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.     

DNA Replication is Required for Tissue-Specific Enzyme Development in Ascidian Embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stages that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

Kinetic behavior of 30S rRNA intermediate labeled in developing embryonic cells of Xenopus laevis

In Xenopus neurula cells, "30S" RNA was found to be labeled with 3H-uridine after a relatively short labeling period. Results obtained from cumulative labeling and pulse-labeling and chase experiments with cells from late gastrulae, yolk plug-stage embryos, and neurulae showed that the 30S RNA is an intermediate in rRNA processing and is derived from 40S pre-rRNA and processed to 28S rRNA. The half-life of the 30S rRNA intermediate was about 7.5 min or less at the three stages examined.     

DNA replication is required for tissue-specific enzyme development in ascidian embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stage that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

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.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

Freezing tolerance in embryos and spermatozoa of the sea urchin

Late embryos of the sea urchin survive freezing, at least for a short period of time, at ?196 °C in the presence of a cryoprotectant. The freezing tolerance in glycolated embryos is greater in advanced developmental stages. High rates of both cooling and warming during a freezethaw sequence were more dangerous in one-cell embryos than in late ones. Both ethylene glycol and DMSO exerted a significant protection against freezing injury on embryos in all the stages after fertilization, but not on unfertilized egg cells. Cryopreservation of sea urchin sperm at ?196 °C in the presence of 1.5 m ethylene glycol has been achieved for 3 days. The fertilizability of eggs inseminated by frozen-thawed sperm was more than two-thirds. Nearly all the eggs thus fertilized developed to normal gastrulae.     

Activation of mitochondrial DNA synthesis during loach embryogenesis

Mitochondria isolated from unfertilized loach (Misgurnus fossilis) eggs incorporate 3H-dTTP at a low rate (0,01 pmoles 3H-dTTP-mg of mitochondrial protein/1 hr incubation). After fertilization the rate of 3H-dTTP incorporation into DNA of mitochondria isolated from embryos of different developmental stages increases exponentially, doubling each 7 hours, and attains the maximum to 35 hour of development. This stage corresponds to the beginning of movement. DNA synthesis in mitochondria from unfertilized eggs resembles repair; as early as 6 hours after fertilization the labeling pattern of mt-DNA is of replicative type. This replicative type of labeling is observed throughout all early development. Activation of mt-DNA biosynthesis in the course of early development is not accompanied by any changes of DNA polymerase activity in mitochondrial extracts or in mitochondrial lysates.     

Translational activation of maternal mRNA encoding the heat-shock protein hsp90 during sea urchin embryogenesis

Changes in protein synthesis induced by heat shock of Strongylocentrotus purpuratus gastrulae were analyzed bt two-dimensional electrophoresis. Hyperthermia induces the synthesis of polypeptides having molecular masses of 90, 70, 50, 40, and 38 kDa. One of these, hsp90, appears as a pair of polypeptides which comigrates with proteins synthesized at normal temperature in eggs and embryos; these comigrating spots produce indistinguishable patterns upon electrophoretic analysis of partial V8 protease digests, indicating that hsp90 is synthesized throughout embryogenesis. The relative rate of incorporation of methionine into hsp90 is low in eggs and zygotes, but increases abruptly in morulae, constituting a rare and striking change in protein synthesis during early development. Cell-free translation analyses indicate that most of the mRNA encoding hsp90 resides in the pool of free ribonucleoprotein particles in eggs and early embryos, but shifts to polysomes by the 64-cell stage while remaining constant in mass. Thus the increase in synthesis of hsp90 appears to be via the selective activation of translation of a stored maternal mRNA. The shift of hsp90 mRNA to polysomes is accompanied by polyadenylation. Heat shock of eggs or zygotes did not result in translational activation of hsp90 mRNA. The sea urchin hsp90 doublet of spots comigrates with hsp90 induced by heat shock of chicken embryo fibroblasts, a conserved protein abundant in many cells of a variety of species.     

