Surface localization of the sea urchin egg receptor for sperm

The sea urchin egg receptor for sperm is thought to be involved in species-specific sperm-egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the high Mr (ca. 350 K) sperm receptor protein which copurified with egg plasma membrane-vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the high Mr receptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein.     

Molecular identification of a hyperpolarization-activated channel in sea urchin sperm

Sea urchin eggs attract sperm through chemotactic peptides, which evoke complex changes in membrane voltage and in the concentrations of cyclic AMP, cyclic GMP and Ca2+ ions The intracellular signalling pathways and their cellular targets are largely unknown. We have now cloned, from sea urchin testis, the complementary DNA encoding a channel polypeptide, SPIH. Functional expression of SPIH gives rise to weakly K+-selective hyperpolarization-activated channels, whose activity is enhanced by the direct action of cAMP. Thus, SPIH is under the dual control of voltage and cAMP. The SPIH channel, which is confined to the sperm flagellum, may be involved in the control of flagellar beating. SPIH currents exhibit all the hallmarks of hyperpolarization-activated currents (Ih), which participate in the rhythmic firing of central neurons, control pacemaking in the heart, and curtail saturation by bright light in retinal photoreceptors. Because of their sequence and functional properties, Ih channels form a class of their own within the superfamily of voltage-gated and cyclic-nucleotide-gated channels.     

Structural and molecular characterization of dynein in a gall-midge insect having motile sperm with only the outer arm

The dipteran Monarthropalpus flavus possesses a peculiar sperm axoneme, characterized by multiple rows of microtubular doublets linked by the outer dynein arms only, lacking any equivalent of the central pair/radial spoke complex. The structure of these dynein molecules was studied by electron microscopy (EM). Using the quick-freeze, deep-etch method of EM, they were found to be similar to outer dynein arms described previously. Two globular "heads," each subdivided by a cleft, are clearly discernible. "Stalks" extend from proximal head to contact the B-tubule of the adjacent doublet. Unlike the situation in vertebrate sperm, the stalks sometimes branch into two thinner strands that contact the B-tubule at different sites. Treatment of demembranated sperm cells with ATP and vanadate induces conformational changes in the dynein outer arms. These are interpreted as the result of rotation of the dynein head with respect to what is observed in axonemes in rigor condition (after ATP depletion). SDS-PAGE indicates that the high-molecular-weight complement of this molecule comprises a single heavy chain. Specific dynein heavy chain-related DNA sequences corresponding to the catalytic-phosphate binding region were amplified by RT-PCR. Only one axonemal dynein sequence was identified among all amplified fragments. Southern blot analysis performed on genomic DNA using this sequence as a probe identified two hybridizing genes, only one of which is able to encode a functional product. Thus, genetic analysis indicates that this axonemal outer arm dynein is a homodymer of a single heavy chain subunit. In vivo, spermatozoa of this species are stored in a rolled configuration in female spermatheca, where they move rapidly with a wave-like motion. This movement could not be reproduced in vitro, except when spermatozoa were constrained in a bent configuration by some mechanical impediment. We propose that, in the absence of both the central pair/radial spoke complex and the inner arms, a curvature-dependent activation acts to trigger motility in these spermatozoa.     

The role of the dynein stalk in cytoplasmic and flagellar motility

We have recently identified a microtubule binding domain within the motor protein cytoplasmic dynein. This domain is situated at the end of a slender 10-12 nm projection which corresponds to the stalks previously observed extending from the heads of both axonemal and cytoplasmic dyneins. The stalks also correspond to the B-links observed to connect outer arm axonemal dyneins to the B-microtubules in flagella and constitute the microtubule attachment sites during dynein motility. The stalks contrast strikingly with the polymer attachment domains of the kinesins and myosins which are found on the surface of the motor head. The difference in dynein's structural design raises intriguing questions as to how the stalk functions in force production along microtubules. In this article, we attempt to integrate the myriad of biochemical and EM structural data that has been previously collected regarding dynein with recent molecular findings, in an effort to begin to understand the mechanism of dynein motility.     

