THE SYNTHESIS OF 5S RNA AND ITS REGULATION DURING EARLY SEA URCHIN DEVELOPMENT

De novo synthesis of 5S RNA and of transfer RNA (tRNA) has been demonstrated previously to occur by mid-cleavage (128-cell stage) in sea urchin embryos (24). The present study focused on determining more precisely the time of onset of activity of the genes for 5S RNA and for tRNA during sea urchin embryogenesis by preloading the GTP precursor pools of unfertilized eggs. The results showed that newly-made 5S RNA and tRNA could be detected as early as the 32-cell stage. In order to determine whether newly-synthesized 5S RNA accumulates coordinately during development with newly-made 26S (34) and 18S ribosomal RNAs (rRNAs), the relative rates of accumulation of these three RNA molecules were measured and compared at each of several stages of sea urchin embryogenesis. In contrast to the coordinated accumulation of newly-synthesized 26S and 18S rRNAs, newly-made 5S RNA accumulated in excess at the mesenchyme blastula (9-fold excess), midgastrula (5-fold excess) and prism (3-fold excess) stages. The 5S RNA/26S RNA molar ratios only approached unity in advanced (48 hr) plutei. The non-coordinated accumulation of newly-made 5S RNA with that of 26S and 18S rRNAs suggests that the accumulation of these newly-synthesized RNAs is differentially regulated during early sea urchin development.     

MITOCHONDRIAL DNA REPLICATION IN SEA URCHIN OOCYTES

Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [³H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.     

Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division.     

Synthesis of rabbit globin in a cell-free protein synthesis system utilizing sea urchin egg and zygote ribosomes

Reports of the reduced ability of sea urchin egg ribosomes to participate in synthetic mRNA-directed protein synthesis have fostered the suggestion that the low protein synthesis rate of eggs is due to ribosome-associated inhibitors. To test this hypothesis with a natural message, we have isolated 80S ribosomes and microsomal ribosomes of sea urchin eggs and zygotes and compared their activity at synthesizing protein from rabbit α and β globin mRNA in a Krebs II ascites tumor cell-free system. Both egg and zygote 80S ribosomes responded to added mRNA and were shown to synthesize complete α and β globin chains by CM-cellulose chromatography. In most cases, the activity of the egg ribosomes was in comparable instances higher than the zygote ribosomes. Attempts to determine the cause of this difference indicated that it was not a function of K⁺ or Mg²⁺ concentration, type of tRNA used, or ribosomal wash proteins. From these studies it is apparent that sea urchin egg ribosomes are functional at a level equivalent to or better than zygote ribosomes, and it appears that the lack of protein synthetic activity in unfertilized eggs is not due to the presence of a population of inhibited ribosomes.     

Kohamaic acid A,a novel sesterterpenic acid,inhibits activities of DNA polymerases from deuterostomes

We previously found and isolated a novel natural product, designated kohamaic acid A (KA-A), which inhibited the first cleavage of fertilized sea urchin eggs. In this paper, we report that this compound could selectively inhibit the activities of DNA polymerases (pol. alpha, beta, gamma, delta and epsilon ) only from species in the deuterostome branch in the animal kingdom, like sea urchin, fish and mammals, but not from protostomes including insects (fruit fly, Drosophila melanogaster) and mollusks (octopus and oyster). Inhibition of deuterostome DNA polymerases was dose dependent. IC(50) values for DNA polymerases of mammals and fish occurred at approximately 5.8-14.9 microM and those of sea urchin at 6.1-30.3 microM. In the sea urchin DNA polymerases, the activities of the replicative DNA polymerases such as alpha, delta and epsilon were more strongly inhibited than that of the repair-related pol. beta. KA-A is an inhibitor of replicative DNA polymerases from the deuterostome species, and subsequently, the inhibition of the first cleavage of fertilized sea urchin eggs might occur as a result of the suppression of DNA replication.     

Quantitative Measurement of Rates of 5S RNA and Transfer RNA Synthesis in Sea Urchin Embryos

Embryonic differentiation is believed to be due to a programmed expression of genes, which includes their time of activation, sequence of appearance, and amount transcribed into the immediate gene product, RNA. Differential synthesis of the major RNA classes, such as the ribosomal RNAs (28S, 18S, 5S) and transfer RNA (tRNA), characterizes many animal developing systems, including the sea urchin embryological system. Previous work has shown that the genes for 5S RNA and tRNA are active during early cleavage in sea urchin embryos. The present study focused on quantitatively measuring and comparing the rate of 5S RNA and tRNA synthesis in cleavage, early blastula, and early pluteus embryos of Arbacia punctulata. At each stage, embryos were labeled for 3 h with [8-³H]-guanosine. Total cellular RNA was extracted using the cold (4°C)-phenol-sodium dodecyl sulfate method and purified (LiCl-soluble) RNA preparations were fractionated by electrophoresis on 10% polyacrylamide gels. The amount of 5S RNA and tRNA synthesized at each stage was calculated from the radioactivity coincident with the 5S RNA and with the tRNA absorbance peaks (A_{260 nm}) on each gel, from the known guanosine monophosphate (GMP) compositions of sea urchin 5S RNA and tRNA and from the average specific radioactivity of the GTP precursor pool during each 3 h labeling period. The results showed that on a per embryo basis the rates of 5S RNA and tRNA synthesis increased slightly (about 1.4-fold) from cleavage through pluteus stages, while on a per cell basis the rates declined severalfold (about 3-fold) during embryogenesis. The rates of 5S RNA and tRNA synthesis determined here parallel previously-reported levels of RNA polymerase III in sea urchin embryos, suggesting that cellular levels of RNA polymerase III may exert some positive control over 5S RNA and tRNA synthesis during sea urchin embryogenesis.     

