Nucleic Acid and Histone Synthesis by Ethanol-Treated Cleavage-Arrested Sea Urchin Embryos

Abstract. It has been found that fertilized sea urchin eggs prevented from normal cleavage by solutions of isosmotic ethanol in sea water are able to complete some cellular and molecular aspects of the normal developmental program that are observed in control cultures. In both treated and control cultures, the type of RNA transcribed changes at 24 h (early gastrula) in favor of higher molecular weight rRNA. Ultrastructural studies reveal the presence of nucleoli in ethanol-treated as well as control embryos. The type of H1 histone synthesized also shifts at 24 h in favor of a higher molecular weight H1 in both ethanol-treated and control embryos. Replication of DNA proceeds at a slower rate in ethanol-treated embryos than in controls, resulting in DNA/embryo values in ethanol which are 20–30% of control values after 24 h. The results relate to the problem of differentiation without cleavage, and the role of normal partitioning, cell-cell interaction, and DNA synthesis in triggering the sequence of events in the developmental program.     

Action of ethanol on different skull and brain parameters in the chick embryo

Fifty microliters of ethanol diluted in 50 microl of distilled water were injected into the air chamber of chick eggs immediately before their incubation, and modifications in a series of parameters were recorded. The somatic weight of the ethanol-treated embryos was lower compared with control and vehicle-administered embryos during days 13, 15, 17, and 19 of incubation, but was the same on day 21. The brain weight was lower in the ethanol-treated embryos on all the days studied (days 13, 15, 17, 19, and 21 of incubation). Skull measurements showed that the transverse anteroposterior and sagittal diameters were significantly smaller in ethanol-treated embryos compared with control and vehicle-administered embryos on days 17, 19, and 21 of incubation.     

Ethanol exerts differential effects on high affinity choline uptake in neuron-enriched cultures from 8-day-old chick embryo cerebral hemispheres

Neuronal-enriched cultures were prepared from 8-day-old chick embryo cerebral hemispheres and exposed to ethanol (50 mM) from day 4 to 8 in culture. At day 8, both control and ethanol-treated cultures were processed for [³H]choline uptake in situ. Uptake was performed on cultures containing either Na⁺-plus or Na⁺-free (Li⁺) HEPES buffer. Total choline uptake as well as Na⁺-dependent and Na⁺-independent choline uptake were calculated. The K_m and V_max were calculated using the Lineweaver-Burke analysis. Our analysis of the data revealed that ethanol-treated cultures exhibited two values for V_max, one similar to that found in control cultures and one significantly lower than controls. No differences were observed in K_m values between control and ethanol-treated cultures. We interpret the low V_max to represent a population of cholinergic neurons which have been arrested at an immature stage as a result of ethanol insult.     

Discontinuous DNA replication in developing sea urchin embryos.

Embryos of the sea urchin, Hemicentrotus pulcherrimus, growing synchronously and entering the third “S” phase at 120 min after fertilization, were pulse-labeled with [³H]thymidine for various periods ranging from 15 sec to 20 min, and the size of nascent DNA was analyzed by centrifugation in an alkaline sucrose gradient. It was found that pulse-labeling for 15 sec gave rise to a sedimentation profile with a major radioactivity peak at the position corresponding to a molecular weight of 4 × 10⁴ daltons. One-minute of labeling, however, gave a major radioactive band around the position corresponding to 1.4 × 10⁶ daltons. Upon increasing the labeling time, the radioactivity peaks or bands shifted toward the increasing molecular weights. Finally, most of the radioactive DNA was found to sediment at the bottom when the embryos were exposed to [³H]thymidine for 15 min or longer. The time span of the S phase in the cleavage embryos was about 15 min. The results of pulse and chase experiments also supported the discontinuous mechanism of DNA replication in the cleavage embryos.     

Effects of Chronic Ethanol Administration on Rat Brain Phospholipid Metabolism

Alterations in brain phospholipid metabolism were observed after chronic ethanol administration for 16 days to developing rats. Animals were injected intraperitoneally with 32Pi 16 h prior to killing. Overall uptake of 32Pi by brain did not differ between the control and ethanol-treated groups, which were killed 2 h and 24 h after the last ethanol feeding. Except for an increase in the labeling of myelin after ethanol treatment, the amount of radioactivity recovered in the synaptosomal-mitochondrial and plasma membrane fractions of control and ethanol-treated groups was not different. Relative to the radioactivity of phosphatidylcholines, which indicated no change, there were increases (20-44%) in labeling of ethanolamine plasmalogens, phosphatidic acids, and phosphatidylinositols in cortical synaptosomes from the 2-h ethanol-treated group. In the plasma membrane fractions, however, increases (9-14%) in labeling of phosphatidylserines and phosphatidylinositols were observed in both 2- and 24-h ethanol-treated groups. In both membrane fractions, there was an obvious increase (44-86%) in labeling of polyphosphoinositides at 24 h after withdrawal from ethanol. Results thus indicate an adaptive increase in the biosynthesis of ethanolamine plasmalogen and brain acidic phospholipids due to chronic ethanol administration. Furthermore, the increase in labeling of polyphosphoinositides in the 24-h withdrawal group may reflect the hypoactivity associated with ethanol withdrawal.     