SCANNING ELECTRON MICROSCOPICAL APPEARANCE OF CELLS FROM XENOPUS LAEVIS EMBRYOS OF DIFFERENT STAGES

The scanning electron microscopical appearances of cells isolated from different regions of Xenopus laevis embryos of different stages, and cultured in vitro have been compared. Blastula inner ectoderm cells initially show filopodia, then become flattened onto the substrate and then form pseudopodia. Blastula outer ectoderm cells are initially similar, but do not form pseudopodia. Most of the ectoderm cells from gastrulae and neurulae are featureless. Endoderm cells from blastulae do not initially form filopodia, but later form pseudopodia. Most of the endoderm cells from gastrulae and neurulae show neither filopodia nor pseudopodia, but in the gastrula some elongated, cylindrical cells are observed. Thus cells change their appearance after the three hour culture period; cells from different regions of embryos of the same stage show different appearances in vitro ; and cells from equivalent regions of embryos of different stages show different behaviours in vitro.     

Fate of amplified nucleoli in Xenopus laevis embryos

We have followed the fate of two components of extrachromosomal nucleoli, amplified ribosomal DNA (rDNA) and 7.5 kb precursor rRNA, during early embryogenesis of Xenopus laevis. Other workers have shown that the amount of amplified rDNA accumulated during oogenesis remains unchanged through the 16-cell stage of embryogenesis. Here we show that as embryonic cleavage continues, the amount of amplified rDNA decreases until it is no longer detectable in the early gastrula embryo. In contrast, the amount of 7.5 kb precursor rRNA in eggs, early cleavage stage embryos, or blastula stage embryos is the same as in oocyte nuclei. Since no rRNA synthesis occurs during these early stages, we conclude that the precursor rRNA sequences synthesized in the oocyte are neither processed nor degraded during early development. The amplified rDNA is not replicated in the early embryo even though the chromosomal DNA of the embryo replicates every 30 min during the first 7.5 hr of embryogenesis. When amplified rDNA is purified and then injected into cleaving embryos, however, we find that it is replicated. This finding suggests that some factor(s) prevents the endogenous amplified rDNA from responding to the cellular replication signals. We show that methylation of cytosine in the rDNA is not related to the DNA's capacity for replication in this system since amplified (unmethylated) and chromosomal (methylated) rDNA are both replicated when injected into embryos. The methylation pattern of these rDNAs appears to be maintained after replication in the embryo.     

Stability of alpha-fetoprotein messenger RNA in mouse yolk sac

Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus. Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.     

Fertilization of sea urchin eggs is accompanied by 40 S ribosomal subunit phosphorylation

After fertilization of sea urchin (Arbacia punctulata) eggs, there is a single prominent alteration in the pattern of protein phosphorylation. In eggs preloaded with ³²PO₄, a 31,000 M_r protein (rp31) becomes labeled within 4 min of sperm addition. A new steady-state level of rp31 labeling is achieved by 11 min. The rate of protein synthesis in sea urchin zygotes also increases at 8–10 min after fertilization. Protein rp31 corresponds to mammalian ribosomal S6 because it cosediments with 40 S subunits on high salt-sucrose gradients, it is similar to the mammalian protein in M_r and charge, and it becomes phosphorylated during an increase in protein synthesis. The specific activity of phosphorylated rp31 (relative to rRNA) is similar between free 80 S monosomes and polysomes, indicating that rp31 phosphorylation is not sufficient for ribosomal activity. A phosphatase, highly specific for rp31, is present in extracts of eggs and very early embryos. This phosphatase becomes inactive at about the same time that the degree of labeling of rp31 increases in embryos. Evidently a control system that maintains a low level of rp31 phosphorylation is active in sea urchin eggs. Inactivation of this system shortly after fertilization leads to the accumulation of phosphorylated ribosomes.     

Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5.

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.     

Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5–8 cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16–32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogenous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles. © 1996 Wiley-Liss, Inc.