A subset of Pr65gag is nucleus associated in murine leukemia virus-infected cells.

Nuclei of cells infected with Moloney murine leukemia virus (MoMuLV) were examined for the presence of gag proteins. This analysis was performed in conjunction with other studies suggesting a possible role for gag proteins in regulating nuclear events relating to processing and/or transport of viral genomic RNA. We detected Pr65gag and a p30-related protein in a nuclear fraction of infected cells. We also found evidence that a highly conserved amino acid sequence, which is shared by p30 and U1 small nuclear ribonucleoprotein 70-kDa protein, is a component of the nuclear targeting sequence for Pr65gag. Immunoelectron microscopy studies with a monoclonal anti-p12 antibody established that approximately 18% of gag-containing proteins of MoMuLV are located in the nucleus. Such gag-containing proteins from a mutant MoMuLV that lacks N-terminal myristic acid had greater affinity for the nucleus, suggesting that fatty acid acylation of Pr65gag plays a role in overcoming the proposed nuclear transport signal. The possible roles that nuclear gag proteins may play in retroviral replication are discussed.

Xlcaax-1 is localized to the basolateral membrane of kidney tubule and other polarized epithelia during Xenopus development.

Xlcaax-1 is a novel, maternally expressed, 110-kDa, CAAX box containing protein that undergoes isoprenylation and palmitoylation through which it associates with the plasma membrane. We report here the cellular and subcellular localization of the xlcaax-1 protein during development of Xenopus laevis. Whole-mount immunocytochemistry and immunoperoxidase staining of tissue sections show that during development the xlcaax-1 protein accumulation is coincident with the differentiation of the epidermis, pronephros, and mesonephros. In the pronephros and mesonephros the xlcaax-1 protein is localized to the basolateral membrane of differentiated tubule epithelial cells. Thus, the xlcaax-1 protein serves as a marker for tubule formation and polarization during Xenopus kidney development. Xlcaax-1 may also be used as a marker for the functional differentiation of the epidermis and the epidermally derived portions of the lens and some cranial nerves. Western blot analysis shows that in the adult the xlcaax-1 protein is most abundant in kidney. Immunogold EM analysis shows that the xlcaax-1 protein is highly enriched in the basal infoldings of the basolateral membrane of the epithelial cells in adult kidney distal tubules. In addition, immunoperoxidase staining of tissue sections detected low levels of xlcaax-1 protein in the epithelial cells of skin, urinary bladder, gall bladder, and parietal glands of the stomach. The localization pattern of xlcaax-1 suggests that the protein may function in association with an ion transport channel or pump.

Elevated beta-cell calmodulin produces a unique insulin secretory defect in transgenic mice.

Transgenic mice with elevated levels of beta-cell calmodulin develop severe diabetes even though pancreatic beta-cells contain reserve levels of insulin. Electron microscopic examination of transgenic pancreas confirmed the presence of abundant insulin secretory granules and failed to reveal obvious morphological abnormalities. These observations suggested that excess calmodulin may specifically impair the secretory process. To directly assess the effect of excess calmodulin on beta-cell function we have isolated pancreatic islets from transgenic animals. Transgenic islets from 6- to 8-day-old mice used 40% less glucose than normal islets and contained 58% of the normal insulin content, 90% of the normal glucagon content, and 5-fold higher levels of calmodulin than islets from control mice of the same age. Parallel perifusions of normal and transgenic islets confirmed that excess calmodulin inhibited glucose-stimulated insulin secretion; first phase secretion was reduced by 60%, and second phase secretion was essentially absent. Static assays were performed to assess the response to other secretagogues. All fuel secretagogues tested were ineffective in stimulating insulin secretion from transgenic islets. Secretion in response to depolarizing levels of potassium was also severely impaired. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine increased transgenic secretion, but not to the level obtained in normal islets. Of the compounds examined, only phorbol 12-myristate 13-acetate and carbachol, two substances thought to act in beta-cells by stimulation of protein kinase-C, produced equivalent secretion in normal and transgenic islets. Phorbol 12-myristate 13-acetate also appeared to restore second phase secretion in transgenic islets. These results indicate that the initial period of calmodulin-induced diabetes is due to a secretory defect. This defect appears to be distal to membrane depolarization and is selective for the second phase of insulin secretion.

