Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA I-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction).     

Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2

Little is known about sperm-binding proteins in the egg envelope of nonmammalian vertebrate species. We report here the molecular cloning and characterization of a recently identified sperm receptor (gp69/64) in the Xenopus laevis egg vitelline envelope. Our data indicate that the gp69 and gp64 glycoproteins are two glycoforms of the receptor and have the same number of N-linked oligosaccharide chains but differ in the extent of O-glycosylation. The amino acid sequence of the receptor is closely related to that of the mouse zona pellucida protein ZP2. Most of the sequence conservation, including a ZP domain, a potential furin cleavage site, and a putative transmembrane domain are located in the C-terminal half of the receptor. Proteolytic cleavage of the gp69/64 protein by a cortical granule protease during fertilization removes 27 amino acid residues from the N terminus of gp69/64 and results in loss of sperm binding to the activated eggs. Similarly, we find that treatment of eggs with type I collagenase removes 31 residues from the N terminus of gp69/64 and has the same effect on sperm binding. The isolated and purified N terminus-truncated receptor protein is inactive as an inhibitor of sperm-egg binding. Earlier studies on the effect of Pronase digestion on receptor activity suggest that this N-terminal peptide may contain an O-linked glycan that is involved in the binding process. Based on these results and the findings on the primary structure of the receptor, a pathway for the maturation and secretion of gp69/64, as well as its inactivation following fertilization, is proposed.     

Human ZP3 restores fertility in Zp3 null mice without affecting order-specific sperm binding

The mammalian zona pellucida surrounding ovulated eggs mediates sperm binding at fertilization, provides a postfertilization block to polyspermy, and facilitates passage of pre-implantation embryos down the oviduct. Although the three zona proteins (ZP1, ZP2, ZP3) are well conserved, mammalian fertilization is relatively specific and human sperm do not bind to the mouse zona pellucida. There are considerable in vitro data that ZP3 acts as a primary sperm adhesion molecule in mice and, by analogy, a similar role has been postulated for human ZP3. Genetically altered mice lacking ZP3 (Zp3(tm/tm)) do not form a zona pellucida and are infertile. To rescue this phenotype, transgenic mice expressing human ZP3 (67% identical to mouse ZP3) were produced and bred with Zp3(tm/tm) null mice. The resultant human ZP3 rescue females had chimeric zonae pellucidae composed of mouse ZP1, mouse ZP2 and human ZP3. Human ZP3 expressed in mouse oocytes had an apparent mass (64 kDa) indistinguishable from native human ZP3 and distinct from mouse ZP3 (83 kDa). Despite the presence of human ZP3, human sperm did not bind to the chimeric zona pellucida, and notwithstanding the absence of mouse ZP3, mouse sperm bound to ovulated eggs in vitro and fertility was restored in vivo. These data have implications regarding the molecular basis of mouse and human sperm binding to their respective zonae pellucidae.     

Human zona pellucida glycoproteins: characterization using antibodies against recombinant non-human primate ZP1, ZP2 and ZP3

Characterization and classification of human zona pellucida glycoproteins is essential to understand the functions of these components during fertilization. To achieve this, antibodies were raised in rabbits against recombinant non-human primate [Bonnet Monkey (Macaca radiata)] zona pellucida proteins, bmZP1, bmZP2 and bmZP3 expressed in Escherichia coli. Antibodies against the three recombinant zona proteins reacted with human zonae as revealed by indirect immunofluorescence. Such antibodies were used as specific probes to further characterize human zona pellucida glycoproteins in Western blot of heat solubilized human zonae pellucidae (hSIZP) resolved by one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Under non-reduced conditions human (h) hZP1, hZP2 and hZP3 resolved as 60, 100 and 53 kDa bands respectively. Under reduced conditions, dominant reactivity of hZP1, hZP2 and hZP3 was localized to 63, 65 and 58 kDa and faint reactivity to 53, 96 and 138 kDa bands respectively. In two-dimensional SDS-PAGE, hZP1 was shown to comprise two chains at 63-58 and 55-45 kDa, each consisting of multiple isomers. hZP2 was less acidic when compared with hZP1 and hZP3 and comprised a major component of 65 kDa and a minor component of approximately 96 kDa. The 65 kDa component displayed a higher degree of charged isomers in comparison with the 96 kDa component. hZP3 comprised a broad band in the range 68-58 kDa. These studies show conclusively that the hZP1 heavy train overlaps with hZP3 and that in previous studies, hZP2 was likely to have been misinterpreted as being hZP1. Our studies failed to distinguish two distinct species of hZP3, unlike previous reports. These studies will further help in our understanding of the nature of human zona pellucida glycoproteins.     

