Synchronization of cell division in eight-cell bovine embryos produced in vitro: Effects of nocodazole

Current methods of arresting and synchronizing cell division have not been very successful and have had few applications in embryo studies. Our objective was to determine the reliability of a metaphase arrest agent, nocodazole, for halting and synchronizing blastomere division in cleavage-stage bovine embryos, and to verify its reversibility and toxicity in vitro. Eight-cell-stage embryos obtained at 58 hr postinsemination were treated with varying concentrations of nocodazole for 12 hr. Treated embryos were assessed for cleavage arrest, chromatin morphology, DNA synthesis, and histone H1 and myelin basic protein (MBP) kinase activity, and were scored for blastocyst formation and hatching rate. They were subsequently fixed to count the number of nuclei. Complete arrest of cell division was observed at concentrations of 0.4 μg ml⁻¹ and above. Removal from nocodazole treatment led to immediate release from cleavage arrest, and was followed by synchronized mitosis, histone H1 kinase deactivation, and reentry into interphase within 3–5 hr. DNA synthesis was reinitiated at 6 hr after release. Although cell numbers and hatching rate decreased, the proportion of embryos reaching blastocyst stage was not significantly affected in nocodazole-treated embryos. It is concluded that nocodazole is a suitable choice for the cell-cycle synchronization of donor embryos for use in studies on the interactions between nucleus and cytoplasm during early embryogenesis. © 1996 Wiley-Liss, Inc.     

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.     

The effects of aphidicolin on morphogenesis and differentiation in the sea urchin embryo

Aphidicolin, an inhibitor of DNA polymerase alpha, blocks DNA synthesis and cell division in sea urchin embryos. The effects of this inhibition appear to be stage dependent. Blastulae treated with aphidicolin before the thickening of the vegetal plate undergo developmental arrest prior to gastrulation. The extent of inhibition of DNA synthesis varies from 60 to 93% in these embryos. However, when aphidicolin is added after the vegetal plate has thickened, development continues normally through pluteus formation, even though DNA synthesis is inhibited by greater than or equal to 90% and cell division has ceased. These observations indicate that, from the vegetal plate stage onward, morphogenesis and overt differentiation are independent of DNA synthesis and cell division.     

Effect of delaying DNA replication on blastocyst formation in the mouse

Abstract. Differentiation in the early mouse embryo involves cellular responses to both spatial and temporal signals. The temporal signals that trigger blastocyst formation, the first differentiative event, are not yet understood, but it has been suggested that the numbers of DNA replications undergone since fertilization might act as the timing mechanism. Preimplantation mouse embryos were treated with aphidicolin, an inhibitor of eukaryotic DNA polymerase a, for eight hours during the S phase of the fourth cleavage division. This treatment produced a delay in cell division but the morphologic event of cavitation, which signals the onset of blastocyst formation, was not delayed. Treated embryos actually cavitated a few hours ahead of control embryos at approximately half the cell number. This result indicates that blastocyst formation is not timed by the number of DNA replicative cycles completed since fertilization, but by some other intrinsic cellular clock.     

Inhibition by aphidicolin of cell cycle progression and DNA replication in sea urchin embryos.

We have recently found that aphidicolin, a tetracyclic diterpene-tetraol produced by several fungi, blocks DNA synthesis of sea urchin embryos by interfering with the activity of DNA polyermase alpha. These cells fail to proliferate in the presence of aphidicolin. In continuation of these studies, we determined the drug-sensitive stage in the first cell cycle of the sea urchin Clypeaster japonicus embryo. In continuous exposure to aphidicolin (2 micrograms/ml) from five minutes after fertilization, mitotic division of the embryo was completely suppressed. Embryos were exposed to the drug at progressively later intervals and their capability for cytokinesis was examined. Evidence was thereby obtained that aphidicolin acts at the S-period to inhibit DNA synthesis resulting in developmental arrest of the embryo.     

The effects of aphidicolin and α-amanitin on DNA synthesis in preimplantation mouse embryos

The effects of aphidicolin and α-amanitin on DNA synthesis by preimplantation mouse embryos were studied. It was found that both blastocyst and 8-cell embryos showed marked inhibition of ³H-thymidine incorporation into DNA by aphidicolin at concentrations of 20–50 μg/ml. However, aphidicolin did not inhibit the conversion of morula embryos to blastocyst embryos, although aphidicolin-treated blastocysts lost their blastocoel and collapsed into a compact form after prolonged exposure to the drug. Both 8-cell and blastocyst embryos were found to be susceptible to inhibition of DNA synthesis by α-amanitin.     

