Proteins of the sea urchin egg vitelline layer

The vitelline layers (VL) of unfertilized sea urchin eggs were isolated, and the diversity of their polypeptide constitutents estimated by two-dimensional polyacrylamide gel electrophoresis. At least 25 components are reproducibly observed. While VL polypeptides are almost certainly synthesized in the growing oocyte, they are not among the more prevalent newly synthesized proteins detected in oocytes that were isolated and labeled in vitro for 4 hr. A set of monoclonal antibodies was raised against VL components and partially characterized. The 31 monoclonals analyzed fell into 11 classes with respect to their avidity for VL proteins solubilized under mild and under strongly denaturing conditions, and to their reactions with surface components of the VLs of living eggs. Fluorescence microscopy showed diverse patterns of surface reactivity when different monoclonal antibodies were compared. Two of the monoclonal antibodies reacted with specific sets of three proteins each on VL protein blots. It is concluded that the VL is a complex structure containing a large number of different polypeptide components, the genes for several of which should now be experimentally accessible.     

A protein from abalone sperm dissolves the egg vitelline layer by a nonenzymatic mechanism

Unfertilized abalone eggs (Haliotis rufescens) possess an elevated fibrous glycoproteinaceous vitelline layer (VL) about 0.6 μm in thickness. Sperm bind to the VL by the tip of a large unreacted acrosome granule. After binding, the tip of the granule opens and the soluble contents are released onto the VL. A hole about 3 μm in diameter then forms in the VL in the area of the discharging acrosome. Ultrastructural observations show the hole to be filled with attenuated VL fibers. The sperm then swims through the hole and interacts with the egg plasma membrane. The soluble contents of abalone acrosomes can be obtained by induction of the acrosome reaction in high-calcium seawater. Two major proteins of subunit molecular weights 13,000 (13K) and 15,000 (15K) are found in the supernatant after removal of the reacted sperm by centrifugation. Gel analysis of whole sperm shows these two proteins are the major components of the cell. The 13K protein can be purified on the basis of its solubility at lower ionic strength. This protein is a potent solubilizer (lysin) of egg vitelline layers. Characterization of the 13K lysin yields an isoelectric point of about 9, basic amino acids accounting for 19.6% of its weight, a negative PAS reaction, a nondenatured-molecular-weight estimate of 17,000, the presence of exposed hydrophobic regions, and a lack of enzyme activity. The lytic action of the 13K protein is rapidly inactivated by boiling, showing that the native conformation is necessary for activity. The lysin does not degrade the macromolecular components of the VL. It does not produce reducing sugars, peptides, lysophosphatides, or SH groups. A turbidometric assay for lysin activity was developed using isolated VLs and 13K lysin. When lysin is added to VLs in seawater the dissolution action occurs for only 15–30 sec before abruptly stopping. Mixing various amounts of lysin with a constant amount of VLs shows that the lysin dissolves VLs by a stoichiometric, noncatalytic (nonenzymatic) mechanism. For example, about 11 μg of lysin are required for the complete dissolution of 63 μg of VL protein (the VL is 36% protein). An identical conclusion was reached by K. Haino-Fukushima (1974, Biochim. Biophys. Acta, 352, 179–191) working with an 8.8K lysin of another archeogastropod, Tegula pfeifferi. Isolated abalone VLs are composed of about five major glycoproteins ranging in molecular weight from 32 to 44K. High ionic strength such as 2 M KCl does not solubilize VLs, but agents which destroy hydrophobic bonds between macromolecules, such as NaSCN, dimethylsulfoxide, and heat, are VL solubilizers. Exposed hydrophobic portions of the lysin might bind to the hydrophobic regions of VL glycoproteins and competitively dissociate the VL fibers from each other, thus, destroying the VL's structural integrity. Stoichiometric mechanisms for making holes in egg investments may be more biologically attractive than enzymatic mechanisms. A stoichiometric reaction would be quickly self-limiting and nondegradative to other cell surface components.     

KDN-glycoprotein: a novel deaminated neuraminic acid-rich glycoprotein isolated from vitelline envelope of rainbow trout eggs

A new acidic glycoprotein containing deaminated neuraminic acid (KDN = 3-deoxy-D-glycero-D-galacto-nonulosonic acid; greater than 50%, w/w) was isolated from vitelline envelope of the unfertilized eggs of rainbow trout (Salmo gairdneri). This glycoprotein is designated as "KDN-glycoprotein" because it contains only KDN but no sialic acid as the acidic carbohydrate moieties. Other major carbohydrate components of KDN-glycoprotein were Gal and GalNAc. Thr and Ala accounted for 71% (mol/mol) of amino acid composition. A possible occurrence of KDN-KDN linkages, i.e. oligoKDN groups has been suggested in the carbohydrate chains presumably linked O-glycosidically to the core protein.     

