Cellular distribution of RNA populations in 16-cell stage embryos of the sand dollar, Dendraster excentricus

RNA was extracted from pure preparations of micromeres and meso-plus macromeres isolated from 16-cell stage embryos of Dendraster excentricus. Molecular hybridization-competition experiments disclosed that the binding of 16-cell stage labeled RNA to denatured sperm DNA was competed equally well by micromere RNA, meso-plus macromere RNA, total 16-cell RNA and unfertilized egg RNA, indicating the egg-type populations were distributed almost equally in the different blastomeres. In contrast, experiments with ³H-RNA extracted from micromeres obtained from pulse-labeled 16-cell stage embryos showed qualitative differences when unfertilized egg RNA and total 16-cell stage RNA were used as competitors. Such differences in RNA populations could not be detected in ³H-RNA isolated from the meso-plus macromere fraction.     

Centrifugal elutriation of large fragile cells: isolation of RNA from fixed embryonic blastomeres.

In order to analyze the RNA populations present in different cells of very early embryos, we have developed a protocol to purify these large blastomeres using counterflow centrifugal elutriation (CCE). This procedure employs ethanol fixation to stabilize the cells against shear forces encountered during CCE. Using this method, we fractionated the three different blastomere types of the 16-cell sea urchin embryo, the micromeres, mesomeres, and macromeres, achieving 96, 94, and 96% mean purities, respectively. We show here that intact RNA is recovered with equal efficiency from each blastomere preparation. Using this method, we have identified several RNAs that are distributed non-uniformly among these cells.     

Similarity of proteins synthesized by isolated blastomeres of early sea urchin embryos

The 16-cell sea urchin embryo has blastomeres of three distinct size classes: micromeres, mesomeres, and macromeres. Each class is already restricted in its developmental fate, micromeres being committed to formation of primary mesenchyme cells. The three classes of blastomeres were isolated in high purity and incubated in [³⁵S]methionine until the next cleavage. Nearly all the radioactive protein was solubilized and subjected to two-dimensional electrophoresis according to O'Farrell. Of approximately 1000 spots resolved, there are no qualitative differences among the three blastomeres. When embryos were labeled between the first and fourth cleavages and blastomeres then isolated, no qualitative differences in protein synthesis were observed. Moreover, there are very few changes when unfertilized eggs are compared to 16-cell embryos. Thus cellular determination during embryonic development is not accompanied by qualitative changes in the distribution within the embryo of abundantly synthesized proteins, virtually all of which are coded for by sequences present in the egg.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

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.     

LOCALIZATION AND SEGREGATION OF MATERNAL RNA'S DURING EARLY CLEAVAGE OF XENOPUS LAEVIS EMBRYOS

The localization and segregation of maternal RNA's during early cleavage of Xenopus laevis embryos were studied. Blastomeres and hemispheres of eggs and early embryos were separated manually and the amounts of ribosomal RNA and poly(A) ⁺RNA extracted from each blastomere and hemisphere were determined by optical density measurement and by ³H-poly(U) hybridization, respectively. It was found that both kinds of the maternal RNA's were more abundant (two-thirds of the total) in the animal hemisphere (cells), while they were evenly distributed between the dorsal and ventral halves. This pattern of localization remained unchanged from the egg to the blastula stage, indicating that these maternal RNA's were segregated into blastomeres quite simply by cell division. Gel electrophoresis showed that the size distributions of poly(A) ⁺RNA and poly(A) sequences obtained from different blastomeres of 8-cell embryos did not differ greatly. It was also found that cytoplasmic polyadenylation of maternal RNA, which occurs during early cleavage and blastulation, took place equally in all regions of the cleaving embryos, suggesting no regional difference in the localization of maternally inherited nonpolyadenylated RNA. These observations are discussed in relation to previous findings on differences along the animal-vegetal and dorsal-ventral axes of the early amphibian embryo.     

Fates of the blastomeres of the 16-cell stage Xenopus embryo

The fate of each of the blastomeres in the 16-cell stage Xenopus embryo which had been carefully selected for stereotypic cleavages was determined by intracellularly marking a single blastomere with horseradish peroxidase and identifying the labeled progeny in the tailbud embryo by histochemistry. Each blastomere populated all three primary germ layers. The progeny of each blastomere were distributed characteristically both in phenotype and in location. For example, most organs were populated by the descendants of particular sets of blastomeres. Furthermore, within an organ the progeny of a single blastomere were restricted to defined spatial addresses. This study describes the fates of identified 16-cell stage blastomeres and demonstrates that they are distinct and predictable if embryos are preselected for stereotypic cleavages.     

Evolutionary modification of specification for the endomesoderm in the direct developing echinoid <Emphasis Type="Italic">Peronella japonica</Emphasis>: loss of the endomesoderm-inducing signal originating from micromeres

We investigated the inductive signals originating from the vegetal blastomeres of embryos of the sand dollar Peronella japonica, which is the only direct developing echinoid species that forms micromeres. To investigate the inductive signals, three different kinds of experimental embryos were produced: micromere-less embryos, in which all micromeres were removed at the 16-cell stage; chimeric embryos produced by an animal cap (eight mesomeres) recombined with a micromere quartet isolated from a 16-cell stage embryo; and chimeric embryos produced by an animal cap recombined with a macromere-derived layer, the veg1 or veg2 layer, isolated from a 64-cell stage embryo. Novel findings obtained from this study of the development of these embryos are as follows. Micromeres lack signals for endomesoderm specification, but are the origin of a signal establishing the oral–aboral (O–Ab) axis. Some non-micromere blastomeres, as well as micromeres, have the potential to form larval skeletons. Macromere descendants have endomesoderm-inducing potential. Based on these results, we propose the following scenario for the first step in the evolution of direct development in echinoids: micromeres lost the ability to send a signal endomesoderm induction so that the archenteron was formed autonomously by macromere descendants. The micromeres retained the ability to form larval spicules and to establish the O–Ab axis.     