The 350-kDa sea urchin egg receptor for sperm is localized in the vitelline layer

Previous studies have established by several methods that the 350-kDa egg receptor for sperm is localized on the plasma membrane-vitelline layer complex of the egg of the sea urchin Strongylocentrotus purpuratus. In addition, it has been found that molecules which are cross-reactive with anti-receptor antibody are present in the cortical granules located at the inner face of the plasma membrane. The objective of this study was to define more precisely the locale of the cell surface receptor. We have found that following fertilization, the immunoreactive receptor initially found on the egg surface moved to the fertilization envelope (FE) and then disappeared in parallel with the loss of sperm binding activity. A cross-linked, high-molecular-weight derivative of soybean trypsin inhibitor (hMW-SBTI) which was unable to pass through the elevating FE blocked the loss of both immunoreactivity and the sperm binding activity of the FE, but did not inhibit the vitelline delaminase activity that has been implicated in FE formation. Western blot analysis following SDS-PAGE of the proteins of the FE isolated in the presence of hMW-SBTI and benzamidine revealed the presence of the 350/320-kDa proteins which cross-reacted with anti-receptor antibody. Experiments in which molecules on the surface of unfertilized eggs were labeled with biotin and traced after FE formation revealed that a significant portion of the 350/320-kDa glycoproteins that were incorporated into the FE originated from the cell surface, rather than from the cortical granules. These findings provide strong evidence that in unfertilized eggs the egg receptor for sperm exists as part of the protein complex known as the vitelline layer which serves as a precursor of the FE. Evidence is presented indicating that some of the receptor in the vitelline layer is cryptic and a possible function for this cryptic form of the receptor is proposed.     

Purealin blocks the sliding movement of sea urchin flagellar axonemes by selective inhibition of half the ATPase activity of axonemal dyneins

Ciliary and flagellar movements are explained by active sliding between the outer doublet microtubules of an axoneme via their inner and outer dynein arms. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, blocked the motility of Triton-demembranated sea urchin sperm flagella within 5 min at concentrations above 20 microM. In a similar concentration range, purealin blocked the sliding movement of the flagellar axonemes in vitro within a few minutes judging from the turbidity measurements. The ATPase activity of axonemes was partially inhibited by purealin in a concentration-dependent manner. The maximum inhibition reached approximately 50% at concentrations above 20 microM, indicating that half the axonemal ATPase activity is sensitive to purealin. Similar results were observed on the ATPase activity of outer-arm-depleted axonemes and that of a mixture of 21S dynein and salt-extracted axonemes. On the other hand, ATPase activity of isolated 21S dynein was not inhibited by purealin. The inhibitory action of purealin on the axonemal ATPases was reversed by dilution of purealin. The effect of purealin on the double-reciprocal plot of the ATPase activity as a function of ATP concentrations showed that the inhibition was not a competitive type. In accord with this finding, purealin did not affect the vanadate-mediated UV photocleavage of axonemal dyneins. These results suggest that purealin binds reversibly to a site other than the catalytic ATP-binding site and inhibits half the ATPase activity of axonemes. Taken together, our results suggest that purealin-sensitive ATPase activity of the dynein arms plays an essential role in generating the sliding movement of flagellar axonemes.     

Purification of EGIP-D-binding protein from the embryos of the sea urchin Anthocidaris crassispina

We report three cases of male breast myofibroblastoma. This uncommon benign tumor arises from breast mesenchyma and is more frequently seen in adult men. Mammographic findings consist of a well-delimited, round to oval dense mass, variable in size but usually 1-4 cm in diameter. No microcalcifications were observed. Ultrasonography confirms the solid nature of the lesion, showing a well-circumscribed, homogeneous, hypoechoic mass, compressible with pressure. Although FNA cytology may support the diagnosis, surgical biopsy should be performed. Tumorectomy is the treatment of choice. To our knowledge, no more than 40 cases of breast myofibroblastoma have been reported. This is the first report in the literature which emphasizes the mammographic and ultrasonographic features of this tumor.     

