Summary. The method for estimation of metabolic turnover by a single embryo at each stage of preimplantation development is based on the incubation of an embryo in the presence of labelled substrate at high specific activity in a miniaturized incubation chamber and the subsequent radioassay of metabolic products. Preliminary tests indicated that the treatment did not affect embryos adversely. Estimates of glycolysis, substrate oxidation and incorporation by mouse embryos throughout the whole of the preimplantation period of development were made. This technique could prove particularly useful for investigating substrate requirements and metabolic turnover in species for which few embryos are available for study.
R. G. WALES
The metabolism of dog spermatozoa in the presence of various ions has been studied. Of the ions tested, potassium had the greatest effect and at least doubled the respiration of washed cells. It did not, however, significantly change aerobic fructolysis. Due to interaction with other diluent components, magnesium ions had variable effects on respiration. Thus magnesium enhanced fructose oxidation in the presence of low levels of potassium but depressed respiration in the presence of low levels of calcium. Calcium improved respiration of washed spermatozoa and caused the accumulation of lactate without any concomitant increase in fructose utilization. Washing the spermatozoa free of their seminal plasma reduced the oxidation of substrates other than fructose. This effect was probably due to the removal of alternative substrates in the seminal plasma. The lactate present in dog semen only accounted for a small proportion of oxygen uptake and other alternative substrates are discussed.
P. QUINN and R. G. WALES
The utilization of energy substrates by preimplantation mouse embryos undergoes several changes during development in vitro (Brinster, 1965a, b) but very little is known about the level of high-energy containing compounds, such as ATP at these stages. In the present study, the content of ATP in freshly collected preimplantation mouse embryos has been measured.
For each estimation, fifty to 150 embryos were collected by flushing the reproductive tracts of superovulated female albino mice with modified Krebs-Ringer bicarbonate medium (Brinster, 1965b). Unfertilized and fertilized ova were removed from the Fallopian tubes 20 to 24 hr after the injection of hcg and the surrounding cumulus cells were removed by incubation in hyaluronidase solution (Brinster, 1965c). Later developmental stages of the embryos were flushed from the Fallopian tubes and uterus at specific times after the injection of hcg. After washing by transfer through 2 ml of fresh medium, the embryos
P. QUINN and R. G. WALES
During the first 3 days of development of the rabbit embryo, carbon fixed from CO2 accumulated in the embryos and culture medium and increased as development proceeded.
Ultracellularly, incorporation of carbon into acid-soluble material accounted for 60 to 85% of the total fixed carbon, with most of the the remaining carbon entering the protein fraction. In the medium, most of the fixed carbon was found in the acid-soluble fraction. Although a similar proportion of embryos developed in either the presence or absence of exogenous energy substrates, the amount of fixed carbon accumulating in both the embryos and culture medium was greater when energy substrates were included in the medium. There was little difference between the amounts of carbon incorporated into embryos cultured in medium containing either glucose or pyruvate plus lactate. In these media, however, the accumulation of fixed carbon in the glucose medium was lower than that in the medium containing pyruvate and lactate. This difference was less marked at the later stages of embryonic development.
Aspartic and glutamic acids were the major compounds containing fixed carbon in the basic portion of the acid-soluble fraction of the embryos. Fixed carbon accumulating in the medium was found in lactate, malate, citrate, pyruvate and, to a lesser extent, acetate.
P. QUINN and R. G. WALES
The development of two-cell, eight-cell and morula stages of the mouse embryo in phosphate-buffered medium incubated aerobically was limited to that which occurred during the initial 24 hr of culture. Fewer embryos developed in phosphate- than in bicarbonatebuffered medium. More two-cell embryos developed in 4 mm- than in 1 mm-phosphate-buffered medium but there was no difference in the number of eight-cell embryos and morulae developing in either concentration. Addition of oxaloacetate or malate to phosphate-buffered media did not improve development. With incubation periods of 2 to 12 hr, two-cell embryos were more susceptible to phosphate than eight-cell embryos.
Culture for 24 hr in phosphate-buffered medium resulted in a reduced incorporation of pyruvate and lactate carbon into the acidsoluble fraction of embryos at all stages of development. In embryos cultured from the two-cell and eight-cell stage, incorporation of substrate carbon into protein and, to a lesser extent, RNA and DNA was also depressed after culture in phosphate-buffered medium. This reduced synthetic activity could result in less ATP utilization and may be responsible in part for the higher levels of ATP found in embryos at these stages after culture in phosphate- as compared to bicarbonate-buffered medium. At the morula stage, macromolecular synthesis was not affected by culture in phosphate-buffered medium and the ATP levels in these embryos were similar to those in embryos developing in bicarbonate-buffered medium. The oxidation of pyruvate by embryos also indicated that the utilization rather than the production of ATP was more affected by culture in phosphate- than in bicarbonate-buffered medium.
P. QUINN and R. G. WALES
The incorporation of substrate carbon from pyruvate and lactate has been measured at all stages of preimplantation development in the mouse embryo using substrates labelled at the C2 position. Embryos were cultured for 24 hr in medium containing pyruvate (0·5 mm) or lactate (5 mm), alone or in combination and the accumulation of label in various intracellular fractions was measured.
The incorporation of both pyruvate and lactate carbon increased with increasing development of the embryos. Before the morula stage, the presence of unlabelled lactate in the medium depressed the incorporation of C2 of pyruvate whereas the opposite effect occurred when embryos were cultured in [2-14C]lactate in the presence of unlabelled pyruvate. The combined incorporation of carbon from both pyruvate and lactate was greater than that from either substrate alone at all stages of development. Lactate contributed a greater proportion to the combined incorporation of substrate carbon than pyruvate at all stages of development. The increased incorporation of substrate carbon into cultured two-cell embryos in the combination of pyruvate and lactate was almost entirely due to the stimulated accumulation of carbon in the protein fraction of the embryos. Incorporation into this fraction contributed proportionately less to the increased total incorporation into the embryos at the later stage of development.