A comparison of protein synthetic patterns in normal and animalized sea urchin embryos

Newly synthesized proteins from normal and animalized sea urchin embryos were compared by the technique of double labeling. Total embryonic protein was solubilized in SDS, urea, and 2-mercaptoethanol. The proteins were examined by coelectrophoresis on an SDS-polyacrylamide gel. The gels were sliced and the radioactivity determined. A standardized ratio of the isotopes served as the basis of comparison. A comparison of newly synthesized proteins from normal embryos 24 and 48 h old showed a shift from larger to smaller molecular weight proteins. Animalized embryos showed a similar shift. When normal and animalized embryos of the same ages were compared, differences were found. The differences were distributed over the entire range of molecular weights. These results show that although differences in protein synthesis between animalized and normal embryos are evident by 24 h, most of the changes in protein synthesis that occur in normal embryos are unaffected by animalization.     

Condensation of DNA by basic protiens does not depend on protien composition

We have studied by electron microscopy the size and morphology of the complexes obtained with different DNAs (between 500 and 5243 base pairs long) and four different proteins: sea urchin histone H1; sea cucumber histone ?₀, chicken erythrocyte histone H5, and clupeine. Surprisingly, the type of protein used has only a marginal influence on the complexes formed. The molecular weight and topology of DNA do not show any influence. The size of the complexes depends strongly on the ratio of positive to negative charges and also on the ionic conditions. Our studies have been mainly carried out at a ratio of 0.4. Under these conditions the average thickness of rods and toroids observed varies between 165 Å at 1.5 mM salt to 290 Å at 100 mM salt, with minor variations around these values depending on the type of DNA and protein used. We conclude that the formation of DNA condensates is mainly determined by a balance of electrostatic and intermolecular forces, the influence of specific interactions is only marginal. This conclusion seems to apply not only to the complexes described here, but also to chromatin fibers and to DNA condensed by low molecular weight counterions and other compounds (polyamines, inorganic ions, ethanol, etc.). © 1994 John Wiley & Sons, Inc.     

Ontogeny of salinity tolerance and hyper-osmoregulation by embryos of the intertidal crabs Hemigrapsus edwardsii and Hemigrapsus crenulatus (Decapoda, Grapsidae): survival of acute hyposaline exposure

The adults of Hemigrapsus edwardsii and Hemigrapsus crenulatus are euryhaline crabs and strong hyper-osmoregulators. Their embryos are carried externally attached to the abdominal pleopods of female crabs, where they are exposed to temporal and spatial changes in salinity associated with their intertidal and estuarine habitats. Although embryos lack the branchial and excretory organs responsible for adult osmoregulation, post-gastrula embryos were highly tolerant of exposure to hypo-osmotic sea water. Detached eggs (embryos+envelopes), of both species, at all developmental stages between gastrulation and hatching, exhibited 80-100% survival for periods up to 96 h in sea water (osmolality, 1050 mmol kg(-1)) and in dilutions to 50%, 10%, and 1%. Cleavage stages were less tolerant of dilution; H. edwardsii, <50% survived 24 h in 10% sea water; H. crenulatus <50% survived 6 h in 10% sea water. Post-gastrulation stages strongly hyper-osmoregulated but cleavage stages were hyper-osmoconformers (maintaining internal osmolality approximately 150 mmol kg(-1) above external). Osmoregulatory capacity was reduced just prior hatching, particularly in H. crenulatus, although salinity tolerance remained high. Gastrulation therefore marks a critical stage in the ontogeny of osmoregulation and salinity tolerance. Total Na+/K(+)-ATPase activity increased greatly during embryogenesis of H. crenulatus (undetectable in blastulae; gastrulae 0.31+/-0.05 pmol P(i) embryo(-1) min(-1); pre-hatching 16.4+/-1.0 pmol P(i) embryo(-1) min(-1)). Na+/K(+)-ATPase activity increased in embryos exposed to dilute sea water for 24 h implicating regulation of this transporter in a short-term acclimation response.     

