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.     

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.     

Shifts in ATP synthesis during preimplantation stages of mouse embryos.

The actual and potential activities of the cyochrome system were studied in cleavage-stage mouse embroys. Activities were determined by assaying embroys for total ATP and the rates of [32-P]ATP synthesis both before and after their incubation in medium supplemented either with an energy coupling site inhibitor (antimycin, amytal or cyanide) or with the FADH-linked substrate, succinate. The data indicate that there are three major shifts in the mode of ATP production during preimplantation stages: the first, between the two-cell and late four-cell stages; the second, between the eight-celland late morula stages; and the third, between the late morula and late blastocyst stages. These data are discussed in relation to studies on the energy metabolism of cleavage and blastocyst stage mouse embryos.     

Quantitative determination of the pentose phosphate pathway in preimplantation mouse embryos

A quantitative calculation was made of the pentose phosphate pathway (PPP) activity in preimplantation mouse embryos from the 2-cell through the late blastocyst stage. This activity varied with development and showed repeated high and low values. Peak activities occurred at both the 2-cell (15.8%) and compacted morula (13.6%) stages, with lowest activity at the development of the late blastocyst (3.2%). The metabolic effectors dimitrophenol (DNP) and phenazine ethosulfate (PES) had opposite effects on PPP activity. Dinitrophenol, although stimulating total CO2 production, virtually eliminated PPP activity while PES stimulated the pentose cycle activity 6-fold. These results indicated that the PPP was under metabolic control and that the embryos had a potential for much larger PPP activities. There was no correlation between the C-1/C-6 ratio obtained from the metabolism of [1-14C] and [6-14C] glucose and calculated PPP activities. A metabolic incubation chamber was devised for these experiments that exhibited certain unique features, including continuous collection of 14CO2 and 3H2O. Single embryos were placed in the chamber and sampled momentarily for metabolic activity. Subsequently, such embryos were successfully transferred to pseudopregnant recipients.     

Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos

The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.     

Metabolism in preimplantation mouse embryos

The ability of preimplantation mouse embryos to utilize glucose oxidatively is controlled, in part at least, at the level of glycolysis. Various experimental observations are reviewed that indicate the regulatory mechanism in delayed implanting blastocysts involves the classic negative allosteric feedback of high levels of ATP on phosphofructokinase while the situation in 2-cell embryos appears to be more complicated. That is, in addition to the usual negative effect of ATP and citrate on phosphofructokinase, there appears to be a modification of hexokinase that prevents phosphorylation of adequate amounts of glucose and results in low levels of fructose-6-phosphate at the 2-cell stage and consequently there is a failure to release the inhibition of phosphofructokinase even if ATP and citrate levels decrease. Although both types of embryos have limited glycolytic activity, they do have adequate capacity for citric acid cycle activity and oxidative phosphorylation, and are able to maintain a high ATP : ADP. It is argued, therefore, that the reduced levels of macromolecular synthesis characteristic of 2-cell and delayed implanting blastocysts are not due to restricted energy substrates or regulatory controls on glycolysis and a subsequent low energy state. On the contrary, it seems that the reduction in oxidative utilization of glucose in these situations is a result of diminished energy demand because of the low level of synthetic activity. The potential significance of this relationship between energy production and utilization in terms of potential regulatory mechanisms in preimplantation embryos is discussed.     

Effects of paternal heat stress on the in vivo development of preimplantation embryos in the mouse

The objective of this study was to examine the effect of paternal heat stress on the in vivo development of preimplantation embryos in the mouse. Synchronised B6CBF1 female mice were mated either to a control male mouse or to one that had been exposed at 7, 21 or 35 days previously, for 24 h to an ambient temperature of 36+/-0.3 degrees C and 66+/-5.6% relative humidity. Embryos were collected from the oviducts of mice at 14-16 h, 34-39 h or 61-65 h after mating or from the uterus at 85-90 h after mating and their developmental status was evaluated morphologically. The number of cells within blastocysts was also determined using bisbenzimide-propidium iodide staining. Paternal heat stress 7 days before mating reduced the proportion of embryos developing from 4-cell (4-C) to morulae (M), hatched blastocysts, total blastocysts and the number of inner cell mass (ICM) and trophectoderm (TE) cells in the blastocyst. Paternal heat stress 21 days prior to mating reduced the proportion of 2-C and 4-C to M embryos with no embryos developing to blastocysts. There were also increases in the number of 1-C and abnormal embryos recorded at this time. Paternal heat stress 35 days before mating decreased the proportion of 2-C embryos, expanded blastocysts and ICM and TE cells in the blastocyst. These results support previous work demonstrating that both the sperm in the epididymis and germ cells in the testis are susceptible to damage by environmental heat stress, with spermatocytes being the most vulnerable. This study also demonstrates that subtle effects on the male such as a short exposure to elevated environmental temperatures can translate to quite profound paternal impacts on early embryo development.     

Effect of RU486 on different stages of mouse preimplantation embryos in vitro

17 beta-Hydroxy-11 beta(4-dimethylaminophenyl)-17 alpha-(1-propynyl)estra-4, 9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 degrees C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 micrograms/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 micrograms/mL culture medium (p less than 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 microgram/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 micrograms/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.     

Expression of a male-specific factor on various stages of preimplantation bovine embryos

Four-cell to blastocyst stage bovine embryos were collected from superovulated donors and cultured for 90 min in Ham's F-10 medium (HF-10) containing 10% (V/V) absorbed anti-histocompatibility (H)-Y antiserum. Embryos were then washed 3 times and placed in HF-10 supplemented with 10% (V/V) fluorescein isothiocynate (FITC)-conjugated goat anti-mouse gamma globulin. After an additional wash, embryos were placed in fresh drops of HF-10, individually evaluated at 200 X magnification, and classified as either fluorescent (H-Y-positive) or nonfluorescent (H-Y-negative). Embryos were then placed in drops of HF-10 containing 14% vinblastin and cultured for 4-6 h. Embryos were coded and individually karotyped, and the sex chromosomes were identified. H-Y antigen was detected as early as the eight-cell stage, but not at the four-cell stage. Seventy-nine percent of fluorescent embryos and 89% of nonfluorescent embryos were XY and XX, respectively. Another experiment was carried out in which H-Y antigen was detected on intact inner cell masses (ICM) isolated by immunosurgery from expanded blastocysts that also had been assayed for H-Y antigen. Eighty-eight and 92%, respectively, of ICM classified as fluorescent or nonfluorescent had been scored the same as intact blastocysts. It is concluded from these data that H-Y antigen can be detected on eight-cell to blastocyst stage bovine embryos. There appears to be a localization of detectable antigen in the area of the ICM at the expanded blastocyst stage. Detection of H-Y antigen is an effective, noninvasive method for identification of the sex of preimplantation bovine embryos.     

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)     

Fucosylated glycoconjugates in mouse preimplantation embryos

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.