Extracellular Coat Research Articles

Fertilization envelope in diapause eggs of Pontella mediterranea (crustacea, copepoda)

The sequence and timing of morphological changes during envelope formation was followed in diapause eggs of Pontella mediterranea (Crustacea, Copepoda). The multilayer coat enveloping these eggs resulted from the exocytosis of 4 types of cortical vesicles that sequentially released their contents in the perivitelline space. These included small high-density vesicles (hDV) with electron-dense material, vesicles (V) with dense ring granules and a uniform matrix contained within the same compartment, large high-density (HDV) vesicles, and large moderately dense (MDV) vesicles. All of these cortical vesicles were present in newly spawned, fertilized eggs. Their exocytosis resulted from egg activation. One of these cortical vesicles (V) was similar in morphology to the intracisternal granules precursors of endogenous yolk. Intracisternal granules, characteristic of previtellogenic oocytes of many crustaceans, were present in previtellogenic oocytes of P. mediterranea but disappeared in later stages of oocyte development once yolk formation was completed. We discuss the role of cortical vesicles in the formation of the complex extracellular coat enveloping copepod diapause eggs.

Transgenic mouse eggs with functional hamster sperm receptors in their zona pellucida.

Sperm receptors are located in the mammalian egg extracellular coat, or zona pellucida. Mouse and hamster sperm receptor glycoproteins, mZP3 (83 x 10(3) M(r)) and hZP3 (56 x 10(3) M(r)), respectively, have very similar polypeptides (44 x 10(3) M(r); 81% identical) that are glycosylated to different extents. Purified mZP3 and hZP3 can bind to mouse sperm, prevent them from binding to eggs and induce them to undergo exocytosis, the acrosome reaction, in vitro. A DNA construct that placed the hZP3 gene under the control of mZP3 gene 5'-flanking sequence was used in this report to produce two mouse lines that harbored the foreign sperm receptor transgene. In both lines, the transgene was expressed only by growing oocytes, at a level comparable to that of the endogenous mZP3 gene, and the developmental pattern of transgene expression resembled that of the mZP3 gene. In addition to mZP3, transgenic mouse oocytes synthesized and secreted a glycoprotein indistinguishable from hZP3, and incorporated both glycoproteins into a mosaic zona pellucida. Importantly, hZP3 purified from such zonae pellucidae exhibited both sperm receptor and acrosome reaction-inducing activities in vitro and, following fertilization of transgenic mouse eggs, was inactivated. These results demonstrate that a biologically active foreign sperm receptor can be synthesized and secreted by transgenic mouse oocytes, assembled into a mosaic zona pellucida, and inactivated following fertilization as part of the secondary block to polyspermy.

Isolation and biochemical characterization of two isoforms of a boar sperm zona pellucida-binding protein

Protein-carbohydrate complementarity has been recognized as a general mechanism of gamete recognition and adhesion in the process of fertilization throughout the whole animal kingdom. It appears that carbohydrate-binding molecules on the anterior sperm head surface mediate the binding of the male gamete to certain glycoconjugates present on the egg's extracellular coat. Subtle differences in protein and carbohydrate conformation may confer to this interaction a species-specific character. The mechanism responsible for gamete recognition is, however, poorly understood. A step in its elucidation is the characterization of the complementary molecules on the egg and sperm surfaces. With this aim we report here the isolation and partial structural characterization of two isoforms of a zona pellucida-binding protein (which we call AWN-1 and AWN-2) from boar spermatozoa, including partial sequence determination, assignment of disulphide bonds and identification of an N-terminal blocking group. AWN-1 and AWN-2 were isolated from acid extracts of washed ejaculated sperm and were present in seminal vesicle secretions, but absent in samples of epididymal fluid, suggesting a seminal vesicle origin for these sperm proteins. No analogous protein sequence could be found in the MIPS data bank, indicating that the AWN proteins may belong to a novel mammalian protein family involved in fertilization.

Mouse gamete adhesion molecules.

