Methylmercury teratogenesis in the killifish, Fundulus heteroclitus.

Exposure of developing eggs of the killifish, Fundulus heteroclitus, to 0.03 or 0.04 mg/l of methylmercuric chloride resulted in a variety of abnormalities. Percentage of axis formation was reduced somewhat, and many embryos developed cyclopia or intermediate conditions leading to cyclopia, reflecting interference with induction of the forebrain. Defects in the cardiovascular system also appeared in the form of failure of the heart to differentiate properly into chambers. The heart was a thin, feebly beating tube, incapable of causing the blood to circulate. Other tissues, however, continued developing fairly normally, and embryos showed spontaneous movement comparable to controls. Embryos with severe cardiovascular or optic defects did not hatch. Upon hatching, some embryos which had previously appeared normal were found to have skeletal malformations in the form of vertebral bends or the inability to uncurl from the position which they had while still inside the chorion. Exposure to the toxicant for shorter periods of time (6, 12, or 24 hours) reduced the incidence of abnormalities. The second day of development was found to be the most sensitive period.     

Non-Coordinated Synthesis of RNA's in Pre-Gastrular Embryos of Xenopus Laevis

The rates of syntheses of 18S and 28S rRNA, 5S RNA, capped mRNA and 4S RNA were determined in isolated cells from pre- and post-gastrular embryos of Xenopus laevis. The rate of rRNA synthesis per nucleolated cell Mas about 0.2 pg/hr, or about 5.5 × 10⁴ molecules/hr at the blastula stage, and this value remained constant in later stages. At the blastula stage, about 30 molecules of 5s RNA, 10 molecules of capped mRNA and 900 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA. These values were all greatly reduced during the gastrula stage, and at the neurula stage, one molecule each of 5S RNA and capped mRNA and 10 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA.     

VEGETALIZATION OF SEA URCHIN LARVAE INDUCED WITH cAMP PHOSPHODIESTERASE INHIBITORS

Treatment of sea urchin embryos with cAMP phosphodiesterase (PDE)-inhibitors such as caffeine (4×10⁻³ M), theophylline (8×10⁻³M), or nicotinamide (10⁻²M), at the morula stage for only a couple of hours, yields vegetalized larvae. Most of the embryos treated with these reagents before the morula stage develop to blastulae filled with mcsenchyme-like cells. Almost all embryos at the blastula stage develop normally even if they are treated with a PDE-inhibitor for a considerable period. The rate of ³H-valine incorporation into protein in the morulae is reduced by caffeine and theophylline, but does not decrease in the presence of nicotinamide. Actinomycin D cancels the vegetalizing effect of PDE-inhibitors on the morulae.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) activates the maternal program of apoptosis shortly after MBT in Xenopus embryos

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) mRNA in 1- and 2-cell stage Xenopus embryos induces cell autonomous dissociation at the late blastula stage and developmental arrest at the early gastrula stage. The induction of cell dissociation took place "punctually" at the late blastula stage in the SAMDC-overexpressing cells, irrespective of the stage of the microinjection of SAMDC mRNA. When we examined the cells undergoing the dissociation, we found that they were TUNEL-positive and contained fragmented nuclei with condensed chromatin and fragmented DNA. Furthermore, by injecting Xenopus Bcl-2 mRNA together with SAMDC mRNA, we showed that SAMDC-overexpressing embryos are rescued completely by Bcl-2 and becometadpoles. These results indicatethat cell dissociation induced by SAMDC overexpression is due to apoptotic cell death. Since the level of S-adenosylmethionine (SAM) is greatly reduced in SAMDC-overexpressing embryos and this induces inhibition of protein synthesis accompanied by the inhibition of DNA and RNA syntheses, we conclude that deficiency in SAM induced by SAMDC overexpression activates the maternal program of apoptosis in Xenopus embryos at the late blastula stage, but not before. We propose that this mechanism serves as a surveillance mechanism to check and eliminate cells physiologically damaged during the cleavage stage.     

