Fertilization in the sea urchin, Strongylocentrotus purpuratus is blocked by fluorescein dyes.

Fertilization in gametes of the sea urchin Strongylocentrotus purpuratus was reversibly inhibited by several analogs of the anionic dye fluorescein. The dyes acted very rapidly and were effective when added before or several seconds after insemination. Eggs and sperm did not appear to be irreversibly modified by incubation in seawater solutions containing tetraiodofluorescein (erythrosin B). Sperm binding to the vitelline layer was also inhibited by erythrosin B, but required concentrations greater than that necessary to block fertilization. The ability of the compounds to block fertilization was a function of the particular fluorescein derivative used and its concentration. The concentration required to inhibit fertilization in 50% of the eggs was related to dye lipid solubility. The dyes may inhibit fertilization by preventing gamete membrane fusion.     

Purification of the sperm-binding factor from the egg of the sea urchin,Hemicentrotus pulcherrimus

The substance which seems to be responsible for the sperm-binding at fertilization was successfully purified from unfertilized eggs of the sea urchin, $\text{Hemicentrotus pulcherrimus}$. It completely cancelled the fertilizing capacity only of homologous sperm without reducing their motility. The antiserum against this substance made only homologous eggs incapable of binding sperm. The methods employed for purification were (1) extraction by urea, (2) fractionation by calcium acetate, (3) salting-out by ammonium sulfate, (4) gel filtration and (5) ion-exchange chromatography. This substance was electrophoresed on cellulose-acetate strip as a single band which was stained with Amido Black, and could not be split by 6 M guanidine hydrochloride.     

Sea urchin fertilization envelope: Uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Strongylocentrotus purpuratus embryos

The sea urchin fertilization envelope (FE) is an extraembryonic coat which develops from the egg vitelline envelope (VE) and the secreted paracrystalline protein fraction of the cortical granules at fertilization. The FE undergoes further developmental changes postinsemination which are characterized by changes in envelope permeability, solubility in reducing and denaturing solvents, and morphology. We have developed a procedure to uncouple cortical granule exocytosis from assembly of the paracrystalline protein fraction onto the VE template. Egg suspensions were inseminated in normal seawater and diluted into Ca²⁺- and Mg²⁺-free seawater at 15 sec postinsemination. Phase-contrast and electron microscopic observations showed that the embryos formed a normally elevated, extremely thin envelope through which the cortical granule exudate permeated. Secretion studies showed that eggs which were diluted into divalent ion-free seawater postinsemination secreted as much protein into the surrounding seawater as eggs which had their VEs removed prior to the experiment. We have termed the envelope elevated in divalent ion-free seawater the VE1 and we believe that it is the VE structural component of the FE based on its thickness and morphology. VE1s were isolated by gentle physical means and the preparations appeared to be greater than 80% pure based on radioactive mixing experiments and on malate dehydrogenase and glucose-6-phosphate dehydrogenase marker studies. VE1s were at least 80% soluble based on extraction of radioiodinated preparations with reducing and denaturing solvents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of VE1s showed eight major polypeptides which ranged from 30,500 to 270,000 in molecular weight.     

Timing the early events during sea urchin fertilization

To determine precisely the timing, duration, and sequences of the earliest events during sea urchin (Lytechinus variegatus) fertilization, the bioelectric recordings of microelectrode-impaled eggs were electronically superimposed, by video mixing, over the microscopic differential interference contrast image of the same egg at insemination. Videotape analysis, utilizing a slow-motion analyzer, demonstrates that the successful sperm triggers the bioelectric membrane potential reversal within 3.36 +/- 3.02 sec (0.72-9.76 sec range; sigma = 23 eggs) of sperm-egg attachment. This sperm, actively gyrating about its attachment site, is indistinguishable from the other, unsuccessful sperm until 12.66 +/- 2.72 sec (6.72-16.60 sec range; sigma = 15) later when the sperm tail ceases its beating and sperm incorporation ensues. The cortical granules begin to discharge, and the fertilization coat starts to elevate at the fusion site at 20.79 +/- 3.18 sec (13.62-26.08 sec range; sigma = 12) after the onset of the fertilization potential, i.e., an average of about 8 sec after the cessation of sperm-tail motility during incorporation. In most cases, the bioelectric responses starts within 7 sec of sperm adhesions; if the data are analyzed excluding the few slow cases, the fertilization potential is found to start 1.93 sec (+/- 1.28 sec) after sperm attachment. These results indicate that the first successful sperm triggers the fast block to polyspermy within 3.4 sec, perhaps as quickly as 1.9 sec, of sperm-egg adhesion, about 13 sec before the first morphological indication of fertilization, and about 21 sec before the characteristic elevation of the fertilization coat responsible for the late block to polyspermy.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

