Energy metabolism in late preimplantation rat embryos

The consumption of pyruvate and glucose, and the production of lactate, by single preimplantation embryos, was measured using a noninvasive technique. Embryos were cultured in 300-500-nl microdrops, for 8-12 h at a time, from Day 4 to Day 6 after mating, when they developed from the 8-cell stage to expanded blastocyst. Pyruvate was the predominant substrate at the 8-cell/morula stage; glucose uptake exceeded that of pyruvate after the onset of blastocoel formation. Lactate production increased in parallel with glucose consumption. For most stages, approximately 100% of the glucose uptake was accountable for by lactate production and in some cases an additional source of lactate must be postulated. Culture in vitro had little effect on lactate production, although a lower level of metabolism was observed compared with fresh blastocysts. Rat embryos were capable of developing to blastocysts in the absence of glucose, when lactate production was greatly reduced.     

Oxidative and conjugative metabolism of diethylstilbestrol by rabbit preimplantation embryos

Five- and six-day-old rabbit preimplantation embryos were found to be capable of metabolizing [3H]diethylstilbestrol (DES) in vitro. Based on the analysis of the metabolites formed during a 24-hr incubation period we conclude that these early stage embryos do have active monooxygenase and conjugative activity. The monooxygenase seems to be specific to this early stage of embryonic development.     

Enzymes of glycogen metabolism and related metabolites in preimplantation mouse embryos

Preimplantation mouse embryos were individually analyzed for glycogen phosphorylase, P-glucomutase, UDPG, UTP, ATP, and the sum of other nucleotide triphosphates (i.e., GTP + CTP). UDPG changes during starvation and refeeding were also determined. Phosphorylase activity was exceedingly low at the two-cell stage and rose eightfold by the morula stage. P-glucomutase was 2000 times more active than phosphorylase in two-cell embryos and fell progressively to about half the initial level by the eight-cell stage. UDPG was highest in one-cell embryos, fell to less then 20% by the two-cell stage, then recovered to about a 35% level at later stages. The ATP to UTP ratio varied from about 5:1 at the earliest stages to about 3:1 in eight-cell and older embryos. GTP plus CTP was 50% higher than UTP at the one-cell stage but was equal to UTP or lower thereafter. The results combined with earlier data from several laboratories indicate that (1) up to the morula stage the embryo can make glycogen but has difficulty using it because of insufficient glycogen phosphorylase and (2) UDPG and glucose-6-P levels are poorly coordinated, probably due to difficulty (or control) at the UDPG pyrophosphorylase step.     

Influence of environmental factors on the metabolism of glucose by preimplantation mouse embryos in vitro

The metabolism of glucose by late preimplantation mouse embryos was studied in a variety of media whose composition had been changed to reflect the environmental conditions in the uterus more closely than do standard culture media. The effects of combinations of energy substrates, the presence or absence of amino acids and the level of potassium in the medium were investigated. The use of energy substrates for in vitro culture at levels present in the uterine environment resulted in rates of synthesis and degradation of glycogen pools similar to those obtained using standard in vitro culture conditions but elevated incorporation into non-glycogen macromolecules. Amino acids influenced the metabolism of glucose by limiting the entry of glucose carbon into the non-glycogen macromolecular pool and directing more glucose into the synthesis of acid-soluble glycogen. Increasing the K+ concentration to 60 mM in the culture medium caused a small but significant increase in the number of eight-cell embryos degenerating during culture for 24 h but the metabolism of glucose was unaffected over this time. At the time of morula transformation to the blastocyst this level of potassium ions suppressed glycogen synthesis by 50% over 5 h but did not affect its turnover during chase culture. It is concluded that factors other than those studied here contribute to the maintenance of the low glycogen levels found in uterine embryos.     

