Disruption of circulation by ethanol promotes fetal alcohol spectrum disorder (FASD) in medaka (Oryzias latipes) embryogenesis

Japanese medaka (Oryzias latipes) embryos exposed to ethanol have developed craniofacial, cardiovascular and skeletal defects which can be compared with the phenotypic features of fetal alcohol spectrum disorder (FASD) observed in human. The present experiment was designed to show that the disruption in circulation by ethanol during embryogenesis is a potential cause of FASD. Fertilized eggs were exposed to ethanol (0, 100 and/or 400 mM) for 24 or 48 h at various developmental stages (Iwamatsu stages 4-30) and were analyzed at 6 day post fertilization (dpf). It was observed that controls and the embryos exposed to 100 mM ethanol were in circulating state; however, a significant number of embryos of stages 4-24 exposed to 400 mM ethanol had disrupted circulation. Compared to controls, protein and RNA contents were significantly reduced in non-circulating embryos. Lipid peroxidation (LPO) analysis was made at 3, 6, 24, 48, 96 and 144 hour post fertilization (hpf). LPO was increased with the advancement of morphogenesis; however, ethanol or the circulation status had no effect. We further analyzed alcohol dehydrogenase (Adh 5 and adh8) and aldehyde dehydrogenase (Aldh9A and Aldh1A2) enzyme mRNAs in the embryos exposed to 400 mM ethanol for 24 h. A developmental stage-specific reduction in these enzyme mRNAs by ethanol was observed. We conclude that ethanol-induced disruption in circulation during embryogenesis is a potential cause of the development of FASD features in medaka.     

Patterns of protein synthesis and metabolism during sea urchin embryogenesis

We have analyzed the patterns of protein synthesis in developing embryos of the sea urchin Strongylocentrotus purpuratus by two-dimensional gel electrophoresis. There was an increase in the number of proteins detectably synthesized during development, as well as significant changes in relative rates of synthesis involving approximately 20% of the nearly 900 newly synthesized polypeptides. The majority of these changes were increases rather than decreases in synthesis; about half were of at least 10-fold, while a few were of more than 100-fold. Very few changes were detected upon fertilization and during the first several hours of development, while about 60% of the changes detected occurred between the hatching and the beginning of invagination. An analysis of proteins detected by silver staining indicated that most remained nearly constant in mass during embryonic development, but several increased or declined substantially. Many proteins present in eggs were not detectably synthesized in either eggs or embryos.     

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.     

Protein synthesis in microsurgically produced androgenetic and gynogenetic mouse embryos

Summary In order to compare paternal and maternal gene activity at the protein synthesis level during early development, androgenetic and gynogenetic mouse embryos were experimentally produced by microsurgically removing either the female or the male pronucleus from fertilized mouse eggs. The resulting haploid eggs were diploidized in a medium containing cytochalasin B and then cultured under normal conditions to the blastocyst stage. Protein synthesis was analyzed at different stages of preimplantation development using 2-dimensional polyacrylamide gel electrophoresis. Both types of uniparental embryos synthesized a similar set of proteins independent of whether the paternal or the maternal genome was present. The isodiploid embryos expressed a protein pattern that corresponded remarkably to normal embryos at the subsequent cleavage stage. This temporal change is probably due to the fact that the operated haploid eggs were kept overnight in cytochalasin B in order to allow chromosomal replication to occur without cell division, and the resulting eggs therefore corresponded to normal 2-cell embryos with respect to karyokinesis but differed as far as cytokinesis was concerned. Several 2-cell specific proteins appeared in these isodiploid eggs and, similarly, following their first cleavage some 4-cell specific proteins were detected in 2-cell androgenetic and gynogenetic embryos. The discordance between nuclear and cellular division, which was retained through the 4-cell stage, however disappeared during subsequent cleavage divisions. At the blastocyst stage, both kinds of uniparental embryos showed a similar protein pattern compared to normal embryos. Our data suggest that some stage-specific proteins are synthesized during preimplantation development and correspond to nuclear rather than cellular divisions.Some of these results were presented at the 13th Annual Meeting of the Union of Swiss Societies of Experimental Biology in Lausanne, March 1981 (Petzoldt et al. 1981)     