A putative precursor to the major yolk protein of the sea urchin

We detected by electrophoresis, several glycoproteins in the eggs of three species of sea urchin. The major glycoprotein band disappears as development of the embryo proceeds. This protein is enriched in the yolk fraction obtained by zone sedimentation in 2.5–30% sucrose gradients. A fractionally larger glycoprotein has been found to be the major protein in the coelomic fluid of male and female gravid sea urchins. Partial proteolysis peptide mapping shows that the major coelomic fluid protein and the major yolk protein are related, presumably in a precursor-product relationship.     

RNA synthesis in unfertilized sea urchin eggs

We have examined the synthesis of messenger-like RNA in unfertilized sea urchin eggs. Most of the RNA synthesized is restricted to the nucleus and sediments from 16 to 30S. A small fraction can be isolated from the postmitochondrial supernatant and displays a sedimentation profile typical of embryonic mRNA with peaks at 9 and 18S. This cytoplasmic RNA is largely present as free RNPs and we estimate that less than 20% of the RNA is in polysomes. The RNA made in the egg is unstable and reaches a steady state with a half-time of about 30 min. We have examined the accumulation of RNA in the egg and have calculated a rate of synthesis of 1.4 × 10^?14 g of RNA/min/egg which is similar, on a per-nucleus basis, to that found in the just-fertilized egg and very early embryo. It is approximately 10 times greater than the rate of RNA synthesis in the blastula nucleus. We estimate that the RNA synthesized by the unfertilized egg amounts to a maximum of 3 × 10^?13 g of potential mRNA at the time of fertilization, or 10–15% of its immediate needs. This RNA cannot account for the increase in protein synthesis that occurs after fertilization, which must be the result of the translation of another population of more stable egg or oogenic mRNA that is kinetically distinct from the RNA we have measured. The steady-state level of labeled RNA present in the egg does not change upon fertilization until after the first cleavage, at about 2.5 hr after fertilization. Thus the RNA synthesis that occurs in the just-fertilized zygote appears to be merely a continuation (at least quantitatively) of the RNA synthesis taking place in the egg.     

Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos

We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

Chicken fumarase. I. Purification and characterization.

Fumarase from chicken heart is purified 400 times from the crude muscle extract. The isolation procedure includes ammonium sulfate fractionations, Bio-Gel P-300 column chromatography and electrofocusings on pH-gradients from pH 3 to 10 and from pH 7 to 9. Chicken fumarase behaves as an homogeneous protein in sedimentation, diffusion and electrofocusing studies; the protein possesses a single amino-terminal residue: lysine. The analysis of the CD and ORD spectra suggests the presence of 60-65 p. cent of alpha-helix, 0 - 5 p. cent of beta-structure with the remaining portions of the protein in an unordered conformation. Chicken fumarase is found to be composed of 4 subunits of identical molecular weight (51.000) and devoid of disulfide bridges. Finally, the physicochemical properties of chicken fumarase are compared with those of the porcine enzyme.     

Developmental time,cell lineage,and environment regulate the newly synthesized proteins in sea urchin embryos

Strongylocentrotus purpuratus embryos were fractionated into two cell populations of defined lineages at times corresponding to two critical developmental events: determination (16-cell stage) and early differentiation (mesenchyme blastula). The 16-cell stage blastomeres, labeled with [³⁵S]methionine, exhibited identical protein synthesis patterns by fluorography, and this pattern was not significantly altered by cell separation. In comparing the proteins of the mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography of newly synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm cells. Additionally, in contrast to the earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially altered by cell separation. The ability to alter protein synthesis in response to environmental factors may be a further demonstration of the differentiation of these cells.     

