Genetic differences in glucose phosphate isomerase activity among mouse embryos

We have compared mouse embryos of three heterozygous, congenic genotypes (with high, medium and low levels of oocyte-coded glucose phosphate isomerase (GPI-1) activity respectively) to test whether 1) the survival time of oocyte-coded GPI-1 activity in the early embryo is affected by its activity level in the oocyte and 2) whether embryo-coded GPI-1 is detected earlier in embryos that inherit low levels of oocyte-coded GPI-1. The oocyte-coded GPI-1 was entirely GPI-1A allozyme in the high and medium groups but was the less stable GPI-1C allozyme in the low group. We determined total GPI-1 activity and the ratio of different GPI-1 allozymes in early embryos and calculated the activity of oocyte-coded and embryo-coded GPI-1. In all three groups, the oocyte-coded enzyme activity remained at a more or less constant level for the first 21 1/2 days. Some oocyte-coded GPI-1 remained in 4 1/2 day embryos from the high and medium groups but was gone by 5 1/2 days. Very little remained in 4 1/2 day embryos that inherited low levels of a less stable form of the enzyme (GPI-1C allozyme). Despite a 4- to 5-fold difference in initial oocyte-coded GPI-1 activity, no differences were seen among the three genotypically distinct groups of embryos in the time of activation of the embryonic Gpi-1s genes. The embryo-coded GPI-1 was first detectable in 3 1/2 day compacted morulae in all three groups. The level of oocyte-coded GPI-1, in the high group, when embryo-coded GPI-1 was first detected was higher than the level in the low group at any stage prior to detection of embryo-coded GPI-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of specific genes in early mouse embryos blocked by cytochalasin

Summary Mouse embryos at the two cell stage derived from C57BL/6 × C3H/Aa F₁-females heterozygous at the X-linked phosphoglycerate kinase locus (Pgk-1) were cultured continuously in the presence of cytochalasin B or D. Further cleavage of the two cell embryos was thus prevented and the embryos became polyploid during culture. The onset of expression of the maternally inherited Pgk-1 gene and of the paternally inherited glucosephosphate isomerase (Gpi-1) gene was determined in these polyploid embryos by cellulose acetate gel electrophoresis of single embryos. In contrast to euploid preimplantation embryos developing normally in utero or in culture without cytochalasins, expression of maternal Pgk-1 was never observed at days 4 and 5 of gestation in polyploid two cell embryos, showing that the Pgk-1 allele on the maternally inherited X chromosome is not activated independently of cytokinesis and morphogenesis. Expression of paternally derived Gpi-1, however, occurred in cleavage blocked embryos von day 5 of development. This may indicate that the activation of two genes which are both expressed during preimplantation development and which both code for glycolytic enzymes, is initiated by different signals.     

Death of mouse embryos that lack a functional gene for glucose phosphate isomerase

A null allele of the Gpi-1s structural gene, that encodes glucose phosphate isomerase (GPI-1; E.C. 5.3.1.9), arose in a mutation experiment and was designated Gpi-1sa-m1H. The viability of homozygotes has been investigated. No offspring homozygous for the null allele were produced by intercrossing two heterozygotes, so the homozygous condition was presumed to be embryonic lethal. Embryos were produced by crossing Gpi-1sa/null heterozygous females and Gpi-1sb/null heterozygous males. Homozygous null embryos were identified at different stages of development by electrophoresis and staining either for GPI-1 alone or GPI-1 plus phosphoglycerate kinase (PGK) activity. At 6 1/2 and 7 1/2 days post coitum homozygous null embryos were present at approximately the expected 25% frequency (37/165; 22.4% overall) although at 7 1/2 days the homozygous null embryos tended to be small. By 8 1/2 days most homozygous null embryos were developmentally retarded and had not developed significantly further than at 7 1/2 days; some were dead or dying. By 9 1/2 days the homozygous null conceptus was characterised by a small implantation site that contained trophoblast and often a small amount of extraembryonic membrane. Surviving trophoblast tissue was also detectable at 10 1/2 days. Previous studies have shown that oocyte-coded GPI-1 persists only until 5 1/2 or 6 1/2 days. Survival of homozygous null embryos to 7 1/2 or 8 1/2 days and survival of certain extraembryonic tissue to 10 1/2 days suggests that the homozygous null condition may not be cell-lethal although it is certainly embryo-lethal. Mutant cells that are deficient in glycolysis may use the pentose phosphate shunt to bypass the block in glycolysis created by the deficiency of glucose phosphate isomerase, and/or might be rescued by the transport, from the maternal blood, of energy sources other than glucose (such as glutamine). Either strategy may only permit slow cell growth that would not be adequate to support normal embryogenesis. Transport of maternal nutrients would be more efficient to the trophoblast and extraembryonic membranes and this may help to explain why these tissues survive for longer than the embryo itself. The morphological similarity between homozygous nulls and androgenetic conceptuses, where the trophoblast also survives better than the embryo, is discussed.     

