THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.     

CHANGE IN THE ACTIVITY OF ARYLESTERASE IN SEA URCHIN EGGS FOLLOWING FERTILIZATION

The activity of arylcsterase in sea urchin eggs ( Anthocidaris craxsispina ), increases at 5 min after fertilization to about 1.5-fold that in unfertilized eggs, and decreases at 15 min to a lower level than that in unfertilized eggs. Then the activity of the enzyme increases again. The enzyme activity in unfertilized eggs is enhanced by either fructose 1, 6-diphosphate (FDP) at concentrations between 4 and 10 μM, or guanosine 3', 5'-cyclic monophosphalc (cGMP) at concentrations between 0.1 and 0.3 μM. The activity is detectable in the crude microsomal fraction and also in the supernatant fraction obtained from sea urchin egg homogenates by centrifugation at 105,000 × g for 2 hr.     

Maternal exposure to estradiol and endocrine disrupting compounds alters the sensitivity of sea urchin embryos and the expression of an orphan steroid receptor

Endocrine disrupting compounds (EDCs) are known to affect reproduction and development in marine invertebrates. In previous work, we have shown that developing sea urchin embryos were sensitive to estradiol and estrogenic EDCs at environmentally relevant concentrations in a tamoxifen-sensitive manner (Roepke et al. 2005. Aquat Toxicol 71:155-173). In this study, we report the effects of maternal exposure to EDCs on embryo sensitivity and regulation of an orphan steroid receptor in sea urchin eggs. Maternal exposures were conducted by injecting female Strongylocentrotus purpuratus sea urchins initiating oogenesis with two concentrations of estradiol, octylphenol, tributyltin and o, p-DDD for 8 weeks with an induced spawning before and after the injection cycle. Developing embryos were less sensitive to estradiol following maternal exposure to estradiol, octylphenol and DDD. The steroidogenesis inhibitor, spironolactone, and the aromatase inhibitor, formestane, affected normal sea urchin development with EC50 values of 18 and 2 microM, respectively. Binding of estradiol was demonstrated in homogenates supernatants of sea urchin embryos by filtration centrifugation and column chromatography, but saturation was not reached until 4-6 hr and was highly variable. Analysis of eggs from pre- and post-injection spawns using real-time Q-PCR for the mRNA of an orphan steroid receptor, SpSHR2, shows that receptor mRNA increased in eggs with estradiol, octylphenol and tributyltin but decreased with DDD. RIA showed that estradiol may be present during gastrulation. In summary, maternal exposure to estradiol and EDCs alters embryo sensitivity and regulates the expression of an orphan steroid receptor in the egg.     

Rho-kinase in sea urchin eggs and embryos

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.     

Extracellular Ca2+ influx is crucial for the early embryonic development of the sea urchin Echinometra lucunter

The involvement of Ca(2+) in the activation of eggs and in the first steps of the embryonic development of several species is a well-known phenomenon. An association between Ca(2+) sources with the fate of the blastopore during embryonic development has been investigated by several authors. Ca(2+) influx mediated by voltage-gated channels and Ca(2+) mobilization from intracellular stores are the major sources of Ca(2+) to egg activation and succeeding cell divisions. Studies on sea urchins embryonic development show that intracellular Ca(2+) stores are responsible for egg activation and early embryogenesis. In the present work we investigated the involvement of extracellular Ca(2+) in the first stages of the embryonic development of the sea urchin Echinometra lucunter. Divalent cation chelators EDTA and EGTA strongly blocked the early embryonic development. Adding to this, we demonstrated the involvement of voltage-gated Ca(2+) channels in E. lucunter embryogenesis since Ca(2+) channel blockers powerfully inhibited the early embryonic development. Our data also revealed that Ca(2+) influx is crucial for embryonic development during only the first 40?min postfertilization. However, intracellular Ca(2+) remains mandatory to embryonic development 40?min postfertilization, seen that both the intracellular Ca(2+) chelator BAPTA-AM and calmodulin antagonists trifluoperazine and chlorpromazine inhibited the first stages of development when added to embryos culture 50?min postfertilization. Our work highlights the crucial role of extracellular Ca(2+) influx through voltage-gated Ca(2+) channels for the early embryonic development of the sea urchin E. lucunter and characterizes an exception in the phylum Echinodermata.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