Inhibition of cysteine protease activity disturbs DNA replication and prevents mitosis in the early mitotic cell cycles of sea urchin embryos

Recent findings suggested that the role of cysteine proteases would not be limited to protein degradation in lysosomes but would also play regulatory functions in more specific cell mechanisms. We analyzed here the role of these enzymes in the control of cell cycle during embryogenesis. The addition of the potent cysteine protease inhibitor E64d to newly fertilized sea urchin eggs disrupted cell cycle progression, affecting nuclear as well as cytoplasmic characteristic events. Monitoring BrdU incorporation in E64d treated eggs demonstrated that DNA replication is severely disturbed. Moreover, this drug treatment inhibited male histones degradation, a step that is necessary for sperm chromatin remodeling and precedes the initiation of DNA replication in control eggs. This inhibition likely explains the DNA replication disturbance and suggests that S phase initiation requires cysteine protease activity. In turn, activation of the DNA replication checkpoint could be responsible for the consecutive block of nuclear envelope breakdown (NEB). However, in sea urchin early embryos this checkpoint doesn't control the mitotic cytoplasmic events that are not tightly coupled with NEB. Thus the fact that microtubule spindle is not assembled and cyclin B-cdk1 not activated under E64d treatment more likely rely on a distinct mechanism. Immunofluorescence experiments indicated that centrosome organization was deficient in absence of cysteine protease activity. This potentially accounts for mitotic spindle disruption and for cyclin B mis-localization in E64d treated eggs. We conclude that cysteine proteases are essential to trigger S phase and to promote M phase entry in newly fertilized sea urchin eggs.     

The appearance of beta-1,3-glucanase in hatching embryos of the sea urchin, Echinometra vanbrunti.

Two characteristics of fertilizing sea urchin eggs are the elevation of a fertilization membrane and the excretion of a β-glucanase. Of 13 species tested, one species, Echinometra vanbrunti, lacks both characteristics. No β-glucanase exists in the eggs and cleavage stages. However, β-glucanase appears at hatching and is secreted to the sea water during and after the hatching period. The enzyme may function in the hatching process. The hypothesis is presented that the β-glucanase excreted by eggs of other sea urchin species may function in the elevation of the fertilization membrane.     

The isolation and measurement of tRNAmeti using RNA/DNA hybridization

A pBR322 plasmid containing the initiator tRNAmet gene of Xenopus (pt145 - donated by Stuart Clarkson) will specifically bind to mouse initiator tRNAmet (tRNAmeti) when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band (95%) and a minor band. The hybridizing tRNA has been identified as initiator tRNAmet by RNA sequencing. Hybridization of tRNAtotal to another plasmid containing the Xenopus gene for tRNAasn results in two bound species with different electrophoretic mobilities than the tRNA bound to the initiator tRNAmet gene. pt145 has been used to measure the steady state concentration of initiator tRNAmet in the uninduced and erythroid Friend cell, and in the unfertilized egg and 21 h blastula of the sea urchin. Initiator tRNAmet represents 0.91% and 0.52% of the tRNA populations extracted from uninduced and erythroid Friend cells, respectively. Based upon the total tRNA content per cell, there is a 3.8 fold decrease in initiator tRNAmet per cell during erythroid differentiation. tRNA extracted from unfertilized eggs and 21 h blastula of the sea urchin both have 0.5% of total tRNA as initiator tRNAmet (approximately 1.5 pg).     

Spindle pole centrosomes of sea urchin embryos are partially composed of material recruited from maternal stores

The spindle poles of fertilized sea urchin eggs have commonly been modeled as being derived from the centrosomes of the fertilizing spermatozoon. Boveri's theory of fertilization, proposed at the turn of the century, states that the maternal centrosome is suppressed or inactivated during oogenesis and that the sperm centrosome is functionally dominant. In support of this proposal, more recent studies have shown that the sperm imports a determinant that is involved in centrosomal replication. Examination of sea urchin zygotes immunofluorescently labeled with a new anti-centrosomal antibody by quantitative confocal laser-scanning microscopy shows, however, that spindle pole centrosomes are not exclusively paternal structures, but additionally contain material derived from maternal pools. Furthermore, this maternal centrosomal material is divided among daughter blastomeres during cleavage. It therefore appears that although the sperm centrosome plays a dominant role in organizing the spindle poles, much of the centrosomal material within the spindle poles of the zygote is actually recruited from preexisting egg cytoplasmic stores. These data indicate that centrosomes of sea urchin embryos are biparentally derived, composite organelles.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

AN INDIRECT METHOD TO ASSAY FOR MITOTIC CENTERS IN SAND DOLLAR (DENDRASTER EXCENTRICUS) EGGS

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C¹⁴-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication.     