Muscle cell differentiation in ascidian embryos analysed with a tissue-specific monoclonal antibody

Utilizing a muscle-specific monoclonal antibody (Mu-2) as a probe, we analysed developmental mechanisms involved in muscle cell differentiation in ascidian embryos. The antigen recognized by Mu-2 was a single polypeptide with a relative molecular mass of about 220 X 10(3). It first appeared at the early tailbud stage and continued to be expressed until the swimming larva stage. There were distinct and separate puromycin and actinomycin D sensitivity periods during the occurrence of the antigen, suggesting the new synthesis of the polypeptide by developing muscle cells. Embryos that had been permanently arrested with aphidicolin in the early cleavage stages up to the 32-cell stage did not express the antigen. DNA replications may be required for the antigen expression. Embryos that had been arrested with cytochalasin B in the 8-cell and later stages developed the antigen, and the number and position of the arrested blastomeres exhibiting the differentiation marker almost corresponded to those of the B4.1-line muscle lineage. Furthermore, in quarter embryos developed from each blastomere pair isolated from the 8-cell embryo, all the B4.1 as well as a part of b4.2 partial embryos expressed the antigen, while the a4.2 and A4.1 partial embryos did not show the antigen expression. These results may provide further support for the existence of cytoplasmic determinants for muscle cell differentiation in this mosaic egg.     

Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere.

The objective of this study was to establish a rapid and reliable PCR method for the sexing of 8- to 16-cell stage bovine embryos. The BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male- and bovine-specific DNA, respectively. But the unequal number of copies of these two repetitive sequences required some modification of the multiplex PCR method. In consecutive and multiplex PCR, the first 10 PCR cycles were done with male-specific primer followed by an additional 23 cycles with bovine-specific primer. In this PCR method, the appearance of male- and bovine-specific bands was independent of the DNA concentration. This PCR method was applied successfully using groups of 8, 4, 2, and 1 blastomeres dissociated from the embryos, and the sexing efficiency was 100.0, 96.3, 94.3 and 92.1%, respectively. The coincident rate of sex determination between biopsied single blastomere and matched blastocyst was 90.0%. Therefore the developmental potential from 8- to 16-cell stage embryos to the blastocyst stage was not significantly different (P>0.2) for intact embryo (42.3%) than for demi-embryos (53.8%), suggesting that trauma to the demi-embryo caused by single-blastomere aspiration using a bevelled micropipette was very small. In conclusion, we developed a rapid (within 2 hours) and effective PCR method for the sexing of 8- to 16-cell stage bovine embryos using a single blastomere.     

Changes in the Pattern of Intercellular Junctions during Early Embryogenesis of the Starfish, Asterias amurensis

Changes in the cellular adhesion pattern during the early embryogenesis of a starfish Asterias amurensis were examined using carboxyfluorescein (CF) dye as a probe. CF that was injected into one of the blastomeres at the 2- or 4-cell stage was in all cases restricted to the progeny cells of the CF-labelled blastomere. With the advancement of gastrulation, however, the injected dye was distributed not only to the progeny of the labelled blastomere, but also to cells that originated from non-injected blastomeres. At the beginning of mesenchyme cell release, the injected dye spread uniformly to most cells comprising the embryo. When one of the blastomeres situated in the vegetal hemisphere of an 8-cell embryo was labelled, the resulting embryo showed more intense fluorescence in the cells surrounding the archenteron than in the ectodermal layer, suggesting that the cells in ectodermal layer became associated more intimately or earlier than those surrounding the archenteron. Likewise, in double embryos formed by combining two denuded eggs, in which one egg had been labelled with CF, dye spread was observed when the ectodermal layer began to expand. The intercellular spread of CF dye in starfish embryo suggests that there is a dramatic change in the cellular adhesion pattern during the course of gastrulation.     

Intravital selection of early mouse embryos by sex and karyotypic characteristics

The possibility of live karyotyping by a single blastomere isolated at the 2-, 4-, and 8-cell stage has been investigated. It is expedient to use to this end a single blastomere isolated from a 4-cell embryo. Three rest blastomeres formed normal morulae or blastocysts upon cultivation during 44 or 68 hrs. When the isolated blastomeres were cultivated for 14-16 hrs in a medium 0.5 micrograms/ml colcemide, 97% of blastomeres were at the metaphase stage and 72% of chromosome plates were suitable for karyotyping. The prospects of the method proposed in experimental embryology and for selection of the early embryos of farm animals by sex are discussed.     

Sexing using single blastomere derived from IVF bovine embryos by fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) is a sensitive technique for molecular diagnosis of chromosomes on single cells and can be applied to sex determination of embryos. The objective has been to develop an accurate and reliable bovine Y chromosome-specific DNA probe in order to sex biopsed blastomeres derived from IVF bovine embryos by FISH. Bovine Y chromosome-specific PCR product derived from BtY2 sequences was labeled with biotin-16-dUTP (BtY2-L1 probe), and FISH was performed on karyoplasts of biopsed blastomeres and matched demi-embryos. Our FISH signal was clearly detected in nuclei of blastomeres of male embryos. FISH analysis of bovine embryos gave high reliability (96%) between biopsied blastomeres and matched demi-embryos. These results indicated that the BtY2-L1 bovine Y chromosome-specific FISH probe was an effective probe for bovine embryo sexing, and the FISH technique of probe detection could improve the efficiency and reliability.     

Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos

We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).