Improvement in development of porcine embryos reconstituted with cells from blastocyst-derived cell lines and enucleated oocytes by optimization of reconstruction methods

The present study was conducted to establish the most suitable system for producing porcine reconstructed embryos by transferring cells from blastocyst-derived cell lines into enucleated oocytes. When the cells were fused to preactivated metaphase II oocytes, or the cells and arrested metaphase II oocytes were fused in medium without CaCl(2) and MgSO(4), the percentages (43-53%) of fused embryos were significantly lower than those (72-79%) produced by fusing the cells to arrested metaphase II oocytes in medium containing CaCl(2) and MgSO(4). High productive efficiency (7%) of blastocysts was obtained when reconstituted embryos produced by the last method were activated again at 3 hours after fusion (F/A --> Activation). Pronuclear formation was observed in 80-91% of the reconstructed embryos produced by F/A --> Activation, with no significant differences between different culture periods in the medium containing cytochalasin B. When cultured in the medium containing cytochalasin B for 0-1 h, almost all (83-85%) the embryos had one pronucleus and one polar body. However, the number of embryos with two pronuclei and no polar bodies was increased significantly by culturing in the medium containing cytochalasin B for 2-4 h. The cleavage rate (34-48%) of reconstructed embryos was not affected by the presence of cytochalasin B for 2 h after activation. However, the percentage of embryos that developed to the blastocyst stage was significantly higher in the presence (23%) than absence (5%) of cytochalasin B. The results indicate that F/A --> Activation and cytochalasin B treatment are effective for the production of porcine embryos reconstituted with cells from blastocyst-derived cell lines and enucleated oocytes.     

Requirement of cell division for muscle actin expression in the primary muscle cell lineage of ascidian embryos

The role of cell division in the expression of muscle actin and its relationship to acetylcholinesterase (AChE) development was examined in cleavage-arrested embryos of the ascidian Styela. Muscle actin expression was detected by two-dimensional gel electrophoresis of radioactively labelled proteins and by in situ hybridization with a cDNA probe, whereas AChE activity was assayed by enzyme histochemistry. In the majority of cases, muscle actin expression was first detected in embryos arrested after the 16-cell stage. Some embryos showed muscle actin expression after arrest at the 8-cell stage, however, muscle actin mRNA did not accumulate in embryos arrested at earlier cleavages. The cells that expressed muscle actin in 8- to 64-cell cleavage-arrested embryos belonged to the primary muscle lineage; secondary muscle cell precursors did not express muscle actin. Zygotic muscle actin mRNA appeared to accumulate with myoplasmic pigment granules in the perinuclear region of cleavage-arrested embryos, suggesting that the myoplasm may have a role in the organization of muscle cells. In contrast to muscle actin, AChE was detected in a small proportion of embryos treated with cytochalasin as early as the 1- or 2-cell stage, and most embryos treated with cytochalasin at later cleavages expressed this enzyme in some of their cells. Most primary muscle lineage cells expressed both muscle actin mRNA and AChE, however, some cells expressed only muscle actin mRNA or AChE. The results suggest that at least three cleavages are required for muscle actin expression and that muscle actin and AChE expression can be uncoupled in cleavage-arrested embryos.     

Studies on the Cytoplasmic Determinant for Muscle Cell Differentiation in Ascidian Embryos: An Attempt at Transplantation of the Myoplasm

The 8-cell stage embryos of the ascidian Halocynthia roretzi which had been prevented from undergoing further divisions by continuous treatment with cytochalasin B could develop histospecific muscle acetylcholinesterase in two blastomeres (B4.1 and B4.1 cells). If the cytoplasm of a B4.1 or B4.1 cell was transplanted by microinjection into either an A4.1 or A4.1 cell of recipient embryos and the transplanted embryos were permanently cleavage-arrested with cytochalasin B, a few eventually developed AChE in three blastomeres instead of in just the two blastomeres found in cleavage-arrested control embryos. Judging from the relative positions of the blastomeres, the third AChE-producing cells appeared to be the A4.1 or A4.1 cells injected with the cytoplasm of B4.1 or B4.1. Although the success rate was considerably low, this result might indicate the presence in the cytoplasm of a determinant for the muscle-specific enzyme development.     

Isolation of cDNA Clones for Epidermis-Specific Genes of the Ascidian Embryo

The present investigation was conducted to isolate cDNA clones that correspond to epidermis-specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one-celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)⁺ RNA from cleavage-arrested embryos and analysis of the products by two-dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis-specific mRNAs in cleavage-arrested embryos. A cDNA library was constructed from arrested one-celled embryos. Differential screening of the library with a total cDNA probe from cleavage-arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage-arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole-mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.     

