Total ATP, ATP/ADP ratios, the rates of synthesis and turnover of ATP, and the level of cyanide inhibition of ATP synthesis were determined for 2-cell, 4-cell, 8-cell, late-morula and late-blastocyst mouse embryos. The results show that from the 2-cell stage to the late-blastocyst stage there are progressive decreases in total ATP and in the ATP/ADP ratios. These are accompanied by increases in the rates of ATP turnover as well as in the percentage of inhibition of ATP synthesis by cyanide. These data are discussed in relation to results from other metabolic studies on mouse cleavage-staged embryos and from studies describing configurational changes in the ultrastructure of mitochondria at these developmental stages. It is postulated that the mitochondrial ultrastructural changes during cleavage reflect differences in the levels of oxidative phosphorylation during specific metabolic steady states.

Numerous studies have indicated increasing synthetic activity in progressively older cleavage-staged mouse embryos. It has been shown by autoradiographic studies (Mintz, 1964) that protein synthesis occurs in mouse cleavage-staged embryos. Using biochemical techniques, it has been demonstrated that this synthesis is in progress as early as the 2-cell stage and that there are significant rate increases occurring between each of the later cleavage stages (Tasca & Hillman, 1967, 1970). Additionally, both light-(Mintz, 1964; Monesi & Salfi, 1967) and electron-microscopy autoradiographic studies (Hillman & Tasca, 1969) have demonstrated increasing RNA synthesis during the preimplantation stages. RNA synthesis, like protein synthesis, begins as early as the 2-cell stage with a threefold increase in total synthesis occurring between the late 2-cell and 8-to 16-cell stages and a fourfold increase between the 8-16 and morula stage (Tasca & Hillman, 1970). Several other studies have defined the specific types of RNA being synthesized at these early embryonic stages (Ellem & Gwatkin, 1968; Woodland & Graham, 1969; Piko, 1970; Hillman & Tasca, 1969; Tasca & Hillman, 1970; Daentl & Epstein, 1971; Epstein & Daentl, 1971; Knowland & Graham, 1972). All of the metabolic studies to date therefore indicate increasing synthetic rates of both nucleic acids and protein. Although both of these syntheses are energy-requiring processes, there have been no direct studies on energy generating systems in mouse embryos. It has been suggested, on the basis of 14CO2 production (Brinster, 1967) and O2 consumption (Mills & Brinster, 1967), that the TCA cycle is functional at all cleavage stages, its activity increasing at the later cleavage stages. Also, Thomson (1967) found that relatively low concentrations of two respiratory chain inhibitors (cyanide and 2,4-dinitrophenol) blocked development of 2-cell embryos, indicating the necessity of activity of the cytochrome system at this early cleavage stage. These observations suggest that oxidative phosphorylation is occurring by way of the TCA cycle and the cytochrome system during the preimplantation stages. It is feasible therefore that the increased respiration in mouse embryos, as noted by Brinster, is associated with increasing cytochrome activity, producing increased amounts of high-energy-bonded ATP, a main energy source for cellular synthetic processes. The rate of ATP synthesis via the cytochrome system is determined by the rate and direction of electron transport, which in turn is controlled either directly (Chance & Hagihara, 1961) or indirectly (Atkinson, 1965, 1966) by the ATP/ADP+ P ratio. A high total ATP and ATP/ADP ratio is related to a lowenergy state (i.e. low levels of cytochrome activity and low levels of ATP synthesis) whereas reverse conditions, low ATP and ATP/ADP ratios, result in high electron transport activity and high rates of ATP synthesis. Therefore one would expect the increased respiration and increased macromolecular synthesis to be associated with decreasing total ATP and ATP/ADP ratios together with increasing ATP synthesis and turnover. The present investigation was undertaken to determine: (1) total ATP and ATP/ADP ratios during successive cleavage stages; (2) rates of ATP synthesis and turnover during the preimplantation stages, a period of increasing synthetic processes; and (3) the efficiency of electron transport at all cleavage stages.

Supply and culture of embryos

The mice used in the present experiments were from a randomly breeding closed colony of Swiss Albino mice. Eightto ten-week-old female mice were superovulated by intraperitoneal injections of 10 i.u. of pregnant mare serum gonadotropin (PMSG, Ayerst), followed 48 h later by 10 i.u. human chorionic gonadotropin (HCG, Organon) (Edwards & Gates, 1959). Following the second injection, each female was placed with a single male overnight. The females were checked for the presence of copulation plugs the next morning and the pregnant females designated as being in day 0 of pregnancy. Twenty-four hours later, 2-cell embryos were flushed from the oviducts with Brinster’s medium (BMOC-3, 1970 modification, Grand Island Biological Company). These embryos were either assayed immediately for ATP or were placed into culture (Brinster, 1963) until they reached the desired cleavage stage (4-cell, 8-cell, late morula (LM), late blastocyst (LB)). The embryos developed normally up to the late blastocyst stage, prior to the time of hatching from the zona pellucida. Other embryos were allowed to develop in vivo, removed at specific times and their stage of development determined. There appeared to be no difference in the rate of cleavage, determined by cell count, between those embryos developing in vitro and those developing in vivo.

