Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

12S and 9S RNA species. Localization and fate during development of Strongylocentrotus purpuratus eggs

Polyacrylamide gel electrophoresis of total RNA obtained from Strongylocentrotus purpuratus eggs and embryos have shown this material to contain a 12S and 9S RNA species, among others. These two species, found during early development of these cells, were not present in late prism or plutei stages. The RNA was extracted by cold phenol deproteinization in the presence of bentonite and precipitated by alcohol. RNAse, obtained from the 140 000 g egg-supernatant was tested against total RNA extracted from unfertilized eggs, blastulae, gastrulae and plutei embryos. The amount of nucleotides liberated was identical in all cases. Egg-RNAse, in the presence of plutei RNA, did not give rise to the 12S and 9S RNA species.     

Phospholipid synthesis in cleaving sea urchin eggs: Model for specific membrane assembly

Fertilization of Strongylocentrotus purpuratus eggs stimulates uptake of choline 4-fold; phosphorylation of intracellular choline is increased >20-fold at the same time. Incorporation, predominantly into phosphatidylcholine, is stimulated >10-fold. The figures for Lytechinus pictus eggs are similar.The rate at which phosphatidylcholine becomes labeled in S. purpuratus and L. pictus embryos is relatively low and, in contrast to DNA synthesis, increases only 3- to 4-fold between first cleavage and the hatching blastula stage. There is no indication of phospholipid turnover during this time. Inhibition of DNA synthesis by hydroxyurea reduces the incorporation of choline into phosphatidylcholine without affecting the phosphorylation of free choline.It is concluded that the assembly of new membranes during cleavage is accompanied by physiologically significant de novo synthesis of phospholipid. This system therefore appears to lend itself well to further studies on the biogenesis of specific membranes.     

Commitment to vegetalized development in sea urchin embryos

Summary Strongylocentrotus purpuratus embryos were reared in 0.025 M LiCl, which causes commitment to vegetalized development within 5 h after treatment begun at fertilization. Treated and control embryos were labelled with³⁵S-methionine for 3 h intervals from 2–14 h, solubilized, and subjected to 2-dimensional polyacrylamide gel electrophoresis. Comparison of autoradiographs of the gels, in which over 400 proteins can be detected, indicate that while LiCl treatment causes a short delay in the initiation or cessation of synthesis of a few proteins, no qualitative or major quantitative differences can be detected between control and treated embryos. Normal gastrulae and vegetalized exogastrulae labelled 38 h after fertilization have several differences in patterns of protein synthesis. We conclude that the early determinative events involved in vegetalization are not reflected in detectable differences in the pattern of protein synthesis.     

Tuning sperm chemotaxis by calcium burst timing

Marine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca²⁺-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca²⁺ increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca²⁺ fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca²⁺ fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca²⁺ fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca²⁺ fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general.     

Simultaneous synthesis,translation, and storage of mRNA including histone mRNA in sea urchin eggs

The kinetics of accumulation of RNA labeled with uridine and the time course of change in the specific activity of the UTP pool were used to estimate the rate constants for synthesis and decay of RNA synthesized in unfertilized eggs of the sea urchin Lytechinus pictus. The rate of synthesis per haploid genome is similar to that in embryos. Most of the RNA is turning over with a half-life of about 5 hr, and an average of 11 pg of newly synthesized RNA accumulates at steady state. About 3.7% of the RNA in the polysomes of the egg is newly synthesized and this RNA has the heterogeneous size distribution expected for mRNA. Thus most, probably all, of the mRNA translated in the egg is also synthesized in the egg. Little, if any, of the RNA synthesized in the egg enters polysomes following fertilization. Thus the egg synthesizes a population of mRNA which is unstable and translated, but it also contains a more stable, untranslated population of previously synthesized, stored mRNA, which is translated only after fertilization. Since the two populations of mRNA code for the same abundant proteins (Brandhorst, B. P. (1976). Develop. Biol., 52, 310–317), there is a temporal separation in the metabolism and function of coexisting mRNA molecules of identical coding sequence. Among the mRNAs synthesized and translated in the egg are histone mRNAs having the same electrophoretic mobilities and rates of synthesis per genome as those synthesized in rapidly cleaving embryos. Thus the synthesis, entry into the cytoplasm, and translation of histone mRNA are not restricted to the S phase of the cell cycle or the period of cell division.     