Alanine, glutamic acid and aspartic acid accounted for most of the carbon incorporated into the basic compounds of the acid-soluble fraction of the embryos. Labelled malate and citrate were found in the acidic compounds of this fraction when two-cell embryos were cultured in [2-14C]pyruvate. After the eight-cell stage, labelled lactate, as well as malate and citrate, accumulated in embryos cultured in medium containing pyruvate, lactate or their combination. Labelled pyruvate only accumulated in these embryos during culture in media containing [2-14C]pyruvate alone or [2-14C]lactate and unlabelled pyruvate.
P. QUINN and R. G. WALES
Preimplantation mouse embryos were cultured for varying times in the presence of different energy substrates. The levels of ATP were then measured and compared to those of freshly collected embryos.
There were no differences in the levels of ATP between embryos cultured from the two- or eight-cell stage to the morula stage and freshly collected embryos at similar stages of development, while embryos which were cultured to the blastocyst stage had more ATP than freshly collected blastocysts. Both eight-cell embryos and morulae which were cultured for 24 to 48 hr in medium containing pyruvate plus lactate had greater amounts of ATP than embryos cultured in either energy substrate-free medium or medium containing glucose.
At the one- and two-cell stage, a combination of pyruvate and lactate was more effective than either energy substrate alone in maintaining the levels of ATP during a 6-hr culture period. As development progressed, the higher levels of ATP in embryos cultured in medium containing glucose as compared to the levels in embryos cultured in energy substrate-free medium indicated that glycolysis played an increasingly important rôle in maintaining the levels of ATP.
The level of ATP in embryos cultured for up to 48 hr beyond the morula stage increased to values equivalent to those at the one- and two-cell stage. However, the ratio of ATP to ADP in morulae and blastocysts was considerably lower than this ratio at the one- and two-cell stage. It is suggested that this high ATP to ADP ratio in one- and two-cell embryos may limit the rate of glycolysis during early development.
P. QUINN and R. G. WALES
A positive correlation was found between the levels of ATP in two-cell embryos from random-bred mice and the proportion of these embryos which developed to the blastocyst stage during culture in vitro. The better development of hybrid one-cell embryos to blastocysts than that of random-bred one-cell embryos may also be related to the higher amounts of ATP found in hybrid two-cell embryos cultured from the one-cell stage compared to those in embryos of the random-bred strain. The relationship between the ATP content of embryos and the proportion of embryos developing in vitro appeared to be different between the two groups of mice and was altered after culture in vitro.
The levels of ATP in two-cell embryos from a random-bred strain and F1 hybrid mice cultured from the one-cell stage in medium supplemented with glucose and serum albumin were higher than the levels of ATP in embryos cultured in glucose-free medium containing less albumin. During culture of hybrid one-cell embryos in the medium containing glucose and albumin, an effect of a low O2 tension in the atmosphere on ATP levels became apparent only after the morula stage.
R. G. WALES and D. G. WHITTINGHAM
The accumulation and utilization of substrate carbon by two-cell mouse embryos incubated in media containing combinations of lactate and pyruvate were studied. The results were consistent with utilization of the two substrates by common metabolic pathways. The intracellular accumulation of substrate carbon was greater when pyruvate as well as lactate was present in the medium. The evidence also suggests that pyruvate is more effective in maintaining the activity of the tricarboxylic acid cycle than lactate alone.
Fractionation of substrate carbon accumulated in embryos during a 30-min incubation period indicated that approximately 10% of this carbon had entered the acid-insoluble (protein) fraction. This accumulation was increased substantially when a combination of lactate and pyruvate, rather than either substrate alone, was present in the incubation medium.
The major organic acids found to accumulate intracellularly from substrate were malate and citrate. The intracellular accumulation of lactate was confined to embryos incubated in medium containing this substrate. Base-containing compounds accounted for a substantial portion of the substrate carbon in embryos. Glutamic acid was the major amino acid identified in this component. Alanine accumulated from pyruvate or its combination with lactate but not from lactate alone. Small amounts of aspartate accumulated from both substrates.
The effect of the interaction of lactate and pyruvate on the development of two-cell mouse embryos to the blastocyst stage was confirmed. Although pyruvate was effective over a limited range of concentration, the effective range of concentration of lactate was not as limited. The present studies suggest that the beneficial effect of combinations of lactate and pyruvate could be due to a stimulation of the metabolism of the embryos and a more favourable level of macromolecule synthesis.
R. G. WALES and I. G. WHITE
Data are presented on the chemical composition of the three fractions of dog semen. Sodium is the main cation present in all fractions; potassium, magnesium and calcium occur in much lower concentration. Chloride is the main anion. All fractions are characterized by low concentrations of total reducing sugar, fructose, lactic acid and citric acid. Orcinol reactive carbohydrate is present in significant amounts and much is acid insoluble and probably bound to protein.
The sperm-bearing fraction is characterized by a high concentration of total phosphorus and the acid insoluble fraction is mainly confined to the spermatozoa. The high concentration of acid soluble phosphorus in this fraction is partly due to the appreciable amounts of glyceryl-phosphorylcholine present.
Analyses of seminal carbohydrate and protein indicate inherent differences between dogs in the composition of semen.