Total ATP content

Fifty embryos were removed from culture at random times during each cleavage stage and assayed for total ATP content using luciferin-luciferase assay. The protocol followed for this assay was a modification of two previously published methods (Epel, 1969; Stanley & Williams, 1969). Each group of embryos was collected in 10 μ1. Brinster’ s medium, in 0·4 ml centrifuge tubes, and 20 μ I cold 0·5 M perchloric acid (PCA) added. The contents were frozen, at −70°C, and thawed twice, and allowed to stand for 20 min at 4°C. Each tube’s contents were neutralized by adding 10 μ I of a solution of 4M-K2CO3 and 1 M triethanolamine (TEA) buffer pH 8·0 (1:2·5, v/v), which precipitated the PCA as a potassium salt. (The presence of PCA in the solution interferes with the assay for ATP (Stanley & Williams, 1969).) The tube was centrifuged at 20000g for 10 min, and the supernatant fraction added to 1 ml of 0·05 M TEA buffer (pH 7·4) in a scintillation vial. One hundred μ I of luciferin-luciferase solution (Sigma) was added to the vial and counted immediately on a Packard Tricarb 3380 liquid scintillation counter preset for 3H. Each vial was counted four times, for 6 sec each time. The second reading was used in determining total ATP content (Stanley & Williams, 1969). To obtain a calibration curve, a series of known quantities (pmoles) of ATP were treated and counted concurrently with the extracted embryonic ATP. In this way pmoles of embryonic ATP could be determined for each embryonic stage. A minimum of six determinations was made for each preimplantation stage. A luciferin-luciferase assay of Brinster’s medium showed no detectable ATP content.

ATP/ADP ratio

Total ATP was determined by luciferin before and after the addition of phosphoenolpyruvate and pyruvate kinase (PK) to staged embryos. Pyruvate kinase in the presence of Mg2+ converts phosphoenolpyruvate and ADP to pyruvate and ATP, the conversion of ADP to ATP occurring in a 1:1 ratio (Conn & Stumpf, 1966). Thirty embryos at each stage were homogenized in 0·5 M PCA, and the homogenate neutralized as for total ATP content. Twenty μ I of 0·045 M phosphoenolpyruvate and 10 μ I of a solution containing 300 i.u. of pyruvate kinase (Calbiochem), 0·05 M TEA buffer, pH 7·4, and 0·01M-MgCl2 were added to the centrifuge tube. The tube was allowed to stand at room temperature for 15 min, then centrifuged at 20000 # for 10 min. The supernatant solution was added to a scintillation vial containing 1 ml of 0·05 M TEA buffer, pH 7·4, and heated to 80°C for 10 min to stop the enzymic reaction. The vial was cooled to room temperature and the contents assayed for total ATP by the luciferin method. The number of counts in each sample was compared to similarly prepared samples of staged embryos without pyruvate kinase. The difference in the number of counts between the two samples gave the pmoles of ADP per embryo converted to ATP. The pyruvate kinase reaction favors the production of pyruvate from phosphoenolpyruvate. The reverse reaction is extremely small especially when the phosphoenolpyruvate and PK is in excess, the case of the present procedure. The limiting factor becomes the amount of ADP in the sample.

Labelling of embryos with32PO4

Thirty to 50 staged embryos were incubated for varying lengths of time (see specific procedures below) in 0·05 ml Brinster’s medium containing approximately 150000 counts 32P-labelled H2PO4 (HCl-free) per 10 μ1 of medium. The 32P was obtained from ICN (specific activity, 285 Ci/mg) and was diluted with medium to the appropriate concentration. The exact counts in the medium were determined by spotting a 10 μ1 aliquot of medium on a piece of filter paper which was placed into a scintillation vial and then dried. Ten ml of toluenebased Omnifluor scintillation fluid was added and the sample counted.

Calculation of pmoles of AT32P and 32PCf

  1. AT32P. The conversion of counts in a sample to pmoles of AT32P was calculated according to the formula of Francis, Mulligan & Wormall (1959):

The molar concentration of inorganic phosphate in radioactive Brinster’s medium was determined by the assay method of Lowry & López (1946). The corrected counts (per emb./h) of the sample were found by subtracting background from the raw counts and dividing by the number of embryos per sample. The background for all studies of AT32P was obtained by counting only scintillation fluid.

(2) 32PO4. A time-course study was done to determine when 32PO4 reached equilibrium within the embryos. For these studies, staged embryos were incubated in 32PO4 medium for 60 min. Samples of 50 embryos were removed, at 15 min intervals, from the radioactive medium. The embryos were then quickly washed four times, collected in 10 μ 1 of the final wash, placed into an 80°C oven and dried. The pmoles of embryonic phosphate were calculated from the sample counts minus background using equations (1) and (2).

