Nucleic acid and histone synthesis by ethanol-treated cleavage-arrested sea urchin embryos

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.     

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.     

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.     

Ethanol induces cell death by activating caspase-3 in the rat cerebral cortex

Ethanol has long been implicated in triggering apoptotic neurodegeneration. We examined the effects of ethanol on the rat brain during synaptogenesis when a spurt in brain growth occurs. This period corresponds to the first 2 postnatal weeks in rats and is very sensitive to ethanol exposure. Ethanol was administered subcutaneously to 7-day- postnatal rat pups by a dosing regimen of 3 g/kg at 0 h and again at 2 h. Blood ethanol levels peaked (677+/-16.4 mg/dl) at 4 h after the first ethanol administration. The cerebral cortexes of the ethanol-treated group showed several typical symptoms of apoptosis such as chromosome condensation and disintegration of cell bodies. Activated caspase-3 positive cells were found in the cortex within 2 h of the first injection, and reached a peak at 12 h. In addition, TUNEL staining revealed DNA fragmentation in the same regions. These results demonstrate that acute ethanol administration causes neuronal cell death via a caspase-3-dependent pathway within 24 h, suggesting that activation of caspase-3 is a marker of the developmental neurotoxicity of ethanol.     

Dynamics of morphofunctional organization and developmental potential of mouse embryos in the state of "two-cell block in vitro"

Vital observation in combination with electron microscopy and immunocytochemistry was used for studying structural organization and developmental potential of BALB/c mouse embryos after cleavage cessation at a two cell stage caused by the "two-cell block in vitro" phenomenon. Modification of structure and viability of embryos was followed for 2 days from the time of cleavage arrest. Several hours before cleavage arrest, changes in mitochondrian distribution were noticed in embryos, no other disturbances in structural organization of blastomeres being obvious. Embryos, whose development was arrested for 24 h, remained viable and demonstrated some morphological changes similar to those seen in normally developing embryos of the same age. Towards the end of a 48 h block period some embryos died, the surviving embryos remained morphologically intact and metabolically active. At the same time, the nuclei of the latter frequently displayed chromatin condensation near the nuclear membrane, which is similar to the pattern of chromatin reorganization in the nuclei of early apoptotic cells. Our results support a concept on the "two-cell block in vitro" phenomenon as a specific functional state of embryos, and well compare with data on a partial realization by blocked embryos of the developmental program.     

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.     

Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).     

Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.     

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.     

The role of lysyl oxidase and collagen crosslinking during sea urchin development

Lysyl oxidase, the only enzyme involved in collagen crosslinking, is shown to be present in embryos of the sea urchin Strongylocentrotus purpuratus. The enzyme specific activity increases over six-fold during development, showing the greatest rise during gastrulation and prism larva formation. The enzyme is inhibited by the specific inhibitor, beta-aminoproprionitrile (BAPN). Continuous BAPN treatment of S. purpuratus and Lytechinus pictus embryos from late cleavage stages onward increases the amount of noncrosslinked collagen present in prism larvae. When BAPN is added at the 128- or 256-cell stage it causes developmental arrest at the mesenchyme blastula stage. Embryos can be maintained in the arrested state for at least 96 h and will resume normal development and morphogenesis following BAPN removal. If BAPN is added after the mesenchyme blastula stage, it has little adverse effect on development; consequently nonspecific toxic effects of the drug are unlikely. The results suggest that lysyl oxidase and collagen crosslinking play a vital role in primary mesenchyme migration, gastrulation, and morphogenesis during sea urchin development and indicate that BAPN may be very useful in studying the extracellular matrix-cell interactions at the cellular and molecular level.     

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).     

The embryonic development of Xenopus laevis under a low frequency electric field

The aim of this study was to determine the effects of a low frequency electric field on the early embryonic development of frogs. The embryos of African clawed toads, Xenopus laevis, were exposed to a 20-μA electric current during the cleavage stages. The developmental processes of embryos during and after electric field exposure were monitored for teratogenic effects. All the embryos continuously exposed to the electric field died without undergoing any developmental processes. However, when the embryos were exposed to the electric field for 20-min periods (four times/over 2 d), the embryos developed into both normal tadpoles (70 %) and malformed tadpoles with light edema, reduced pigmentation, or axial anomalies, such as crooked tails. After exposure, the control embryos were at development stage 35.5 (2 d 2 h), while the normal embryos of the assay group were at developmental stage 41(3 d 4 h). There was a 1 d 2 h difference between the two developmental stages, revealing the importance of that time period for embryogenesis. In conclusion, the effects of electric current on Xenopus embryos are dependent on the initial developmental stage and the duration of exposure.