Synthesis of histone mRNA sequences in isolated nuclei of cleavage stage sea urchin embryos

A qualitative assay for detection of histone mRNA sequences in nuclear RNA was developed using actinomycin D-CsCl gradients to separate histone DNA from bulk DNA by differences in buoyant density. A significant amount of RNA synthesized in vitro in isolated nuclei from early blastula stage sea urchin embryos hybridized coincident with the histone DNA satellite, and this hybridization was competed out by unlabeled “9S” polysomal RNA purified from embryos at the same stage of development. The biogenesis of these histone mRNA sequences appeared similar as observed during in vivo and in vitro synthesis. Nuclear RNA from embryos pulse labeled in vivo was found to lack histone sequences, suggesting a rapid exit time for these sequences from the nucleus. Attempts to study the exit of histone sequences from isolated nuclei labeled in vitro also suggested a rapid exit time for histone sequences. The histone sequences were synthesized to a much lesser extent in isolated nuclei from late blastula stage embryos, as anticipated from the much reduced amount of histone mRNA labeled on polysomes at this stage.     

Essential role of the yolk syncytial layer for the development of isolated blastoderms from medaka embryos

To investigate a possible role of the yolk syncytial layer (YSL) in the development of the medaka embryo, blastoderms were isolated at different stages of embryogenesis either with or without the layer and were incubated in a culture medium. The blastoderms from cleavage stage embryos (stage 8–9), in which the YSL had not yet formed, developed into an irregular mass of cells. But some of the blastoderms isolated with the YSL from the blastula embryos (stage 10) developed into embryo-like structures with apparent body axes and contained differentiated organs, such as the eye, ear, contractile heart, yolk sac-like sphere and posterior body trunk with notochord. The proportion of such explants increased as the developmental stage proceeded. However, the proportion was much smaller when blastoderms were isolated at the blastula stage without the YSL. These results suggest that the YSL is essential for the development of embryonic structures. At stage 12 (early gastrula), the frequency of formation of such structures was the same among blastoderms with or without the YSL, so that these embryos are apparently committed for pattern formation.     

Effects of 5 azacytidine on DNA methylation and early development of sea urchins and ascidia

5-azacytidine (5-azaCR), an analogue of cytidine, inhibits nuclear DNA methylation in early sea urchin embryos. This inhibition is specific and dose-dependent. Exposure of sea urchin embryos at any stage between one-cell and blastula, to micromolar quantities of 5-azaCR invariably inhibits development beyond the blastula stage. In a substantial number of embryos arrested at the blastula stage, spicule formation proceeds although other morphological differentiation is lacking. No significant effect on development is seen if sea urchin embryos are exposed to 5-azaCR at post-blastula stages. 5-azaCR also inhibits the development of a mosaic egg such as the ascidian Phallusia mammilata at the blastula stage, indicating that both regulative (sea urchin) and mosaic (ascidian) embryos respond more or less similarly to 5-azaCR treatment.     

The factors that promote the development of symmetry properties in aggregates from dissociated echinoid embryos

Summary Embryos of Hemicentrotus pulcherrimus at the 16 cell, 400 cell or mesenchyme blastula stage of development were dissociated into single cells. The cells were reaggregated, and the development of individual aggregates was monitored. Only aggregates from 16 cell embryos developed into pluteus-like larvae with radial or bilateral symmetry. When embryos at these three developmental stages were incompletely dissociated so that there were mixtures of single cells and groups of undissociated cells, the percentage of aggregates from 16 cell embryos that developed in a pluteus-like manner was greater than in aggregates from completely dissociated 16 cell embryos. Also a small percentage of aggregates from 400 cell embryos now developed into pluteus-like larvae. In both of these experiments small aggregates tend to develop in a more normal manner than larger aggregates.In order to test the role of undissociated cells in promoting pluteus-like development in aggregates from incompletely dissociated blastula stage embryos, pieces of intact animal, lateral, or vegetal blastula wall were grafted to aggregates formed from completely dissociated embryos. While each kind of graft improved the ability of the aggregate to develop in a pluteus-like manner, grafts of vegetal blastula wall were most effective. In an aggregate, a graft differentiates according to its presumptive fate and influences the cells of the aggregate to differentiate in an appropriate manner. The ability of the graft to influence the development of the other cells in the aggregate depends on the developmental stage of the cells that make up the aggregate and the size of the aggregate.     

