Egg Coat Research Articles

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.

Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N-acetylglucosaminidase found in cortical granules was identified as beta-hexosaminidase B, the beta, beta homodimer. Inhibition of N-acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase-binding site, accounting for the block in sperm binding to the zona pellucida.

Glycosyltransferases as cell adhesion molecules

Specific glycosyltransferases are believed to be present on the cell surface, where they may function as cell adhesion molecules by binding oligosaccharide substrates on adjacent cell surfaces or in the extracellular matrix. The best studied of these is β1,4-galactosyltransferase, which mediates sperm binding to the egg coat and selected cell interactions with the basal lamina. Molecular models have recently been presented to account for the expression of β1,4-galactosyltransferase on the cell surface, thus enabling one to alter its surface expression and to examine the consequences on fertilization and development.

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.

S20.11 Comparative study of the O-linked carbohydrate chains released from the jelly coat of amphibian eggs

S20.11 Comparative study of the O-linked carbohydrate chains released from the jelly coat of amphibian eggs

High-Molecular-Weight Protease Complexes (Proteasomes) of Sperm of the Ascidian, Halocynthia roretzi: Isolation, Characterization, and Physiological Roles in Fertilization

It has been proposed that a chymotrypsin-like enzyme plays a key role in sperm penetration through the vitelline coat of eggs of the ascidian, Halocynthia roretzi . In this report, two kinds of chymotrypsin-like enzymes were isolated from spermatozoa of H. roretzi. Both enzymes were characterized as high-molecular-weight protease complexes that comprised several different subunits and that showed several different protease activities. Both showed immunoreactivity with anti-proteasome antibody. One showed properties characteristic of the 20 S proteasome and its molecular mass was 630 kDa, whereas the other protease complex of 930 kDa showed inhibitor susceptibility different from that of the 20 S proteasome. Upon the sperm reaction which is induced by treatment with high pH, the proteasomes were found to be released from sperm and they had the activity to digest the vitelline coats. These results suggest that the proteasomes are exposed extracellularly when sperm contact with the egg coat and may function as lysins in the fertilization process.

Botulinum ADP-Ribosyltransferase C3 Induces Elevation of the Vitelline Coat of Ascidian Eggs

Botulinum exoenzyme C3 ADP-ribosylates a 23 kDa protein of unfertilized eggs of the ascidian, Halocynthiaroretzi. Microinjection of C3 into the eggs induced elevation of the egg vitelline coat. Co-injection of heparin or EGTA with C3 inhibited the inducing effect of C3. The vitelline coat of eggs which had been previously co-injected with heparin and C3 was elevated by addition of calcium ionophore, but not by insemination. C3 also induced an increased formation of inositol 1,4,5-triphosphate (IP3) in ascidian egg membranes. Thus the ADP-ribosylation of small GTP-binding protein by C3 seems to be responsible for elevation of the vitelline coat of ascidian eggs through IP3 formation and intracellular calcium mobilization.

Vitelline coat of Unio elongatulus egg: I. Isolation and biochemical characterization

In this study we isolated and purified the vitelline coat (vc) of Unio elongatulus eggs in order to investigate its protein and carbohydrate composition. SDS dissolved up to 80% of the vitelline coat protein content whereas 100 mM Ammonimum acetate (AA) at pH 11 and 1 mM lithium diiodosalicylate (LISH) dissolved only 40-50%. The ability of extremes of pH or LIS to solubilize the vitelline coats on eggs was then investigated. The results showed that pH from 7 to 11 progressively dissolved the vitelline coats without gross damage to the oocytes. SDS-PAGE of the solubilized material revealed only two components corresponding to the main components revealed by SDS-PAGE of the isolated vcs. These peptides have an apparent MW of 220 and 180 kD, are ConA positive, and seem to be connected to each other to form polycomponents. The latter feature is suggested by the electrophoretic pattern of the solubilized material under nondenaturing conditions.

Specific binding of acrosome-reaction-inducing substance to the head of starfish spermatozoa.

In the starfish, spermatozoa undergo the acrosome reaction upon encountering the jelly coat of eggs. A highly sulphated glycoprotein in the jelly coat is called acrosome-reaction-inducing substance (ARIS) because it is the key signal molecule to trigger the acrosome reaction. The activity of ARIS is mainly attributed to its sulphate and saccharide residues. The extremely large molecular size and species-specific action of ARIS suggest the presence of a specific ARIS receptor on the sperm surface, but no experimental evidence for the receptor has been presented. We therefore measured specific binding of ARIS and its pronase digest (P-ARIS), which retains the full activity of ARIS, to homologous spermatozoa by using fluorescein-isothiocyanate-labelled ARIS and 125I-labelled P-ARIS, respectively. The spermatozoa had the ability to bind ARIS, as well as P-ARIS, specifically. The binding was species-specific and mostly localised to the head region of spermatozoa. Scatchard plot analysis indicated the presence of one class of ARIS receptor on the surface of acrosome-intact spermatozoa. Furthermore, the specific binding of P-ARIS to the anterior region of sperm heads was microscopically confirmed by using P-ARIS conjugated to polystyrene latex beads with intense fluorescence. It is concluded that starfish spermatozoa have a specific receptor for ARIS on the surface of the anterior region of heads.

