Response to heat shock of different sea urchin species

It is demonstrated that sea urchin embryos of the species Sphaerechinus granularis are able to respond to heat shock by producing heat shock proteins at the same stage as embryos of Paracentrotus lividus, i.e. after hatching. Arbacia lixula embryos are able to synthesize heat shock proteins already at the stage of 64-128 blastomeres. Embryonic survival is observed if the embryos are heated at the stages at which they can synthesize the heat shock proteins. The inhibition of the bulk protein synthesis after heating at 31 degrees C is never less than 50%.     

Does protein synthesis decline in lithium-treated sea urchin embryos because RNA synthesis is inhibited?

Vegetalization of sea urchin embryos by Li⁺ is characterized by rates of protein synthesis which are normal during cleavage, and decline after hatching. This paper tests the hypothesis that Li⁺ interferes with RNA synthesis during cleavage, resulting in the decline in protein synthesis at hatching when newly synthesized mRNA becomes critical for further normal development. Treatment with Li⁺ does cause a decline in the incorporation of [³H]guanosine into RNA. However, this decline could be accounted for by reduced uptake of the labeled precursor with a concomitant reduction in precursor pool specific activity. Therefore, reduced protein synthesis after hatching in Li⁺-treated embryos cannot be accounted for by a comparable reduction in RNA synthesis.     

Salinity sensitivity of early embryos of the Antarctic sea urchin, <Emphasis Type="Italic">Sterechinus neumayeri</Emphasis>

Embryos and larvae of the Antarctic sea urchin, Sterechinus neumayeri, have received considerable experimental attention assessing impacts of low temperature on development; however, salinity effects are not well documented because heretofore, the Antarctic coastal marine environment has been remarkably stenohaline. In this study, subtle decreases of 2 and 4 parts per 1,000 in standard salinity were tested to see if the developmental rate of S. neumayeri embryos would be impacted by a potential hyposmotic stress. At 30 psu, significantly fewer embryos (2 individuals out of 198 tested) reached morula stage by 36 h post-fertilization in comparison embryos in control treatments at 34 psu. Antarctic sea urchins are an important component of marine environments due to their grazing activities. Reductions in larval recruitment success due to the influx of freshwater from melting ice shelves resulting from global climate change could have far-reaching impacts on benthic ecosystem structure in Antarctica.     

Effects of seawater acidification on cell cycle control mechanisms in Strongylocentrotus purpuratus embryos

Previous studies have shown fertilization and development of marine species can be significantly inhibited when the pH of sea water is artificially lowered. Little mechanistic understanding of these effects exists to date, but previous work has linked developmental inhibition to reduced cleavage rates in embryos. To explore this further, we tested whether common cell cycle checkpoints were involved using three cellular biomarkers of cell cycle progression: (1) the onset of DNA synthesis, (2) production of a mitotic regulator, cyclin B, and (3) formation of the mitotic spindle. We grew embryos of the purple sea urchin, Strongylocentrotus purpuratus, in seawater artifically buffered to a pH of ～7.0, 7.5, and 8.0 by CO(2) infusion. Our results suggest the reduced rates of mitotic cleavage are likely unrelated to common cell cycle checkpoints. We found no significant differences in the three biomarkers assessed between pH treatments, indicating the embryos progress through the G(1)/S, G(2)/M and metaphase/anaphase transitions at relatively similar rates. These data suggest low pH environments may not impact developmental programs directly, but may act through secondary mechanisms such as cellular energetics.     

Inhibition of translation and modification of translation factors during apoptosis induced by the DNA-damaging agent MMS in sea urchin embryos

Translational control was investigated in sea urchin eggs and embryos in response to the DNA-damaging agent methyl methanesulfonate (MMS). We have shown in this report that exposure of sea urchin embryos to MMS induces drastic effects on protein synthesis activity, and on translation factors level, integrity and post-translational modifications. In response to the treatment of embryos by the DNA-damaging agent MMS, protein synthesis is inhibited independently of the translation inhibitor 4E-BP and in correlation with phosphorylation of the translation factor eIF2alpha subunit. Furthermore, a low molecular weight form of translation initiation factor eIF4G is detected correlatively with MMS-induced apoptosis. We propose that modifications of translation factors play an important role in protein synthesis modulation that occurs during DNA-damage induced apoptosis.     