Selective identification of the paternal mitochondrion in living sea urchin eggs and embryos by chlorotetracycline

When sea urchin eggs are pretreated with fluorescent chelate probe chlorotetracycline (CTC) and then fertilized with unlabeled sperm, a small, brightly fluorescent particle resembling the mitochondrion of free-swimming sperm both in size and fluorescent staining characteristics appears in the egg cytoplasm. This particle first appears near the base of the insemination cone and, like the paternal mitochondrion identified in previous ultrastructural studies, remains closely associated with the male pronucleus during its microtubule-dependent migration toward the egg center. These similarities strongly suggest that the fluorescent particle observed in the cytoplasm of living, CTC-pretreated sea urchin eggs is, in fact, the mitochondrion of the fertilizing sperm.     

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.     

Ultrastructural localization of intracellular calcium stores by a new cytochemical method

We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods.     

The sea urchin multicatalytic protease: purification, biochemical analysis, subcellular distribution, and relationship to snRNPs

We have purified and extensively characterized a 19-S particle from sea urchin eggs. This particle is the sea urchin homologue of the "prosome", a particle originally identified in duck erythroblasts. We now show that these sea urchin prosomes contain multiple proteolytic activities. As shown for analogous particles from other cells, these particles hydrolyze synthetic substrates containing neutral hydrophobic or basic amino acids at the carboxy terminus of the synthetic peptides. They contain 16-20 small proteins ranging in molecular weight from 20,000 to 32,000. Peptide mapping shows that most of the polypeptides are unique, however, three exist in two isoelectric forms. We have investigated the possible function of the sea urchin multicatalytic proteases (MCPs) by determining their subcellular distribution, their relationship to egg snRNPs, and their possible role in translational repression. There are almost as many MCPs (2 x 10(8] as ribosomes (6.6 x 10(8] or mRNPs (1.8 x 10(7] per egg. This suggests that like ribosomes, the MCPs are stored in the egg for use during later development. We find that a substantial proportion of egg MCPs move into nuclei by the late blastula stage. Using a specific antibody against one of the sea urchin MCP proteins and antibodies against U1-U6, La, and Ro RNPs, we show that the sea urchin particle is distinct from these RNPs, although the anti-U1-U6 RNP antibody cross-reacts with a single MCP protein. In addition, the sea urchin MCP appears to be associated with a large structure in the cytoplasm of unfertilized eggs and is released under the same conditions that activate egg mRNPs in vitro.     

Cleavage inhibition in marine eggs by puromycin and 6-dimethylaminopurine

The possibility is discussed that puromycin may not inhibit cleavage in marine eggs solely by inhibiting protein synthesis. It is suggested that part of the effect of puromycin is through 6-dimethylaminopurine (DMAP) (the purine component of puromycin) a potent cleavage inhibitor which appears to enhance protein synthesis in sea urchin eggs.     

Multiple levels of regulation of protein synthesis at fertilization in sea urchin eggs

Fertilization of sea urchin eggs results in a large stimulation of protein synthesis. This increase in protein synthesis is mediated by the mobilization of stored maternal mRNA (mRNPs) into polysomes, but the details of the molecular mechanisms which regulate this process are not well understood. Using a sea urchin egg cell-free translation system, evidence has been obtained which indicates that the capacity to initiate protein synthesis on new mRNAs is limited. Addition of exogenous mRNAs failed to stimulate overall protein synthesis, whereas supplementing the system with a nuclease-treated reticulocyte lysate, an S-100 supernatant fraction, or purified eIF-2 stimulated nearly twofold. In addition, the levels of 43 S preinitiation complexes containing a 40 S ribosomal subunit and methionyl-tRNA were increased at pH 7.4 compared to pH 6.9, or when reticulocyte S-100 was added. However, other experiments showed clearly that mRNA availability may also regulate translation in the sea urchin egg. Sea urchin lysates only stimulated poorly the nuclease-treated reticulocyte lysate system, and the mRNPs in the sea urchin lysate did not bind to reticulocyte 43 S preinitiation complexes. Since purified sea urchin egg mRNA was active in both assays, the bulk of sea urchin mRNA must be masked in the egg, and remain masked in the in vitro assays. Thus, protein synthesis appears to be regulated at both the level of mRNA availability and the activity of components of the translational machinery.