Stage-specific gene expression in asynchronous tetraploid mouse embryos formed by fusion of blastomeres and fertilized eggs.

Asynchronous tetraploid mouse embryos were generated by electrofusion of fertilized eggs with blastomeres from different cleavage stages. The majority of the cytoplasm was always contributed by the egg. The best development was observed when eggs were fused with 2-cell blastomeres. Both genomes became active in fusion embryos (at least the genes for glucose phosphate isomerase did). Stage-specific protein synthesis seemed to be more adjusted to the developmental stage of the egg's than of the blastomere's genome, but at the 2-cell stage both contributed slightly differently to the protein patterns. Also, the time range of the first appearance of the stage-specific embryonic antigen SSEA-1 was wider in fusion embryos than in controls. It seems that the two genomes are not completely synchronized in these tetraploid embryos, a further indication that, in the mouse, the cytoplasm of fertilized eggs might not be compatible with older embryonic nuclei.

Developmental profile of glucose phosphate isomerase allozymes in parthenogenetic and tetraploid mouse embryos.

Glucose phosphate isomerase (GPI) allozymes were compared in eggs and embryos of the mouse strains C57BL/6-JHan (GPI-1BB) and 129/Sv (GPI-1AA) under different experimental conditions. The quantitative differences in eggs of the two strains disappeared by the blastocyst stage at day 4 to 5, both in fertilized and diploid parthenogenetic embryos. The degree of degradation of oocyte-coded enzyme molecules and the activation of the embryonic genome for GPI appeared to be equivalent in parthenogenetic embryos from heterozygous females when only one or other maternal allele type remained in the egg after meiosis. Also in tetraploid embryos, generated by electrofusion of homozygous fertilized eggs from the two strains, both genomes seemed to be activated at the same time at day 4; here, however, the GPI-1BB allozyme remained predominant up to day 6.

Survival of maternal mRNA in anucleate and unfertilized mouse eggs.

Unfertilized mouse eggs were parthenogenetically activated in vitro and then bisected. Anucleate fragments were aged in vitro, and their protein synthesis was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proteins were compared with those which were synthesized by aging unfertilized eggs and those which were translated in vitro from mRNA extracted from the unfertilized eggs. Normally cleaving parthenogenetic eggs served as controls. Cytoplasts and unfertilized eggs synthesized considerable quantities of protein after 2 days in culture. The protein patterns of cytoplasts and unfertilized eggs shifted in this time mainly within a group of proteins with a molecular mass of about 35 kDa. This shift was also seen in controls between day 1 and 2 but was delayed in unfertilized eggs. There was no clear appearance of new proteins in aging cytoplasts, which might have indicated a selective activation of maternal mRNA at a certain time after the activation stimulus, nor was such a change apparent in unfertilized eggs. The survival of maternal allozymes of glucose phosphate isomerase was tested in cytoplasts derived from fertilized eggs. The allozymes remained active during 4 days of aging and did not change their quantitative correlation.

Ultrastructure, protein synthesis and secretion of day-6 rabbit blastocysts cultured in a chemically defined, protein-free medium.

Day-6 rabbit blastocysts were cultured in Ham's F10 medium supplemented with polyvinylpyrrolidone as a macromolecular component, for 4 to 12 h. The integrity of the blastocyst cells was demonstrated by electron microscopy. Expansion and biosynthesis of proteins and of DNA were studied after culturing in the presence of 35S-methionine and 3H-thymidine. Polyvinylpyrrolidone did not interfere with the subsequent protein analysis, which was performed by two dimensional gel electrophoresis followed by silver staining and fluorography. More than 600 labelled proteins were found in the blastocyst tissue, many of them were also present in the blastocyst fluid and in the blastocyst coverings. Several proteins seemed to be produced for incorporation into the blastocyst coverings; others, only detected in the culture medium, might have been synthesized for secretion into the environment.

In vitro survival of cells derived from isodiploid uniparental half embryos of the mouse after aggregation with normal embryos.

Fertilized mouse eggs, heterozygous for two allozymes of glucose phosphate isomerase (GPI) were bisected, and the resulting half eggs were diploidized with cytochalasin B. After separate aggregation with normal embryos carrying a third allozyme of GPI, the resulting chimaeras were kept in culture up to 10 days. The majority grew out on the culture dish during this period. By GPI analysis, 7.7% of the embryos were found to be chimaeric. Both types of uniparental cells, from gynogenetic and from androgenetic half eggs, were capable of surviving in chimaeras in vitro. These results are comparable with published data obtained by using uniparental embryos generated by micromanipulation.