DNA replication is required for tissue-specific enzyme development in ascidian embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stage that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

DNA Replication is Required for Tissue-Specific Enzyme Development in Ascidian Embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stages that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

Establishment of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

In an attempt to estimate the number of pigment precursor cells in sea urchin embryos, DNA synthesis and cell divisions were blocked with aphidicolin from various stages of development. Interestingly, pigment cells differentiated on a normal time schedule, even if the embryos were treated from late cleavage stages on. In most of the embryos treated from 10 h on, 10-15 pigment cells differentiated. Thereafter, the number of pigment cells in the aphidicolin-treated embryos further increased, as the initiation of the treatment was delayed. On the other hand, total cell volumes in the pigment lineage, calculated from the averaged number and diameter of differentiated pigment cells, were almost the same irrespective of the time of the initiation of aphidicolin treatment. This indicated that the increase in the number was caused by divisions of the pre-existing cells in the pigment lineage. Thus, the founder cells that exclusively produce pigment cells could be identified. They are nine times-cleaved blastomeres and specified by 10 h post-fertilization. The obtained results also clarified the division schedule in the pigment lineage; the founder cells divide once (10th) until hatching, and divide once more (11th) by the end of gastrulation.     

The fate of DNA originally existing in the zygote nucleus during achromosomal cleavage of fertilized echinoderm eggs in the presence of aphidicolin: microscopic studies with anti-DNA antibody

In fertilized echinoderm eggs, the male and female pronuclei fuse to form the diploid nucleus even in the presence of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase-alpha. The subsequent first cleavage is independent of chromosomes but dependent on spindle and amphiaster. The fate of DNA originally existing in the fused nucleus during achromosomal cleavage of fertilized sea urchin and starfish eggs induced by aphidicolin was determined using antidenatured DNA antibody. The nucleus is not formed in the divided daughter cells at the two-cell stage but the nuclear-envelope-free chromatin mass which is unassociated with mitotic apparatus remains in the center region of embryos, especially near the first cleavage furrow. These results indicate that the condensed and nonreplicated chromatin can not be associated with spindle and asters in the presence of aphidicolin.     

Relationships between DNA synthesis and mitotic events in fertilized sea urchin eggs: aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule organizing centers, but not cycles of microtubule assembly

The synthesis of DNA in fertilized eggs of the American Gulf Coast sea urchin Lytechinus variegatus is 90% inhibited in the presence of 5.0 micrograms/ml aphidicolin. This inhibition may be imposed immediately upon addition of aphidicolin to the external medium when embryos are in "S" phase. Observations of living embryos with Nomarski optics and time-lapse video microscopy reveal that when eggs are fertilized and cultured in the continuous presence of aphidicolin, nuclear envelope breakdown, chromosome condensation, and cytokinesis are inhibited. All other post-fertilization events observable with this technique, including the assembly and disassembly of a bipolar spindle, proceed in the presence of aphidicolin. Antitubulin immunofluorescence microscopy of aphidicolin-arrested embryos demonstrates that microtubules attempt to assemble a mitotic apparatus at the first cell cycle; the arrested intact zygote nucleus is embedded within this bipolar structure. Subsequent cycles of microtubule assembly and disassembly proceed roughly on schedule with later division cycles, but the microtubule organizing centers (MTOC's) are unable to duplicate properly and irregular monasters are observed. If aphidicolin is added to embryos after the first DNA synthetic period, nuclear envelope breakdown, chromosome condensation, and cytokinesis proceed for that cycle and the embryos arrest at the two-cell stage. These results suggest that the direct inhibitory effects of aphidicolin may well be limited to the synthesis of DNA, which itself regulates nuclear cycles independently from the subsequent generation of mitotic poles, and that cytoplasmic clocks regulate microtubule assembly cycles but not the configuration of microtubule arrays.     