Characterization and representative structures of N-oligosaccharides bound to apolipoprotein H

We studied the structure of N-linked carbohydrates bound to apolipoprotein H by a combination of two methods which make use of lectins. Digoxigenin-labelled lectins are used for the structural characterization of carbohydrate chains of glycoproteins. Concanavalin A lectin affinity chromatography was used to analyse apolipoprotein H according to the characteristics of its carbohydrate chain inner to sialic acid residues. Our results from digoxigenin-labelled lectins analysis showed that apolipoprotein H gave positive bands to SNA, DSA, GNA, PNA and AAA lectins. Apolipoprotein H gave a negative band when reacted with MAA lectin. When we applied apolipoprotein H onto the Concanavalin A lectin column no detectable amounts of protein were eluted with Concanavalin A buffer. After adding a buffer with low sugar concentration (10 mM glucoside) a large amount of apolipoprotein H was recovered. These molecules of apolipoprotein H weakly bound to the lectin. When a higher sugar concentration (500 mM mannoside) was added most of the sample applied was eluted. These molecules of apolipoprotein H firmly bound to the column having high affinity for the lectin. These results combined with those coming from the digoxigen-labeled lectins method enable us to understand the inner structure of carbohydrate chains with their outer branches. Molecules of apolipoprotein H which weakly bind to Concanavalin A could bear complex N-glycans organized in biantennary or truncated hybrid structures. Firmly bound apolipoprotein H referred to molecules rich in N-glycan hybrid structures. They have an outer branch belonging to the high mannose carbohydrate chains which explain the ability to bind to the column and an other main branch bearing the sequence galactose beta-(1-4)-N-acetylglucosamine beta-(1-2) mannose. Galactose could be the terminal sugar or, alternatively, be masked with sialic acid alpha-(2-6) terminally linked.     

The P2Y1 receptor is necessary for adenosine 5''-diphosphate-induced platelet aggregation

Population control has become a major problem in many wildlife species. Fertility control through immunocontraception has been proposed as a method for reducing population size. We have tested the concept that immunocontraception can be achieved with a recombinant ectromelia virus expressing an ovary-specific antigen, the mouse zona pellucida 3 (ZP3) glycoprotein. Female mice infected with the recombinant virus produced autoimmune antibodies against ZP3 and were infertile for 5-9 mo after infection. For almost half the infertile mice, immunity to ZP3 was associated with a disruption of ovarian follicular development and the depletion of mature follicles without observable oophoritis. Mice returned to fertility as the anti-ZP3 antibody level in the serum decreased. Reinfection of the mice with the recombinant virus boosted the anti-ZP3 response and restored infertility.     

Electroencephalographic changes in experimental autoimmune myasthenia gravis

A procedure for the determination of activity and linkage specificity of sialidases is described. The sialoglycoprotein fetuin is coated onto a microtiter plate and incubated with sialidases from different sources. Enzymatic activities and linkage specificities are then determined by a sandwich method which measured the binding of different lectins to fetuin. The lectins used were peanut agglutinin (PNA) from Arachis hypogaea, which binds specifically the galactose beta-1-3-N-acetylgalactosamine structures that are unmasked following sialidase treatment of fetuin, the lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for alpha-2-6 and alpha-2-3 bound sialic acids, respectively, and the slug agglutinin from Limax flavus (LFA) that is specific for N-acetyl and N-glycolyl neuraminic acids. Increased PNA and decreased LFA, SNA, and MAA lectin binding correlated with sialidase-induced desialylation of the substrate. In this report, the assay was used to determine the activities and specificities of influenza, Vibrio cholerae, and Arthrobacter ureafaciens sialidases.     