Gross synthesis of ATP

Embryos were incubated in 32PO 4-containing medium for 90 min, removing approximately 50 embryos at 15 min intervals. Following the desired labelling period, each group of embryos was quickly washed, collected in 10 μ of nonradioactive medium and placed into a 0·4 ml centrifuge tube. Ten μ 1 cold PCA was added to each tube, the contents mixed on a Vortex, and then allowed to stand at 4°C for 20 min. Five μ 1 of 0-01 M carrier ATP was added, the contents twice frozen at -70°C and thawed, and again mixed on a Vortex for 20 sec. The tubes were centrifuged for 10 min at 20000 g, the supernatant solutions were spotted on PEI-cellulose TLC plates and chromatographed two-dimensionally (Randerath & Randerath, 1967). The spots, identified by the fluorescence of the carrier ATP, were cut out, placed into a scintillation vial containing 10 μ l of scintillation fluid, and counted. Background was obtained by counting a vial containing only scintillation fluid. To serve as controls, either 10 μ1 of the last wash or 10 μ1 of the radioactive medium was added to 5 μ1 of ATP carrier and chromatographed. In these controls the numbers of counts in

ATP turnover

Approximately 100 embryos at each cleavage stage were incubated in radioactive medium for 1 h as described above. Following this incubation the embryos were quickly washed. One-half of the embryos were collected in 10 μ1 medium and were immediately added to 10[A 0·5M cold PCA. The other half of the sample was reincubated in non-radioactive medium for an additional 10 min, collected in 10 μ 1 of medium and added to 10 μ of 0·5M PCA. The ATP was extracted from both groups of embryos and chromatographed as described above. The difference between the numbers of counts in the two samples, before and after reincubation, was used to determine the uncorrected rate of ATP turnover at each developmental stage.

Net synthesis of ATP

To determine the net synthesis of ATP the pmoles of ATP accumulated after a 1 h labelling period were corrected for the loss of counts resulting from the turnover of labelled ATP during this same time period. The reaction for the production of ATP can be represented by the equation

The rates of synthesis (Lx) and turnover (Æ2) of ATP can be calculated from the equations

Equations (4), (5) and (6) (Francis, Mulligan & Wormall, 1959) were modified for use in the present studies. In (5), x represents the gross amount of ATP remaining after a 10 min chase. The natural log of this number is multiplied by 6 to convert the 10 min reading to 1 h. The k2from (5) is used to calculate the rate of synthesis (k1 in (6), where y is the rate of gross synthesis of ATP in one hour. To compare k2 with k1k2 has to be multiplied by the total ATP determined by luciferin assay. These equations are valid provided that the total embryonic ATP and phosphate remain relatively constant during the 1 h treatment, and that there is no great lag in the transport of 32PO4 in or out of the cell. These requirements are met in mouse embryos (see Results below).

Total ATP content and gross synthesis of ATP in the presence of cyanide

Embryos at each cleavage stage were incubated either in non-radioactive medium containing 10−4M cyanide for 10 min or in radioactive medium containing 10−4M cyanide for 60 min. This concentration of cyanide has been shown to be the LD50 dosage after 24 h incubation at the 2-cell stage (Thomson, 1967). Following incubation, the total ATP of the non-labelled embryos was determined by using the luciferin-luciferase method, while the effect of cyanide on gross ATP synthesis in the labelled sample was determined using the technique described above.

Statistical studies

Standard errors of the mean are included in the text where applicable. Significant differences between means (P<0·05) were determined by the Student t-test.

Total ATP

Two-cell embryos contain the highest content of ATP (Fig. 1). Total ATP per embryo decreases significantly at each successive cleavage stage with the greatest absolute decrease occurring between the 2-cell (1·47 ± 0·07 (s.E.) pmoles) and 4-cell (0·99 ± 0·01 pmoles) stages. Lesser but significant decreases occur between later developmental stages: between the 8-cell (0·91 ± 0·01 pmoles) and late morula (0·65 ± 0·01 pmoles) and between the late morula and late blastocyst (0·43 ± 0·01 pmoles) stages. The smallest decrease, in both absolute and percentage amounts, occurs between the 4and 8-cell stages.

Fig. 1

Changes in the total ATP level during early development. At each cleavage stage total ATP was assayed by luciferin (○ —○). The effect of cyanide on total ATP was determined after incubating embryos in 10−4M cyanide for 10 min before being assayed by luciferin (×— × ). LM = late morula; LB = late blastocyst.

Fig. 1

Changes in the total ATP level during early development. At each cleavage stage total ATP was assayed by luciferin (○ —○). The effect of cyanide on total ATP was determined after incubating embryos in 10−4M cyanide for 10 min before being assayed by luciferin (×— × ). LM = late morula; LB = late blastocyst.

Effect of cyanide on total ATP

A 10 min treatment with 10−4M cyanide results in a pattern of increasing loss of total ATP, at each older cleavage stage up to but not including the late blastocyst stage when the loss appears to level off (Fig. 1). There is a slight reduction in total ATP at the 2-cell stage (from T47 ± 0·07 to T37 ± 0-06 pmoles), a 40% loss at 4-cell (from 0-99 ± 0·01 to 0·59 ± 0·01 pmoles), a 42% decrease at the 8-cell (from 0·91 ± 0·01 to 0·53 ± 0·02 pmoles), a 51% decrease at late morula (from 0·65 ± 0·01 to 0·32 ± 0·02 pmoles), and a 26% loss at the late blastocyst stage (from 0·43 ± 0·01 to 0·32 ± 0·02 pmoles).