Increased association of synaptosome-associated protein of 25 kDa with syntaxin and vesicle-associated membrane protein following acrosomal exocytosis of sea urchin sperm

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated integral membrane protein expressed almost exclusively in neuronal and neuroendocrine tissues. This protein forms a ternary complex with vesicle-associated membrane protein (VAMP) and syntaxin, which is thought to regulate the fusion of plasma and vesicle membranes during exocytosis. We report the identification of SNAP-25 expressed in sea urchin sperm. Sea urchin SNAP-25 shares greater identity with mammalian SNAP-25 than with mammalian SNAP-23, a ubiquitously expressed homologue believed to regulate membrane fusion in non-neuronal tissues. Sea urchin sperm contain a single exocytotic vesicle, the acrosomal vesicle, whose contents are exposed during the acrosome reaction. Fusion of the plasma membrane with the acrosomal vesicle membrane at multiple points (vesiculation) results in the release of SNAP-25 with the shed acrosome reaction vesicles. A complex containing SNAP-25, syntaxin, and VAMP is present in sperm, as detected by affinity chromatography and immunoprecipitation. Although this complex is present prior to the acrosome reaction, the amount of complex increases over 4-fold following acrosomal exocytosis. These findings support the involvement of SNAP-25 in the invertebrate sperm acrosome reaction, possibly through increased association with VAMP and syntaxin driving the fusion of plasma and acrosomal membranes.     

Changes in the topography of the sea urchin egg after fertilization

Rats of various postnatal ages were utilized to study developmental changes in the distribution of Na, K and H2O between the various compartments of the lateral ventricular plexus (LVP). During the 3 weeks after birth, as the LVP grows from 0.5 to 0.8 mg, there is a significant increase in plexus K which is accompanied by a progressive decrease in Na and H2O. Also, during this postnatal period the decrease in [3h]inulin space in the plexus is proportional to the decrease in the Na space. Between 3 weeks and adulthood, the [3h]inulin and Na spaces are both augmented to a similar extent; moreover, during this same period of development there is a trebling of the residual [51cr]erythrocyte volume. Despite the substantial changes in the volume of the extracellular fluid and of the residual blood in the plexus with age, the calculated concentrations (mEquiv./kg H2O) of choroid cell Na (30-35) and K (145-155) are similar for all ages investigated. The derived data for cellular ionic concentration, together with the analysis of the ionic concentration gradients (cerebrospinal fluid/plasma H2O), suggest that the transport mechanism which translocates Na and K across the choroidal membrane is operative as early as 3-4 days postnatal. The important role of the choroid plexus in central nervous system homeostasis is discussed in relation to the developing brain.     

The coordination of centrosome reproduction with nuclear events of the cell cycle in the sea urchin zygote

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1-cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Delta-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase-anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary "licensing" event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.     

The nucleus negatively controls the synthesis of mitochondrial proteins in the sea urchin egg

Enucleation of Paracentrotus lividus eggs, followed by parthenogenetic activation induces a sharp increase in the synthesis of mitochondrial proteins as shown by electrofluorography after in vivo labeling with radioactive amino acids. These results further substantiate the hypothesis that the cell nucleus negatively controls mitochondrial replication in the sea urchin egg.     

Effects of extracellular ATP on hair cells isolated from the guinea-pig semicircular canals

Using stereological methods, two cerebral cortical areas from AIDS brains were investigated. Neuronal density, profile area of neurons, and perikaryon volume fraction were measured and compared to age-matched control brains. In the fronto-orbital cortex (area 11) of AIDS brains, a significant loss of neurons was seen. The perikaryon volume fraction was likewise decreased. The size of neurons did not differ between control and AIDS brains. In patients with clinical signs of progressive dementia and in brains with human immunodeficiency virus (HIV)-specific neuropathology (HIV-leukoencephalopathy and/or HIV-encephalitis) as compared to patients lacking these features, a small decrease in neuronal density was noted but this difference did not reach the level of statistical significance (P = 0.16). In the superior parietal lobule (area 7) of AIDS brains, no loss of nerve cells was noted. AIDS patients with progressive dementia and brains with HIV-specific neuropathology showed no difference in neuronal densities as compared to those without such features. We conclude that the fronto-orbital cortex, in contrast to the parietal cortex, is mainly damaged in AIDS brains. Neuronal loss was not significantly correlated with development of dementing symptoms and of HIV-specific neuropathology.     

