Isolation of a glycopeptide fraction from the surface of the sea urchin egg that inhibits sperm-egg binding and fertilization.

The role of cell surface glycoproteins of the sea urchin egg in binding sperm has been examined by studying the biological activity of glycopeptides derived from these glycoproteins. Glycopeptides were produced from egg surface glycoproteins by Pronase digestion. After fractionation by gel filtration the glycopeptides were tested for their ability to inhibit the binding of sperm to eggs, presumably by competing with the egg surface glycoproteins for binding sites on the sperm. One glycopeptide fraction with an apparent molecular weight of approximately 6,000 was found to be a potent inhibitor of sperm-egg binding, as well as fertilization, even at nanomolar concentrations. This activity was heat stable and exerted its effect against the sperm and not the egg. Experiments with a radiolabeled form of the glycopeptide fraction directly demonstrated that at least one component of it bound to sperm. Specific binding of the radiolabeled glycopeptide occurred only to acrosome-reacted sperm. Because the isolated glycopeptide fraction has many of the characteristics that one would expect of a biologically active fragment of an egg surface receptor for sperm, these findings are consistent with the idea that one or more glycoconjugates on the surface of the egg are involved in sperm binding.

Isolation and partial characterization of the plasma membrane of the sea urchin egg.

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.

Phorbol ester treatment stimulates tyrosine phosphorylation of a sea urchin egg cortex protein.

Fertilization of the sea urchin egg results in the phosphorylation, on tyrosine, of a high molecular weight protein localized in the egg cortex. In the present study, treatment of unfertilized eggs with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulated tyrosine phosphorylation of the high molecular weight cortical protein to levels three- to fivefold higher than that occurring in response to fertilization. Experiments using agents that inhibit the egg Na+/H+ exchange system or mimic the fertilization-induced shift in cytoplasmic pHi, suggest a signal transduction pathway in which protein kinase C activates the egg Na+/H+ exchange system and the resultant cytoplasmic pHi shift promotes tyrosine phosphorylation of the high molecular weight cortical protein.

Fertilization triggers activation of Fyn kinase in the zebrafish egg.

Fertilization results in the tyrosine phosphorylation of several egg proteins and studies have shown that tyrosine protein kinase activity is required for successful fertilization. The Fyn protein kinase has been detected in eggs of the sea urchin, frog and rat, although measurement of fertilization-induced changes in Fyn kinase activity have only been successful in the sea urchin system. The present study demonstrates the presence of Fyn kinase in the zebrafish egg and the stimulation of this enzyme at fertilization. Activation of Fyn was detected as early as 30 seconds post-fertilization and increased approximately six-fold by 2 minutes post-insemination. The activation of Fyn in the zebrafish egg required sperm and was not observed in spontaneously activated eggs.

Fertilization promotes selective association of the Abl [correction of AbI] kinase with the egg cytoskeleton.

Fertilization of the sea urchin egg is known to involve activation of a variety of protein kinases including at least one protein tyrosine kinase. In the present study, fertilization was found to stimulate the association of the Abl [correction of AbI] protein kinase with the detergent-insoluble egg cytoskeleton. Increased levels of Abl protein and of protein tyrosine kinase activity were detected in cytoskeleton preparations made as early as 5 to 15 min after insemination. Immunofluorescence localization demonstrated that the Abl kinase becomes associated primarily with the cortical cytoskeleton of the fertilized egg. A separate, 57 kDa protein tyrosine kinase did not associate with the cytoskeleton indicating that fertilization results in the selective association of the Abl kinase with the cortical cytoskeleton.

Seborrheic keratoses from the genital region may contain human papillomavirus DNA.

Sections from paraffin-embedded tissues of lesions interpreted as seborrheic keratoses localized to the pubic, genital, and crural regions were assayed for the presence of human papillomavirus types 6, 11, 16, 18, and 33 using DNA amplification followed by specific hybridization. Lesions with the histologic characteristics of condyloma were excluded from the study. Human papillomavirus DNA sequences were found in 24 (42%) of 57 seborrheic keratosis-like lesions from the genital region. No human papillomavirus DNA was detected in 27 control specimens that represented a variety of other processes occurring in this area. We conclude that human papillomavirus infection cannot be excluded in genital seborrheic keratoses.

Differential phosphorylation of a 57-KDa protein tyrosine kinase during egg activation.