ATP/ADP ratio

There is a progressive decrease in the ATP/ADP ratio as the embryo develops from the 2-cell to the late blastocyst stage (Fig. 2). A 2-cell stage embryo has 10·5 + 0·54 times more ATP than ADP, whereas the 4-cell embryo contains only 5·8 ± 0·29 times more ATP than ADP. Although there is only a small decrease in total ATP between the 4- and 8-cell stages (Fig. 1), the ATP/ADP ratio drops from 5-8 ± 0·29 to 2·5 ± 0·14 reflecting a twofold increase in ADP between these two stages. At the late morula stage the ATP/ADP ratio decreases to 1·8 ± 0·10, and at the late blastocyst stage to 1·3 ± 0·06.

Fig. 2

ATP/ADP ratios in preimplantation mouse embryos. Total ATP was measured with luciferin in comparable samples before and after pyruvate kinase treatment. The increase in ATP after enzyme action reflects the amount of ADP converted to ATP.

Fig. 2

ATP/ADP ratios in preimplantation mouse embryos. Total ATP was measured with luciferin in comparable samples before and after pyruvate kinase treatment. The increase in ATP after enzyme action reflects the amount of ADP converted to ATP.

Equilibrium of’A2PO4

Because of the size of the cleavage-staged mouse embryos it is extremely difficult to directly measure inorganic phosphate pool size by any established method. The pool size can, however, be estimated by determining the equilibrium level of 32PO4 at each stage. Embryonic 32PO4 reaches equilibrium after 30 min incubation in 32PO4 containing medium. The time needed to reach equilibrium is the same for all preimplantation stages. This equilibrium level is maintained for at least 30 additional minutes under culture conditions. The 2-cell stage reaches equilibrium at 1·22 pmoles of phosphate and remains at this level through the 4-cell stage (Fig. 3). The accumulation increases and equilibrates at 4*5 and 6·5 pmoles of 32PO4 in the 8-cell and morula stages respectively (Fig. 3). By the late blastocyst stage the equilibrium level drops to 2·0 pmoles 32PO4.

Fig. 3

Equilibrium of 32PO< in cleavage-staged embryos.

Fig. 3

Equilibrium of 32PO< in cleavage-staged embryos.

Gross synthesis of ATP

Embryos at each preimplantation stage were incubated for varying time periods in 32PO4 containing medium and were then assayed for the incorporation of radioactivity into AT32P (Fig. 4). The data from this time course study show that there is no significant incorporation into ATP for the first 15 min of incubation at any cleavage stage. Equilibrium of 32PO4 incorporation into ATP is reached after 60 min of incubation at every stage examined. The concentration reached at 60 min is constant for at least an additional 30 min. In order to determine whether the long labelling periods necessary for AT32P accumulation to reach equilibrium were detrimental to development, embryos at each stage were incubated for 90 min in radioactive medium and reincubated in nonradioactive medium. There was no effect of the radioactivity on the further development of these embryos up to hatching from the zona pellucida regardless of the stage at which the embryos were treated.

Fig. 4

Time course study of 32PO4 into ATP. Fifty staged embryos were incubated for varying lengths of time in 32PO4 medium. The ATP was extracted and chromatographed two-dimensionally on PEI-cellulose TLC. The ATP carrier spot was cut out and counted. The counts were converted to pmoles ATP/emb./h. ○ –○,2-cell stage; ×. –×, 4-cell stage; △ –△, late morula stage; □–□, late blastocyst stage

Fig. 4

Time course study of 32PO4 into ATP. Fifty staged embryos were incubated for varying lengths of time in 32PO4 medium. The ATP was extracted and chromatographed two-dimensionally on PEI-cellulose TLC. The ATP carrier spot was cut out and counted. The counts were converted to pmoles ATP/emb./h. ○ –○,2-cell stage; ×. –×, 4-cell stage; △ –△, late morula stage; □–□, late blastocyst stage

Because a plateau of the accumulation of the isotope into ATP is reached at 60 min, this length of incubation time in radioactive medium was used for all cleavage-staged embryos in determining the gross synthesis of ATP. Each stage was assayed at least five times, and the cpm were converted to pmoles ATP/ emb./h (Fig. 5). There are significant increases in the gross synthesis of ATP between the 2-cell, 4-cell and 8-cell embryos, as well as between the late morula and late blastocyst stages. A significant decrease in gross synthesis is found between the 8-cell and late morula stage. There is a 55% increase from the 2-cell (0·22 ± 0·01 pmoles) to the 4-cell (0·49 ± 0·02 pmoles) and a 22% increase between the latter and the 8-cell (0·63 ± 0·06 pmoles) stage in ATP accumulated after 1 h. Between the 8-cell and the late morula, the gross synthesis of ATP decreases from 0·63 ± 0·06 to 0·27 ± 0·01 pmoles per embryo, a decrease of 57%. The gross synthesis increases from 0·27 ± 0·01 to 0·34 ± 0·01 pmoles by the late blastocyst stage, an increase of 21%.