Partial characterization of a nuclear proteolytic activity from fertilized sea urchin eggs

The nuclei from fertilized sea urchin eggs, obtained 80 min after fertilization, contains a neutral proteolytic activity. Optimal action on casein was observed at pH 7-8 and a Km value of 1.2 mg/ml was determined for this substrate. The proteolytic activity was stimulated 1.5 fold by the addition of 3 M urea and decreased at higher urea concentrations. NaCl and CaCl2 were inhibitory whereas MgCl2 increased the enzyme activity. Isolated histones from sea urchin sperms, and especially histones H1, H2A, H2B and H3, were degraded by the nuclear activity. A partial inhibition of histones degradation was caused by sodium bisulfite and NaCl. The proteolytic activity was found associated to the chromatin of fertilized sea urchin eggs.     

ANTIGENS IN EGGS AND DEVELOPMENTAL STAGES OF THE SEA URCHIN : II. Localization

Homogenates of fertilized eggs of the sea urchin Paracentrotus lividus were fractionated by differential centrifugation. In addition, whole eggs were fragmented, on a preparative scale, by centrifugation in sea water-sucrose gradients. The fractions and fragments were subsequently assayed for their content of soluble protein antigens described in an earlier publication. Relative concentrations of antigen present in quantitatively isolated cell fractions were estimated by graded antiserum absorption in combination with agar-diffusion technique. Two of six antigens were found to be associated mainly with the low speed sediments. Treatment of the various sediments with hypotonic medium and results obtained with fragmented eggs suggested that these two antigens and possibly a third were probably located in the yolk granules. The other antigens were more evenly distributed among the low speed sediments and the non-sedimented part of the cytoplasm. Only one of the antigens was consistently associated with the microsomal fraction.     

A conserved region in the sea urchin U1 snRNA promoter interacts with a developmentally regulated factor.

The expression of the sea urchin L. variegatus U1 snRNA gene is temporally regulated during embryogenesis. Using a microinjection assay we show that a region between 203 and 345 nts 5' of the gene is required for expression. There are four conserved regions between two sea urchin species in the 345 nts 5' to the U1 gene. One region, located at about -300, binds a protein factor which is present in blastula but not gastrula nuclei. Three other potential protein binding sites within the first 200 nts 5' to the gene have been identified using a mobility shift assay and/or DNase I footprinting. Two of these regions bind factors which are not developmentally regulated and one binds a factor which is developmentally regulated. It is likely that the factor which binds at -300 is involved in expression and developmental regulation of the sea urchin U1 snRNA gene.     

Effect of millimeter waves on the early development of the mouse and sea urchin embryo]

The action of nonthermal electromagnetic radiation (EMR) of the millimeter range on the early development of murine and sea urchin embryos was investigated. An MRTA-01E-03 generator with a frequency of 54-78 GHz and radiation intensity of 0.06 mWt/cm2 was used. The embryos were irradiated during 30 min at the stage of two blastomeres. The number of murine embryos that reached the blastocyst stage increased (up to 97.3% in comparison with 87.5% in control). The total time of cultivation up to the blastocyst stage was also shorter (72 h) than in control (96 h). The irradiation had effect on the development of sea urchin embryos only if embryos with a weakened viability were tested. The results indicate that millimeter electromagnetic radiation has a stimulating effect on the early development of embryos, increasing the resistance of embryos to unfavorable environmental conditions.     

O-glycosylhydrolases of embryos of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> and effect of some natural substances on their biosynthesis

Embryos of the sea urchin Strongylocentrotus intermedius have been found to contain o-glycosyl hydrolases: highly active 1,3-β-D-glucanase and β-D-mannosidase as well as a lower activity of β-D-glucosidase and β-D-galactosidase. Dynamics of changes of the enzyme activities has been studied at various stages of the sea urchin development. There has also been studied effects of some substances (natural fucoidans, β-1.3; 1.6-glucans formed by enzymatic synthesis as well as protein inhibitor of marine mollusc endo-1,3-β-D-glucanases) on development of the embryos and biosynthesis of 1,3-β-D-glucanase and α-D-mannosidase.     