Spawning Mode and Reproductive Output of the Tropical Cephalopod Idiosepius pygmaeus

Reproductive biology of the tropical sepioid cephalopod Idiosepius pygmaeus was investigated in wild specimens and in individuals maintained in aquaria through the adult life span. This species produced multiple egg batches over 80% of the observable adult weight range, indicating a coordination of reproductive and somatic growth. Reproductive output was consistent within, but variable between, individuals. Oocyte synthesis and maturation occur continuously after sexual maturation has been reached. Senescence and death are not related to an exhaustion of reproductive potential. On average, captive female I. pygmaeus with access to unlimited food produced 640 eggs in 11 batches over 18 d. When reproductive output was expressed as a ratio of dry female body weight, on average, specimens had incorporated five times their body weight into eggs and egg coatings. Under food stress, captive specimens laid fewer eggs but maintained egg size and periodicity of egg laying. Food stress had no effect on either laying duration or weight at death. This study provides further evidence that terminal spawning modes are not ubiquitous amongst cephalopods.

1H-and 13C-n.m.r. spectroscopy of 2-oxo-3-deoxy-D-glycero-D- galactononulosonic acid-containing oligosaccharide-alditols bearing Lewis X, Lewis Y and A-Lewis Y determinants isolated from the jelly coat of Pleurodeles waltl eggs.

Three acidic oligosaccharide-alditols carrying Lewis X, Lewis Y and A-Lewis Y determinants were isolated from the jelly coat of Pleurodeles waltl eggs. These compounds possess the following structures. Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3[2-oxo-3-deoxy-D- glycero-D-galactononulosonic acid (KDN)alpha 2-6] GalNAc-ol; Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(KDN alpha 2-6) GalNAc-ol and Fuc alpha 1-2(GalNAc alpha 1-3)Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(KDN alpha 2-6)GalNAc-ol. The complete 1H-n.m.r.-spectrum assignment for the three compounds and the 13C-n.m.r. analysis of the A-Lewis Y determinant-containing heptasaccharide are reported.

Xenopus laevis sperm proteins, previously identified as surface proteins with egg coat binding capability, are indeed histone H4, histone H3, and sperm specific protein SP2

Recently, four Xenopus sperm proteins thought to be involved in binding to the egg envelope were identified (Lindsay and Hedrick, J. Exp. Zool., 245:286-293, '88). We have studied the three more abundant ones of apparent molecular weight of 14, 19, and 25 kd in SDS-PAGE. We have shown that these proteins are indeed nuclear basic proteins: the 14 kd is the histone H4, the 19 kd is the histone H3, and the 25 kd is the sperm-specific protein SP2.

Control of the fertilization process by the egg coat: How does it work in humans

Control of the fertilization process by the egg coat: How does it work in humans

Primary structure of neutral and acidic oligosaccharide-alditols derived from the jelly coat of the Mexican axolotl. Occurrence of oligosaccharides with fucosyl(alpha 1-3)fucosyl(alpha 1-4)-3-deoxy-D-glycero-D-galacto-nonulosonic acid and galactosyl(alpha 1-4)[fucosyl(alpha 1-2)]galactosyl(beta 1-4)-N-acetylglucosamine sequences.

Six major neutral and acidic oligosaccharide-alditols were prepared from the jelly coat of Mexican axolotl eggs. These compounds were demonstrated to contain 3-deoxy-D-glycero-D-galacto-nonulosonic acid (dNloA) and L-fucose (Fuc). The structures of the six major oligosaccharides were established as follows: [sequence: see text]

A novel saccharide structure, Xyl 1→3 Gal 1→ (SO 3−)3, 4 Fuc→, is present in acrosome reaction-inducing substance of the starfish, Asterias amurensis

The jelly coat of echinoderm eggs contains a glycoconjugate, acrosome reaction-inducing substance (ARIS), that is essential for triggering the acrosome reaction in homologous spermatozoa. In the starfish, Asterias amurensis, ARIS is a sulfated glycoprotein of an apparent molecular size of greater than 10(7). Since its biological activity is dependent mostly on its sugar moiety, oligosaccharides liberated by hydrolysis with 10 mM H2SO4 for 60 min at 100 degrees C from pronase digests of ARIS (P-ARIS) were chemically analyzed. The main oligosaccharide purified by high-performance anion-exchange chromatography was determined to be Xyl1----3Gal1----(SO3-)3,4Fuc by compositional analysis and FAB mass spectrometry. This structure indicates that ARIS possesses a novel saccharide chain having sulfated fucose as an internal residue.

Complementarity between sperm surface beta-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding.