Neuroblast cell death in ovo and in culture: Interaction of ethanol and neurotrophic factors

We used two experimental paradigms to examine the influence of the neurotrophins, NGF, EGF, and bFGF on normal neuroblast survival and also after ethanol insult. In the first paradigm, chick embryos received in ovo at embryonic day 1 and 2 (E1 and E2) saline (control) ethanol (10mg/50 l/day), NGF (50 ng/50 l/day), or EGF (25 ng/50 l/day), or ethanol+NGF or EGF. At E3, cultures were prepared from whole embryos separately from each group. At C2, all cultures were labeled with [³H]thymidine and assessed for effects or neuronal survival. In the second paradigm, cultures were prepared from 3-day-old whole embryos and at CO, cultures were treated with either ethanol (50 mM) alone, NGF (50 ng/ml) alone, EGF (25 ng/ml) alone, bFGF (50 ng/ml) alone, or were treated concomitantly with ethanol plus one of the neurotrophins; control had only the culture medium, DMEM+5% FBS. We obtained the following findings. 1) Cultures derived from embryos treated with either of the three neurotrophins exhibited a higher neuronal survival as compared to controls (1st paradigm). 2) The survival-promoting effect was also observed when the neurotrophins were added directly to the cultures (2nd paradigm). 3) As reported previously, cultures derived from ethanol-treated embryos exhibited a marked decline in neuronal survival as compared to controls. 4) All three neurotrophins attenuated the decline in neuronal survival produced by ethanol. The rescuing effects of the neurotrophins support our early hypothesis that ethanol administration during early neurogenesis interferes with microenvironmental trophic signals essential for neuroblast survival and differentiation.     

Histone synthesis by cleavage arrested sea urchin eggs.

A change in Hl histone synthesis occurs in blastulae, from Hlm to a faster moving Hlg in acrylamide gel electrophoresis. The experiments below indicate that this shift occurs in the absence of normal cleavage. Hybrid embryos containing paternal Hl histone markers along with homospermic embryos were studied. Both were labeled with L-[3H]lysine. Some cultures were kept at 11 degrees C to inhibit cleavage. It was found that Hlm and Hlg are synthesized sequentially in time by embryos grown at 20 degreet C as well as by those grown at 11 degrees C. The hybrid data establish that Hlm is translated at least in part from mRNA newly transcribed from paternal DNA. This observation also holds for cleavage inhibited hybrid embryos. Hlg is made by both hybrid and homospermic embryos during the later phases of development at both 11 and 20 degrees C. These results confirm and extend those of Seale et al. (1973), Ruderman et al. (1974) and Easton et al. (1974).     

Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos

DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development.     

The response of circulating parameters of bone mineral metabolism to ethanol- and EDTA-induced hypocalcemia in the rat

The mechanism of the acute hypocalcemia that follows acute ethanol administration has not been established. Measurements of parathyroid hormone (PTH) performed during this hypocalcemia reveal conflicting results. We compared the response of ionized calcium (Ca²⁺), immunoreactive PTH and bone Gla protein (BGP) after ethanol- and EDTA-induced hypocalcemia. 103 male Sprague Dawley rats each weighing approximately 300 g received ethanol and 100 rats of similar weight received EDTA. In each of these studies the animals were divided into experimental and control groups. The ethanol-treated rats received ethanol, 2 g/kg body weight, by ip injection and the EDTA-treated rats received 100 mg EDTA/kg body weight by im injection. Controls received normal saline by the corresponding route of administration. Rats were sacrificed at 0, 30, 60, 90, 180 and 360 min for the measurement of the above parameters. In both experimental groups Ca²⁺ levels were significantly reduced to the same degree by 30 min with return to control values by 360 min. There was no significant difference in immunoreactive PTH, and BGP between control and ethanol-treated groups. In the EDTA-treated rats, however, PTH values were significantly increased at 30 (P < 0.005) and BGP at 60 and 90 minutes (P < 0.005) vs. control. Therefore acute ethanol administration appears to blunt the PTH response to hypocalcemia. A direct inhibitory effect of ethanol on osteoblast function ie BGP production cannot be excluded. In addition, PTH may stimulate BGP.     

Characterization of a new variant DNA (cytosine-5)-methyltransferase unable to methylate double stranded DNA isolated from the marine annelid worm Chaetopterus variopedatus.

The enzyme S-adenosylmethionine-DNA (cytosine-5)-methyltransferase has been identified, first time for invertebrates, in embryos of the marine polychaete annelid worm Chaetopterus variopedatus. The molecule has been isolated from embryos at 15 h of development. It is a single peptide of about 200 kDa molecular weight, cross-reacting with antibodies against sea urchin DNA methyltransferase. The enzymatic properties of the molecule are similar to those of Dnmt1 methyltransferases isolated from other organisms, but with the peculiarity to be unable to make 'de novo' methylation on double stranded DNA.     