Glucocorticoid regulation of rat liver urate oxidase.

Urate oxidase, an enzyme involved in purine catabolism, comprises the crystalline core of rat liver peroxisomes. An affinity-purified monospecific antibody was developed to study the expression of urate oxidase protein levels. Immunoreactive urate oxidase was not detectable in prenatal liver; however, it is present at low levels after birth until approximately day 15 (postnatal age); expression sharply increases just prior to day 20, after which the enzyme is maintained at adult levels. This pattern of expression was similar to that of another peroxisomal enzyme, catalase; these developmental increases reflect the increase in peroxisomal number. Administration of exogenous glucocorticoid hormone to 10-day-old rats resulted in a precocious rise (2.5-fold) in urate oxidase levels. Adrenalectomy at 10 days of age did not cause decreased levels in the fourth week of life. In adult animals, while exogenous glucocorticoid administration did not influence urate oxidase levels, adrenalectomy at 60 days of age decreased urate oxidase levels to 40 percent of control levels. Subsequent administration of exogenous glucocorticoid hormone restored urate oxidase to normal levels. Parallel studies of catalase levels indicate that this glucocorticoid-sensitive response is not generalized for all peroxisomal proteins. Our results suggest that peroxisomes proliferate during early postnatal development, but after this process is complete, the biogenesis of individual peroxisomal proteins may be independently regulated.

Hormonal responsiveness by immature rabbit uterine epithelial cells polarized in vitro.

This paper reports the development of an in vitro cell culture system of polarized uterine epithelial (UE) cells from the immature rabbit. UE cells from immature rabbit were cultured on EHS (Engelbreth-Holm-Swarm) matrix-coated semipermeable filter inserts in a serum-free, phenol red-free defined medium. The cells in primary culture attached, proliferated, formed monolayers, and exhibited structural and functional polarity. Structural differentiation was validated by ultrastructural features, such as apical microvilli, intercellular junctions, desmosomes, and polar organization of apical vs. basal membrane domains. Trans-monolayer epithelial resistance, a reflection of functional tight junctions, preferential basal uptake of [35S]methionine, polarized distribution of labeled secretory proteins, and increased secretory activity marked by preferential apical secretion (greater than 90%) of proteins were indices of functional polarity. With the development and maintenance of polarized state by the UE cells, some of the newly synthesized proteins were sorted and vectorially secreted into their distinct compartments. De novo synthesis and exclusive apical secretion of uteroglobin (a progesterone-induced protein in vivo) by the polarized UE cells occurred in response to progesterone treatment in vitro. The hormone responsiveness was maintained for a prolonged period under these conditions. A culture system in which the UE cells maintain functional and morphological polarity and sustain their hormonal responsiveness facilitates the study of regulation of specialized functions by the UE cells that are regulated by steroid hormones and their interactions with the other uterine cell types.

Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells.

The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000-60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the Mr 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the beta subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperature-dependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoelectron microscopy. Treatment of mKSA cells with 10 microM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control.

Biogenesis of the endoplasmic reticulum in activated B lymphocytes: temporal relationships between the induction of protein N-glycosylation activity and the biosynthesis of membrane protein and phospholipid.

