close apposition to the outer surface of the oocyte. The presence of subcortical vescicles containing a fuzzy electron-dense material and their opening into the oocyte surface parallels the formation of VCPM. Numerous microvillar-like structures emerge from the oocyte surface. When the VCPM completely surrounds the oocyte the microvilli are withdrawn. An overall increase of VCPM parallels the growth of the oocyte. The next step in the differentiation of the vitelline coat consists in the packing of the constituent fibrils in a dense layer at its outer surface, i.e. the one in contact with the follicle cells. At this time the VC is penetrated by microvilli protruding both from the oocyte and follicle cells. The VC reaches its final structure and thickness at the time the test cells are extruded into the perivitelline space. The participation of the follicle cells in VC organization is also discussed. [ABSTRACT FROM AUTHOR]

Spawned ascidian oocytes are surrounded by a membrane called the chorion (or vitelline coat) and associated with two populations of maternally-supplied cells. Outside the chorion are follicle cells, which may affect the buoyancy of eggs. Inside the chorion are test cells, which during oogenesis provision the egg and which after fertilisation contribute to the larval tunic. The structure of maternal cells may vary between species. The model ascidian Ciona intestinalis has been recently split into two species, currently named type A and type B. The ultrastructure of extraembryonic cells and structures from type A embryos has been reported. Here we describe the ultrastructure of follicle and test cells from C. intestinalis type B embryos. Test cells are about 5 µm in diameter and line the inside of the chorion of developing embryos in a dense sheet. Follicle cells are large (> 100 µm long) and spike-shaped, with many large vesicles. Terminal electron dense granules are found towards the tips of spikes, adjacent to cytoplasm containing numerous small electron dense bodies connected by filaments. These are probably vesicles containing material for the terminal granules. Removal of maternal structures and cells just after fertilisation, as commonly used in many experiments manipulating C. intestinalis development, has been reported to affect embryonic patterning. We examined the impact of this on embryonic ectoderm cells by scanning electron microscopy. Cells of embryos that developed without maternal structures still developed cilia, but had indistinct cell boundaries and a more flattened appearance than those that developed within the chorion.

Vitelline coats (VC) were isolated from the eggs of Bufo japonicus, and were added with sperm in reconstituted salt solution, which mimics the physiological role of jelly envelopes, to determine the rates of sperm binding per unit area (0.2 mm 2 ) of VC. The rate of sperm binding to VC from uterine eggs was high, but was low to VC from coelomic eggs and eggs activated in 1/20 De Boer's solution (DB) and moderately low to VC from eggs activated in DB. The binding rate increased when VC from coelomic eggs were treated with extracts of the pars recta portion of the oviduct. The sperm that bound to VC were not acrosome-reacted and their binding to VC required both a low salinity, assuring motility of sperm, and sufficiently high levels of Ca 2+ and Mg 2+ . The rate of sperm binding was reduced by either coexisting solubilized VC materials, periodate-oxidation of VC or the pretreatment of VC with Fab fragments of anti-VC antibodies, which reacted mostly to carbohydrate residues of VC glycoproteins. Sperm-VC binding assays in combination with gel-filtrated VC components revealed that the fractions containing 36-39 kDa components were most effective both in inhibiting the binding and in neutralizing the antibody induced inhibition of binding. These results indicate that carbohydrate moieties in 36-39 kDa glycoproteins of VC, exposed as a result of hydrolysis by the oviducal pars recta protease, are involved in binding with fertilizing sperm.

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.