Complementary adhesion molecules are located on the surface of mouse eggs and sperm. These molecules support species-specific interactions between sperm and eggs that lead to gamete fusion (fertilization). Modification of these molecules shortly after gamete fusion assists in prevention of polyspermic fertilization. mZP3, an 83,000-Mr glycoprotein located in the egg extracellular coat, or zona pellucida, serves as primary sperm receptor. Gamete adhesion in mice is carbohydrate-mediated, since sperm recognize and bind to certain mZP3 serine/threonine- (O-) linked oligosaccharides. As a consequence of binding to mZP3, sperm undergo the acrosome reaction, which enables them to penetrate the zona pellucida and fertilize the egg. A 56,000-Mr protein called sp56, which is located in plasma membrane surrounding acrosome-intact mouse sperm heads, is a putative primary egg-binding protein. It is suggested that sp56 recognizes and binds to certain mZP3 O-linked oligosaccharides. Acrosome-reacted sperm remain bound to eggs by interacting with mZP2, a 120,000-Mr zona pellicida glycoprotein. Thus, mZP2 serves as secondary sperm receptor. Perhaps a sperm protease associated with inner acrosomal membrane, possibly (pro)acrosin, serves as secondary egg-binding protein. These and, perhaps, other egg and sperm surface molecules regulate fertilization in mice. Homologous molecules apparently regulate fertilization in other mammals.

The Physiological Effect of Aureococcus anophagefferens ("brown tide") on the Lateral Cilia of Bivalve Mollusks.

The "brown tide" alga, Aureococcus anophagefferens, had no effect on the activity of the lateral cilia of isolated gills of the bivalve mollusks Argopecten irradians, Geukensia demissa, and Mya arenaria. A. anophagefferens caused a significant decrease in the activity of lateral cilia of five other bivalve mollusks, Crassostrea virginica, Ostrea edulis, Mercenaria mercenaria, Modiolus modiolus, and Mytilus edulis. Exposure of isolated gills of M. edulis to the water from which A. anophagefferens had been removed, or to polystyrene beads at the same concentration as A. anophagefferens, had no effect upon the activity of the lateral cilia. Thus, the inhibition of the lateral cilia is not caused by a compound excreted into the water, nor is it the result of the high density of cells. The response of lateral cilia to dopamine was identical to the response to A. anophagefferens; lateral cilia that were inhibited by dopamine were also inhibited by A. anophagefferens. Pretreatment of the gills of M. edulis with the dopamine antagonist ergometrine blocked the inhibition of the lateral cilia by both dopamine and A. anophagefferens. A water-soluble inhibitory compound was released from A. anophagefferens by exposing the cells to amylase, and then removing the cells by filtration. The effect of this inhibitory compound was also blocked by ergometrine. We propose that the isolated gills are digesting the extracellular coat of A. anophagefferens releasing a water soluble dopamine-mimetic compound that causes inhibition of lateral cilia.

Embryogenesis and Larval Development of the Asteroid Patiriella regularis Viewed by Light and Scanning Electron Microscopy.

The sea star Patiriella regularis (Verrill, 1867) has indirect development through bipinnaria and brachiolaria larvae. Development of this species is typical of asteroids with planktotrophic larvae and takes 9-10 weeks. The embryos develop through a wrinkled blastula and hatch as early gastrulae. In contrast to most asteroids, a third enterocoel forms on the left side of the stomach of the bipinnaria. This structure gives rise to the left posterior coelom; its significance is discussed. We suggest that this coelom is homologous to the trunk coelom in enteropneust embryology. The surface features of the larvae were examined by scanning electron microscopy. Newly hatched gastrulae are covered by cilia, and the bipinnaria have bands of cilia that follow the contours of the larval processes. A previously undescribed plug-like structure positioned on the post-oral surface appears to function as a seal for the mouth. Brachiolaria larvae have three brachiolar arms and a centrally located adhesive disc. Each arm is covered by adhesive papillae. Raised epithelial cells that dot the surface of the papillae and adhesive disc may be batteries of secretory cells. The brachiolar arms have an extracellular coat that may serve as a protective cover for the adhesive surfaces. Competent brachiolaria swim along the substratum and exhibit searching behavior with flexure of the median brachium. They settle on the undersides of natural shell substrata and do not respond to a primary algal film. Shade appears to be an important factor in settlement and metamorphosis in P. regularis. Metamorphosis takes 5-6 days, and the post-larvae take up a free existence at a diameter of 450-500 {mu}m. The indirect development of P. regularis contrasts with the lecithotrophic and viviparous modes of development of other Patiriella species and provides the comparative basis to determine the ontogenic changes involved with evolution of direct development in the genus. The use of the divergent life histories of Patiriella as a model system for the study of evolutionary change in development is discussed.