An earlier report from this laboratory documented a substantial increase in the rates of dolichol-linked oligosaccharide intermediate synthesis and protein N-glycosylation in purified murine splenic B lymphocytes (B cells) activated by treatment with bacterial lipopolysaccharide (LPS). In this study the developmental patterns for the induction of lipid-mediated protein N-glycosylation, membrane protein, and phosphatidylcholine (PC) biosynthesis were compared during the proliferative response of B cells to LPS. By electron microscopy it could be seen that a distinct endoplasmic reticulum (ER) network began to develop by 24-48 h after exposure of the purified B cells to LPS. The rate of synthesis of membrane protein increased markedly during the first 10 h after activation, reaching a maximum at 30-40 h. The induction of protein N-glycosylation was delayed slightly relative to membrane protein synthesis, with glycoprotein synthesis increasing sharply approximately 20 h after activation. When phospholipid synthesis was monitored by measuring [CH3-3H]choline incorporation into PC, the rate of labeling increased slowly during the first 35 h, but more substantially between 35 and 90 h. The incorporation of labeled choline into PC was drastically reduced by 5'-deoxy-5'-isobutylthio-3-deazaadenosine, an inhibitor of CDP-choline synthesis, indicating that the incorporation of radiolabeled choline is primarily a measurement of the rate of de novo synthesis of PC. In vitro assays revealed that while choline kinase activity was virtually unchanged, CDP-choline synthetase activity increased gradually throughout the activation period. Diacylglycerol cholinephosphotransferase activity, an ER-associated enzyme, was present at low levels between 0 and 35 h, but increased fivefold between 35 and 90 h. On the basis of the developmental patterns for the rates of protein N-glycosylation, membrane protein insertion, and PC biosynthesis determined by metabolic labeling experiments, we tentatively conclude that all of the ER-associated membrane proteins involved in these biosynthetic processes are not induced concurrently during the activation of B cells by LPS.

Vectorial secretion of prostaglandins by polarized rodent uterine epithelial cells.

Uterine epithelial cells (UEC) isolated from mature mice as well as immature mice and rats were cultured on EHS matrix-coated nitrocellulose filters in order to determine their ability to secrete prostaglandin (PG) F2 alpha and PGE2 in a polarized manner. Ultrastructural analyses were performed to validate the polar nature of mouse UEC and demonstrate the presence of separate apical and basolateral plasma membrane domains. These properties included the presence of tightly juxtaposed lateral membranes, apical microvilli, and a relatively flat basal surface. Biochemical indices of polarity included the preferential (approximately 5:1) basal uptake of [35S]methionine as well as a preferential (approximately 9:1) apical secretion of protein. UEC isolated from mice during the estrous and diestrous stages of the estrous cycle did not differ in their degree of polarity, as measured by these morphological and biochemical indices. UEC of estrous and diestrous mice as well as immature mice and rats preferentially secreted PGF2 alpha to the basal medium to an approximately 4-fold greater extent than to the apical medium. PGE2 was secreted at least 10-fold less than PGF2 alpha, and a preferential basal secretion could not be demonstrated. Polarized UEC accumulated relatively large cellular pools of PGF2 alpha, while nonpolarized cells grown on matrix-coated plastic did not. This difference was reflected by the inability of an inhibitor of PG biosynthesis, indomethacin, to inhibit PGF2 alpha secretion by polarized cells during short (4-h) incubations. In contrast, this drug effectively inhibited secretion in nonpolarized cells or polarized cells incubated with indomethacin for longer (24-h) intervals. Therefore, cellular PGF2 alpha pools apparently support continued secretion of this lipid even when de novo synthesis is transiently inhibited. Preferential basal secretion of PGF2 alpha was due to the polar nature of UEC, since disruption of tight junctions with EGTA modified the basal to apical ratio of PGF2 alpha secretion to near unity. Sodium azide inhibited the secretion of PGF2 alpha, indicating that PGF2 alpha secretion was energy dependent. PGF2 alpha secretion was not coupled to protein synthesis or secretion, since cycloheximide did not inhibit this process in polarized or nonpolarized cells. These studies describe the first evidence for polarized secretion of lipid-derived hormones by epithelial cells. The preferential basal secretion of PGF2 alpha may play an important role in regulating UEC interactions with the underlying stroma.

Loss of yolk platelets and yolk glycoproteins during larval development of the sea urchin embryo.