Accumulation of WGA receptors in the cleavage furrow during cytokinesis of sea urchin eggs

Sea urchin eggs stained with fluorescein-conjugated wheat germ agglutinin (F-WGA) before or after fixation showed a marked accumulation of fluorescence at the cleavage furrow in the first and the second cell divisions. WGA receptors (WGA-binding membrane glycoproteins) were redistributed to the equatorial region through several steps in compressed eggs. Accumulated WGA receptors showed a distribution similar to that of contractile-ring microfilaments throughout most of the steps. Therefore, the former is probably associated with the latter directly or indirectly. Labeling with F-WGA provides a simple method to detect contractile-ring microfilaments in living eggs. Treatment of eggs with colcemid shortly before cytokinesis dispersed the ring-like accumulation of WGA receptors together with contractile-ring microfilaments. This result suggests that microtubule structures, probably asters, are involved in the redistribution of WGA receptors. Cytochalasin B prevented furrowing when it was applied shortly before cytokinesis. While contractile-ring microfilaments showed a spotty distribution in the expected furrow region, WGA receptors were normally redistributed. Furthermore, a higher concentration of the drug allowed the appearance of accumulated WGA receptors in compressed eggs although the development into a ring-like configuration was inhibited. These observations suggest the possibility that the redistribution of WGA receptors is involved in the formation of contractile ring.     

Bottle cells are required for the initiation of primary invagination in the sea urchin embryo

Invagination of epithelial tissue occurs during gastrulation, neurulation, and organogenesis in many organisms. However, the underlying morphogenetic mechanisms of invagination are not understood. To elucidate these mechanisms, we have analyzed the initial invagination of the vegetal plate in the sea urchin embryo, a process termed primary invagination. At the onset of invagination, a ring of cells with highly constricted apices (bottle cells) encircles a group of two to eight round, central cells. To investigate the morphogenetic role of the bottle cells in the process of primary invagination, we have undertaken a series of laser ablation studies in which different proportions of various cell types were ablated and the effects were recorded using 4-D microscopy. Elimination of a 90 degrees-180 degrees arc of bottle cells markedly retards invagination, but only within the ablated region. Ablation of other cell types does not result in a statistically significant effect on primary invagination. These studies indicate that the number and arrangement of the bottle cells are critical factors for proper initiation of invagination. In addition, we have used the perturbing anti-hyalin antibody mAb183 to show that cell attachment to the hyaline layer is necessary for bottle cell formation and the initiation of primary invagination.     

Experimental transmission of the nematode Echinomermella matsi to the sea urchin Strongylocentrotus droebachiensis in the laboratory

Three experiments were conducted to investigate the possibility of maintaining the Echinomermella matsi-Strongylocentrotus droebachiensis system in the laboratory. The experiments were performed by injecting E. matsi larvae taken directly from gravid female nematodes into the mouths of sea urchins. In all experiments, this treatment resulted in a higher infection in treated animals than in unmanipulated controls. The successful establishment of larvae indicates that E. matsi has a monoxenous life cycle. The growth of larvae in experimentally infected hosts was slow, indicating that the generation time of the parasite is of the same magnitude as the life expectancy of the host, 1-2 yr. This slow growth rate suggests that considerable resources will be needed to maintain the system in the laboratory.     

Hox-type and non-Hox homeobox gene sequences in genomic DNA of the sea urchin Holopneustes purpurescens

As a preliminary step in an analysis of Hox gene expression and radial body plan specification in sea urchin development, we amplified partial homeobox sequences in H. purpurescens by PCR using degenerate primers. The primers, HoxE and HoxF (Pendleton et al., 1993), spanned a highly conserved region of 82 nucleotides encompassing amino acids 21-47 of the homeodomain. Seven Hox-type homeobox sequences and two non-Hox homeobox sequences were identified. The seven Hox-type sequences were placed provisionally in Hox paralogous groups, one in paralogous group 3, three in paralogous groups 6-8 and three in paralogous groups 9 13. The non-Hox sequences had similarities with Xlox and Gbx homeobox genes.