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.     

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.     

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.     

Protein synthesis and specific dynamic action in crustaceans: effects of temperature

Temperature influences the specific dynamic action (SDA), or rise in oxygen uptake rate after feeding, in eurythermal and stenothermal crustaceans by changing the timing and the magnitude of the response. Intra-specific studies on the eurythermal crab, Carcinus maenas, show that a reduction in acclimation temperature is associated with a decrease in SDA magnitude, resulting from an increase in SDA duration but a decrease in peak factorial scope (the factorial rise in peak SDA over prefeeding values). Inter-specific feeding studies on stenothermal polar isopods revealed marked differences in SDA response between the Antarctic species, Glyptonotus antarcticus and the Arctic species, Saduria entomon. Compared to S. entomon held at 4 and 13 degrees C, the SDA response in G. antarcticus held at 1 degrees C was characterised by a lower absolute oxygen uptake rate at peak SDA and an extended SDA duration. At peak SDA, whole animal rates of protein synthesis increased in proportion to the postprandial increase in oxygen uptake rate in the Antarctic and the Arctic species. Rates of oxygen uptake plotted against whole animal rates of protein synthesis gave similar relationships in both isopod species, indicating similar costs of protein synthesis after a meal, despite their differences in SDA response and thermal habitat.     

The hierarchy of requirements for an elevated intracellular pH during early development of sea urchin embryos

The intracellular pH (pHi) rises 0.3-0.5 units after fertilization of sea urchin eggs, and this and previous work show this pHi change is necessary for initiating the developmental processes leading to cell division. The experiments described here reveal that while the elevated pHi is permanently required for a normal early development, lowering pHi of embryos after fertilization affects different processes to different extents. Protein synthesis gradually becomes less sensitive to pHi. Karyokinesis proceeds to completion under a low pHi, but is retarded, while cytokinesis is always impaired. These results indicate a hierarchy of requirements for high pHi during early development of sea urchin embryos, with protein synthesis, karyokinesis, and cytokinesis showing, respectively, increasing requirements for an elevated pHi.     

The Effects of Chemical Animalizing and Vegetalizing Agents and of Cell Dissociation on the Synthesis of 5S RNA and Transfer RNA in Cleaving Sea Urchin Embryos

The effect of altering normal cell associations and interactions on the synthesis of 5S RNA and transfer RNA (tRNA) was studied in cleaving embryos of the sea urchin, Arbacia punctulata. Cell interactions were altered: (1) by culturing cleaving embryos in the animalizing agent, Evans Blue, and in the vegetalizing agent, Li⁺ as LiCl and (2) by culturing dissociated cells. Control and experimental embryos each were labeled from 3 h to 6 h post fertilization with [8-³H]-guanosine. Sixteen-cell embryos, whose GTP precursor pools had been preloaded, were dissociated, labeled and cultured under conditions which prevent reaggregation. Quantitative measurements of rates of accumulation of newly synthesized 5S RNA and tRNA showed that these rates are similar in cleaving sea urchin embryos and in corresponding embryos cultured in the presence of Evans Blue and of Li⁺. In addition, cells dissociated from cleavage embryos and maintained under conditions which prevent reaggregation retained the ability to synthesize 5S RNA and tRNA. These results suggest that normal cell associations and interactions are not necessary for the synthesis of 5S RNA and tRNA to occur in cleaving sea urchin embryos.     

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.     

The biology, life cycle and ecophysiology of the Antarctic mite Alaskozetes antarcticus

This paper is dedicated to the late Nigel Bonner, who as Head of the Life Sciences Division at British Antarctic Survey, encouraged and supported this research with his characteristic enthusiasm.
The cryptostigmatid mite Alaskozetes antarcticus (Michael) is a dominant member of many terrestrial communities in the maritime Antarctic, where it survives extreme temperatures, short cold summers, numerous freeze-thaw cycles, desiccating conditions and a limited season for growth and reproduction. However, examination of features of its biology, from morphology, through life-history strategy to physiology, indicate very little specialization to the Antarctic environment. Alaskozetes antarcticus is a herbivore/detritivore, typical of the Cryptostigmata in general, with low feeding and growth rates, long life span and low reproductive output. Physiological specializations exist in the form of low enzyme activation energies and elevated metabolic rates at low temperatures when compared with temperate species, and associated low optimum temperatures for activity, feeding and growth. Growth rates comparable with temperate species are achieved in the field, with an extended life cycle of five years or more as a result of the short growing season, and the ability of all life stages to overwinter equally successfully. Overwintering survival, involving supercooling enhanced by the use of antifreezes such as glycerol, although initially described in Antarctic species, is now known to be characteristic of many temperate relatives, so it is not a specific adaptation to the polar environment. The obvious success of A. antarcticus in maritime Antarctic terrestrial environments must be attributed to a combination of several features characteristic of the Cryptostigmata in general, rather than to specific polar adaptations.     