Histone Analysis During the First Cell Cycle of Development of the Sea Urchin Tetrapygus niger

To obtain information on the contribution of paternal and maternal histone complement to the assembly of sea urchin chromatin after fertilization, we have compared the complete set of histones of zygotes obtained at the beginning of the first S phase those of sperm and unfertilized eggs of Tetrapygus niger . The electrophoretic pattern on acetic acid-urea polyacrylamide gels and the amino acid composition of the histones isolated from zygotes are virtually identical with those isolated from unfertilized eggs. These results strongly suggest that sperm histones must be lost before the initiation of the first replication event. The histones of zygotes obtained after the completion of the first S phase have the same electrophoretic pattern as the proteins isolated from zygotes prior to the first S phase; however, differences were found in their amino acid composition. These results suggest that some new proteins may associate with chromatin during the first replication round in Tetrapygus niger development.     

Structural studies of fertilization-associated carbohydrate-rich glycoproteins (hyosophorin) isolated from the fertilized and unfertilized eggs of flounder, Paralichthys olivaceus. Presence of a novel penta-antennary N-linked glycan chain in the tandem repeating glycopeptide unit of hyosophorin

New glycoproteins of 100-120 kDa were isolated from the unfertilized eggs of flounder, Paralichthys olivaceus. Compositionally indistinguishable glycopeptides of 6 kDa were also purified from the activated or fertilized eggs. These high and low molecular mass glycoproteins are characterized by high (about 85%) carbohydrate content. Although some heterogeneities exist in the amino acid sequences, the 6-kDa glycopeptides (decapeptides with single large N-linked glycan chains), isolated from the fertilized eggs are the repeating units of the high molecular mass glycoproteins. As judged from several distinctive features the 100-120-kDa glycoproteins are apparently major components of cortical alveoli of flounder eggs and are regarded as members of glycoproteins we have defined under the name of "hyosophorin" (Kitajima, K., Inoue, S., and Inoue, Y. (1989) Dev. Biol. 132, 544-553). Composition analysis, Smith degradation, hydrazinolysis-nitrous acid deamination, permethylation analysis, and 400-MHz 1H NMR spectroscopy provided evidence for the structure of a novel penta-antennary glycan chain attached to the repeating unit (decapeptide) of the protein core. The structure thus determined is: (Formula: see text). The presence of a unique class of carbohydrate-rich glycoproteins (H-hyosophorin) in the unfertilized eggs, their conversion to the repeating unit (L-hyosophorin) at fertilization, and the finding of a free glycan chain that was formed by scission between the GlcNAc and Asn residues of L-hyosophorin, in the fertilized eggs including embryos of 4-11-h postinsemination, support the view that these molecules may be important in fertilization and subsequent development.     

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida

Single-step purification of boar sperm P68/62 that is cross-reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68-kDa band reacted with both the 68- and 62-kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm-egg binding in a dose-dependent manner when added exogenously to sperm-egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight-cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two-cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm-ZP binding that is adhesive in nature. Mol. Reprod. Dev. 49:203–216, 1998 © 1998 Wiley-Liss, Inc.     

Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.     

Isolation and characterization of a novel type of sialoglycoproteins (hyosophorin) from the eggs of medaka, Oryzias latipes: nonapeptide with a large N-linked glycan chain as a tandem repeat unit