Expression of glucose phosphate isomerase in interspecific hybrid (Mus musculus x Mus caroli) mouse embryos.

Hybrid Mus musculus x Mus caroli embryos were produced by inseminating M. musculus (C57BL/OlaWs) females with M. caroli sperm. Control M. caroli embryos developed more rapidly than did control M. musculus embryos and implanted approximately 1 day earlier. At 1 1/2 days, both the hybrid embryos and those of the maternal species (M. musculus) had cleaved to the 2-cell stage. By 2 1/2 days some of the hybrids were retarded compared to M. musculus, and by 3 1/2 days most were lagging behind. This is consistent with the idea that the rate of development of hybrid embryos declines once it becomes dependent on embryo-coded gene products. We have used this difference in rate of preimplantation development, between hybrid and M. musculus embryos, to try to determine whether the activation of embryonic Gpi-1s genes, that encode glucose phosphate isomerase (GPI-1), is age-related or stage-related. In control M. musculus embryos (both mated and Al groups), the GPI-1AB and GPI-1A allozyme, indicative of paternal gene expression, were detected in 7 of 9 samples of 3 1/2-day compacted morula stage embryos and were seen in all 19 samples of 3 1/2-day blastocysts. In hybrid embryos, these allozymes were detected 1 day later. They were not detected in any 3 1/2-day samples (12 samples of compacted morulae) but were consistently detected at 4 1/2 days (4 samples of blastocysts and 2 samples of uncompacted morulae). Our interpretation of the results is that gene activation in hybrid embryos is stage-specific, rather than age-specific, and probably begins around the 8-cell stage, with detectable levels of enzyme accumulating later. Analysis of GPI-1 electrophoresis indicated that both the paternal (M. caroli) and maternal (M. musculus) Gpi-1s alleles were equally expressed in hybrid embryos and that the paternally derived allele was not activated before the maternally derived allele.     

Onset of paternal and maternal Gpi-1 expression in preimplantation mouse embryos

The initial activation of the glucose phosphate isomerase gene, Gpi-1, was studied in mouse embryos produced by transplanting pronuclei between two strains of mice differing in alleles for this enzyme. Protein isozymes encoded by the embryonic cell nuclei were first detected on Day 4 of embryogenesis, and the maternal and paternal genes are seen to be activated simultaneously. Comparison of isozymes produced by these nuclear-transfer embryos and by F1 embryos from these two strains suggests the absence of oocyte mRNA for GPI-1 at the time when these genes are first activated. Thus, the GPI-1 present is derived from newly transcribed mRNA contributed by both maternal and paternal genes. The relative proportion of maternal cytoplasmic GPI-1 enzyme declines from Day 3 to Day 6, such that on Day 6, almost no oocyte GPI-1 is detected.     

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.     

Unusual glucose phosphate isomerase isozyme patterns in lymphocytes from two C57BL/6J in equilibrium A/J allophenic mice

Analysis of C57BL/6J in equilibrium A/J allophenic mice for their lymphocyte composition, using H-2 antigens as external markers, and glucose phosphate isomerase (GPI) isozymes as internal markers, has led to the discovery of two unusual mice. Both mice showed heterodimers of GPI isozymes upon electrophoresis of the lymphocyte lysate. Specific anti-H-2 antisera confirmed that the cells of the mice were of C57BL/6J and A/J origin, as expected, but that the "A/J" cells seemed to behave as F1 hybrids containing the Gpi-1a and Gpi-1b alleles. Possible origins of the Gpi-1b allele in the "A/J" cells are discussed.     

Synchronous activation of both parental alleles at the 6-PGD locus of Japanese quail embryos

The gene locus for the enzyme 6-phosphogluconate dehydrogenase belongs to that part of the genome which is activated at the beginning of embryonic development. The present experiment, utilizing three alleles at this autosomally inherited locus of the Japanese quail, was designed to show whether exhaustion of maternally stored 6-PGD is followed by maternally hemizygous de novo synthesis of the same enzyme. 6-PGD phenotypes of early embryos resulting from the mating between a male homozygous for one allele and a female heterozygous for two other alleles were examined by starch gel electrophoresis. The result showed that the maternally stored 6-PGD is exhausted before the twenty-fourth hour of incubation. This is followed by synchronous activation of both parental alleles. Previous studies on the development of various interspecific crosses have revealed that, at all loci studied, the activation of the maternally derived allele preceded that of the paternally derived allele. The present experiment reveals that preferential activation of maternally derived alleles need not be a rule of development.This work was supported in part by a grant (CA-05138) from the National Cancer Institute, U.S. Public Health Service, and in part by a research fund established in honor of General James H. Doolittle. Contribution No. 6-68, Department of Biology, City of Hope Medical Center.     