Effect of fucoidans on the developing embryos of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

Effect of fucoidans from the Far Eastern brown algae Laminaria japonica, L. cichoroides, and Fucus evanescens on developing embryos of the sea urchin Strongylocentrotus intermedius was studied. Fucoidans from these algae were shown to have different (immunostimulating, inhibitory) effects on the developing embryos. All studied fucoidan fractions added at the zygote stage had a stimulatory effect (acceleration of developmental stages and an increase of life spans) on the developing embryos, with differences by their effective concentrations. Many fucoidans added at the late blastula stage did not produce the stimulatory effect. The lowest number of immunostimulators (only a half of the tested substances) were present among fucoidans from L. japonica cichoriodes. The best immunostimulators that increased 2–3 times the life span of the embryos were heteropolysaccharides containing mannose or xylose apart from L-fucose. Some fucoidans at high concentrations produced an inhibitory effect; they were mainly composed of L-fucose or their monosaccharide composition included, apart from fucose, a significant amount of galactose, but no mannose. The method that we used turned out to be sensitive to structural peculiarities of charged polysaccharides, so it most likely can be applied for evaluation of their immunostimulating properties. From two variants of the experiment the most sensitive was the second one, in which polysaccharides were added at the late blastula stage.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 51–58.Original Russian Text Copyright © 2005 by Kiseleva, Shevchenko, Krupnova, Zvyagintseva.     

Auxin-stimulated somatic embryogenesis from immature cotyledons of white clover

Cotyledons from immature embryos of white clover (Trifolium repens L.) cv. Osceola were exposed to 2,4-D or NAA to induce somatic embryogenesis. NAA at 10 or 20 mg 1^–1 was very inefficient at stimulating embryogenesis, while concentrations of 30 or 40 mg 1^–1 resulted in death of the explant tissue. Continuous exposure of cotyledons to 40 mg 1^–1 2,4-D resulted in somatic embryos which were arrested at the globular stage, or which underwent cycles of secondary embryogenesis, never proceeding beyond the globular stage. A 10 day exposure time to 2,4-D at the same concentration led to formation of somatic embryos, most of which had poorly developed cotyledons. Almost 10% of the somatic embryos converted into plants following transfer to medium devoid of growth regulators. Attempts to improve morphology of somatic embryos by using shorter exposure times to 2,4-D at 40 mg 1^–1, or by maintaining the 10 day exposure time while varying the concentration of 2,4-D, were not successful. Plants were obtained from all parents evaluated, although at different frequencies.     

Effects of high gravity on amphibian development.

In order to clarify the possible effects of high gravity environments on eggs and developing embryos, Rana rugosa and Xenopus laevis fertilized eggs and early embryos were raised in 2 G, 5 G, 7 G and 10 G up to the hatched tadpole stage. The results showed that: (1) High gravity significantly retarded the development of eggs and embryos beginning treatment before the blastula stage and induced various abnormalities, including two heads and microcephally suggesting that high gravity is apt to disrupt the animal-vegital axis. On the other hand, embryos beginning treatment after the gastrula stage showed a striking increase in the number of normal-appearing feeding tadpoles. (2) Autopsy revealed that brains, notochords and muscles were reduced in development and differentiation for embryos and tadpoles developed in high gravity. (3) It seems likely that the system for hydrogen peroxide detoxification develops abnormally in high gravity-treated embryos and tadpoles, which probably results in oxidative stress, leading to considerable cell damage.     