Patterns of protein synthesis and metabolism during sea urchin embryogenesis

We have analyzed the patterns of protein synthesis in developing embryos of the sea urchin Strongylocentrotus purpuratus by two-dimensional gel electrophoresis. There was an increase in the number of proteins detectably synthesized during development, as well as significant changes in relative rates of synthesis involving approximately 20% of the nearly 900 newly synthesized polypeptides. The majority of these changes were increases rather than decreases in synthesis; about half were of at least 10-fold, while a few were of more than 100-fold. Very few changes were detected upon fertilization and during the first several hours of development, while about 60% of the changes detected occurred between the hatching and the beginning of invagination. An analysis of proteins detected by silver staining indicated that most remained nearly constant in mass during embryonic development, but several increased or declined substantially. Many proteins present in eggs were not detectably synthesized in either eggs or embryos.     

Isolation and characterization of protease do from Escherichia coli, a large serine protease containing multiple subunits

A new cytoplasmic proteolytic enzyme in Escherichia coli, named protease Do, has been purified to near homogeneity. The enzyme is an endoprotease that degrades casein, denatured bovine serum albumin, and globin but shows little or no hydrolytic activity against insulin, growth hormone, native bovine serum albumin, or a variety of commonly used peptide substrates. The molecular size of the enzyme was large, and it could be isolated in different preparations in either of two forms. One showed a molecular weight of about 500,000 on gel filtration and a sedimentation coefficient of 15.9 S on sucrose gradient centrifugation. The other appeared to be about 300,000 and sedimented at 12.7 S. No interconversion between the two forms and no other difference in the properties was found. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) shows that both forms contain a major 54,000-dalton band and three additional minor polypeptides with molecular weights of 45,000, 44,000, and 42,000. These minor polypeptides appear to result from autolytic degradation of the major protein as demonstrated by peptide mapping with Staphylococcus aureus V8 protease. Thus, protease Do appears to contain a single subunit of 54,000, and can exist either as a decamer or as a hexamer or pentamer. The enzyme is a serine protease. It is sensitive to diisopropyl fluorophosphate (DFP) but not to metal chelating agents, sulfhydryl blocking groups, certain chloromethyl ketones, or various peptide aldehyde inhibitors. The enzyme covalently binds [3H]DFP, and the labeled subunit was visualized on SDS-polyacrylamide gels by fluorography. When cells growing in rich broth enter stationary phase, the relative concentration of protease Do increases more than twofold.     

Purification and properties of protease F, a bacterial enzyme with chymotrypsin and elastase specificities

It has been previously demonstrated that commercial bacterial fibrinolysin (EC 3.4.21.7) selectively cleaves the bond between Met-53 and Ala-54 in ovine prolactin (199 amino acids). A one-step purification procedure on DEAE-cellulose for Protease F, which is the active component of bacterial fibrinolysin, and properties of the purified enzyme are reported. The enzyme is homogeneous as judged by acrylamide gel electrophoresis. Its molecular weight, calculated from gel filtration experiments on Sephadex G-100, is around 13,800. Amino acid analyses do not reveal the presence of any half-cystines. The presence of one tryptophan residue per enzyme molecule was resolved from the fluorescence spectrum. Amino terminal analysis showed that leucine was at the amino terminal position. Protease F hydrolyzes casein and synthetic specific substrates for chymotrypsin and elastase esterases but not for trypsin esterases. It is fully inhibited by phenylmethylsulfonyl fluoride, by chicken ovoinhibitor, and by Chymotrypsin Inhibitor I from potatoes but not by the trypsin-chymotrypsin inhibitors from soybeans and chick peas or by tosyl-L-phenylalanine chloromethyl ketone. The enzyme is stable at room temperature and in the cold, it is not affected by dialysis or by freezing and thawing, but it is inactivated during freeze-drying. The circular dichroism spectra of Protease F indicate an approximate 20% alpha-helix content of the enzyme with a considerable similarity to those of subtilisin, elastase, and beta-trypsin. The relatively low molecular weight of Protease F, the absence of intrachain disulfide bridges, and the fact that it is inhibited by several, but not all, chymotrypsin inhibitors suggest that it may differ phylogenetically from the known serine proteases.     

Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea urchin fertilization

Anti-tubulin immunofluorescence microscopy is used here to demonstrate the configurations of the microtubule-containing structures which participate in the pronuclear movements of sea urchin fertilization. This technique shows that the egg is devoid of microtubules until after the fertilizing sperm is fully incorporated. All the microtubules which appear during the course of fertilization are organized around the base of the sperm head and the sperm aster thus formed behaves in a way that could account for the characteristic motions of the male and female pronuclei as documented by time-lapse video microscopy. Extension of astral microtubules appears to be responsible for the slow (ca. 2.5 μm min^?1) movement of the sperm aster into the cytoplasm of the egg; the rapid (ca. 15 μm min^?1) migration of the female pronucleus to the sperm aster seems to depend on connection of the female pronucleus to microtubules of the sperm aster. Continued extension of astral microtubules after the pronuclei are brought into conjunction can account for the centripetal motion of the paired (or fused) pronuclei and for the positioning of the zygote nucleus in the center of the egg. The behavior of astral microtubules during these motions suggests that they are capable of transmitting both pushing and pulling forces. All the pronuclear movements, and the assembly of detectable microtubules, are sensitive to the microtubule inhibitors griseofulvin and colchicine. Because of this sensitivity, and since all the observable microtubules within the egg during fertilization arise at the sperm aster, it is concluded that the pronuclear movements of fertilization result from the actions of the sperm aster. The pronuclear movements of sea urchin fertilization represent a simple but striking example of microtubule-mediated motility.     

Similarity of proteins synthesized by isolated blastomeres of early sea urchin embryos

The 16-cell sea urchin embryo has blastomeres of three distinct size classes: micromeres, mesomeres, and macromeres. Each class is already restricted in its developmental fate, micromeres being committed to formation of primary mesenchyme cells. The three classes of blastomeres were isolated in high purity and incubated in [³⁵S]methionine until the next cleavage. Nearly all the radioactive protein was solubilized and subjected to two-dimensional electrophoresis according to O'Farrell. Of approximately 1000 spots resolved, there are no qualitative differences among the three blastomeres. When embryos were labeled between the first and fourth cleavages and blastomeres then isolated, no qualitative differences in protein synthesis were observed. Moreover, there are very few changes when unfertilized eggs are compared to 16-cell embryos. Thus cellular determination during embryonic development is not accompanied by qualitative changes in the distribution within the embryo of abundantly synthesized proteins, virtually all of which are coded for by sequences present in the egg.     

An ultrastructural immunocytochemical localization of hyalin in the sea urchin egg

The fertilized sea urchin egg is invested by the hyaline layer, a thick extracellular coat which is necessary for normal development. On the basis of ultrastructural studies and the fact that hyalin is released during the time of the cortical reaction, it has been generally accepted that hyalin is derived from the cortical granules. However, this has never been proven definitely, and recently, it has been reported that hyalin is a membrane and/or cell surface protein. To determine where hyalin is stored, we carried out an ultrastructural immunocytochemical localization of hyalin in the unfertilized egg. Hyalin purified from isolated hyaline layers was used to immunize rabbits. Antisera so obtained were shown to be hyalin specific following absorption with a combination of sea urchin proteins. Immunocytochemical localizations were carried out on sections of Epon-embedded material using protein A-coated gold particles as an antibody marker. Our results demonstrate that, prior to fertilization, hyalin is stored in the homogeneous component of the cortical granule in Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus. Labeling of small cortical vesicles in both unfertilized and fertilized eggs, suggests that these vesicles may contain a secondary reservoir of hyalin.