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.     

The Expression of X-Linked Phosphoglycerate Kinase in the Early Mouse Embryo

Abstract. During early mouse embryogenesis, the activity of X-chromosomally linked maternal and paternal phosphoglycerate kinase (PGK-1) alleles was determined using electrophoretic separation of their gene products and a sensitive fluorometric enzyme assay. In the embryos collected from females homozygous for PGK-1^b mated with PGK-1^a males and vice versa, the paternally derived allozyme was first detected after implantation on day 6. Expression of the maternally inherited allele was studied in embryos from females heterozygous for PGK-1^b and PGK-1^a. From day 1 to day 4, the embryos maintained a constant ratio of enzyme activity of PGK-1B to PGK-1A. Prior to implantation of the embryos between day 4 and day 5, the activity ratio of the two PGK-1 allelic variants changed significantly due to the first appearance of newly synthesized PGK derived from the maternally inherited allele.
Our data demonstrate a temporal difference in the onset of PGK synthesis depending on whether this particular gene product is of maternal or paternal origin. Therefore, we conclude that the maternal PGK-1 locus is already activated during late preimplantation development whereas the paternally inherited gene locus remains silent at the preimplantation stage but is subsequently expressed at approximately the time of X-chromosomal inactivation.     

Expression of glucose phosphate isomerase in interspecific hybrid (Mus musculus × Mus caroli)

Hybrid Mus musculus × Mus caroli embryos were produced by inseminating M. musculus (C57BL/Ola Ws) females with M. caroli sperm. Control M. caroli embryos developed more rapidly than did control M. musculus embryos and implanted approximately 1 day earlier. At 1 1/2 days, both the hybrid embryos and those of the maternal species (M. musculus) had cleaved to the 2-cell stage. By 2 1/2 days some of the hybrids were retarded compared to M. musculus, and by 3½ days most were lagging behind. This is consistent with the idea that the rate of development of hybrid embryos declines once it becomes dependent on embryo-coded gene products. We have used this difference in rate of preim-plantation development, between hybrid and M. musculus embryos, to try to determine whether the activation of embryonic Gpi-1s genes, that encode glucose phosphate isomerase (GPI-1), is age-related or stage-related. In control M. musculus embryos (both mated and Al groups), the GPI-1AB and GPI-1A allozyme, indicative of paternal gene expression, were detected in 7 of 9 samples of 3 1/2-day compacted morula stage embryos and were seen in all 19 samples of 31/2-day blastocysts. In hybrid embryos, these allozymes were detected 1 day later. They were not detected in any 31/2-day samples (12 samples of compacted morulae) but were consistently detected at 4½ days (4 samples of blastocysts and 2 samples of uncompacted morulae). Our interpretation of the results is that gene activation in hybrid embryos is stage-specific, rather than age-specific, and probably begins around the 8-cell stage, with detectable levels of enzyme accumulating later. Analysis of GPI-1 elec-trophoresis indicated that both the paternal (M. caroli) and maternal (M. musculus) Gpi-1s alleles were equally expressed in hybrid embryos and that the paternally derived allele was not activated before the maternally derived allele. © 1992 Wiley-Liss, Inc.     

Expression of paternal glucose phosphate isomerase-1 (Gpi-1) in preimplantation stages of mouse embryos

Electrophoretic variants of glucose phosphate isomerase have been used to study the time of paternal gene activation during early embryogenesis of the mouse. Hybrid embryos obtained from matings of GPI-1A ♀ X GPI-1B ♂ were examined electrophoretically, and assayed for GPI activity during preimplantation stages. The heteropolymeric GPI-1AB band was detected in late blastocysts and all three bands of the hybrid pattern were discernible in samples of expanded blastocysts, day 6. These findings indicate that the Gpi-1 paternal locus is expressed by day 5. Activity levels of GPI were comparable to values reported for G6PD. The activity of GPI was constant for days 1, 2, and 3; however, a marked decrease in activity occurred by day 4. A slight decrease in activity was observed in embryos from days 5 and 6. Our results demonstrate the value of using electrophoretic variants to pinpoint synthesis of new enzyme which may not be reflected in changes in levels of activity.     

The development of XO gynogenetic mouse embryos

Diploid gynogenetic embryos, which have two sets of maternal and no paternal chromosomes, die at or soon after implantation. Since normal female embryos preferentially inactivate the paternally derived X chromosome in certain extraembryonic membranes, the inviability of diploid gynogenetic embryos might be due to difficulties in achieving an equivalent inactivation of one of their two maternally derived X chromosomes. In order to investigate this possibility, we constructed XO gynogenetic embryos by nuclear transplantation at the 1-cell stage. These XO gynogenones showed the same mortality around the time of implantation as did their XX gynogenetic counterparts. This shows that the lack of a paternally derived autosome set is sufficient to cause gynogenetic inviability at this stage. Autosomal imprinting and its possible relation to X-chromosome imprinting is discussed.     