Fertilization results in activation of many protein kinases which function during egg activation. We have used metabolic labelling and immunoprecipitation to study changes in the phosphorylation state of a 57-KDa src-family protein tyrosine kinase during fertilization of the sea urchin egg. The kinase was phosphorylated on serine at all periods studied but it was also phosphorylated transiently on tyrosine at 5 minutes post insemination and then on threonine at 90 minutes after fertilization. These data indicate that the 57-KDa PTK may be under complex regulatory control during the first cell cycle.

Protein tyrosine kinase activity during egg activation is important for morphogenesis at gastrulation in the sea urchin embryo.

Fertilization triggers a series of preprogrammed events functioning to activate egg metabolism, incorporate the paternal genome, and initiate development. The activity of protein tyrosine kinases during egg activation is required for several steps leading to the first cell division. We now present evidence for an additional protein tyrosine kinase-mediated event that occurs between 30 and 45 min after insemination and is not required until gastrulation, which occurs over 24 hr later. Eggs treated with protein tyrosine kinase inhibitors within this window of time cleaved and formed normal blastulae but could not gastrulate or undergo further development to the pluteus stage. These findings provide the first evidence that some of the control mechanisms used in later development are established during a brief period of time in the fertilized egg and require the action of one or more protein tyrosine kinases.

Tyrosine kinase signaling at fertilization.

The unfertilized egg is a highly differentiated cell that retains unlimited developmental potential. The execution of that potential requires signal transduction pathways that release the egg from its quiescent metabolic state, direct the union of the maternal and paternal genome, and initiate a developmental program that will guide embryogenesis. The egg is equipped with an array of cytosolic as well as cell surface receptor protein tyrosine kinases as part of a preassembled signal transduction mechanism. These protein tyrosine kinases have been found to act at several points during this egg activation process, beginning as early as the initial sperm-egg interaction. While many of these kinase functions are common to all cells, several functions unique to fertilization demonstrate the versatility of this class of protein kinases.

Biphasic activation of Fyn kinase upon fertilization of the sea urchin egg.

Fertilization results in the tyrosine phosphorylation of several egg proteins within minutes of sperm-egg binding and inhibitor studies have shown that tyrosine kinase activity is required for many aspects of egg activation. The present study demonstrates the presence of p59c-fyn kinase in the sea urchin egg and examines the effect of fertilization on the activity of this enzyme. Fertilization had little effect on Fyn kinase activity during the first 2 min after insemination; however, activity had increased approximately eightfold between 5 and 15 min postinsemination. This initial, rapid increase in kinase activity was followed by a period of slightly elevated kinase activity, which was two- to threefold higher than that in the unfertilized egg. Bindin, as well as various parthenogenic agents known to activate the calcium- and pH-mediated pathways of egg activation, failed to elicit any change in enzyme activity, indicating that activation of the kinase required sperm-induced egg activation. However, phorbol ester treatment did induce a slow increase in kinase activity within 30 to 60 min of administration. These findings indicate that the p59fyn kinase is activated within minutes of fertilization and may play a role in the egg activation process.

Regulation of tyrosine-specific kinase activity at fertilization.

The sea urchin egg contains a protein kinase which phosphorylates tyrosine residues of endogenous membrane proteins as well as synthetic peptide substrates. Fertilization results in an increase in tyrosine kinase activity which first becomes apparent 20-30 min postinsemination and continues throughout the early cleavage stages. This effect can be duplicated by treating unfertilized eggs with the calcium ionophore A23187. The kinase activity begins to increase about 20 min after addition of the ionophore and continues to increase for at least 1 hr. Both the time course and the extent of kinase activity in ionophore treated eggs closely resemble the effects of fertilization. The concentration of ionophore necessary to induce the increase in enzyme activity (2-5 microM) is also effective in inducing the cortical reaction. Neither A23187 nor calcium has a significant effect on the kinase activity of egg homogenates solubilized in NP40, suggesting that the ionophore affects tyrosine phosphorylation indirectly, possibly acting through other calcium-sensitive enzymes.

Transient nuclear localization of Fyn kinase during development in zebrafish.