Mitochondrial regulation in sea urchins. I. Mitochondrial ultrastructure transformations and changes in the ADP:ATP ratio at fertilization.

Mitochondrial ultrastructural transformations have been examined in intact eggs and embryos from three sea urchins, Arbacia punctulata, Strongylocentrotus purpuratus, and Lytechinus pictus. Following fertilization, naturally occurring ultrastructural transformations (designated as condensed to orthodox) were observed to occur in mitochondria of all three families. The ratios of the soluble ADP and ATP pools were examined in eggs of S. purpuratus before and after fertilization in order to test whether the ultrastructural transformations reflect a decrease in relative size of the ADP pool following fertilization. Our data indicate that there is a decrease in the ADP:ATP ratio at fertilization. These findings and their implications are discussed with respect to presently accepted theories on mitochondrial regulation.     

Sequence analysis and evolution of sea urchin (Lytechinus pictus and Strongylocentrotus purpuratus) histone H4 messenger RNAs

The putative histone H4 (F2a₁) mRNA has been isolated from early blastula Strongylocentrotus purpuratus sea urchin embryos. Nucleotide sequences of oligonucleotides obtained by digestion of this RNA with T₁ ribonuclease have been obtained and many are found to be colinear with the amino acid sequence of histone H4 protein. The sequences obtained from the H4 mRNAs of S. pnrpuratus have been compared with those obtained from Lytechinus pictus (Grunstein & Schedl, 1976). The two mRNAs for this highly conserved protein have undergone considerable divergence of the sort that would be predicted from the degeneracy of the genetic code. 11.5% of the bases have undergone substitution at a rate calculated to be 3 × 10^?9 base changes · codon^?1 · year^?1.     

THE EFFECT OF SOLUBLE EGG JELLY ON THE FERTILIZABILITY OF ACID-DEJELLIED SEA URCHIN EGGS*

Acid-dejellied Lytechinus pictus eggs bind few sperm and show decreased fertilizability. Addition of solubilized egg jelly increases sperm binding and fertilizability, presumably by increasing the frequency of the acrosome reaction. However, dejellied Strongylocentrotus purpuratus bind more sperm and show increased fertilizability in the complete absence of soluble egg jelly. Addition of soluble egg jelly greatly decreases fertilizability in S. purpuratus. Such species differences may be the basis for the controversy between Lillie and Tyler on the one hand, who believed that egg jelly increased egg fertilizability; while Loeb and Hagström on the other hand, believed jelly had no effect on, or actually decreased egg fertilizability. ¹²⁵I-labeling of dejellied S. purpuratus egg surfaces and immunofluorescent studies show that egg jelly persists on the surfaces of acid-dejellied eggs. Egg jelly appears to be a non-removable component of the vitelline layer of this species.     

Initial characterization of sulfated macromolecules in the blastocoels of mesenchyme blastulae of Strongylocentrotus purpuratus and Lytechinus pictus

Sulfated macromolecules have been implicated in many morphogenetic activities, including cell migration and gastrulation in echinoid embryos. Since the blastocoel is a major site for the accumulation of sulfated macromolecules, this study focuses on an initial characterization of sulfated components present in isolated blastocoelic matrix from mesenchyme blastula-stage embryos of Strongylocentrotus purpuratus and Lytechinus pictus. Based on transmission and scanning electron microscopy, the isolated matrix resembles that seen in situ, containing 25- to 30-nm-diameter granular and small fibrous components. Only a small proportion of the labeled material enters 7.5% polyacrylamide gels to separate in a number of species-specific bands larger than 30,000 daltons. A major portion of the label is voided from Sepharose CL-2B columns prepared in dissociative solvent. Based on the chromatographic isolation of digestion products after treatment with chondroitinase AC and ABC, in S. purpuratus about 37% of the label is in dermatan sulfate and 16% in chondroitin 6-sulfate. In contrast, in L. pictus about 25% of the label is in dermatan sulfate with only low levels of chondroitin sulfate. In both species, based on nitrous acid and heparinase sensitivity of Pronase digests prior to chromatography on Sephadex G50, about 13% of the label is in heparan sulfate. A small residual fraction remains uncharacterized.     