Change in phosvitin kinase activity during early development of the sea urchin

Protein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE-cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE-cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X-100 but was extracted by 1 M NaCl.     

Site of nuclear DNA replication in embryonic cells of the sea urchin Strongylocentrotus intermedius]

In the sea urchin embryonic cells, all newly synthesized nuclear DNA (n-DNA) pulse-labeled by 3H-thymidine was found within DNA-membrane complex (DNA-mc) isolated by centrifugation of lysates of nuclei after their treatment with Sarkosyl, Brij-35, or sodium dodecylsulfate through neutral sucrose (10--30%) gradients. This attachment has been shown not to be an artifact due to the unspecific effect of the detergents or the destabilization of the secondary structure of n-DNA because the association of the exogenous 14C-DNA with nuclear membrane and chromatin did not occur during the isolation of the DNA-mc. n-DNA was not replaced from DNA-mc when the latter was isolated in the excess of unlabeled denatured DNA. n-DNA associated with DNA-mc behaved as a precursor of chromosomal DNA. It is suggested that in sea urchin embryonic cells the synthesis of nuclear DNA is carried out by the replicative complex attached to the nuclear membrane.     

Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis.

Poly(A)-binding proteins (PABPs) are the best characterized messenger RNA-binding proteins of eucaryotic cells and have been identified in diverse organisms such as mammals and yeasts. The in vitro poly(A)-binding properties of these proteins have been studied intensively; however, little is known about their function in cells. In this report, we show that sea urchin eggs have two molecular weight forms of PABP (molecular weights of 66,000 and 80,000). Each of these has at least five posttranslationally modified forms. Both sea urchin PABPs are found in approximately 1:1 ratios in both cytoplasmic and nuclear fractions of embryonic cells. Quantification in eggs and embryos revealed that sea urchin PABPs are surprisingly abundant, composing about 0.6% of total cellular protein. This is 50 times more than required to bind all the poly(A) in the egg based on the binding stoichiometry of 1 PABP per 27 adenosine residues. We found that density gradient centrifugation strips PABP from poly(A) and therefore underestimates the amount of PABP complexed to poly(A)+ RNA in cell homogenates. However, large-pore gel filtration chromatography could be used to separate intact poly(A)-PABP complexes from free PABP. Using the gel filtration method, we found that the threefold increase in poly(A) content of the egg after fertilization is paralleled by an approximate fivefold increase in the amount of bound PABP. Furthermore, both translated and nontranslated poly(A)+ RNAs appear to be complexed to PABP. As expected from the observation that PABPs are so abundant, greater than 95% of the PABP of the cell is uncomplexed protein.     

The sea urchin stem-loop-binding protein: a maternally expressed protein that probably functions in expression of multiple classes of histone mRNA

Following the completion of oogenesis and oocyte maturation, histone mRNAs are synthesized and stored in the sea urchin egg pronucleus. Histone mRNAs are the only mRNAs that are not polyadenylated but instead end in a stem-loop which has been conserved in evolution. The 3' end binds the stem-loop-binding protein (SLBP), and SLBP is required for histone pre-mRNA processing as well as translation of the histone mRNAs. A cDNA encoding a 59 kDa sea urchin SLBP (suSLBP) has been cloned from an oocyte cDNA library. The suSLBP contains an RNA-binding domain that is similar to the RNA-binding domain found in SLBPs from other species, although there is no similarity between the rest of the suSLBP and other SLBPs. The suSLBP is present at constant levels in eggs and for the first 12 h of development. The levels of suSLBP then decline and remain at a low level for the rest of embryogenesis. The suSLBP is concentrated in the egg pronucleus and is released from the nucleus only when cells enter the first mitosis. SuSLBP expressed by in vitro translation does not bind the stem-loop RNA, suggesting that suSLBP is modified to activate RNA binding in sea urchin embryos.