Fyn protein tyrosine kinase is present in the unfertilized and fertilized egg, becomes activated within minutes following fertilization, and has been localized to the cortical cytoplasm and spindle apparatus of the zygote. In order to establish the expression pattern of Fyn in the early embryo, we examined the distribution pattern of Fyn by immunofluorescence microscopy. Fyn protein is distributed evenly among cells of the cleavage stage zebrafish embryo and is concentrated in the cortical region of each cell. During blastula and gastrula stages, Fyn was expressed in all cells, however a subpopulation of cells exhibited strong nuclear staining for Fyn. Nuclear Fyn staining was not observed after the gastrula period of development, nor in the adult zebrafish. Immunoprecipitation of Fyn from isolated mid-blastula nuclei confirmed Fyn was present in the nucleus. This is, to our knowledge, the first demonstration of Fyn kinase, which lacks a nuclear localization signal, present in the nucleus. The transient compartmentalization of Fyn in the nucleus could be important in nuclear signaling.

Effects of protein tyrosine kinase inhibitors on egg activation and fertilization-dependent protein tyrosine kinase activity.

Fertilization results in the activation of protein tyrosine kinases within minutes of sperm-egg binding, although the role of the kinase(s) involved is not clear. In the present study, we have treated sea urchin eggs with genistein, as well as other protein tyrosine kinase inhibitors, and have characterized the subsequent effect on fertilization and egg activation. Genistein treatment of sea urchin eggs inhibits the overall fertilization-dependent tyrosine kinase activity as well as the specific phosphorylation of a 350-kDa protein, but it did not inhibit cAMP-dependent kinase and had little effect on protein kinase C at concentrations less than 100 microM. Genistein, erbstatin, and tyrphostin B42 did not inhibit the early events of fertilization such as elevation of the fertilization envelope; however, later events such as pronuclear migration, DNA synthesis, and cell division were inhibited. These results suggest that protein tyrosine kinases activated following fertilization play a role in the later events of egg activation such as the initiation of pronuclear movement and entry into the S phase of the cell cycle.

Identification of an abl-related protein tyrosine kinase in the cortex of the sea urchin egg: possible role at fertilization.

Fertilization results in the tyrosine phosphorylation of several egg proteins within minutes of sperm-egg binding, although the identity of the kinase(s) involved and the mechanism of regulation is not known. In the present study, we have used site-directed antibodies based on the predicted amino acid sequence of a sea urchin egg transcript that shares significant homology with members of the ABL family of protein tyrosine kinases. These antibodies identified a 220-kDa protein kinase, highly enriched in the egg cortex, where it is tightly associated with detergent-insoluble cytoskeletal elements. The enzyme is capable of phosphorylating synthetic peptide substrates which were used to characterize the kinase activity in an immune-complex assay. Measurement of the protein tyrosine kinase activity immunoprecipitated at different times after fertilization revealed that the level of kinase activity is transiently elevated during the first few minutes postinsemination. Western blot analysis indicated that the amount of the 220-kDa protein did not increase significantly during this period, so the increased kinase activity probably results from activation of the enzyme. These in vitro studies indicate that the 220-kDa abl-related kinase is one of the protein kinases activated during fertilization and suggest that it may play a role in the egg activation process.

Characterization of cortical secretory vesicles from the sea urchin egg.

In order to evaluate the potential role of cortical vesicle exocytosis in modifying the egg surface at fertilization, we have begun characterization of the cortical secretory vesicle. Earlier reports (N. K. Detering, G. L. Decker, E. D. Schmell, and W. Lennarz, 1977, J. Cell Biol. 75, 899-914) have described the isolation of an egg cell surface complex which consists of the egg plasma membrane and cortical secretory vesicles. We have now developed a method of dissassembling the cell surface complex and isolating the cortical vesicles. The very low levels of contaminating plasma membrane in this preparation allow the meaningful comparison of plasma membrane and cortical vesicle proteins and lipids. The cortical vesicles were found to be rich in high-molecular-weight PAS-positive proteins. The majority of these glycoconjugates were solubilized by hypotonic lysis of the cortical vesicles and probably represent proteins sequestered inside the intact vesicles. The fatty acid composition of the cortical vesicles was found to be unusually high in arachidonic acid. The fatty acid composition of the cortical vesicles was closely similar to that of the plasma membrane; however, the cortical vesicles were substantially higher in cholesterol content.

Role of the Fyn kinase in calcium release during fertilization of the sea urchin egg.