The fate of the yolk platelets and their constituent yolk glycoproteins was studied in Strongylocentrotus purpuratus eggs and embryos cultured through the larval stage. Previous studies have shown that the yolk glycoproteins undergo limited proteolysis during early embryonic development. We present evidence that the yolk glycoproteins stored in the yolk platelets exist as large, disulfide-linked complexes that are maintained even after limited proteolysis have occurred. We provide additional evidence that acidification of the yolk platelet may activate a latent thiol protease in the yolk platelet that is capable of correctly processing the major yolk glycoprotein into the smaller yolk glycoproteins. Because we previously showed that these yolk glycoproteins are not catabolized during early embryonic development, it was of interest to study their fate during larval development. Using a specific polyclonal antibody to a yolk glycoprotein, we found that both yolk glycoproteins and the yolk platelets disappeared in feeding, Day 7, larval stage embryos, but that starvation did not significantly affect the levels of the yolk glycoproteins. We also found that the yolk glycoproteins reappeared in 30-day-old premetamorphosis larvae.

Development of morphological and functional polarity in primary cultures of immature rat uterine epithelial cells.

The present study describes a culture environment in which luminal epithelial cells isolated from immature rat uteri and cultured on a matrix-coated permeable surface, with separate apical and basal secretory compartments, proliferate to confluence. Subsequently the cells undergo a process of differentiation accompanied by progressive development of functional polarity. Ultrastructural and immunocytochemical evidence verifies the ability of these primary cultures to regain polar organization, separate membrane domains, and form functional tight junctions as demonstrated by the development of transepithelial resistance. The appearance of uvomorulin is restricted to the lateral cell surface. Coordinated indices of functional polarity that develop progressively in post-confluent cultures include the preferential uptake of [35S]methionine from the basal surface and a rise in uterine epithelial cell secretory activity characterized by a progressive preference for apical secretion. The time dependent development of polarity was characterized by differences in the protein profiles of the apical and basolateral secretory compartments. The maintenance of hormone responsiveness by the cultured cells was validated by the secretion of two proteins identified as secretory markers of estrogen response in the intact uterus. The technique of culturing the cells on a matrix-coated permeable surface with separate secretory compartments produces a uterine epithelial cell that morphologically and functionally resembles its in situ equivalent. The culture method and analytical approach used in this present study may be applied to primary cultures of a variety of natural epithelia, which have hitherto proven resistant to more conventional culture methodologies.

Developmental distribution of a cell surface glycoprotein in the sea urchin Strongylocentrotus purpuratus.

Recent studies from this laboratory have shown that an antigen recognized by a monoclonal antibody (MAb 1223) displays a bimodal distribution of expression in development of the embryo of Strongylocentrotus purpuratus. This molecule is specifically localized to the primary mesenchyme cells of the embryo, but is also found within the egg. In the current study, immunoelectron microscopy was used to determine the subcellular distribution of the antigen and to determine its fate during early stages of development of the embryo. In eggs, the epitope recognized by MAb 1223 was localized to the cortical vesicles. Immunoblot analysis of an isolated cell surface complex (CSC) that contained the cortical vesicles revealed the presence of a 130-kDa protein, as well as immunoreactive components of higher molecular weight. Upon fertilization, the antigen was exocytosed from the cortical vesicles and became associated with the hyaline layer, the fertilization envelope, and the plasma membrane. Subsequently, the epitope could be detected within small vesicles and yolk platelets. By 60 min postfertilization, the amount of epitope detected intracellularly or in the perivitelline compartment was greatly reduced. At later stages of development, when formation of the embryonic skeleton occurred, the 1223 antigen was principally localized to the Golgi complex and to the syncytial cell surface of the primary mesenchyme cells. Thus, the results of this study suggest that in S. purpuratus the 1223 antigen is stored and secreted from the cortical vesicles of the egg, degraded after fertilization, and then later expressed on the surface of the primary mesenchyme cells.

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells.

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.

Growth of linear spicules in cultured primary mesenchymal cells of sea urchin embryos is bidirectional.

The growth of spicules of the sea urchin embryo was studied in a simple in vitro system in which primary mesenchymal cells attach to the substrata, migrate and fuse via filopodia, and subsequently deposit CaCO3, which in most cases is in the form of linear rods. The use of autoradiographs following 45Ca2+ pulse-chase labeling revealed that in such a system linear spicules that formed had two focal points of growth. Elongation occurred by addition of approximately equivalent amounts of Ca2+ to both ends of each rod. Multiform spicules having variable numbers of elongation sites (tips) also showed a similar pattern of Ca2+ deposition. Thus, the growth of both linear and relatively complex skeletal forms is apparently accomplished by the same basic mechanism.