Parental influences on expression of glucose-6-phosphate dehydrogenase, G6pd, in the mouse; a case of imprinting

Glucose-6-phosphate dehydrogenase (G6PD) activity was measured in blood from heterozygotes for the normal allele G6pda and the low activity allele G6pda-mlNeu. In adult mice lower activity was found in G6pda/G6pda-mlNeu than in the reciprocal heterozygote G6pda-mlNeu/G6pda (the maternal allele being listed first). Thus, either the paternally derived allele was over-expressed or the maternally derived allele was under-expressed. By contrast, in younger mice the difference in G6PD activity in reciprocal crosses was less marked. The findings are interpreted in terms of differential imprinting of maternally and paternally inherited information. The explanation offered for age related differences is that, as a consequence of imprinting, either the paternal X-chromosome is preferentially reactivated, or cells in which the paternally derived allele is active are at a selective advantage, and proliferate better than those in which the maternally inherited allele is active.     

Transitional hemizygosity of the maternally derived allele at the 6PGD locus during early development of the Cyprinid fish Rutilus rutilus

The activation of individual alleles during early embryogenesis was studied at the 6-phosphogluconate dehydrogenase gene locus of the Cyprinid fish Rutilus by means of starch gel electrophoresis. By using three alleles occurring at this locus, it was possible to discriminate between (1) maternally transmitted gene products stored in the egg cytoplasm, (2) newly synthesized protein of the maternally derived allele in the embryonic genome, and (3) newly synthesized protein of the paternally derived allele. It was found that, until the fifth day of development, maternal products were present in the embryo. By the seventh day after fertilization, these storage products were nearly exhausted, and a hemizygous phenotype for the maternally derived gene became visible. On the eighth day, the patterns of all four allelic combinations of the mating type used were demonstrable in the offspring. The findings suggest that for the alleles used in this study, the maternally derived gene is preferentially activated during embryogenesis.Supported by the Deutsche Forschungsgemeinschaft.     

Gene activation of alcohol dehydrogenase in danio hybrids

The regulation of alleles encoding the enzyme alcohol dehydrogenase (ADH) was investigated in F1Brachydanio hybrids (zebra danio female x spotted danio male) by acrylamide gel electrophoresis. Both parental species showed a single, cathodal band of species-specific ADH. During development at 26 degrees C, hybrid fry showed a preferential activation of the maternally derived Adh allele. It is suggested that the low activity of the paternally derived allele may result from an incompatibility between maternal regulatory factors and the paternal regulative element controlling gene expression.     

Immunochemical differentiation of glucose phosphate isomerase (GPI) allozymes of the mouse

Polyclonal xenoantisera against mouse GPI-1B and GPI-1C were produced in rabbits and analyzed for their ability to recognize allozyme-specific determinants. These studies showed a high degree of serological similarity among the three allozymes of mouse glucose phosphate isomerase (GPI). However, GPI-1B and GPI-1C could be differentiated from GPI-1A as well as GPI-1A and GPI-1B from GPI-1C using quantitative solid-phase immunobinding assays. In addition, polyclonal and monoclonal alloantibodies specific for GPI-1C were produced in BALB/c (Gpi-1a/Gpi-1a) mice. As indicated by immunoblotting data, the allozyme specificity of rabbit antisera and monoclonal alloantibodies against GPI-1C is dependent on the native structure of that allozyme.     

Allocyclic early replicating X chromosome in mice: Genetic inactivity and shift into a late replicator in early embryogenesis

The allocyclic X chromosome in early female mouse embryos undergoes DNA replication either late or early in the S phase. Earlier studies indicated that the early-replicating X chromosome is restricted to the trophectoderm and primitive endoderm cell lineages in which the allocyclic X is almost exclusively paternal in origin. There has been, however, no compelling evidence for the genetic inactivity of the early-replicating X chromosome and a shift from early to late replication or vice versa. The present study employing a combination of 3H-thymidine autoradiography and BrdU labeling-acridine orange fluorescence staining in day-6 female mouse embryos found that the early-replicating X chromosome can change directly into a late-replicating one. The activity state of the early-replicating X chromosome was examined by electrophoretic determination of the X linked enzyme, phosphoglycerate kinase (PGK-1), in tissues isolated from 6.0-day and day-8.5 Pgk-1a/Pgk-1b embryos. Only the maternally derived Pgk-1 allele was expressed in the proximal endoderm and extraembryonic ectoderm of 6.0-day and the chorion of 8.5-day embryos. Thus, the early-replicating, paternally derived X chromosome found in about 70%-80% of the cells in these tissues seems to be repressed like the late-replicating one.