Fig. 5

The gross synthesis and turnover of AT32P. After 1 h incubation in 32PO4 medium, samples of embryos were removed, ATP extracted, and separated by two-dimensional chromatography (○ –○). Additional samples were reincubated in cold medium for 10 min to determine the rate of gross turnover ( × –× ).

Fig. 5

The gross synthesis and turnover of AT32P. After 1 h incubation in 32PO4 medium, samples of embryos were removed, ATP extracted, and separated by two-dimensional chromatography (○ –○). Additional samples were reincubated in cold medium for 10 min to determine the rate of gross turnover ( × –× ).

Turnover of ATP

Fig. 5 also shows the amount of accumulated AT32P remaining in embryos which were incubated in radioactive medium for 1 h and then reincubated in cold medium for an additional 10 min. At each successive cleavage stage there is a progressive increase in the percentage of ATP turnover, i.e. the amount of AT32P present in embryos following reincubation when compared with the gross amount of AT32P in similarly staged embryos prior to reincubation. During the 10 min reincubation period there is no loss of accumulated AT32P at the 2-cell stage, a 10% loss (from 0·49 ± 0·02 to 0·44 ± 0·01 pmoles) at the 4-cell stage, a 19% loss (from 0·63 ± 0·06 to 0·51 ± 0·03 pmoles) at the 8-cell, a 30% loss (from 0·27 ± 0*01 to 0·19 ± 0·01 pmoles) at the late morula stage, and a 47% loss (from 0·34 ± 0·01 to 0-18 ± 0·01 pmoles) at the late blastocyst stage.

Net synthesis and turnover rates of ATP

Data from measurements of 1 h accumulation levels and 10 min turnover values (Fig. 5), were used to calculate the rates of net synthesis (k-1 and rates of turnover (kf) (see Materials and Methods). The net rates of synthesis increases by threefold between the 2- and 4-cell stages (from 0-22 to 0-66 pmoles), and by twofold between the 4- (0-66 pmoles) and 8-cell (1-11 pmoles) stages (Fig. 6). There is a reduction from the 8-cell (Ml pmoles) to the late morula (0-70 pmoles), followed by a large increase between the late morula and late blastocyst (1-38 pmoles) stages. The turnover rate, k2, of ATP increases significantly at successive cleavage stages (Fig. 6). Only at the 2-cell is there no detectable turnover of ATP in 60 min. At the 4- and 8-cell stages the respective kt and k2 values are equivalent. Later stages, late morula and late blastocyst, have a higher rate of turnover (k2,) than synthesis (k1,).

Fig. 6

Changes in net synthesis (ÆJ and net turnover (Æ2) of ATP during early development. Values were calculated from the levels of gross synthesis, turnover, and total ATP (○ – ○, k1, ×–×, k2,). △ – △, Effect of cyanide on ATP synthesis (k1,).

Fig. 6

Changes in net synthesis (ÆJ and net turnover (Æ2) of ATP during early development. Values were calculated from the levels of gross synthesis, turnover, and total ATP (○ – ○, k1, ×–×, k2,). △ – △, Effect of cyanide on ATP synthesis (k1,).

Inhibitory effect of cyanide on ATP

The effect of cyanide on both the net synthesis of ATP and total ATP (Fig. 1) indicates the level of activity of oxidative phosphorylation during early development. Incubation of embryos for 60 min in radioactive medium containing 10 4M cyanide caused a reduction in k2values of 23% (from 0·22 to 0·17 pmoles) at the 2-cell, 65% (from 0·66 to 0·23 pmoles) at 4-cell, 69% (from 1·11 to 0·34 pmoles) at 8-cell and 73% (0·70 to 019 pmoles) at the morula stage, the oldest stage studied (Fig. 6).

The present studies show that 2-cell mouse embryos are characterized by a high total ATP content, a high ATP/ADP ratio and a low level of both net synthesis and turnover of ATP when compared with embryos of later cleavage stages. Beginning at the 4-cell stage, there is a decrease in total ATP and the ATP/ADP ratio, together with increased synthesis and turnover of ATP. Embryos at each successively later cleavage stage contain progressively less total ATP and lower ATP/ADP ratios. At the 8-cell stage the net turnover and net synthesis of ATP are equivalent, whereas at both the late morula and late blastocyst, net turnover exceeds net synthesis. The differences between the k1and k2 values at these latter stages most likely reflect the increasing usage of AT32P in macromolecular synthesis. The amount of AT32P used in these syntheses cannot be determined nor included in the k1 value of AT32P using the present techniques, but would, however, be reflected in the turnover (k2) rates. The turnover rates at the later stages are therefore a better measure of synthesis than are the k1 values. The k1 values thus show increased amounts of ATP synthesis at each successive cleavage stage. Preliminary studies on gross ATP synthesis and turnover have recently been published (Ginsberg & Hillman, 1972). Although there have been no reported studies on rates of ATP synthesis and turnover, or ATP/ADP ratios in mouse embryos, there are two earlier studies reporting luciferin assayed values for total ATP content in cleavage-staged embryos. Our observations agree with the data of Quinn & Wales (1971), who measured total ATP in 2-cell, 8-cell, morula, and both early and late blastocyst mouse embryos. Total ATP was not determined for 4-cell embryos. Although the values for total ATP in the present report are slightly higher than the values reported by Quinn & Wales for correspondingly staged embryos, both reports agree that there are progressive decreases at each successive cleavage stage. Both of these reports disagree with that of Epstein & Daentl (1971), who found no change in total ATP between the 2-cell and late morula stages. It is likely that differences in protocols, such as the removal or non-removal of PCA prior to counting, could result in the discrepancies found among these three reports.

The present data show that the synthesis of ATP is slightly inhibited by cyanide as early as the 2-cell stage and is inhibited in increasingly greater amounts at the older cleavage stages. Since cyanide blocks electron transport at cytochrome a3, these findings indicate that ATP is being synthesized by oxidative phosphorylation and they suggest that the activity of the cytochrome system increases at each cleavage stage. It has been shown that the degree of activity of the cytochromes is directly determined by the relative ratios of mitochondrial ATP, ADP and PO4 (Chance & Hagihara, 1961) and that the cytoplasmic ATP, ADP and PO4 relative ratios regulate the activity of the cytochrome system by controlling the activity of specific regulatory enzymes (Atkinson, 1965). In the present study, the measured total ATP and ATP/ADP ratios reflect the total nucleotide level in the cell, and it is assumed that the levels in the cytoplasm and mitochondria are directly proportional.

The observed increase in ATP synthesis and turnover as well as the observed increased inhibition of ATP synthesis by cyanide and the decreased ATP/ADP ratios were predictable, therefore, in light of the studies of Mills & Brinster (1967) and Brinster (1967), who found that O2 and 14CO2 production from glucose consumption increased in mouse embryos at each successive cleavage stage. Brinster further found (1967) that most of this CO2 was produced in the TCA cycle. From these combined data, Brinster suggested that the TCA cycle was functional and showed increased activity as the embryos advanced from one cleavage stage to another. In support of this hypothesis were the findings of both Brinster (1966) and Epstein, Wegienka & Smith (1969), who reported that malic dehydrogenase (presumably mitochrondrial malic dehydrogenase) activity increased significantly at the 8-cell stage. Additionally, Kramen & Biggers (1971) noted a marked increase in the permeability of cell membranes to TCA intermediates at the 4-cell stage, with increasing rates of uptake in the progressively older cleavage stages.

The results of the present studies, together with those presented above, are evidence for an increase in the activities of both the TCA cycle and the electrontransport mechanism during mouse preimplantation stages. It is probable that these increases can be correlated with the mitochondrial configurational changes which occur in mouse cleavage-staged embryos (Hillman and Tasca, 1969; Stern, Biggers & Anderson, 1971). Piko & Chase (1971) have found that chloramphenicol treatment of cleavage-staged mouse embryos does not stop these mitochondrial transformations. In an earlier study Piko (1970) noted that uniformly labelled [14C]thymidine is incorporated into nuclear DNA but not into mitochondrial DNA, during cleavage. Thus, the normal sequential differentiation does not involve de novo formation of the different-appearing mitochondria but merely the reorganization of the cristae of existing mitochondria. Hackenbrock (1966) has described four basic types of mitochondrial ultrastructural configurations: condensed, intermediate, swollen and orthodox. Either of the extremes of these mitochondria - condensed or orthodox-are present in cells or in a population of isolated mitochondria, depending upon the metabolic steady state of these organelles. Chance & Williams (1955,1956) have defined the criteria of five metabolic steady states. State I has low substrate and O2 level, slow respiratory rate but an O2 consumption level greater than zero; State II is like State I except it has a high substrate level; State III again has an O2 level greater than zero, but has both high substrate and ADP levels and the respiratory rate is fast; State IV has a high substrate level, low ADP level, slow respiratory rate and again an O2 level greater than zero; State V is present under anaerobiosis in which substrate and ATP levels are both high but O2 consumption and respiration rates are equal to zero. Hackenbrock (1966, 1968, 1972) and Hackenbrock, Rehn, Weinbach & LeMasters (1971) have suggested that the ultrastructural configuration of the mitochondria can be correlated with the metabolic steady state of either isolated mitochondria or of mitochondria within intact cells. Condensed mitochondria are described as being electron-dense, and the inner membranes are irregularly folded (similar to those found in 2-cell mouse embryos), whereas orthodox mitochondria contain a much less dense matrix and regularly arranged cristae (similar to those found in later cleavage-staged embryos). In an experiment in which Hackenbrock (1966) placed isolated mitochondria under different but specific sets of conditions for any one of the five metabolic steady states, he found that condensed mitochondria were initially found under those sets of conditions which produced States I, II, III, and IV metabolism. Upon the utilization of endogenous substrate or upon the addition of either substrate and/or ADP, mitochondria underwent oxidative phosphorylation and their configuration changed from condensed to orthodox with only one exception. Under State II conditions, mitochondria were condensed and remained condensed or became intermediate between condensed and orthodox upon the addition of ADP and completion of oxidative phosphorylation.

If one subscribes to the hypothesis that mitochondrial configuration reflects the steady state of metabolism, and specifically the occurrence of oxidative phosphorylation or electron transport within that state, then it follows that the mitochondrial configurational changes which occur within or between cleavage stages can also be correlated with different steady states of metabolism of the embryo. For instance, at the 2-cell stage, when the mitochondria are condensed, total ATP and the ATP/ADP ratio are high, there is little inhibition of ATP synthesis (k1) by cyanide, and O2 consumption (Mills & Brinster, 1967) and 14CO2 production are low (Brinster, 1967), suggesting State I metabolism. Since ADP is low at this state, active phosphorylation would not occur and consequently the mitochondria would not change to the orthodox configuration. At the 4-cell stage, the majority of mitochondria are condensed but there are, at the late 4-cell, some mitochondria with a more orthodox appearance. At this stage, the ATP level as well as the ATP/ADP ratio is lower than at the 2-cell stage. The increased relative amount of ADP could shift the metabolism from State I to State II. In the latter metabolic state the mitochondria are condensed and only partially transform following oxidative phosphorylation. This incomplete transformation could be correlated with the appearance of the few more orthodox appearing mitochondria which are present at the late 4-cell stage. At the late 4-cell and 8-cell stages, the embryos show greater permeability to TCA intermediates (Kramen & Biggers, 1971), suggesting increased substrate levels, and at the 8-cell, ATP is lower, ADP higher and O2 consumption (Mills & Brinster, 1967) greater than at the earlier developmental stages. These conditions are basic for a shift from State II to State III metabolism. The condensed populations of mitochondria in 8-cell and older embryos could indicate State III metabolism alone or could be present together with State IV metabolism, oscillating between the two states within each mitochondrion. State III would be present in those mitochondria with a relatively low ATP/ADP ratio and, upon the phosphorylation of all available ADP, enter State IV. This oscillation between steady states would result in an oscillation between condensed and orthodox configurations and would account for the mixed population of mitochondrial forms found in the older cleavage-staged embryos. Although it is not possible to determine continuing configurational changes by examining fixed sections of embryos at the ultrastructural level, it is possible to show whether or not the embryos do have different steady states of metabolism at different cleavage stages. Each steady state reduces pyridine nucleotides and consumes O2 in specific relative patterns. It is necessary therefore to determine the level of reduction of nucleotides at each cleavage stage, and relate these to the amount of oxygen consumption at corresponding stages in order to determine definitely whether the embryos do in fact demonstrate different metabolic steady states at the different preimplantation stages. These studies are now in progress.

The research was supported by U.S. Public Health Research Grant HD-00827.