Effects of ethanol on the primitive streak stage mouse embryo

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.     

Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo

A fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by CyIIIa actin gene cis-regulatory sequences was injected into unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The distribution of CAT DNA sequences was measured directly by in situ hybridization in squashed 24-hr blastula preparations derived from these eggs. Earlier studies had shown that stable mosaic incorporation of the exogenous DNA occurs during cleavage, after which the exogenous sequences replicate at approximately the pace of the host cell genomes. The fractions of embryonic cells observed in this study to include CAT DNA sequences imply that their stable incorporation into a replicating nuclear form occurs most often in a single cell at the 3rd or 4th cleavage stages, though it may occur as early as 2nd cleavage, or as late as 7th cleavage. Corroborative measurements were carried out by the same method on squashed preparations of embryos at earlier stages, and by in situ hybridizations of CAT mRNA, both in dissociated embryos and in cytological sections of 72-hr pluteus-stage embryos. Hybridizations to CAT mRNA and to CAT DNA were carried out on alternate sections of several embryos. The results confirm unequivocally that although CAT mRNA appears only in the aboral ectoderm in embryos derived from eggs injected with the CyIIIa.CAT fusion gene, the exogenous sequences are indeed present, though silent, in the various other cell types of the late embryo.     

DNA polymerase activity in preimplantation mouse embryos.

DNA polymerase activity was measured in mouse embryos at stages before implantation to determine whether it increases in proportion to the amount of DNA synthesis, as it does in populations of differentiated mammalian cells, or remains constant, as it does in early sea urchin embryos. Total enzyme activity was found to be relatively unchanged following fertilization and in the first few cleavage stages. However, between the 12- and 120-cell (blastocyst) stage, the amount of activity increased by several-fold. These results indicate that the relationship between amount of DNA polymerase activity and DNA synthesis in mouse embryos exhibits two phases: in the early cleavage phase it is similar to that in sea urchin embryos, whereas, in the blastocyst phase, it is similar to that in differentiated mammalian cells.     

Post-replication DNA repair in barley embryos treated with N-methyl-N-nitrosourea

In sterile cultures of free barley embryos, N-methyl-N-nitrosourea (MNU) caused a decrease in the size of both template [¹⁴C]-labeled DNA and of daughter [³H]DNA strands as determined in alkaline sucrose gradients, and inhibited the rate of [³H]thymidine incorporation. In addition, duplexes containing [³H]-daughter DNA analyzed in BND cellulose contained more single-stranded regions in MNU-treated embryos than in the corresponding control. Incubation of MNU-treated embryos in nutrient medium for up to 18 h after the [³H]-labeling permitted the recovery of small-sized daughter DNA to full-sized strands and led to the enhancement of double-strandedness of DNA duplexes containing [³H]-labeled strands. If [³H]-labeling had been carried out 8–10 h after the MNU treatment, the size of daughter DNA, the proportion of double-strandedness and the rate of thymidine uptake into DNA partially increased in comparison with rates observed when labeling had been done just after or 3 h after the MNU treatment, but these variables did not reach the values of the corresponding controls.     

Ethanol-induced cardiac hypertrophy: effects of peripheral sympathectomy

The development of cardiac hypertrophy was examined in rats that had undergone sympathectomy with 6-hydroxydopamine. After 4 days, the rats were given severely intoxicating doses of ethanol or isocalorically paired quantities of maltose-dextrin by intubation at 8-h intervals up to 48 h. The ethanol and sugar intubations were applied in a nutritionally adequate, liquid diet mix. The extent of the peripheral sympathectomy was evident from the absence of detectable quantities of noradrenaline in hearts of animals injected with the neurotoxin and in the reduced levels of excreted noradrenaline. The adrenal medullary catecholamine contents of sympathectomized rats were unchanged in the absence of ethanol; there were reduced quantities of adrenaline in the medullae of rats given ethanol. The adrenal glands of rats given ethanol were larger than those from control animals. Urine samples from sympathectomized and control rats, given ethanol, displayed equivalent increases in excreted adrenaline and noradrenaline. Increases in relative cardiac weight were evident in hearts from sympathectomized animals after 4 days of sympathectomy, and this change reached significance in the hearts from 6-hydroxydopamine-treated rats after a further 2 days on the control diet. Hearts from animals exposed to ethanol showed a marked, rapid development of cardiomegaly; after 24 h there was an increased mass of some 17%, which was sustained over the remaining 24-h period. The proportion of cardiac protein did not differ in the large hearts from ethanol-treated animals and those from their controls, hence myocardial oedema could not account for the increase in weight.(ABSTRACT TRUNCATED AT 250 WORDS)