Program of early development in the mammal: Changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse

The absolute rates of synthesis of specific ribosomal proteins have been determined during growth and meiotic maturation of mouse oocytes, as well as during early embryogenesis in the mouse. These measurements were made possible by the development of a high-resolution twodimensional gel electrophoresis procedure capable of resolving basic proteins with isoelectric points between 9.1 and 10.2. Mouse ribosomal proteins were separated on such gels and observed rates of incorporation of [³⁵S]methionine into each of 12 representative ribosomal proteins were converted into absolute rates of synthesis (femtograms or moles synthesized/hour/oocyte or embryo) by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and embryos (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341–359). Ribosomal proteins were synthesized at all stages of oogenesis and early embryogenesis examined and, while equimolar amounts of ribosomal proteins were found in ribosomes, they were always synthesized in nonequimolar amounts during development. Rates of synthesis of individual ribosomal proteins differed from each other by more than an order of magnitude in some cases. Synthesis of ribosomal proteins accounted for 1.5, 1.5, and 1.1% of total protein synthesis during growth of the oocyte, in the fully grown oocyte, and in the unfertilized egg, respectively. During meiotic maturation of mouse oocytes the absolute rate of synthesis of ribosomal proteins decreased about 40%, from 620 to 370 fg/hr/cell, as compared to a 23% decrease in the rate of total protein synthesis during the same period. On the other hand, during early embryogenesis the absolute rates of synthesis of each of the 12 ribosomal proteins examined increased substantially as compared with those of the unfertilized egg, such that at the eight-cell stage of embryogenesis synthesis of ribosomal proteins (4.17 pg/hr/embryo) accounted for about 8.1% of the total protein synthesis in the embryo. Consequently, while the absolute rate of total protein synthesis increased about 1.5-fold during development from an unfertilized mouse egg to an eight-cell compacted embryo, the absolute rate of ribosomal protein synthesis increased more than 11-fold during the same period. These results seem to reflect the differences reported for the patterns of ribosomal RNA synthesis during early development of mammalian, as compared to nonmammalian, animal species. The results are compared with those obtained using oocytes and embryos fromXenopus laevis.     

Changes in ecdysteroid and juvenile hormone levels in developing eggs of Bombyx mori

It is well-known that insect eggs can contain very high concentrations of ecdysteroids, which undergo drastic changes during embryogenesis. We found that this is equally valid for juvenile hormones. Three juvenile hormone-immunoreactive compounds were observed in developing Bombyx mori eggs. They were assumed to be juvenile hormones 1, 2 and 3 according to their retention time in HPLC. These hormones underwent drastic and sudden changes. In the space of one day their concentration was seen to rise rapidly from an undetectable level up to as high as 4 × 10^?6 micromoles per mg of eggs. Their presence was detected as early as the first day of embryonic development, as well as during the blastokinesis period (day 5 to day 9) and in late embryos (day 12 to day 14). Their relative concentrations varied greatly. On two occasions, day 1 and day 8, all three hormones were simultaneously present. Moreover, juvenile hormone 3 was present during the blastokinesis period, either alone or in combination with hormone 2. The latter was the only hormone present in late embryos, before hatching. Thus, with regard to both ecdysteroids (ecdysone and 20-hydroxyecdysone) and juvenile hormones, each day of embryonic development displayed a different hormonal pattern. These patterns undoubtedly constitute a “hormonal code” of embryogenesis control. While 20-hydroxyecdysone can be assumed to trigger cuticulogenesis in embryos as it does in larvae, the effects of the other hormones as well as their possible interactions are questionable.     

dCMP-aminohydrolase activity during early sea urchin development. An example of negative enzyme control during embryogenesis