Analysis of the polypeptide composition of the cell walls of Neurospora crassa. Similarities with the proteinaceous material secreted by the slime variant

The polypeptide composition of cell walls from the wild-type strain of Neurospora crassa is compared with that of the proteinaceous extracellular coat (PEM) secreted by the slime strain of this fungus. Analyses included determination of the polypeptide pattern by polyacrylamide gel electrophoresis and blotting followed by staining with Concanavalin A and antibodies raised against the overall antigenic components present in either (whole cell walls or PEM) structure. A complex protein assortment was found associated to the walls of the wild type strain. The similarities observed between the polypeptide patterns of the cell walls and PEM, in addition to the immunological cross-reactivity exhibited by some proteinaceous species, suggest a close relationship between PEM and the proteins present in the wild-type cell walls.

Identification and characterization of the major components of the Oncorhynchus mykiss Egg Chorion

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS-PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic-acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the "asparagine-linked" glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS-PAGE. These results suggest that the four main chorion polypeptides might share common structural features.

Genomic organization and polypeptide primary structure of zona pellucida glycoprotein hZP3, the hamster sperm receptor

During the course of fertilization in mammals, free-swimming sperm bind tightly to receptors located in the egg extracellular coat, or zona pellucida. Recently, the hamster sperm receptor, a 56,000 M r zona pellucida glycoprotein called hZP3, was identified and partially characterized (C. C. Moller et al., (1990). Dev. Biol. 137, 276–286). Here, we describe genomic cloning of hZP3, certain organizational features of the hZP3 gene, and primary structures of hZP3 mRNA and polypeptide. The findings are compared with reported results of comparable analyses of the mouse sperm receptor, an 83,000 M r zona pellucida glycoprotein called mZP3. Such comparisons reveal a high degree of conservation of genomic organization and polypeptide structure for the two mammalian sperm receptors, despite the considerable difference in their M rs. These findings are of interest in view of the extremely restricted expression of the ZP3 gene during development and the important role of ZP3 oligosaccharides in gamete adhesion.

Distribution of lectin binding sties and anionic sites in the rat zona pellucida

The zona pellucida (ZP) in mammals is an extracellular coat which surroudns the oocyte during growth, fertilization and early development, consisting of a limited number of glycoproteins. Carbohydrates residues and charge properties of the ZP are believed to have a role in sperm-egg interaction. The purpose of the present study was to furtehr characterize the specific nature and distribution of the carbohydrate residues of the ZP and cumulus cells and anionic residues at the ultrastructural level.We used lectins as probes for specific sugar residues and polycationic colloidal gold (PCG) as a marker for aninonic sites. Immature female rats were induced to superovate. Ovulated, cumulus-oocyte complexes were isolated and fixed with 2.5% Glutaraldehyde, dehydrated and embedded in Araldite or LR-Gold. For light microscopic (LM) histochemistry, 0.5 μm sections were cut, deresined by Na-methoxide and labeled with biotin-derivatized lectins as probes and avidin biotin-peroxidase complex as visualant.

Morphological effects of aspirin and prostaglandin on the canine gastric mucosal surface

Abstract An iodoplatinate technique that selectively stains phospholipid was used to evaluate morphologically the effects of aspirin and 16,16-dimethyl prostaglandin E 2 on canine gastric mucosa in an in vitro Ussing chamber system. The association between these morphological alterations and the changes in mucosal surface hydrophobicity as determined by contact angle analysis was also investigated. Prostaglandin (1 μg/ml) did not alter aspirin-induced cell injury, although it reduced (45%) the detachment of damaged cells from the lamina propria. Mucus release appeared to be stimulated independently both by aspirin and prostaglandin treatment and by the blotting and drying procedure routinely performed before contact angle analysis. When an extracellular mucus gel coat was present, it contained numerous iodoplatinate-reactive vesicles and myelinated or lamellated structures. Iodoplatinate-reactive substances also appeared at the luminal surface of the mucus gel as a filamentous band. This band of reactivity was frequently seen after simultaneous treatment with prostaglandin and aspirin but only sporadically observed after aspirin treatment alone. The hydrophobic nature of the canine gastric mucosa under the above experimental conditions correlated well (r = 0.743, p