Effect of reduced protein synthesis on the cell cycle in sea urchin embryos

We have reinvestigated the existence of cyclical fluctuations of protein synthesis and have examined the effects of reducing it in early embryos of the purple sea urchin, Strongylocentrotus purpuratus. The results show that protein synthesis increases linearly during the first 45-60 minutes after fertilization, then transiently decreases during mitosis, and rises again at first cleavage. Reducing protein synthesis of embryos to 35% its normal value only slightly affects the rate of progression through the cell cycle. It is also shown that the observed retardations of the cell cycle, under depressed protein synthesis, are attributable (by 80%) to a lengthening of the premitotic phase but also, to a lesser extent (20%), to a lengthening of the mitotic phase itself. These results suggest that mitotic proteins, in sea urchin embryos, are stable and little affected by an imposed decrease of protein synthesis during their accumulation phase. This analysis supports the view that specific mechanisms, other than decreased protein synthesis, need be turned on only at appropriate times during the cell cycle in order to explain the destruction or deactivation of mitotic proteins. Finally, a one-dimensional SDS-PAGE analysis of synthesized proteins, labeled with 35S-methionine, reveals the presence of a 50-kDa cyclin showing the expected characteristics of mitotic proteins deduced from our results.     

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.     

Biological weighting functions for DNA damage in sea urchin embryos exposed to ultraviolet radiation

Laboratory experiments examining the effects of ultraviolet radiation (UVR, 290-400 nm) on DNA damage were carried out using the embryos of three species of sea urchins from different habitats; Strongylocentrotus droebachiensis from the Gulf of Maine, Sterechinus neumayeri from the Antarctic, and Evechinus chloroticus from New Zealand. All three species exhibited significant amounts of accumulated DNA damage, measured as cyclobutane pyrimidine dimers (CPD) photoproducts, when exposed to UVR in the laboratory. Biological weighting functions (BWFs) revealed that S. neumayeri has significantly higher sensitivity to UVR-induced DNA damage across most of the UVR spectrum compared to the other two species, and all species were observed to have weightings in the ultraviolet-A (UVA, 320-400 nm) portion of the spectrum. The increased sensitivity to ultraviolet-B (290-320 nm) and UVA in S. neumayeri is correlated with the lowest concentration of UVR absorbing compounds observed in the embryos of the three species of urchin used in this study. Sea urchin embryos and larvae in the respective habitats of the species tested are known to occur within 5 m of the surface of the ocean where both UVB and UVA wavelengths occur. Solar irradiances of UVR at a depth of 5 m, weighted using the urchin DNA damage BWFs, show that E. chloroticus receives the greatest amount of biologically effective UVR despite having the lowest wavelength dependent weightings for DNA damage when compared to the other two species.     

Re-examination of histone changes during development of newt embryos

Embryos of Triturus pyrrhogaster (BOIE) were labeled with Na₂¹⁴CO₃ and the incorporation of radioactivity into histone fractions was determined by the electrophoresis of the acid-soluble protein from isolated nuclei on a polyacrylamide gel with or without Triton X-100. The results supported the previous observation that the content of H1 histone might be low in blastulas and increased during development but they did not confirm the displacement of blastula H1 by other H1 molecular species in later embryos. The rate of H2b or H2a histone synthesis did not change much during development which contrasted sharply with the case of histone synthesis in sea urchin embryos. By changing the label duration or by culturing various durations after the label it was suggested that the histone fractions were synthesized or degraded as a set and any particular fraction that had a markedly long or short life could not be detected. The results were discussed in relation to the possible functions of H1 histone and to the histone synthesis in sea urchin embryos.