In sea urchin, unfertilized eggs have a very high level of dCMP-aminohydrolase (dCMPase) activity, which decreases gradually and at the pluteus stage it is only about a quarter of that found in the unfertilized egg. But in abnormal embryos and in disaggregated cells from embryos, no decrease in the dCMPase activity takes place. To understand the control mechanism involved in this enzyme activity during development, we have analyzed the effect of various drugs which interfere with information transfer, such as actinomycin C, puromycin, 5-azacytidine, 2-thio-uracil and p-fluoro-DL-phenylalanine on dCMPase activity in embryos of Paracentrotus lividus and Sphaerechinus granularis. Among these drugs only actinomycin induces a remarkable increase of the dCMPase activity in embryos with respect to unfertilized eggs. Puromycin has a differential and dose-dependent effect. Other drugs, although they affect normal development and macromolecular synthesis, do not significantly alter the dCMPase activity. On the basis of these results we suggest the presence of a repressor mechanism in the control of dCMP-aminohydrolase level during early embryogenesis of sea urchin.     

Analysis of proteins expressed at the time of murine organogenesis

Two-dimensional electrophoretograms were prepared from wild-type C57BL/6J embryos from day 7.5 through day 9.0 of development. This time period encompasses a critical window of development as the embryo traverses from an egg cylinder through major organogenesis. Consequently, we term this resource MOPED (for mouse organogenesis protein electrophoresis database). By resolving and analyzing the behavior of approximately 1,000 polypeptides per time point, we were able to track many of these polypeptides through this time period in development. Of special note was a burst of induced protein synthesis that was observed in mouse embryos development. Polypeptides observed in mouse embryos that match those identified previously in mouse fibroblasts were noted. Two of them (the intermediate filament-associated protein and tropomyosin-4) were significantly altered in 8.5 day embryos. As more polypeptides are designated, it will be possible to expand the known proteins in the database. MOPED establishes the patterns of synthesis of a large number of polypeptides during a crucial period of development. Thus MOPED is designed to analyze proteins relevant to mouse embryogenesis in the future.     

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin ( ca . 8 × 10⁸ molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus . The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.     

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked xenopus laevis embryos

In the present study we have characterized the synthesis of members of the HSP30 family during Xenopus laevis development using a polyclonal antipeptide antibody derived from the carboxyl end of HSP30C. Two-dimensional PAGE/immunoblot analysis was unable to detect any heat-inducible small HSPs in cleavage, blastula, gastrula, or neurula stage embryos. However, heat-inducible accumulation of a single protein was first detectable in early tailbud embryos with an additional 5 HSPs at the late tailbud stage and a total of 13 small HSPs at the early tadpole stage. In the Xenopus A6 kidney epithelial cell line, a total of eight heat-inducible small HSPs were detected by this antibody. Comparison of the pattern of protein synthesis in embryos and somatic cells revealed a number of common and unique heat inducible proteins in Xenopus embryos and cultured kidney epithelial cells. To specifically identify the protein product of the HSP30C gene, we made a chimeric gene construct with the Xenopus HSP30C coding sequence under the control of a constitutive promoter. This construct was microinjected into fertilized eggs and resulted in the premature and constitutive synthesis of the HSP30C protein in gastrula stage embryos. Through a series of mixing experiments, we were able to specifically identify the protein encoded by the HSP30C gene in embryos and somatic cells and to conclude that HSP30C synthesis was first heat-inducible at the early tailbud stage of development. The differential pattern of heat-inducible accumulation of members of the HSP30 family during Xenopus development suggests that these proteins may have distinct functions at specific embryonic stages during a stress response.     

Bombyx acid cysteine proteinase

Summary

Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.     

Silkworm egg proteins at the germ-band formation stage and a functional analysis of BmEP80 protein

The patterning of embryos in early stages is a critical process for embryo development. In order to understand the molecular mechanism of early embryogenesis in silkworm, 2-DE combined with MALDI-TOF-MS technologies were used to analyze the proteins from diapause-destined eggs at the germ-band formation stage. From over 1000 spots, 93 were selected for analysis and data were obtained from 59 revealing 42 proteins. Gene Ontology annotation showed these proteins were involved in several biological processes at the germ-band formation stage, including cell stress response and protein folding, cell growth and migration, termination of diapause, and nutrition storage. Prominent among them was a new 80 kDa protein, named Bombyx mori egg protein 80 (BmEP80). BmEP80 was a component of the eggshell which was secreted by follicle cells during the late vitellogenesis stage to early choriogenesis stage (FCs −5 to +10). It disappears during early embryogenesis and RNAi against it resulted in the collapse of eggs, thus it is likely that BmEP80 is a new component of the silkworm vitelline membrane.     