Structural and functional relationships between mouse and hamster zona pellucida glycoproteins

The hamster egg's extracellular coat, or zona pellucida, consists of three glycoproteins, designated hZP1, hZP2, and hZP3, that exhibit extensive heterogeneity on SDS-PAGE. hZP1 is a relatively minor component of hamster zonae pellucidae, as compared with hZP2 and hZP3. In the presence of reducing agents, hZP1, 200,000 apparent M r, migrates on SDS-PAGE with an apparent M r of 103,000. This suggests that hZP1, like mouse ZP1, is composed of two polypeptides held together by intermolecular disulfides. When purified hamster ZP glycoproteins were tested at relatively low concentrations in an in vitro competition assay, employing either hamster or mouse gametes, only hZP3 (56,000 apparent M r) exhibited sperm receptor activity (i.e., inhibited binding of sperm to eggs). Thus, apparently hZP3 is the hamster counterpart of mouse ZP3, the mouse egg receptor for sperm. Furthermore, at relatively high concentrations, solubilized hamster egg ZP preparations induced both hamster and mouse sperm to undergo the acrosome reaction in vitro. hZP3 is encoded by a relatively abundant ovarian mRNA that is detected by a mouse ZP3 cDNA probe and is the same size, about 1.5 kb, as mRNA encoding the mouse sperm receptor, ZP3 (83,000 apparent M r). Like mouse ZP2, hZP2 undergoes limited proteolysis following artificial activation of hamster eggs in vitro. Results of in vitro assays employing intact eggs and isolated zonae pellucidae demonstrate that hamster eggs possess a ZP2-proteinase which has a substrate specificity similar to that of the mouse enzyme. These observations are discussed in terms of structural and functional relationships that may exist between hamster and mouse zona pellucida glycoproteins.

Profile of a mammalian sperm receptor1,2

Complementary molecules on the surface of eggs and sperm are responsible for species-specific interactions between gametes during fertilization in both plants and animals. In this essay, several aspects of current research on the mouse egg receptor for sperm, a zona pellucida glycoprotein called ZP3, are addressed. These include the structure, synthesis, and functions of the sperm receptor during oogenesis and fertilization in mice. Several conclusions are drawn from available information. These include (I) ZP3 is a member of a unique class of glycoproteins found exclusively in the extracellular coat (zona pellucida) of mammalian eggs. (II) ZP3 gene expression is an example of oocyte-specific and, therefore, sex-specific gene expression during mammalian development. (III) ZP3 is a structural glycoprotein involved in assembly of the egg extracellular coat during mammalian oogenesis. (IV) ZP3 is a sperm receptor involved in carbohydrate-mediated gamete recognition and adhesion during mammalian fertilization. (V) ZP3 is an inducer of sperm exocytosis (acrosome reaction) during mammalian fertilization. (VI) ZP3 participates in the secondary block to polyspermy following fertilization in mammals. (VII) The extracellular coat of other mammalian eggs contains a glycoprotein that is functionally analogous to mouse ZP3. The unique nature, highly restricted expression, and multiple roles of ZP3 during mammalian development make this glycoprotein a particularly attractive subject for investigation at both the cellular and molecular levels.

Quantitative energy-filtered image analysis in cytochemistry: II. Morphometric analysis of element-distribution images

A combination of energy-filtered electron microscopy (EFEM) and an image-analysis system (IBAS/2000) is used for a morphometric analysis of chemical reaction products in cells. Electron energy-loss spectroscopic element-distribution images are acquired from cytochemical reaction products in a variety of cellular objects: (1) colloidal thorium particles in extra-cellular coat material, (2) iron-containing ferritin particles in liver parenchymal cells, (3) barium-containing reaction products in endoplasmic reticulum stacks, (4) elements present in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. Areas or area fractions are determined from such element-distribution images by application of an objective image segmentation method. By superposition of two or more element-distribution images, mutual element relations are qualitatively established in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. By comparing electron spectroscopic images (ESI) with element-distribution images, the mutual contrast per elemeny relations are quantitatively investigated. The obtained gain in resolution in such electron energy-loss spectroscopic element-distribution images will be explained and discussed.