Inhibition of the synthesis of glycosphingolipid by a ceramide analogue (PPMP) in the gastrulation of Bufo arenarum

In the present study the role of glycosphingolipids (GSL) in amphibian development was investigated. We analysed the de novo synthesis of neutral GSL and gangliosides through the initial stages of Bufo arenarum embryo development and their participation during gastrulation using 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), a potent inhibitor of glucosylceramide synthase. Ganglioside synthesis began at the blastula stage and reached a maximum during gastrulation (stages 10-12) while neutral GSL synthesis showed a slight gradual increase, the former being quantitatively more significant than the latter. Ganglioside synthesis was reduced by 90% while neutral GSL synthesis was inhibited by 65% when embryos at blastula stage were cultured for 24 h in 20 microM PPMP. The depletion of GSL from amphibian embryos induced an abnormal gastrulation in a dose-dependent manner. We found that PPMP had a pronounced effect on development since no embryos exhibited normal gastrulation; their developmental rate either slowed down or, more often, became totally arrested. Morphological analysis of arrested embryos revealed inhibition of the gastrulation morphogenetic movements. Analysis of mesodermal cell morphology in those embryos showed a severe decrease in the number and complexity of cellular extensions such as filopodia and lamellipodia. Mesodermal cells isolated from PPMP-treated embryos had very low adhesion percentages. Our results suggest that glycosphingolipids participate in Bufo arenarum gastrulation, probably through their involvement in cell adhesion events.     

The formation of primordial germ cells from germline cells in spherical embryos derived from the blastodisc of 2-cell embryos in goldfish, Carassius auratus

The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nos1 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.     

六价铬在斜带石斑鱼胚胎中的生物累积及其对igf2、glut2和pparg基因表达的影响

以斜带石斑鱼(Epinephelus coioides)为研究对象,探讨了水中不同浓度梯度六价铬(0、0.20、0.60、1、5、10、20、40、60和80 mg/L)在胚胎发育不同阶段(桑葚期、囊胚期、原肠末期、脑泡形成期和心脏跳动期)的生物累积及其对细胞生长基因igf2和营养代谢基因glut2、pparg的mRNA表达的影响。结果表明,胚胎中Cr⁶⁺生物累积量和吸收率随暴露浓度的增加而增加,且吸收率随胚胎发育时期的延伸而降低。在桑葚期时,斜带石斑鱼胚胎吸收Cr⁶⁺能力最强,是心脏跳动期的20倍。在桑葚期、囊胚期和原肠末期, Cr⁶⁺对胚胎中igf2和glut2基因mRNA表达水平有显著促进作用(P<0.05),但对pparg基因mRNA表达水平有显著抑制作用(P<0.05)。在原肠末期和脑泡形成期, Cr⁶⁺对胚胎中pparg基因mRNA表达水平有显著促进作用(P<0.05)。研究表明水中Cr⁶⁺暴露在斜带石斑鱼胚胎发育过程中有明显的生物累积,且对细胞生...     

Determination of optimal conditions for physical and chemical inactivation of nuclei for obtaining haploid and anuclear loach (Misgurnus fossilis L.) embryos]

The doses of X-irradiation sufficient for obtained truly haploid and anuclear embryos of the loach were determined. Mitomycin C in concentrations arresting the development at the late blastula stage (100-500 mcg/ml) was tested for the purposes of chemical enucleation of embryos. The data obtained allowed to recommend the following doses of X-irradiation to obtained the 100% inactivation of one or both the paternal genomes and haploid and anuclear embryos of the loach: 40 kr for eggs and 60 kr for testes. Mitomycin in the concentrations tested did not cause the 100% enucleation of embryos.     

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.     