Teratocarcinoma stem cells and early mouse embryos contain only a single major lamin polypeptide closely resembling lamin B

The nuclear lamina in adult mammalian somatic cells is composed of three major proteins, lamins A, B, and C. The expression of these proteins during the differentiation of teratocarcinomas and mouse embryogenesis is described. Embryos up to day 8 of gestation and embryonal carcinoma (EC) cells express only a single lamin species closely resembling, if not identical to, lamin B. Lamins A and/or C were detected in fertilized eggs, but disappear during the first 2-4 cleavage divisions, only reappearing in 8 day post-implantation embryos. These two lamins are absent from EC cells, but are strongly expressed in some of their derivatives. These results show that cells of the early mouse embryo do not have a functional requirement for lamins A and C and imply that the structural organization of the nucleus may change fundamentally during embryogenesis.     

Modulation of calmodulin levels, calmodulin methylation, and calmodulin binding proteins during carrot cell growth and embryogenesis.

Carrot cell cultures were used to study the dynamics of calmodulin protein levels, calmodulin methylation, and calmodulin-binding proteins during plant growth and development. Comparisons of proliferating and nonproliferating wild carrot cells show that, while calmodulin protein levels does not vary significantly, substantial variation in post-translational methylation of calmodulin on lysine-115 is observed. Calmodulin methylation is low during the lag and early exponential stages, but increases substantially as exponential growth proceeds and becomes maximal in the postexponential phase. Unmethylated calmodulin quickly reappears within 12 h of reinoculation of cells into fresh media, suggesting that the process is regulated according to the cell growth state. Calmodulin and calmodulin-binding proteins were also analyzed during the formation and germination of domestic carrot embryos in culture. Neither calmodulin methylation nor calmodulin protein levels varied significantly during somatic embryogenesis. However, upon germination of embryos, the level of calmodulin protein doubled. By calmodulin overlay analysis, we have detected a major 54,000 M(r) calmodulin-binding protein that also increased during embryo germination. This protein was purified from carrot embryo extracts by calmodulin-Sepharose chromatography. Overall, the data suggest that calmodulin methylation is regulated depending upon the state of cell growth and that calmodulin and its target proteins are modulated during early plant development.     

Endoplasmic reticulum associated glucose-6-phosphatase activity is developmentally regulated and enriched in microsomes of endo/mesoderm in sea urchins

Summary Glucose-6-phosphatase (G-6-Pase) activity was analyzed during early embryogenesis of the sea urchinS. purpuratus. This activity is detected in very low levels in unfertilized eggs and early embryos but is present at high levels in preparations of endoplasmic reticulum (microsomes) from gastrula stage embryos. The approximately eight-fold increase in the relative activity of G-6-Pase associated with the ER occurs abruptly during a 12 h interval at gastrulation, and thereafter remains at a level comparable to that found in mammalian liver microsomes. The enzyme activity associated with the ER of gastrula stage embryos was completely eliminated from the microsomal pellet when cell lysates were first treated with non-ionic detergent. Analysis of germlayer tissues from late stage pluteus embryos revealed that G-6-Pase activity was more highly enriched in microsomes of endo/mesoderm tissues as compared to microsomes from ectoderm. The increase in ER associated G-6-Pase activity during embryonic development and its enriched activity in the ER of endo/mesoderm, as well as the observation that the signal recognition particle becomes associated with the ER at gastrulation (Le Blanc and Infante 1989), opens the question that this cellular organelle may be differentiating during embryogenesis in sea urchins.     