Characterization of a proteinaceous extracellular coat synthesized by the ?slime? variant of Neurospora crassa

Cells of the “slime” strain of Neurospora crassa synthesize a coherent extracellular material which remains attached to the cell surface, but is released into the liquid medium by shaking. The material was purified and studied by different criteria. By electron microscopy it appears as long wavy sheets which strongly bind concanavalin A, but not wheat germ agglutinin, and maintain their integrity in the absence of structural polysaccharides. Analysis of the purified material revealed that it was free of contaminating membranes; it contained more than 70% protein, 1% neutral sugars (glucose, mannose, fucose and galactose), less than 2% lipids and ca. 4% not-characterized hexosaminelike compounds. Its polypeptide pattern as determined by PAGE was complex. The significance of this material is discussed.

Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20.

Immunization of male and female animals with extracts of whole sperm cells is known to cause infertility. Also, men and women who spontaneously produce antisperm antibodies are infertile but otherwise healthy. Although the critical sperm antigens are unknown, these observations have led to the proposal that sperm proteins might be useful in the development of a contraceptive vaccine. The guinea pig sperm surface protein PH-20 is essential in sperm adhesion to the extracellular coat (zona pellucida) of the egg, a necessary initial step in fertilization. Here, we report that 100% effective contraception was obtained in male and female guinea pigs immunized with PH-20. Antisera from immunized females had high titres, specifically recognized PH-20 in sperm extracts, and blocked sperm adhesion to the egg zona pellucida in vitro. The contraceptive effect was long-lasting and reversible: immunized females, mated at intervals of six to fifteen months after immunization, progressively regained fertility.

Primary structure of the mouse sperm receptor polypeptide determined by genomic cloning.

The mouse sperm receptor, a glycoprotein called ZP3, is synthesized and secreted by growing oocytes. It is present in more than a billion copies in the unfertilized egg's extracellular coat, or zona pellucida. We have cloned and characterized a region of the mouse (CD-1) genome that spans 10 kilobases of the ZP3 locus. The genomic clones described encompass the entire ZP3 coding region, which contains eight exons. The exons were identified, mapped, and sequenced, yielding the entire primary structure of the ZP3 polypeptide chain (424 amino acids; Mr, 46,300), which includes a 22-amino acid signal sequence. In addition, sequencing of genomic clones has revealed some unusual features of ZP3 mRNA and a region just downstream of the ZP3 gene.

Characterization of the rabbit sperm membrane autoantigen, RSA, as a lectin-like zona binding protein

Adhesion between spermatozoa and the egg's extracellular coat, the zona pellucida, involves the sperm's zona binding proteins (ZBP) and their interaction with the carbohydrate residues of the zona. To investigate this interaction in more detail, a purified nonenzymatic ZBP, the rabbit sperm membrane autoantigen, RSA, was used. RSA-zona binding was demonstrated on nitrocellulose blots and by using the Denny-Jaffe crosslinking reagent which identified an 87,000 molecular weight zona component as the ligand for RSA. The RSA-zona binding was of high affinity with a dissociation constant of 5.6 × 10 −13 M. Furthermore, the binding of capacitated spermatozoa to intact zona was inhibited in the presence of RSA. Characterization of the RSA-zona interaction with a variety of simple and complex carbohydrates indicated that the sulfated, complex carbohydrates fucoidin, dextran sulfate, chondroitin sulfate B, and heparin strongly inhibited RSA-zona binding while chondroitin sulfates A and C, cholesterol-3-sulfate, and monosaccarides such as galactose inhibited RSA-zona binding only weakly. It is concluded that RSA functions as a sperm lectin-like molecule to bind the spermatozoon to the zona pellucida.

Identification of a secondary sperm receptor in the mouse egg zona pellucida: Role in maintenance of binding of acrosome-reacted sperm to eggs

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polycolonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.

Eggs, Sperm, and Sugar. A Recipe for Fertilization

The extracellular coat surrounding unfertilized mammalian eggs contains receptors that sperm recognize and bind in a species-specific manner during fertilization. These sperm receptors are glycoproteins that regulate interactions between eggs and sperm both before and after fertilization. Recent evidence suggests that it is certain of the sperm receptor's oligosaccharides that mediate gamete recognition and binding during mammalian fertilization.