Oxidative metabolism of energy substrates by preimplantation mouse embryos in the presence of platelet-activating factor

The effects of exogenous platelet-activating factor (PAF, 0.0186-18.6 microM) on the production of CO2 from [U-14C]glucose and [1-14C]lactate by mouse embryos in vitro were investigated. Two-cell embryos displayed significant dose-dependent responses for both energy substrates. The maximal response was observed at 9.3 microM-PAF for glucose metabolism and 1.86 microM-PAF for lactate with increases of 62% and 18%, respectively, over control treatments. After culture from the 2-cell stage for 72 h in the presence of PAF, the resulting blastocysts exhibited a significant dose-dependent increase in metabolism of lactate. It was also apparent that such embryos were not desensitized to PAF as demonstrated by a further enhancement of the metabolic response after re-exposure to PAF. The specificity of action of PAF was confirmed by the absence of any effect on the oxidative metabolism of glucose by lyso-PAF (a catabolite of PAF) over a concentration range of 0.0202-20.2 microM and by the demonstration that SRI 63-441 (a PAF-receptor antagonist) significantly reduced the amount of CO2 produced from glucose in response to 9.3 microM-PAF and abolished the effect on lactate metabolism in response to 1.86 microM-PAF. These results demonstrate a specific, direct influence of exogenous PAF on the oxidative metabolism of glucose and lactate by the preimplantation mouse embryo and suggest an autocrine role for embryo-derived PAF in early pregnancy.     

Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo

This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity.     

Handling of actinomycin D by preimplantation mouse embryos

A protein isolated from serum is required in the cell suspension for the chemotactic response of normal mouse peritoneal macrophages to complement-derived C5a. Macrophage stimulating protein (MSP) has a molecular weight of 100000 and an isoelectric point of 7.0. It is resistant to changes in pH over a range of 1.3–11, is heat labile especially after partial purification and does not survive proteolytic enzyme attack. Binding to ConA Sepharose suggests that it contains a carbohydrate moiety. Its concentration in normal serum is very low and it is detectable only by virtue of a sensitive bioassay. An upper limit has been estimated at 75 ng/ml; the actual concentration may be considerably lower.     

Effects of in vitro fertilization conditions on preimplantation development and quality of pig embryos

The present study was to investigate the effects of in vitro fertilization conditions on in vitro development and structural integrity of pig embryos. Porcine oocytes matured in vitro were co-incubated with four different spermatozoa concentrations (0.6 x 10(5), 1.2 x 10(5), 2.5 x 10(5) and 5 x 10(5) cells/ml) for 6 h, and at a spermatozoa concentration (1.2 x 10(5) cells/ml) for 2, 4 and 6 h, respectively. Spermatozoa penetration and blastocyst formation were observed at 10 and 144 h post insemination, respectively. The allocation of a blastocyst to inner cell mass (ICM) and trophectoderm (TE) cells was determined by using a differential staining method. Polyspermy frequency increased with increasing spermatozoa concentrations. The spermatozoa-oocyte co-incubation period of 2 h provided for decreased in vitro development rate than 4 and 6 h groups (P < 0.05), although no difference was detected in polyspermy frequency between spermatozoa-oocyte co-incubation periods. Interestingly, blastocysts derived from the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml) had significantly fewer ICM cell nuclei as compared with those groups with lesser spermatozoa concentrations (0.6 x 10(5) and 1.2 x 10(5) cells/ml). There was no difference in the structural integrity of blastocysts among the co-incubation periods. Blastocysts derived from respective experiments were individually classified into three groups (I: <20%; II: 20-40% and III: >40%) based on the ratio of ICM to total cells. Proportion of blastocysts in Group II, with a presumptive normal range of structural integrity, was slightly decreased in the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml). The results indicate that the spermatozoa concentration during in vitro fertilization may be important for developmental competence and quality of pig 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.     

Synthesis of H-2 antigens by preimplantation mouse embryos

An enzyme-linked immunosorbent assay (ELISA), which utilized anti-H-2 monoclonal antibody, was used to detect H-2 antigens on preimplantation mouse embryos. All embryonic stages studied, including unfertilized eggs and 1-cell, 2-cell, 8-cell, and blastocyst-stage embryos, showed the presence of H-2 antigens. To prove that the H-2 antigens were not cytophilically adsorbed to the embryos, blastocysts were treated with papain to strip off the H-2 antigens, and then the embryos were further incubated to allow the H-2 antigens to regenerate. After a 3-h incubation time, 60% of the H-2 antigens on the embryos had reappeared, proving that the H-2 antigens were synthesized by the embryos themselves.     

Electrophoretic analysis of proteins synthesized by preimplantation mouse embryos

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ¹⁴C/³H ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ¹⁴C/³H incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.