Protein tyrosine kinase activity has been implicated as part of the signaling mechanism leading to the sperm-induced calcium transient following fertilization. In the present study, we have tested the role of the Fyn kinase in triggering the calcium transient by microinjecting domain-specific fusion proteins encoding regions of Fyn sequence as inhibitors of Fyn function in vivo. A fusion protein encoding the SH2 domain of Fyn caused an increase in the latent period between sperm-egg fusion and the beginning of the calcium transient and reduced the amplitude of the calcium signal. A fusion protein encoding the U + SH3 domains also caused a small increase in the latent period. Microscopic examination revealed that a large percentage of eggs injected with the U+SH3 or SH2 domains became polyspermic as a result of the delayed block to polyspermy. Affinity experiments demonstrated that the U+SH3 and SH2 domains of Fyn were capable of forming a stable complex with phospholipase Cgamma from the sea urchin egg. The results suggest that the Fyn kinase participates in the signaling events leading up to the calcium transient and may directly regulate phospholipase Cgamma activity at fertilization.

Fine structural localization of Concanavalin A binding sites on hamster spermatozoa.

The plasma membrane of epididymal spermatozoa of the golden hamster (Mesocricetus auratus) exhibits morphological differences over various parts of the head and tail as detected by air-dried replicas and freeze-etching techniques. In an attempt to ascertain whether any topographical differences exist in the number or distribution of carbohydrate moieties associated with the cell surface, cells were labeled with Concanavalin A and marked with hemocyanin. It was found that while the plasma membrane over the acrosomal region differed from that of the postacrosomal region in membrane components revealed by freeze fracturing, there was no apparent difference in the distribution or density of Con A binding sites detectable by hemocyanin localization. The tail regions exhibited differences in both fracture face appearance and the distribution of detectable carbohydrate moieties. It was also found that binding sites for Concanavalin A exist on the inner and outer acrosomal membranes in addition to those on the plasma membrane.

Quantitation of a src-like tyrosine protein kinase during fertilization of the sea urchin egg.

Fertilization of the sea urchin egg is known to involve an increase in overall protein tyrosine kinase activity which precede the first cell division. In order to determine the types of tyrosine kinases that are involved in fertilization, we have used immunological and other criteria to identify a c-src related protein kinase in eggs of the sea urchin L. variegatus. Using an immune complex assay, we have measured the level of this c-src related protein kinase during fertilization and early embryonic development. Fertilization results in a decrease in the c-src kinase detectable by this technique suggesting that c-src does not contribute to the fertilization induced increase in protein tyrosine kinase activity.

Protein tyrosine phosphorylation in response to fertilization.

The sea urchin egg contains one or more protein tyrosine kinases which are active during the response of the egg to fertilization. In the present study, we have used an antibody specific for phosphotyrosine to determine which egg proteins are phosphorylated on tyrosine in response to fertilization. Analysis of immunoblots prepared from fertilized and unfertilized eggs revealed that fertilization results in a major increase in the phosphotyrosine content of a 350-kDa egg protein. Increased phosphorylation of this protein was detected as early as 1 min after fertilization, at which time it represented the most prominent phosphotyrosine containing protein in the egg. Tyrosine phosphorylation of this protein was transient however, and after 5 min post-insemination, the protein was dephosphorylated or otherwise degraded. Egg membrane proteins of approximately 40, 75, and 145 kDa were also found to act as substrates for protein tyrosine kinases in vitro, but did not exhibit significant changes in phosphotyrosine content during egg activation.

Synthesis of a novel class of sulfated glycoproteins in embryonic liver and lung.

Slices from various organs and tissues of 14-day-old chick embryos synthesize a novel class of glycoproteins containing sulfated oligosaccharide chains that are not released from the polypeptides under conditions that cleave O-glycosidically linked chains. Of the tissues examined, embryonic lung and liver were the most active in incorporating [35S]O4 into these oligosaccharide chains, which were analyzed as glycopeptides following Pronase digestion. These low molecular weight sulfated glycopeptides were resistant to several chemical and enzymatic treatments that degrade a variety of high molecular weight sulfated glycosaminoglycans. The incorporation of both [3H]glucosamine and [35S]O4 into the glycopeptides was inhibited by tunicamycin, an antibiotic which inhibits the N-glycosylation of proteins. These observations, coupled with the finding that these chains were resistant to beta elimination, suggest that this novel type of sulfated oligosaccharide is N-glycosidically linked to protein. The sulfated glycopeptides from chick embryonic lung were characterized as containing terminal fucose and sialic acid residues as well as O-sulfated N-acetylglucosamine residues. Comparable studies with slices of adult liver and lung indicated that they were far less active in the synthesis of this class of sulfated glycopeptides, even though these organs, like those from the embryo, actively synthesize sulfated glycosaminoglycans.