Immunological identification of zona pellucida 2 (ZP2) protein in human oocytes

The ZP2 protein is a zona pellucida glycoprotein that plays a major role in fertilization. It mediates secondary binding of spermatozoa and is one of the proteins that are involved in zona 'hardening'. ZP2 proteins were identified in various mammalian zonae pellucidae. Their primary structures are highly conserved as revealed by cDNA cloning. Antisera were used against synthetic peptides generated either against a ZP2 amino acid that is homologous in human and mouse ZP2 amino acid sequences (AS ZP2-20) or antibodies against a synthetic human ZP2 peptide (AS ZP2-26). Immunoblots showed that antiserum AS ZP2-20 and AS ZP2-26 strongly recognized human ZP2 protein with an apparent molecular mass of about 72 kDa; both antisera reacted with a minor immunoreactive polypeptide at 96 kDa. In human ovary sections, both antisera revealed immunoreactivity to human zonae pellucidae. Immuno-electron microscopy demonstrated an equal distribution of ZP2 throughout the human zona pellucida. Considerable amounts of immunoreactive material were observed in the ooplasm; some ramification-like extensions of zona pellucida antigen were found close to cells surrounding the oocyte. Our results indicate that antisera against synthetic ZP2 peptides can be used as specific markers for the identification of ZP2 protein in human oocytes.     

Low blastocyst formation rates in day-2 fertilized oocytes

PURPOSE: To examine the blastocyst formation rates of day-2 fertilized oocytes. METHODS: A retrospective study of the outcomes/blastocyst formation of day-2 fertilized oocytes was undertaken. RESULTS: Fertilization rates of day-1 and -2 oocytes by intra-cytoplasmic sperm injection were similar. The development frequencies to four cells were similar. However, the blastulation rates were significantly lower from the day-2 fertilized eggs. The fertilization rates from day-2 conventional in vitro fertilization reinsemination were lower than the fertilization rates of day-1 oocytes. The blastulation rates from day-2 fertilized eggs were also lower than the rates from day-1 fertilized eggs in the in vitro fertilization group. CONCLUSIONS: Fertilization is not a good indicator to predict the viability of fertilized oocytes. Day-2 fertilized oocytes had significantly lower blastocyst formation rates than the rates from day-1 fertilized oocytes.     

Characterization of the binding of recombinant mouse sperm fertilin alpha subunit to mouse eggs: evidence for function as a cell adhesion molecule in sperm-egg binding

Fertilin (previously known as PH-30) is a sperm protein that is a candidate molecule for mediating the binding and fusion of the sperm and egg plasma membranes. Fertilin is a heterodimer, with a beta subunit that has a region of homology to the disintegrin family of integrin ligands and an alpha subunit that has a region of homology to viral fusion peptides. It has been hypothesized that fertilin beta and alpha subunits mediate the interactions between sperm and egg plasma membranes, namely, binding and fusion, respectively. To address this hypothesis and to examine specifically the role of fertilin alpha in fertilization, we have expressed the predicted extracellular domain of mouse fertilin alpha as a bacterial fusion protein with maltose-binding protein. This fusion protein (hereafter referred to as recombinant fertilin alpha-EC) binds to the microvillar region of zona pellucida (ZP)-free eggs, the region of the membrane to which sperm bind. This binding is reduced in the absence of divalent cations and is supported by Ca2+, Mg2+, or Mn2+. Eggs that have been treated with chymotrypsin bind less recombinant fertilin alpha-EC than do untreated eggs, suggesting that a chymotrypsin-sensitive binding site for recombinant fertilin alpha-EC is present on egg surfaces. Binding to eggs is also affected by the method used to remove the ZP. Finally, recombinant fertilin alpha-EC inhibits the binding of sperm to eggs during in vitro fertilization of ZP-free eggs. These data are the first evidence to suggest that fertilin alpha can function as a cell adhesion molecule during fertilization, mediating the binding of sperm and egg plasma membranes.     

Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3

The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zona pellucida (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZP1-3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the mZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous (mZP3 +/-) or homozygous (mZP3-/-) for the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female mZP3 +/- mice are as fertile as wild-type animals, but their eggs have a thin ZP (approximately 2.7 microns thick) as compared to the ZP (approximately 6.2 microns thick) of eggs from wild-type animals. On the other hand, female mZP3-/- mice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated mZP3-/- females. Light micrographs reveal that development of ovarian follicles is often retarded in mZP3-/- mice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in mZP3-/- females. It seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells.     

Regulation of the polyspermy block in the mouse egg: maturation-dependent differences in cortical granule exocytosis and zona pellucida modifications induced by inositol 1,4,5-trisphosphate and an activator of protein kinase C

Germinal vesicle (GV)-intact fully grown mouse oocytes do not undergo cortical granule (CG) exocytosis in response to A23187 treatment, whereas metaphase II (MII)-arrested eggs do. This differential response may reflect the development of the ability of the egg to undergo CG exocytosis, which is responsible for the biochemical modification of the glycoprotein ZP2 in the zona pellucida. Accordingly, we compared in these two stages the ability of 12-O-tetradecanoyl phorbol 13-acetate (TPA) or inositol 1,4,5-trisphosphate (IP3) to promote CG exocytosis and/or the ZP2 to ZP2f conversion; these agents are known to stimulate early events of mouse egg activation. TPA (10 ng/ml) treatment for 60 and 120 min resulted in a 25% and 52% CG loss in GV-intact oocytes and a 38% and 76% loss in MII eggs, respectively; fertilization resulted in a CG loss of approximately 70-80%. Although a similar extent of ZP2 to ZP2f conversion was observed in oocytes and eggs after a 120-min TPA treatment (approximately 70-80%), a greater extent of conversion was observed in oocytes after a 60-min treatment (80% for oocytes, 50% for eggs). Microinjection of IP3 (final concentration 1 microM) into MII eggs resulted in an extent of ZP2 conversion similar to that observed following fertilization, whereas little conversion occurred in GV-intact oocytes similarly injected. These results indicate that a protein kinase C sensitivity develops prior to meiotic maturation, whereas responsiveness to IP3 develops after maturation has resumed. We propose that the regulatory mechanism involving an IP3-mediated calcium release is deficient in GV-stage oocytes.     

The molecular genetics of the zona pellucida: mouse mutations and infertility

The zona pellucida is an extracellular matrix surrounding growing oocytes, ovulated eggs and the preimplantation embryo. After mediating the relatively species-specific events of fertilization, the zona pellucida provides a post-fertilization block to polyspermy and protects the growing embryo as it passes down the oviduct. The genes that encode the three zona pellucida proteins (ZP1, ZP2, ZP3) have been characterized in mouse and human. The ability to genetically manipulate the zona pellucida genes in mouse models has enhanced our knowledge of zona pellucida structure and function in vivo and may translate into a better understanding of human fertility.     

Sperm and its soluble extract cause transient increases in intracellular calcium concentration and in membrane potential of sea urchin zygotes

Fertilization is known to initiate a transient increase of intracellular calcium concentration and an accompanying change of membrane potential in sea urchin eggs. If the fertilization membrane and hyaline layer are removed, sperm can again enter fertilized eggs (refertilization). We have found that Ca2+ and voltage transients were repeatedly induced in fertilized eggs during refertilization. Similar changes were also obtained by external application of a soluble extract of sperm to fertilized eggs. This sperm extract caused no changes in unfertilized eggs. The active factor in the sperm extract survives heating (100 degrees C, 10 min) and incubation with pronase. Its molecular weight is less than 1300.     

Synthesis of artificial N-glycopolypeptides carrying N-acetyllactosamine and related compounds and their specific interactions with lectins

Artificial N-glycopolypeptides carrying N-acetyllactosamine (LacNAc) or related compounds were synthesized. First, sugars were converted into their corresponding beta-glycosylamines with ammonium hydrogen carbonate. Then, the beta-glycosylamines were condensated with the carboxyl groups of poly(L-glutamic acid). N-Glycopolypeptides with different degrees of substitution of sugars were isolated by passage through a column of Sephadex G-25. These synthetic polymers were used as model compounds in the analysis of oligosaccharide-lectin interactions. Interactions with some lectins were investigated by agar-gel double-diffusion tests and in terms of inhibition of hemagglutination. A glycopolypeptide substituted with LacNAc reacted with Erythrina cristagalli agglutinin (ECA), peanut (Arachis hypogaea) agglutinin (PNA), Ricinus communis agglutinin-120 (RCA120), wheat germ (Triticum vulgaris) agglutinin (WGA) lectins, which recognize either galactosyl or N-acetylglucosamine (GlcNAc) residues. Other synthetic glycopolymers carrying N-acetylisolactosamine, GlcNAc, N,N'-diacetylchitobiose, or N,N', N"-triacetylchitotriose also reacted with WGA, and these last two polymers inhibited hemagglutination most. Of these five glycopolypeptides, only the one substituted with LacNAc reacted with ECA. These sugar-substituted glycopolypeptides interacted specifically with the corresponding lectins, no matter how much shorter the sugar side chains of the glycopolymers were than those of natural glycoproteins.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 microns during maturation and increased to 106 microns after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination.     

Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for immunocontraception. Studies on this potential use can be facilitated by the availability of recombinant proteins. A cDNA lambda gt11 library was constructed using poly(A)+ mRNA isolated from bonnet monkey (Macaca radiata) ovaries and was screened for bonnet monkey ZP1 using a 404-basepair (bp) human ZP1 fragment (nucleotides 818-1221) as probe. Bonnet monkey ZP1 cDNA comprises 1617 nucleotides and encodes a polypeptide of 539 amino acid residues that share 92.0% identity with human ZP1. The major difference between bonnet monkey ZP1 and human ZP1 is the deletion of a 28-amino acid domain (amino acid residues 100-127 corresponding to human ZP1). An internal fragment (1317 bp) of bonnet monkey ZP1, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by polymerase chain reaction. The amplified Sac I and Kpn I restricted fragment was cloned in a frame downstream of the T5 promoter under the lac operator control for expression in the pQE-30 vector. Recombinant ZP1 (r-ZP1) was expressed as a polyhistidine fusion protein in Escherichia coli strains SG13009[pREP4] and ompT and Ion protease-deficient BL21 (plysS). SDS-PAGE analysis and immunoblotting with a murine monoclonal antibody, MA-410 (raised against porcine ZP3alpha--a homologue of bonnet monkey ZP1--and cross-reactive with bonnet monkey zona pellucida), revealed major bands of 51 and 40 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Immunization of male rabbits with r-ZP1 purified on nickel-nitrilotriacetic acid (NTA) resin under denaturing conditions and of female rabbits with r-ZP1 conjugated with diphtheria toxoid-generated antibodies reactive with r-ZP1 in ELISA. Moreover, immune sera, when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The information on the sequence of bonnet monkey ZP1 and the availability of the recombinant protein will help toward better understanding and evaluation of the contraceptive potential of homologous immunization in a nonhuman primate model.     

Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus laevis eggs

As a step toward defining in molecular terms the sperm-triggered block to polyspermy reaction established by the egg at fertilization, vitelline (VE) and fertilization (FE) envelopes were isolated from eggs of the Sounth African clawed toad Xenopus laevis and some of their physicochemical properties determined. Envelopes were isolated after lysis of the fertilized or unfertilized eggs by sieving techniques; isolated envelopes retained their in situ morphology as determined by electron microscopy. The isolated envelopes had different solubility properties and, in general, VE was more readily dissolved by aqueous solvents than FE, although both could be completely dissolved by detergents or chaotropic agents. Changes in envelope solubility correlated with the progression of the cortical reaction implicating a role for cortical granule material in modifying the solubility properties of the envelope. The VE and FE were composed of protein and carbohydrate with no lipid components detected. As determined by immunodiffusion experiments, the FE contained the same antigens as the VE plus components derived from the cortical granules and the innermost jelly layer, J. The macromolecular composition of the envelopes was determined by sodium dodecyl sulfate gel electrophoresis. The VE contained at least 11 glycoproteins with molecular weights ranging from 125 000 to less than 16 000 with two components (40 000 and 33 000) accounting for almost two-thirds of the total stainable material. The FE contained ten glycoproteins that had the same molecular weights as those in the VE. One glycoprotein component underwent a reduction in molecular weight from 77 000 to 67 500 when the VE was converted to the FE. This molecular weight change was interpreted as the probable result of limited proteolysis. In addition, the FE gel electrophoresis patterns contained macromolecular components derived from the cortical granules and jelly layer, J, consistent with the immunodiffusion experiments. These components were absent when the FE was prepared in the absence of Ca2+, suggesting a role for Ca2+ in binding the VE, cortical granules, and J components together. We concluded that the conversion of the glycoproteinaceous VE to FE at fertilization is caused by interaction of the VE with components from the cortical granules and jelly layer J. These interactions are of both a chemical and physical nature.     

Cytochalasin B-induced triploidy in mouse oocytes fertilized in vitro

Mouse eggs and spermatozoa were treated in various ways with 5 or 10 mug cytochalasin B/ml. The fertilization rate in vitro was reduced by treatment with the drug but 80-90% of the eggs fertilized were triploid. Many of the experimental eggs were penetrated by one or more spermatozoa but remained unfertilized (75% compared with 9% in control eggs). It is suggested that cytochalasin B weakens the zona reaction and interferes with fusion of gametes but does not prevent the block to polyspermy.     

Binding of antibiotics to glycoproteins of the vitelline and fertilization envelopes of cherry salmon eggs

The binding of antibiotics (gentamicin, oleandomycin and chloramphenicol) to vitelline and fertilization envelopes and their extracts was investigated by immunohistochemical and immunocytochemical techniques and immunoblot analysis using mature and artificially activated eggs of the fish Oncorhynchus masou. Binding of antibiotics was detected in the vitelline and fertilization envelope outermost layers, the fertilization envelope inner surface and cortical alveolus exudates, with differences in immunoreactive intensity and deposition. The fertilization envelope outermost layer had the capacity to bind much greater amounts of the antibiotics than the vitelline envelope outermost layer. The greater capacity was caused by the deposition of cortical alveolus exudates, which were known to be responsible for functional roles of protection against bacteria, fungi and noxious materials. Treatment of the vitelline and fertilization envelopes with neuraminidase markedly reduced the binding of gentamicin and chloramphenicol but slightly increased that of oleandomycin; binding of the latter to the vitelline and fertilization envelope outermost layers was considerably reduced after treatment with alpha-fucosidase. Treatment of the two envelopes with alpha-mannosidase, beta-galactosidase or beta-D-glucosaminidase did not cause any alteration in immunoreactive intensity or number of immunoreactive deposits. Immunoblot analysis of the vitelline or fertilization envelope extracts indicated that many of the antibiotic-binding substances were glycoproteins, and several major bands were bound by all three antibiotics. These results suggest that the vitelline or fertilization envelopes may have the ability to protect the egg itself, or the embryo, respectively, by trapping antibiotics, and the trapping may be related to the presence of carbohydrate moieties, such as sialyl or fucosyl residues.