We found a novel type of sialoglycoprotein (SGP) with apparent molecular mass ranging from 15,000 to 100,000 Da in the unfertilized eggs of the medaka fish, Oryzias latipes. From fertilized eggs we isolated the corresponding sialoglycopeptides of apparent molecular weight 7000. The amino acid and carbohydrate compositions of these glycoproteins and glycopeptides are very similar, if not identical, and they contain 90%, by weight, of carbohydrate, the predominant sugars being Gal, GlcNAc, and NeuAc. The chemical and physical data indicate that 15- to 100-kDa SGPs are made up of tandem repeat structures whose repeating unit is 7-kDa sialoglycopeptide, and, upon fertilization, higher molecular weight SGPs undergo proteolytic depolymerization to the least structural unit, 7-kDa sialoglycopeptide. As is the case with polysialoglycoproteins (PSGP) found in salmonid fish eggs, a novel family of sialoglycoproteins has been proven to be a major component of cortical alveoli of medaka eggs, namely, hyosophorin. However, we found that they differ markedly from PSGPs (salmonid fish egg hyosophorins) in terms of the carbohydrate composition. The chemical composition and the results of Smith degradation indicate that SGP contains one large N-linked glycan chain per repeat unit. We have determined the amino acid sequence of 7-kDa sialoglycopeptide: Asp-Ala-Ala-Ser-Asn*-Gln-Thr-Val-Ser, where * indicates the asparagine residue to which a large glycan chain consisting of Fuc2Man3Gal15GlcNac9NeuAc6 is attached. The direct experimental evidence for the presence of a polyprotein structure suggests that the covalent nature of the higher molecular weight SGPs should be expressed as [Asp-Ala-Ala-Ser-Asn*-Gln-Thr-Val-Ser]N, where N = 2 to 14 but for the major fraction N = 12.     

Electrophoretic analysis of the stored histone pool in unfertilized sea urchin eggs: quantification and identification by antibody binding

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.     

Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein

In this investigation, the interaction of mouse sperm with unfertilized eggs and embryos, solubilized zonae pellucidae isolated from eggs and embryos, and purified zona pellucida glycoproteins ZP1, 2, and 3 (J. D. Bleil, and P. M. Wassarman, (1980b) Dev. Biol. 76, 185-202) has been examined in vitro by light and electron microscopy. The experiments described were carried out in order to determine the temporal sequence of events during sperm-egg interaction in vitro and to identify the component(s) of zonae pellucidae responsible for inducing mouse sperm to undergo the acrosome reaction. "Pulse-chase" analysis of the sequence of sperm-egg interactions revealed that mouse sperm first "attach" loosely and then "bind" tightly to the unfertilized egg's zona pellucida. Binding of sperm to egg zonae pellucidae is followed by induction of the acrosome reaction. Induction of the acrosome reaction can be mediated by the zona pellucida, since solubilized zonae pellucidae isolated from unfertilized eggs were found to be just as effective as the calcium ionophore A23187 in inducing the reaction in vitro. Furthermore, ZP3 purified from zonae pellucidae isolated from unfertilized eggs, but not from two-cell embryos, was also just as effective as either solubilized zonae pellucidae from eggs or ionophore A23187 in inducing the acrosome reaction. ZP1 and 2 from both eggs and embryos, and ZP3 from embryos, had little effect on the extent of the acrosome reaction as compared to control samples. The results of these and other experiments (J. D. Bleil, and P. M. Wassarman, (1980b) Cell 20, 873-882) strongly suggest that, at least in vitro, mouse sperm recognize and bind to ZP3 of egg zonae pellucidae, and that such binding leads to the induction of the acrosome reaction. Modification of ZP3 following fertilization eliminates sperm binding to zonae pellucidae and, consequently, induction of the acrosome reaction is precluded.     

Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs. Zonae pellucidae of mouse eggs are composed of three different glycoproteins, designated ZP1, ZP2 and ZP3, having apparent molecular weights of 200,000, 120,000 and 83,000, respectively Bleil and Wassarman, 1978, Bleil and Wassarman, 1980a, Bleil and Wassarman, 1980b. In this investigation, ZP1, ZP2 and ZP3 were purified from zonae pellucidae isolated individually from unfertilized mouse eggs and 2-cell embryos. Each of the glycoproteins was then tested for its ability to interfere with the binding of sperm to eggs in vitro. Solubilized zonae pellucidae isolated from unfertilized eggs, but not from 2-cell embryos, reduced binding of sperm to as little as 10% of control values. Similarly, ZP3 purified from zonae pellucidae of unfertilized eggs reduced the binding of sperm to eggs in vitro to an extent comparable to that observed with solubilized zonae pellucidae. On the other hand, ZP3 purified from zonae pellucidae of 2-cell embryos had no significant effect on the extent of sperm binding, consistent with the inability of solubilized zonae pellucidae from 2-cell embryos to affect sperm binding. In no case did purified ZP1 and ZP2 interfere significantly with the binding of sperm to eggs in vitro. These results suggest that ZP3 possesses the receptor activity responsible for the binding of sperm to zonae pellucidae of unfertilized mouse eggs. Fertilization apparently results in modification of ZP3 such that it can no longer serve as a receptor for sperm.     

Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro.

Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39 degrees C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0.2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30-40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.     

Assisted fertilization by zona drilling: a mouse model for correction of oligospermia

A micromanipulation apparatus was used to produce holes in the zonae pellucidae of unfertilized mouse oocytes. A microneedle loaded with acid Tyrode's solution was brought into contact with the zona surface, and positive flow was used in conjunction with mechanical pressure to cause a localized dissolution of the zona. Treated eggs were then fertilized in vitro in comparison with control cells. The zona drilling procedure decreased the sperm count required to achieve fertilization by a factor of approximately 100. The rate of polyspermy in zona-drilled oocytes was not greater than in controls, and oocytes fertilized after drilling, when implanted into pseudopregnant foster females, developed to term at the same rate as controls. The results demonstrate that zona drilling is a safe, effective method of increasing the efficiency of fertilization in vitro and may be useful both in agriculture and medicine for conferring fertility upon males with low sperm counts.     

Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilized oocytes (8.50 +/- 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 +/- 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 +/- 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 +/- 74.1 nmol/egg in unfertilized eggs, 146.0 +/- 50.0 nmol/egg in DSH eggs and 39.7 +/- 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilization. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 +/- 1.5 nmol/egg in unfertilized oocytes and 10.1 +/- 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 +/- 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilization but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.     

Detection of differences in surface carbohydrates of unfertilized and fertilized golden hamster eggs using potato agglutinin

Changes in the surface carbohydrate moieties of zonae pellucidae and naked vitelli isolated from unfertilized and fertilized hamster eggs were examined through potato agglutinin (STA)-mediated agglutination and binding of FITC-STA as a marker. The results indicated that the stereochemical properties of the saccharide residues within STA-binding sites on the outer surface of the zona pellucida, inner region of the zona pellucida, and the surface of the ooplasmic membrane were different in unfertilized and fertilized eggs, that part of the differences between the two kinds of eggs could be visualized with FITC-STA and that, on the basis of these data, if we use STA as a probe to analyze the surface change following fertilization, we may be able to analyze these changes to a substantial degree.     

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.     

Technical and physiological aspects associated with the lower fertilization following intra cytoplasmic sperm injection (ICSI) in human

The fertilization rates with ICSI range from 30% to 70% and suggest that, despite injecting sperm into mature oocytes, significant fertilization failure still occurs in humans. The objective of this study was to determine technical and physiological factors which may contribute to lower fertilization following ICSI. Eggs that failed to show two pronuclei (PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). The eggs were analyzed by staining with DNA fluorochromes, Hoescht 33258 and DAPI. The extent of sperm head as well as maternal chromatin decondensation in unfertilized ICSI eggs was determined by high resolution fluorescence microscopy. The average fertilization rate (FR) from all ICSI cycles in these two groups was 45%. The FR in Groups A and B were 35% and 59%, respectively (P < 0.05). In Group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In Group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin while 45% were partially decondensed. Sperm chromatin was not detected in 24% of all unfertilized eggs studied. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause for failure of fertilization rates in ICSI cycles. The increased percentage of eggs undergoing sperm head decondensation may be related to subtle changes in technique as experience is gained over time. The failure of sperm head decondensation in some of the ICSI eggs may be associated with cytoplasmic immaturity but not nuclear maturity.     

Ultracytochemical Modifications of Surface Carbohydrates in Fertilized Eggs of the Common Carp

The surface of the plasma membrane of unfertilized and fertilized carp eggs was examined by four cytochemical techniques, colloidal thorium, colloidal iron, ruthenium red and phosphotungstic acid stainings, to determine the carbohydrate moieties. The surface of the plasma membrane of unfertilized eggs stained only with colloidal iron, which was heterogeneously deposited: no deposits were seen on the plasma membrane near overlying cortical alveoli. In fertilized eggs, the membrane was stained by all four methods. These ultracytochemical modifications of the surface of the plasma membrane may be caused by participation of the limiting membranes of secretory organelles, probably by turnover of the inner surface of the limiting membranes. Neuraminidase treatment of fertilized eggs eliminated the deposits of colloidal iron on the surface of the plasma membrane and caused an increase in stainability with ruthenium red. Treatment with neuraminidase or trypsin prevented the staining with phosphotungstic acid.     

Human sperm do not bind to rat zonae pellucidae despite the presence of four homologous glycoproteins

The specificity of sperm-egg recognition in mammals is mediated primarily by the zona pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62-70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals.