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P. J. QUINN

Summary.

The methyl green assay method has been used to determine the activity of DNase I and DNase II in ram, bull, human, dog and rabbit seminal plasma. The activity of DNase II was higher except in the ram. The DNase I activity of ram seminal plasma was inhibited by low concentrations of citrate, zinc and cetyltrimethylammonium bromide ; this detergent also caused a reduction of DNase II activity. There was a stimulation of DNase I activity by 10 mm-citrate but not by concentrations less than 2 mm. Addition of antibiotics or shaking of ram seminal plasma with toluene did not affect the activity of either DNase. After deep freezing of ram semen there was a decrease of DNase I activity in the plasma and an increase of DNase I and II activity in extracts obtained from disintegrated spermatozoa. No significant change in DNase activity could be detected in the plasma of ram semen which had previously been subjected to cold shock.

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C. O'Neill and P. Quinn

Summary. Uterine flushings from artificially 'pseudopregnant', pseudopregnant and pregnant mice and those with 'diapausing' embryos were tested for their effect on [3H]uridine incorporation by mouse blastocysts. An inhibitor of [3H]uridine incorporation was detected in the uterine fluid of the mice with diapausing embryos and the activity of the inhibitor was significantly reduced 6·25 h after an injection of oestrogen. This reduction of the inhibitory activity was dependent on the presence of blastocysts in utero, since a similar reduction did not occur in uterine fluids of pseudopregnant mice. The results support the suggestion that 'delayed' implantation in mice is caused by the presence of inhibitors of blastocyst metabolism and that activation, after an increase in oestradiol, is due to an embryo-dependent loss of activity of the inhibitors.

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P. QUINN and R. G. WALES

Summary.

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.

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C. O'Neill and P. Quinn

Summary. Culture of mouse blastocysts in medium supplemented with uterine flushings from mice at random stages of the oestrous cycle resulted in a depression of [3H]uridine incorporation. This depression was maintained for up to 12 h, but by 24 h of culture, inhibition of uterine incorporation was no longer apparent. The loss of inhibition was due to a change in the activity of the flushings and not to a change in the ability of blastocysts to respond to the inhibitory influence. The inhibition of [3H]uridine incorporation was maintained for at least 24 h when blastocysts were transferred every 6 h to fresh uterine flushings.

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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

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P. QUINN and R. G. WALES

Summary.

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.

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P. QUINN and R. G. WALES

Summary.

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.

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P. QUINN and R. G. WALES

Summary.

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.

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P. QUINN and R. G. WALES

Summary.

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.

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P. J. QUINN and I. G. WHITE

Summary.

Cold-shocking and deep-freezing bull and ram semen caused an influx of sodium to and an efflux of potassium and magnesium from the spermatozoa. The movement of these cations was accentuated when the semen was diluted 1 : 8 in sodium phosphate buffers. Calcium is actively accumulated in the spermatozoa of these two species after cold-shocking.

No significant changes in the cation content of human, dog, rabbit or fowl spermatozoa were found after cold-shocking. There was no great disturbance of the cation status of human and fowl spermatozoa, even after deep-freezing.

Lecithin prevented calcium accumulation in bull and ram spermatozoa on cold-shocking, and glycerol also had this effect on cold-shocked bull spermatozoa. An egg-yolk-citrate/glycerol-fructose diluent protected bull and ram spermatozoa to a considerable extent from the influx of sodium and the efflux of potassium that occurs after deep-freezing to −79°C.