Heparin/heparan sulfate is involved in attachment and spreading of mouse embryos in vitro.

The involvement of embryonic cell surface proteoglycans in the attachment and outgrowth of cultured mouse embryos has been investigated. Several lines of evidence indicate that periimplantation stage blastocysts express heparin/heparan sulfate proteoglycans on their cell surfaces that can mediate embryo attachment and trophoblast outgrowth on a variety of matrices. First, in the presence of soluble heparin, the rate at which embryos attach and outgrow on laminin, fibronectin, or monolayers of uterine epithelial cells is reduced considerably. In the case of fibronectin, the rate of outgrowth in the presence of the heparin is slower than in the presence of the Arg-Gly-Asp-Ser-containing peptide that is recognized by a fibronectin receptor. Embryos also attach and exhibit a limited ability to outgrow on platelet factor IV, a heparin binding protein that does not possess the additional binding domains of laminin or fibronectin. Attachment on platelet factor IV is inhibited by heparin. Second, cell surface digestion of attachment-component embryos with heparinase, but not chondroitinase ABC, slows the rate of outgrowth on tissue culture plates in the presence of serum. Third, selective staining for sulfated molecules on the trophectoderm surface of periimplantation stage embryos indicates that such molecules are abundant and uniformly distributed on these cell surfaces. Last, heparin/heparan sulfate proteoglycans are detected as major cell surface components of embryos using vectorial labeling with lactoperoxidase and Na125I. Collectively, these data indicate that heparin/heparan sulfate-bearing molecules have a direct role in attachment and outgrowth of implantation stage blastocysts.

Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro.

An in vitro culture system for primary mesenchyme cells of the sea urchin embryo has been used to study the cellular characteristics of skeletal spicule formation. As judged initially by light microscopy, these cells attached to plastic substrata, migrated and fused to form syncytia in which mineral deposits accumulated in the cell bodies and in specialized filopodial templates. Subsequent examination by scanning electron microscopy revealed that the cell bodies and the filopodia and lamellipodia formed spatial associations similar to those seen in the embryo and indicated that the spicule was surrounded by a membrane-limited sheath derived by fusion of the filopodia. The spicules were dissolved from living or fixed cells by a chelator of divalent cations or by lowering the pH of the medium. However, granular deposits found in the cell bodies appeared relatively refractory to such treatments, indicating that they were inaccessible to agents that dissolved the spicules. Use of rapid freezing and an anhydrous fixative to preserve the syncytia for transmission electron microscopy and X-ray microprobe analysis, indicated that electron-dense deposits in the cell bodies contain elements (Ca, Mg and S) common to the spicule. Examination of the spicule cavity after dissolution of the spicule mineral revealed openings in the filopodia-derived sheath, coated pits within the limiting membrane and a residual matrix that stained with ruthenium red. Concanavalin A--gold applied exogenously entered the spicule cavity and bound to matrix glycoproteins. Based on these observations, we conclude that components of the spicule initially are sequestered intracellularly and that spicule elongation occurs in an extracellular cavity. Ca2+ and associated glycoconjugates may be routed in this cavity via a secretory pathway.

Developmental expression of a cell-surface protein involved in calcium uptake and skeleton formation in sea urchin embryos.

The developmental expression of a cell-surface protein involved in Ca2+ accumulation and skeleton formation in sea urchin embryos has been studied. In Strongylocentrotus purpuratus, this protein is present in the egg and in all cell types of the early embryo. After gastrulation, its synthesis and expression are restricted to the skeleton-forming primary mesenchyme cells. In Lytechinus pictus, the protein cannot be detected in eggs or in embryos until the mesenchyme blastula stage. Hybrid embryos demonstrate a pattern of expression indistinguishable from that of the species contributing the maternal genome, which suggests that early expression of the protein in S. purpuratus embryos is due to utilization of maternal transcripts from the egg. Later expression of this protein in primary mesenchyme cells is the result of cell-type-specific synthesis, likely encoded by embryonic transcripts. This cell-type-specific expression in primary mesenchyme cells correlates temporally with Ca2+ accumulation during skeleton formation in the embryo.