Protein synthesis in embryonic tissues during mouse postimplantation development

Mouse embryos of the NMRI strain between the 7th and 9th day of gestation were isolated from the uterus and dissected into the various tissue derivatives in order to investigate newly synthesized proteins during morphogenesis. The day 7 embryo was fragmented into trophoblast and ectoplacental cone, distal and proximal endoderm, extraembryonic and embryonic ectoderm. The day 8 and day 9 embryos were divided into trophoblast and placental anlage, yolk sac, amnion, and allantois, as well as cranial, central, and caudal embryonic tissue. The intact embryos were incubated in Dulbecco's minimum essential medium in the presence of 35S-methionine for 4 h, then dissected into the various fragments, and further processed for two-dimensional gel electrophoresis. Protein synthesis of the isolated tissue derivatives was analyzed and compared for the three developmental stages. Concerning the proteins with isoelectric points in the range of 4.5 to 8.0 and molecular weight ratio (M(r)) values between 20,000 and 200,000, we found several significant quantitative and qualitative differences in the various tissue fragments. In addition, we observed further quantitative and qualitative differences in protein synthesis during the postimplantation period investigated. We propose that the differences reflect some of the cell lineage- and developmental stage-specific changes in gene expression during early mammalian differentiation.     

Experimental changes in the amount of maternally stored ribosomes affect the translation efficiency of ribosomal protein mRNA in Xenopus embryo

The amount of maternal free ribosomes in developing Xenopus embryos has been experimentally modified; an increase was obtained by microinjection of purified ribosomes into fertilized eggs, and a decrease was induced by treatment with a drug which reduces the amount of free ribosomes. The effect of this manipulation on the partition of the ribosomal protein mRNA (rp-mRNA) was analyzed during embryo development; it was observed that when ribosomes available for translation are in excess, polysome loading with rp-mRNA decreases. Conversely, when ribosomes are scarce, polysome loading of rp-mRNA increases. These experiments, which artificially stress events observed in the course of development, indicate that there is a relationship between the availability of ribosomes in the cells and the utilization of rp-mRNA for synthesis of ribosomal proteins, as already suggested by previous observations on r-protein synthesis during embryogenesis.     

Biosynthesis of an acidic galactan and sulfated glycosaminoglycans during embryonic development of the mollusc Pomacea sp.

The synthesis of acidic polysaccharides by eggs of Pomacea sp. at different stages of development was studied. By day 3 after oviposition an acidic galactan became apparent. This compound reaches its maximum concentration by day 6 and then slowly decreases in concentration, no longer being detectable by day 12. Among the sulfated glycosaminoglycans, chondroitin sulfate is the first to appear (day 10), followed by heparan sulfate and other sulfate glycosaminoglycans, which were synthesized in large amounts until hatching (around day 15). Each one of the compounds was purified and characterized by chemical analysis and enzymatic degradation. The synthesis and characterization of the sulfated glycosaminoglycans was confirmed by the use of radioactive sulfate applied to intact eggs at different stages of development. These studies suggest that the main difference between vertebrate and mollusc development regarding the acidic polysaccharides is that hyaluronic acid seems to be absent during early mollusc development. It is proposed that the acidic galactan may be synthesized from the neutral galactan, already present in the eggs in high amounts, and may replace this glycosaminoglycan in the mollusc embryogenesis.     

Exogastrulation and interference with the expression of major yolk protein by estrogens administered to sea urchins

Although estrogens have been detected in some echinoderm species, their role is not clearly understood; so we examined the effects of estrogens administered to sea urchin embryos and larvae. A typical malformation was exogastrulation, induced by the exposure to ethynylestradiol (EER) in a defined period of 12 h from 12 h after fertilization (HAF). Morphogenesis for gastrulation was delayed in the treated embryos: protrusion of the archenteron started at 30 HAF when gastrulation had already finished in normal embryos. Exogastrulation induced by EER was cancelled by the antiestrogen chemical, ICI182,780. Feeding larvae were less sensitive to estrogens than those in early embryogenesis and, at certain concentrations, developed without abnormal morphology. The effect of estrogens was examined at the level of gene expression of the major yolk protein (MYP). MYP expression started during the larval stage and was suppressed by estrone at the six-armed stage, but not by β-estradiol, and in later stage larvae, the expression was not affected by treatment with either estrogen. Estrogens affect sea urchins in the early stage of embryogenesis, leading to abnormal morphogenesis and interference with gene expression.