Diisopropylfluorophosphate inhibits acetylcholinesterase activity and disrupts somitogenesis in the zebrafish.

Acetylcholinesterase (AChE) activity, localized histochemically, appeared in the nuclei of presumptive somitic mesodermal cells prior to the onset of somitogenesis. AChE activity appeared in a rostro-caudal sequence, in cells located the equivalent of five somite lengths caudal to the last formed somite. To investigate whether AChE activity was required for somitogenesis, several inhibitors of AChE activity were tested for their ability to block somitogenesis. Diisopropylfluorophosphate (DFP), a broad spectrum inhibitor of serine proteases and related enzymes, was the only AChE inhibitor tested that disrupted somitogenesis. Gastrulae at 50% epiboly exposed continuously to DFP at concentrations between 40 microM and 90 microM completed epiboly, but exhibited a dose-dependent decrease in the number of somites formed, and a parallel decrease in the caudal extent of somite innervation, by 24 hours post-fertilization (h). Fifteen somite (15h) embryos exposed to DFP at the ED50 of 70 microM for 3 hours, followed by recovery to 24h, developed abnormal somites. Approximately five normal somites formed after drug treatment before the first abnormal somite formed. The abnormal somites corresponded in location to that area of the presumptive somitic mesoderm that would have initiated AChE activity while the DFP was present. While exposed to 70 microM DFP, presumptive somites formed and motoneurons extended processes that had initiated AChE activity at the time of treatment with DFP, although at a slower than normal rate. However, embryos exposed to 1 mM DFP for 30 minutes at both the 5 and 15 somite stages, followed by recovery to 24h, developed the normal number of somites but were reduced in the caudal extent of somite innervation, and occasionally developed abnormal primary motoneurons. As with the abnormal somites, the abnormal motoneurons would have initiated AChE activity while the DFP was present. Presumptive somitic mesoderm unable to initiate AChE activity due to inhibition by DFP developed abnormally. While the effects of DFP are not limited to inhibiting AChE, the data support the "clock and wavefront" model proposed for somite formation, and support the hypothesis that AChE activity has a role in somitogenesis in zebrafish.     

Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.     

Regulation of stage-specific gene expression during early mouse development: effect of cytochalasin B and aphidicolin on stage-specific protein synthesis in mouse eggs

Mechanisms regulating stage-specific translation in mouse embryos were studied by inhibitor experiments. When fertilized eggs were continuously treated with cytochalasin B, cleavage was prevented, whereas karyokinesis proceeded, resulting in protein synthesis patterns changing stage-specifically as in control embryos through preimplantation development. When fertilized eggs were continuously exposed to aphidicolin, cleavage and DNA synthesis were inhibited, thus keeping their protein synthesis at the level of fertilized eggs with few new polypeptides appearing after one day. The next day these eggs stopped translation almost completely. Stage-specific translation therefore might be controlled by nuclear replications rather than by cytoplasmic clock.     

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.     

Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division

Summary The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.Some of these results were presented at the IX Congress of the International Society of Developmental Biologists in Basle, Switzerland, August 28–September 1, 1981     

Expression of epidermis-specific antigens during embryogenesis of the ascidian, Halocynthia roretzi

We have produced two monoclonal antibodies (Epi-1 and Epi-2) which specifically recognize epidermal cells and their derivative, the larval tunic, of developing embryos of the ascidian Halocynthia roretzi. The antigens, examined by indirect immunofluorescence staining, first appear at the early tailbud stage and are present until at least the swimming larval stage. There were distinct and separate puromycin and actinomycin D sensitivity periods for each antigen. Aphidicolin, a specific inhibitor of DNA synthesis, prevented the appearance of each antigen when embryos were exposed to the drug continuously from cleavage stages. These results suggest that the antigens are synthesized during embryogenesis by developing epidermal cells and that several rounds of DNA replication are required for the antigen expression. Early cleavage stage embryos, including fertilized but unsegmented eggs, in which cytokinesis had been blocked with cytochalasin B expressed the antigens, and blastomeres exhibiting the antigens were always of the epidermis lineage. In partial embryos produced by four separated blastomere pairs of the 8-cell embryos, the expression of antigens was seen only in those developed from the animal blastomere pairs, which are progenitors of epidermal cells. These observations indicate that differentiation of epidermal cells in ascidian embryos takes place in a typical "mosaic" fashion.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

Genesis of Epidermal Action Potentials in Cytokinesis Arrested Xenopus Embryos

When Xenopus embryos were treated continuously with cytochalasin B (3–10 μg/ml) from the 8 cell stage, cleavage arrested embryos in various degrees were observed. In 3–5 μg/ml cytochalasin B, cytokinesis was inhibited at the midblastula stage and pigment granules remained at the cell cortex of the animal pole. These cells showed epidermal like action potentials when the control embryos (St. 26/28) generated epidermal action potentials. In 5–7 μg/ml cytochalasin B, furrows, following their formation at early cleavage stages, regressed and no further cleavage from the 16 cell stage to morula stage took place. The pigment granules were dispersed throughout the interior of the cytoplasm. These cells showed no epidermal action potentials. Thus, it is considered that cytokinesis per sé , following the midblastula stage, is not a prerequisite for the genesis of epidermal action potentials, and that chronological times corresponding to the tailbud larva stage and a stable structure of the cellular cortex are required to bring about these potentials.     

Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis

We have studied the effect of maturation-promoting factor (MPF) on embryonic nuclei during the early cleavage stage of Xenopus laevis development. When protein synthesis is inhibited by cycloheximide during this stage, the embryonic cell cycle arrests in an artificially produced G2 phase-like state, after completion of one additional round of DNA synthesis. Approximately 100 nuclei can be arrested in a common cytoplasm if cytokinesis is first inhibited by cytochalasin B. Within 5 min after injection of MPF into such embryos, the nuclear envelope surrounding each nucleus disperses, as determined histologically or by immunofluorescent staining of the nuclear lamina with antilamin antiserum. The breakdown of the nuclear envelope occurs at levels of MPF comparable to or slightly lower than those required for oocyte maturation. Amplification of MPF activity, however, does not occur in the arrested egg as it does in the oocyte. These results suggest that MPF can act to advance interphase nuclei into the first events of mitosis and show that the nuclear lamina responds rapidly to MPF.     

M-phase regulation of the recruitment of mRNAs onto polysomes using the CDK1/cyclin B inhibitor aminopurvalanol

Translation under the control of the universal cell cycle regulator CDK1/cyclin B was investigated during the first cell cycle in sea urchin embryos. The CDK1/cyclin B inhibitor aminopurvalanol arrested embryos at the G2/M transition. Polysomal mRNAs were purified from control and arrested embryos, and screened for specific mRNA recruitment or release at M-phase by subtractive hybridization. The polysomal repartition of clones issued from this screen was analyzed. Three specific mRNAs were selectively recruited onto polysomes at M-phase. Conversely, two other specific mRNAs were released from polysomes. The isolation of these translationally regulated mRNAs gives now important tools for insights into the regulation of protein synthesis by the cell cycle regulator CDK1-cyclin B.     

Late G1 amino acid restriction point in human dermal fibroblasts

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.     

Morphogenesis and regulated gene activity are independent of DNA replication in Xenopus embryos.

Xenopus embryos were transferred into media containing aphidicolin at late blastula, mid-gastrula, and early neurula stages. In each case, embryos continued to differentiate in the absence of DNA replication. When the inhibitor was added at late blastula, embryos continued to develop for about 8 h. However, when aphidicolin was added at the early neurula stage, development could be seen for up to 40 h after addition. The influence of replication on embryonic gene activity was studied by RNA blot analysis. Of the genes we examined only histone gene expression was down regulated by the addition of aphidicolin. The expression of various embryo-specific genes was unaffected by the lack of DNA synthesis. Even after several hours of treatment with aphidicolin, replication-inhibited tailbud and tadpole stages showed the same levels of specific mRNAs as control embryos containing 4-5 times more DNA. We conclude that morphogenesis and embryo-specific gene activity are independent of both DNA replication and a precise amount of DNA per embryo.     

Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools

The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.     

Quantitative measurement by microdensitometry of tyrosinase (Dopa oxidase) development in whole small ascidian embryos

Summary Embryos of the ascidian, Ciona intestinalis, were fixed in either cold (5° C) 70% ethanol or cold absolute methanol during their tyrosinase development phase and incubated in buffered (pH 7.2) solutions of the enzyme substrate l-dihydroxyphenylalanine. Optical density of the reaction product (melanin) was measured in the whole small embryos at 450 nm with a Vickers M85 scanning and integrating microdensitometer. The frequency distribution of the reaction density in embryos of a population was Gaussian, and the mean optical density in embryo samples (N=25) increased linearly with incubation time when a saturation level of substrate was used. Absolute optical density units of dopa oxidase activity in embryos increased linearly in proportion to the development time preceding melanin granulogenesis thereby suggesting that the enzyme activity measured by this procedure is proportional to the amount of tyrosinase present. Since this developmental increase in activity was blocked by treatment of the embryos with puromycin, an inhibitor of protein synthesis, the change is apparently caused by new enzyme synthesis. The microdensitometry assay also confirmed results obtained previously with a radiometric assay: embryos cleavage-inhibited at 7 h development time with cytochalasin B to produce giant melanocytes developed only the same amount of enzyme activity as control embryos.