Despite its importance, the molecular basis of mammalian gamete recognition has remained unclear. The enzyme beta-1,4-galactosyltransferase (Gal-transferase) has been viewed traditionally as a biosynthetic component of the Golgi complex, but is also found on the surface of many cells where it can bind its specific glycoside substrate on adjacent cell surfaces or in the extracellular matrix. In mouse it has been suggested that Gal-transferase on the sperm head mediates fertilization by binding oligosaccharide residues in the egg coat, or zona pellucida, and that the ability of the zona pellucida to bind sperm is conferred by oligosaccharides of the ZP3 glycoprotein. However, it has not been confirmed that Gal-transferase and ZP3 are in fact complementary gamete receptors whose interaction mediates sperm-egg binding. Here we show that mouse sperm Gal-transferase specifically recognizes those oligosaccharides on ZP3 that have sperm-binding activity, but does not interact with other zona pellucida glycoproteins. In contrast, all zona pellucida glycoproteins are recognized by non-sperm Gal-transferase, demonstrating a more stringent substrate specificity for the sperm enzyme. This interaction is required for sperm-egg binding because blocking or removing the binding site for Gal-transferase on ZP3 inhibits its ability to bind sperm. After the release of the sperm acrosome, the transferase relocalizes to a new membrane domain where it can no longer bind to ZP3, which is consistent with the inability of acrosome-reacted sperm to bind ZP3 or to initiate binding to the zona pellucida. Following fertilization, ZP3 is modified by egg cortical granule secretions so that it loses sperm receptor activity, which can be accounted for by a selective loss of its binding site for sperm Gal-transferase. These results show that sperm surface beta-1,4-galactosyltransferase and the egg-coat glycoprotein ZP3 are complementary adhesion molecules that mediate primary gamete binding in the mouse.

A histological study of the accessory reproductive organs of female Loligo forbesi (Cephalopoda: Loliginidae)

In Loligo forbesi Steenstrup, the female reproductive system consists of the ovary and accessory reproductive organs which include the oviducal gland, the nidamental gland, the accessory nidamental gland and seminal receptacle. Histological studies were made on the accessory reproductive organs of female L. forbesi. The various changes observed during maturation are described and the functional significance discussed. The secretions produced by the oviducal gland and nidamental gland apparently form the egg coats. The seminal receptacle serves to store spermatozoa after mating. The function of the accessory nidamental gland is unknown.

The Involvement of Sperm and Egg Glycosidases in Animal Fertilization

A number of glycosidases have been detected in the seminal fluid of the male genital tract of mammals and in sperm and eggs of many invertebrate and vertebrate animals. Although some of these enzymes have been isolated and characterized, the elucidation of their presumed function in the fertilization process proved to be rather difficult. In relatively recent investigations evidence was presented that glycosidases are involved in binding of sperm to the acellular egg-coats of ascidian and mouse eggs, a step that is indispensable for successful fertilization. Furthermore, hyaluronidase and possibly β-N-acetylglucosaminidase from mammalian sperm are claimed to be involved in the penetration of the acellular egg coats, particularly through the cumulus oophorus. The precise mechanism of sperm penetration and whether it can be applied to fertilization in all mammals is still debated. Ascidians and mice are so far the only animals from which evidence exists that egg-bound glycosidases might be involved in the slow block to polyspermy.In summary, good evidence exists that glycosidases play an essential role in gamete interactions, particularly in the interaction of the sperm cell with the acellular egg matrix.

Self-Nonself Recognition Activity Extracted from Self-Sterile Eggs of the Ascidian, Ciona intestinalis: (ascidian/fertilization/allo-recognition/acid extract/HPLC).

Eggs of the hermaphrodite, self-sterile ascidian, Ciona intestinalis, were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non-autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self-nonself recognition activity. This activity was heat-stable and insensitive to trypsin, but was destroyed by V-8 protease and α-glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo-recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm-egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc-enriched nonspecific inhibitor of sperm-egg binding, which could be the primary effector of self-incompatibility, and Glx-enriched modulators, which serve as acceptors of allo-sperm. The cooperative interactions of these components may be responsible for the diversity of allo-recognition in Ciona gametes.

Current Concepts in Gamete Receptors for Fertilization in Mammals

This chapter discusses the current information available on gamete receptors involved in interactions, as well as immunobiology of gamete interaction during fertilization in mammals. The progress in biochemical analysis of sperm receptors on Zona Pellucida (ZP) is more rapid than in egg receptors on sperm. This is because of the fact that improved methods are now available to obtain large amounts of homogenous preparations of ZP. The binding and fusion of male and female gametes during fertilization in most cases are species specific and are important to establish the diploid complement required for development of an organism. Fertilization in mammals follows a series of defined events that occur in succession before fusion of gametes occurs. With the advancements in technology for study of reproduction such as hybridoma, monoclonal antibodies (mAbs), complimentary DNA (cDNA), immunoflourescence, autoradiography, and other refined biochemical techniques, a recurring theme has emerged in gamete interaction in different mammalian species in which carbohydrate binding proteins of the sperm surface have been involved in gamete recognition by binding with high affinity and specificity to complex glycoconjugates of the egg coat.