RNA synthesis and coding capacity of polyadenylated and nonpolyadenylated mRNA from cultures of differentiating Drosophila melanogaster myoblasts

We have investigated the synthesis and coding capacity of RNA isolated from cultures of differentiating Drosophila embryonic muscle cells. We find that following muscle cell fusion, the sedimentation profile of newly synthesized polyadenylated RNA becomes somewhat lighter. In vitro translation products analyzed by two-dimensional gel electrophoresis indicate that the coding capacity of translatable myogenic mRNA changes during differentiation. A group of several muscle-specific proteins (including the contractile proteins) is translated only from mRNA isolated after the initiation of fusion. This pattern coincides with proteins synthesized in vivo during differentiation. Additionally, we find that polyadenylated and nonpolyadenylated myogenic mRNA from a given developmental stage in culture have extremely similar coding potentials.     

Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urchin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60° C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5°C; S. franciscanus 51% and 3.5° C; Lytechinus pictus 12% and 7.5° C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06–0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.Also staff member, Carnegie Institution of Washington, Washington, D.C. 20015     

FLAGELLAR MOTILITY IS NOT INVOLVED IN THE INCORPORATION OF THE SPERM INTO THE EGG AT FERTILIZATION*

Cinemicrography of sea urchin fertilization reveals that the fertilizing sperm is one of the first sperm to attach to the egg. Just before the cortical reaction the fertilizing sperm ceases motility and then is incorporated into the egg without flagellar beating. The rate of incorporation is 5–11 μm/sec and is constant. Lytechinus pictus sperm rendered immotile by azide treatment can bind to and fertilize eggs but binding, and therefore fertilization, is blocked by azide treatment of Strongylocentrotus purpuratus gametes.     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

Studies on the interactions of sperm with the surface of the sea urchin egg

We have examined the relationship between sperm adhesion and fertilization in the cross species insemination of Arbacia punctulata eggs by Strongylocentrotus purpuratus sperm. As previously reported (Kinsey et al., 1980) the addition of S. purpuratus egg jelly results in induction of the acrosome reaction in sperm and significant numbers of S. purpuratus sperm adhere to A. punctulata eggs. However, in the absence of S. purpuratus egg jelly, S. purpuratus sperm fail to bind to A. punctulata eggs. Although at least 200 S. purpuratus sperm bind to an A. punctulata egg in the presence of S. purpuratus jelly, less than 8% of the eggs are fertilized. The adhesion of S. purpuratus sperm meets the same functional criteria as homologous A. punctulata sperm-egg adhesion. Electron microscopy shows that S. purpuratus sperm that have undergone the acrosome reaction adhere to A. punctulata eggs by their bindin-coated acrosomal process in a manner that is morphologically identical to that observed with homologous A. punctulata sperm. We have also compared the ability of S. purpuratus and A. punctulata sperm to fuse and fertilize with A. punctulata eggs after removal of the vitelline layer. Using high levels of sperm of either species, heterologous as well as homologous fertilization is readily detectable. Under these conditions, where stable binding is not demonstrable, there is no difference in the ability of S. purpuratus and A. punctulata sperm to fertilize A. punctulata eggs. These observations suggest that the failure of S. purpuratus sperm to fertilize A. punctulata eggs under normal conditions may be due to their inability to penetrate the vitelline layer so that they can fuse with the egg plasma membrane. In relation to the possible mechanism of vitelline layer penetration, we have also investigated the mode of action of chymostatin, an inhibitor of chymotrypsin that has been reported to inhibit fertilization of sea urchin eggs (Hoshi et al., 1979). Our findings suggest that the fertilization inhibitory activity of chymostatin is not related to its antichymotrypsin activity. Rather, it appears that this inhibition is due to the induction of an abnormal acrosome reaction in sperm that precludes formation of the acrosome process.     

The glutathione thiol-disulfide status in the sea urchin egg during fertilization and the first cell division cycle

The intracellular levels of GSH, GSSG, and protein-glutathione disulfide (protein-SSG) have been measured in the eggs and developing embryos of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus. Total cellular glutathione is maintained in a very highly reduced state during these initial stages of development. Thus for unfertilized eggs of L. pictus the results (μmol/g dry weight) were 11 ± 1 for GSH, 0.02 ± 0.01 for GSSG, and 0.07 ± 0.02 for protein-SSG. No significant change in these values was observed upon fertilization of the eggs or during the first cell division cycle. The values obtained with S. purpuratus were somewhat greater, but were also found to exhibit no significant variations upon fertilization or cell division. These observations indicates that changes in the total cellular glutathione thiol-disulfide status are not involved in the control mechanisms which operate during fertilization or the first cell division cycle in the sea urchin egg.