Atkinson
,
D. E.
(
1965
).
Biological feedback control at the molecular level
.
Science, N.Y
.
151
,
851
857
.
Atkinson
,
D. E.
(
1966
).
Regulation of enzyme activity
.
A. Rev. Biochem
.
35
,
85
123
.
Brinster
,
R. L.
(
1963
).
A method for in vitro cultivation of mouse ova from two-cell to blastocyst
.
Expl Cell Res
.
32
,
205
207
.
Brinster
,
R. L.
(
1966
).
Malic dehydrogenase activity in the preimplantation mouse embryo
.
Expl Cell Res
.
43
,
131
135
.
Brinster
,
R. L.
(
1967
).
Carbon dioxide production from glucose by the preimplantation mouse embryo
.
Expl Cell Res
.
47
,
271
277
.
Chance
,
B.
&
Hagihara
,
B.
(
1961
).
Direct spectroscopic measurements of interaction of components of the respiratory chain with ATP, ADP, phosphate and uncoupling agents
.
Proc. 5th Int. Congr. Biochem., Moscow
5
,
3
32
.
Chance
,
B.
&
Williams
,
G. R.
(
1955
).
Respiratory enzymes in oxidative phosphorylation
.
J. biol. Chem
.
217
,
409
427
.
Chance
,
B.
&
Williams
,
G. R.
(
1956
).
The respiratory chain and oxidative phosphorylation
.
Adv. Enzymol
.
17
,
65
134
.
Conn
,
E. E.
&
Stumpf
,
P. K.
(
1966
).
Outlines of Biochemistry
, 2nd ed.
New York
:
John Wiley
.
Daentl
,
D. L.
&
Epstein
,
C. J.
(
1971
).
Developmental interrelationships of uridine uptake, nucleotide formation and incorporation into RNA by early mammalian embryos
.
Devi Biol
.
24
,
428
442
.
Edwards
,
R. G.
&
Gates
,
A. H.
(
1959
).
Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotropins
.
J. Endocr
.
18
,
292
304
.
Ellem
,
K. A. O.
&
Gwatkin
,
R. B. L.
(
1968
).
Patterns of nucleic acid synthesis in the early mouse embryo
.
Devi Biol
.
18
,
311
330
.
Epel
,
D.
(
1969
).
Does ADP regulate respiration following fertilization of sea urchin eggs?
Expl Cell Res
.
58
,
312
319
.
Epstein
,
C. J.
&
Daentl
,
D. L.
(
1971
).
Presursor pools and RNA synthesis in preimplantation mouse embryos
.
Devi Biol
.
26
,
517
524
.
Epstein
,
C. J.
,
Wegienka
,
E. A.
&
Smith
,
C. W.
(
1969
).
Biochemical development of preimplantation mouse embryos: In vivo activities of fructose 1,6-diphosphate aldolase, glucose 6-phosphate dehydrogenase, malate dehydrogenase, and lactate dehydrogenase
.
Biochem. Genet
.
3
,
271
281
.
Francis
,
G. E.
,
Mulligan
,
W.
&
Wormall
,
A.
(
1959
).
Isotopic Tracers
, 2nded.
University of London: Athlone Press
.
Ginsberg
,
L.
&
Hillman
,
N.
(
1972
).
ATP synthesis in normal and mutant cleavage-staged mouse embryos
.
Genetics
71
,
Suppl. 3
,
S 20
.
Hackenbrock
,
C. R.
(
1966
).
Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria
.
J. Cell Biol
.
30
,
269
297
.
Hackenbrock
,
C. R.
(
1968
).
Ultrastructural bases for metabolically linked mechanical activity in mitochondria. IL Electron transport-linked ultrastructural transformations in mitochondria
.
J. Cell Biol
.
37
,
345
369
.
Hackenbrock
,
C. R.
(
1972
).
Energy-linked ultrastructural transformations in isolated liver mitochondria and mitoplasts
.
J. Cell Biol
.
53
,
450
465
.
Hackenbrock
,
C. R.
,
Rehn
,
T. G.
,
Weinbach
,
E. C.
&
Lemasters
,
J. J.
(
1971
).
Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell
.
J. Cell Biol
.
51
,
123
137
.
Hillman
,
N.
&
Tasca
,
R.
(
1969
).
Ultrastructural and autoradiographic studies of mouse cleavage stages
.
Am. J. Anat
.
126
,
151
174
.
Knowland
,
J.
&
Graham
,
C.
(
1972
).
RNA synthesis at the two-cell stage of mouse development
.
J. Embryol. exp. Morph
.
27
,
167
176
.
Kramen
,
M. A.
&
Biggers
,
J. D.
(
1971
).
Uptake of tricarboxylic acid cycle intermediates by preimplantation mouse embryos in vitro
.
Proc. natn. Acad. Sci. U.S.A
.
68
,
2656
2659
.
Lowry
,
O. H.
&
López
,
J. A.
(
1946
).
The determination of inorganic phosphate in the presence of labile phosphate esters
.
J. biol. Chem
.
162
,
421
428
.
Mills
, Jr.,
R. M.
&
Brinster
,
R. L.
(
1967
).
Oxygen consumption of preimplantation mouse embryos
.
Expl Cell Res
.
47
,
337
344
.
Mintz
,
B.
(
1964
).
Synthetic processes and early development in the mammalian egg
.
J. exp. Zool
.
157
,
85
100
.
Monesi
,
V.
&
Salfi
,
V.
(
1967
).
Macromolecular synthesis during early development in the mouse embryo
.
Expl Cell Res
.
46
,
632
635
.
Piko
,
L.
(
1970
).
Synthesis of macromolecules in early mouse embryos cultured in vitro: RNA, DNA, and a polysaccharide component
.
Devi Biol
.
21
,
257
279
.
Piko
,
L.
&
Chase
,
D.
(
1971
).
Mitochondrial differentiation in the early mouse embryo: the effect of ethidium bromide and chloramphenicol
.
Cell Biol
.
51
,
443
(abstr.).
Quinn
,
P.
&
Wales
,
R. G.
(
1971
).
Adenosine triphosphate content of pre-implantation mouse embryos
.
J. Reprod. Fert
.
25
,
133
135
.
Randerath
,
K.
&
Randerath
,
E.
(
1967
).
Thin-layer separation methods for nucleic acid derivatives
.
Meth. Enzym
.
12
,
323
347
.
Stanley
,
P. E.
&
Williams
,
S. G.
(
1969
).
Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme
.
Analyt. Biochem
.
29
,
381
392
.
Stern
,
S.
,
Biggers
,
J.
&
Anderson
,
E.
(
1971
).
Mitochondria and early development of the mouse
.
J. exp. Zool
.
176
,
179
192
.
Tasca
,
R. J.
&
Hillman
,
N. W.
(
1967
).
Uptake and incorporation of H3-uridine and C14-leucine in preimplantation mouse embryos: Effects of inhibitors on RNA and protein synthesis
.
Am. Zool
.
1
,
170
(Abstr.).
Tasca
,
R. J.
&
Hillman
,
N.
(
1970
).
Effects of actinomycin D and cycloheximide on RNA and protein synthesis in cleavage stage mouse embryos
.
Nature, Lond
.
225
,
1022
1025
.
Thomson
,
J. L.
(
1967
).
Effect of inhibitors of carbohydrate metabolism on the development of preimplantation mouse embryos
.
Expl Cell Res
.
46
,
252
262
.
Woodland
,
H. R.
&
Graham
,
C. F.
(
1969
).
RNA synthesis during early development of the mouse
.
Nature, Lond
.
221
,
327
331
.