Characterization of yolk platelets isolated from developing embryos of Arbacia punctulata.

Yolk platelets, a major organelle of sea urchin eggs and embryos, were isolated from Arbacia punctulata and biochemically characterized over the course of development to the pluteus stage. Fractionation by sucrose gradient centrifugation revealed yolk platelets in two major density classes. The low-density yolk platelet fraction could be obtained as a very homogeneous preparation and was highly enriched in acid phosphatase activity, while depleted of mitochondrial (cytochrome c oxidase) and plasma membrane (phosphodiesterase) marker enzymes. The chemical composition of low-density yolk platelets prepared from eggs and embryos at various stages of development remained unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and protein. However, analysis of the major yolk platelet glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a number of stage-specific changes. These glycoproteins were found to be major glycoproteins of crude embryo lysates and were predominantly of the polymannose, N-linked type. The predominance of polymannose-type glycoproteins in yolk platelets was further demonstrated by their staining with concanavalin A-colloidal gold in Lowicryl-embedded sections of embryos. These studies represent the first systematic biochemical characterization of intact yolk platelets and the changes in them during early embryonic development.

Characterization of the Strongylocentrotus purpuratus egg cell surface receptor for sperm.

In earlier studies from our laboratory, the intact sperm receptor was partially purified from Strongylocentrotus purpuratus crude egg membranes, but due to its insolubility, it was not possible to purify it to homogeneity. Nonetheless, this receptor preparation bound with species specificity to acrosome-reacted sperm, thereby inhibiting fertilization. Antibodies against the partially pure receptor inhibited fertilization in S. purpuratus (but not Arbacia punctulata) by coating the egg surface, indicating the presence of binding sites that can be species-specifically recognized by both sperm and antibody molecules. Recently we were able to further purify and characterize the receptor from S. purpuratus eggs. Chaotropic agent solubilization of the receptor prepared from crude egg membranes yielded a very high molecular weight glycoconjugate that had many of the properties of a proteoglycan. The receptor interacted with binding in an in vitro assay and bound with species specificity to acrosome-reacted sperm to inhibit fertilization. Unfortunately, this receptor preparation was soluble only in certain chaotropic agents. Exhaustive Pronase digestion of the intact receptor yielded a soluble high-molecular-weight (greater than 10(6)) polysaccharide that was virtually devoid of protein. This glycosaminoglycan-like fragment was highly sulfated, and contained fucose, galactosamine, and iduronic acid. The fragment inhibited fertilization, but did not do so with species specificity. Recently, soluble molecules with receptor activity were generated by treating intact dejellied eggs with trypsin. These proteolytically derived molecules contained (on a weight basis) approximately equal amounts of protein and carbohydrate. Importantly, they inhibited fertilization with species specificity. The results suggested that the binding activity was conferred by the polysaccharide component of the receptor and that the intact receptor and the tryptic fragments contained structural elements in the polypeptide chain necessary for species recognition.

A monoclonal antibody inhibits calcium accumulation and skeleton formation in cultured embryonic cells of the sea urchin.

The assembly of the spicules (primitive skeleton) of the sea urchin embryo is being studied in primary mesenchyme cells cultured in vitro. A monoclonal antibody (1223) has been prepared that inhibits the deposition of CaCO3 into the spicules. This antibody reacts with a 130,000 Mr cell-surface protein that is concentrated on the surface of approximately 5% of the cells of dissociated gastrula stage embryos. When primary mesenchyme cells in the embryo or cells cultured in vitro are examined, the 1223 antigen is detected on the surface of the cells and on the extracellular material associated with the spicule. We conclude that the 1223 antibody recognizes a cell-surface protein that plays an essential role in spicule formation.