Involvement of Glycoproteins in the Development of Early Mouse Embryos: Effect of Tunicamycin and α, α' Dipyridyl in vitro

Early mouse embryos grown in tissue culture were treated with tunicamycin, an inhibitor of protein glycosylation or with αα' dipyridyl, an inhibitor of collagen secretion. Neither treatment blocked development of cleavage stage embryos nor did either interfere with blastocyst formation, hatching, or adhesion to the substratum at low concentrations. However, both treatments caused marked and specific changes in the morphology of the blastocyst outgrowth. Treatment of embryos with tunicamycin caused severe deterioration of the trophoblast layer and subsequent disintegration of the inner cell mass. Tunicamycin completely inhibited the incorporation of mannose into proteins. Treatment with αα' dipyridyl caused dose dependent retardation of the inner cell mass while the trophoblast cells were virtually unaffected. These alterations in morphogenesis occurred only in embryos treated at the blastocyst stage or later in development. Changes caused by α,α' dipyridyl could be partially reversed by addition of collagen to the culture. These findings might indicate the involvement of extracellular matrix macromolecules in embryonic organization.     

Lectin-induced abnormalities of mouse blastocyst hatching and outgrowth in vitro

We have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachment, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL-1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin-1 (LTA-1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded blastocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their environment.     

Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos.

We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p less than 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Aphidicolin on Arylsulfatase Gene Expression in Sea Urchin Embryos

Expression of the arylsulfatase (Ars) gene in sea urchin embryos begins just before hatching and ceases at the pluteus stage. Initiation of the Ars gene expression is inhibited by aphidicolin, which inhibits DNA synthesis without arresting the total RNA synthesis. Based on these finding it is supposed that DNA replication is a prerequisite for initiation of the Ars gene expression in developing sea urchin embryos.     

Studies on differentiation without cleavage in Chaetopterus: effects of inhibition of DNA synthesis with aphidicolin

The effects of aphidicolin - a powerful inhibitor of DNA polymerase alpha and of DNA replication - on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.     

Action site of the lethal Ay gene in the mouse embryo.

We investigated the lethal effect of Ay gene in embryos at the preimplantation stage in vitro. First, the development until the blastocyst stage and the division of individual cells from 8-cell stage embryos were examined. No difference in development was detected between embryos from the experimental cross (Ay/a x Ay/a) and those from the control cross (a/a x a/a). Therefore, it seems that the abnormality of the Ay/Ay embryo does not appear until blastocyst formation in vitro. We subsequently examined the hatching from zona pellucida of the blastocysts. The hatching ratio of the embryos from the experimental cross was significantly lower than that of the control crosses (Ay/a x a/a, a/a x a/a: p < 0.05). Our observation indicates that deficiency of the Ay/Ay embryo can be detected in vitro at hatching. In order to elucidate the mechanism of the gene action of the Ay, we attempted to rescue the lethal embryos from decreased hatching ratio in vitro. When dbcAMP at the concentration of 1 mM was added to the culture medium, the hatching ratio of blastocysts from the experimental cross increased until the level of those from the control crosses. Since this result indicates that the cAMP content in Ay homozygote seemed to be lower than those in a/a and Ay/a, the cAMP content in individual blastocyst was quantified. It is found that Ay homozygosity was associated with lower level of cAMP. When adenylate cyclase was activated by forskolin and cholera toxin, the hatching ratio was increased. These results seem to suggest that Ay homozygote embryos possess a defect in signal transduction system mediated by adenylate cyclase during hatching.     

Studies in vitro on mouse-egg radiosensitivity from fertilization up to the first cleavage

Super-ovulated eggs from the Balb/c strain were incubated, at various times after injection of HCG, in Whitten's medium containing tritiated thymidine. They were fixed on the following day at the 2-cell stage and prepared for autoradiography. On the basis of the results, pregnant mice were irradiated with various doses of X-rays at 15 h post HCG (fertilization), 19 h (phonuclear stage before DNA synthesis), 24 h (maximal DNA synthesis) and 27.5 h (DNA synthesis completed). On the day following irradiation, embryos were collected and classified into incleaved or 2-cell embryos, and development of the 2-cell embryos was followed in culture.

Irradiation was most effective when administered at 19 h after injection of HCG. Such a treatment increased the mortality before the first cleavage and, thereafter, from the 8-cell (100 rad) or morula stage (25, 50 rad). Blastocyst hatching and implantation were also impaired. Irradiation at other times was much less harmful for the embryos, which died mainly from the blastocyst stage. Finally, radiosensitivities of the mouse zygote at the various times studied can be estimated as follows: fertilization, + + +; pronuclear stage before DNA synthesis, + + + + +; maximal DNA synthesis, +; DNA synthesis terminated, + +.     

Genetic and environmental determinants of interferon-tau secretion by in vivo- and in vitro-derived bovine blastocysts

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-tau secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo's ability to develop to the blastocyst stage and of subsequent interferon-tau secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-tau. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-tau was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-tau by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.