Polysaccharides sulfated at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus

Based on the fact that the development of sea urchin embryos is arrested at the blastula stage in sulfate-free sea water (SFSW), we attempted in the present study to elucidate the nature of sulfated polysaccharides (PSs) which appear at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus. Electrophoretic analysis of PSs prepared from embryos at different developmental stages revealed that three kinds of PSs (3A, 3B, 3C) appear de novo at the gastrula stage, and that these PSs are not found in embryos at the hatching blastula stage, nor are they found in permanent blastula reared in SFSW. These, three PSs were mostly of extracellular matrix origin. Among them, 3C was identified as dermatan sulfate on the basis of its electrophoretic mobility and sensitivity to enzymatic digestion. 3A and 3B remained to be identified. Further, a plausible precursor of 3C, which was sulfated under normal conditions, was detected as 6D in the embryos reared in SFSW. Autoradiographic analysis using [35S]sulfate revealed that these three PSs, accounted for more than 90% of [35S]sulfate incorporated into the acid PS fraction during gastrulation.     

Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro.

Curly tail (ct/ct) mouse embryos, which have a genetic predisposition for neural tube defects (NTD), were grown in culture from the 2-5 somite stage, before the initiation of neurulation, up to the 22-24 somite stage, when closure of the anterior neural tube is normally complete. The embryos were cultured in whole rat serum or in extensively dialysed serum supplemented with glucose, amino acids, and vitamins, with inositol omitted or added at concentrations of 2, 10, 20, and 50 mg/l. Two strains were used as controls; CBA mice, which are related to curly tails, and an unrelated PO stock. It was found that ct/ct embryos were particularly sensitive to inositol deficiency; both they and the CBA embryos showed a similar high incidence of cranial NTD after culture in inositol deficient medium (12/17 and 11/18, respectively). Furthermore, the lowest dose of inositol had no effect on the frequency of head defects in ct/ct mice, though it halved the incidence in CBA embryos. With higher inositol concentrations, the majority of ct/ct embryos completed head closure normally, and their development was generally similar to that obtained in whole serum. PO embryos showed a lower proportion (5/19) of cranial NTD in the inositol deficient medium than the other two strains, and this was further reduced by even the lowest inositol dose.     

Different aggregation properties of sea urchin embryonic cells at different developmental stages. II. Stage specific response to fibronectin and collagen

Fibronectin and collagen were added to cells dissociated from embryos at the blastula and at the 16 cell stages. Both molecules stimulate aggregation of cells dissociated from blastula but they do not affect aggregation of blastomeres dissociated from the 16 cell stage. The stage-specific response to fibronectin and collagen appears to be related to the onset of new functional role(s) of the two molecules at the blastula stage.     

Rates of histone synthesis during early development of Rana pipiens

Newly synthesized histones have been extracted from Rana pipiens oocytes or cleaving embryos previously injected with [³H]lysine or [³H]arginine. The radioactive proteins were fractionated by cation-exchange chromatography and electrophoresis on acid/urea or SDS-polyacrylamide gels; histones were identified by coelectrophoresis with authentic markers. From percentage total incorporation in the putative histones, and absolute rates of lysine or arginine incorporation, rates of histone synthesis were estimated. Rates of histone synthesis in two-cell embryos were at least 10-fold higher than in maturing oocytes. Between the two-cell and blastula stages, the rate increased an additional threefold, from about 1200 pg hr^?1 per embryo to about 4500 pg hr^?1 per embryo. While all histone classes are synthesized during cleavage, synthesis of the various classes is not coordinated; histones are not synthesized in the same relative proportions at which they are found in blastula chromatin. The synthesis of histone H4 in particular is barely detectable during cleavage. This, and other observations, suggested the existence of cytoplasmic histone pools. In approaching the possible existence of histone pools, the amount of H4 present in oocytes was determined. Oocytes contain about 74 ng of H4, an amount sufficient to allow development to the blastula stage. These data are compared to those reported by others on histone synthesis during cleavage in Xenopus.