Influence of embryo size on lactate dehydrogenase isozyme expression in giant mouse chimaeras.

Chimaeras consisting of 2, 4, 8 and up to 16 mouse embryos were formed at day 3 and cultured in vitro up to day 8. The influence of size and cell number on the activation of the embryonic genome for LDH-5 was studied in non-attaching giant chimaeric blastocysts and compared to control embryos implanting in vivo or growing out in vitro. Day 5 chimaeric blastocysts consisting of 4 to 8 embryos had a similar size as early in vivo egg cylinders, but still expressed exclusively LDH-1 as normal preimplantation embryos do. Only during further ageing of non-attaching chimaeric blastocysts, a few samples were positive for LDH-5. There is no convincing evidence that the experimental increase of the cell number triggers a premature activation of the embryonic LDH genes. These results are discussed in comparison to other possible mechanisms regulating stage-specific activation of the embryonic genome.

The effect of the nucleocytoplasmic ratio on protein synthesis and expression of a stage-specific antigen in early cleaving mouse embryos.

The nucleocytoplasmic ratio of fertilized mouse eggs was manipulated by removing or injecting cytoplasm by micropipette, and bisection of denuded eggs to obtain both pronuclei in one half of the eggs cytoplasm. The experimental eggs were capable of cleavage to the morula stage and, in some instances, developed to the blastocyst stage similar to unmanipulated eggs. The removal of large quantities of cytoplasm by micropipette and injecting them into a recipient egg did not provide sufficient numbers of viable eggs, whereas transfer of smaller quantities (about a quarter of the cytoplasm) was less deleterious, at least for recipient eggs. However, the alteration of the nucleocytoplasmic ratio by this method was not of the correct magnitude for the purpose of this experiment. Therefore, bisection was the preferred method whereby the nucleocytoplasmic ratio was doubled. This resulted in both pronuclei residing in one half of the egg's cytoplasm. Half eggs with one pronucleus (haploid) but retaining a nucleocytoplasmic ratio similar to unmanipulated control eggs served as additional controls for the bisection experiments. Protein synthesis was analysed by two-dimensional gel electrophoresis, showing that the 2-cell- and 4-cell-stage bisected embryos with double and normal nucleocytoplasmic ratio expressed equivalent protein synthesis patterns as control embryos of the same stage. Likewise, the stage-specific surface antigen SSEA-1 did not appear before the 6- to 8-cell stage. Also in cytoplasm transfer experiments, there was no indication that altering the nucleocytoplasmic ratio in either direction changed the timing of stage-specific gene expression. These results support the idea that stage-specific gene activity during early mouse cleavage might proceed in parallel to DNA replication cycles and is independent of the nucleocytoplasmic ratio.

Expression of two surface antigens and paternal glucose-phosphate isomerase in polyploid one-cell mouse eggs.

Cleavage in 1-cell mouse eggs was suppressed by continuous treatment with cytochalasin B for 3 or 4 days. During this period the eggs became highly polyploid and showed gene products not found before the 6- to 8-cell stage in control embryos. These polyploid 1-cell eggs (a) reacted with two monoclonal antibodies (anti-SSEA-1 and ECMA 7), which recognize stage-specific surface antigens, and (b) expressed the paternal form of the enzyme glucosephosphate isomerase. The molecular development of early mouse embryos, therefore, seems to progress stage specifically even without cleavage occurring.

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.

Spontaneous parthenogenesis in Mus musculus: comparison of protein synthesis in parthenogenetic and normal preimplantation embryos.

In preimplantation stages of normal and spontaneously activated parthenogenetic embryos of the LT/Sv mouse strain, protein synthesis was analyzed by using two-dimensional polyacrylamide gel electrophoresis. Fertilization and parthenogenetic activation cause similar changes polypeptide synthesis when compared with those of unfertilized eggs. The overt developmental delay of early parthenotes, which is probably due to an initial retarded activation in comparison with normal fertilization, is documented molecularly by a similar delay in their protein synthesis pattern. These differences are clearly visible at the two-cell stage but gradually disappear during further cleavage. The basic protein patterns of normal and parthenogenetic embryos are remarkably similar up to the blastocyst stage. However, quantitative differences occur in all preimplantation embryos analyzed and become more distinct at the blastocyst stage. In addition, only minor qualitative changes appear during late preimplantation. These alterations in protein synthesis may reflect at the molecular level early events in abnormal development of parthenotes. Our biochemical results are discussed in context with biological experiments rescuing parthenogenetic LT/Sv embryos by chimera formation.