atp metabolism in cleavage-staged mouse embryos · the molar concentratio o inorganif nc phosphat...

/ . Embryol. exp. Morph. Vol. 30, J, pp. 267-282, 1973 2 6 7Printed in Great Britain

ATP metabolism in cleavage-stagedmouse embryos

By L. GINSBERG1 AND N. HILLMAN1

From the Department of Biology, Temple University, Philadelphia

SUMMARY

Total ATP, ATP/ADP ratios, the rates of synthesis and turnover of ATP, and the levelof cyanide inhibition of ATP synthesis were determined for 2-cell, 4-cell, 8-cell, late-morulaand late-blastocyst mouse embryos. The results show that from the 2-cell stage to thelate-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 percentageof inhibition of ATP synthesis by cyanide. These data are discussed in relation to results fromother metabolic studies on mouse cleavage-staged embryos and from studies describingconfigurational changes in the ultrastructure of mitochondria at these developmental stages.It is postulated that the mitochondrial ultrastructural changes during cleavage reflectdifferences in the levels of oxidative phosphorylation during specific metabolic steadystates.

INTRODUCTION

Numerous studies have indicated increasing synthetic activity in progressivelyolder cleavage-staged mouse embryos. It has been shown by autoradiographicstudies (Mintz, 1964) that protein synthesis occurs in mouse cleavage-stagedembryos. Using biochemical techniques, it has been demonstrated that thissynthesis is in progress as early as the 2-cell stage and that there are significantrate 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 preimplantationstages. RNA synthesis, like protein synthesis, begins as early as the 2-cell stagewith a threefold increase in total synthesis occurring between the late 2-cell and8- to 16-cell stages and a fourfold increase between the 8-16 and morula stage(Tasca & Hillman, 1970). Several other studies have denned the specific typesof 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).

1 Authors' address: Department of Biology, Temple University, Philadelphia, Pa. 19122,U.S.A.

268 L. GINSBERG AND N. HILLMAN

All of the metabolic studies to date therefore indicate increasing syntheticrates of both nucleic acids and protein. Although both of these syntheses areenergy-requiring processes, there have been no direct studies on energy generatingsystems in mouse embryos. It has been suggested, on the basis of 14CO2 pro-duction (Brinster, 1967) and O2 consumption (Mills & Brinster, 1967), that theTCA cycle is functional at all cleavage stages, its activity increasing at the latercleavage stages. Also, Thomson (1967) found that relatively low concentrationsof two respiratory chain inhibitors (cyanide and 2,4-dinitrophenol) blockeddevelopment of 2-cell embryos, indicating the necessity of activity of the cyto-chrome system at this early cleavage stage. These observations suggest thatoxidative phosphorylation is occurring by way of the TCA cycle and thecytochrome system during the preimplantation stages. It is feasible there-fore that the increased respiration in mouse embryos, as noted by Brinster, isassociated with increasing cytochrome activity, producing increased amountsof high-energy-bonded ATP, a main energy source for cellular syntheticprocesses.

The rate of ATP synthesis via the cytochrome system is determined by therate and direction of electron transport, which in turn is controlled eitherdirectly (Chance & Hagihara, 1961) or indirectly (Atkinson, 1965, 1966) by theATP/ADP + P ratio. A high total ATP and ATP/ADP ratio is related to a low-energy state (i.e. low levels of cytochrome activity and low levels of ATPsynthesis) whereas reverse conditions, low ATP and ATP/ADP ratios, result inhigh electron transport activity and high rates of ATP synthesis. Therefore onewould expect the increased respiration and increased macromolecular synthesisto be associated with decreasing total ATP and ATP/ADP ratios together withincreasing ATP synthesis and turnover.

The present investigation was undertaken to determine: (1) total ATP andATP/ADP ratios during successive cleavage stages; (2) rates of ATP synthesisand turnover during the preimplantation stages, a period of increasing syntheticprocesses; and (3) the efficiency of electron transport at all cleavage stages.

MATERIALS AND METHODS

Supply and culture of embryos

The mice used in the present experiments were from a randomly breedingclosed colony of Swiss Albino mice. Eight- to ten-week-old female mice weresuperovulated by intraperitoneal injections of 10 i.u. of pregnant mare serumgonadotropin (PMSG, Ayerst), followed 48 h later by 10 i.u. human chorionicgonadotropin (HCG, Organon) (Edwards & Gates, 1959). Following the secondinjection, each female was placed with a single male overnight. The femaleswere checked for the presence of copulation plugs the next morning and thepregnant females designated as being in day 0 of pregnancy. Twenty-four hours

ATP metabolism in mouse embryos 269later, 2-cell embryos were flushed from the oviducts with Brinster's medium(BMOC-3, 1970 modification, Grand Island Biological Company). Theseembryos were either assayed immediately for ATP or were placed into culture(Brinster, 1963) until they reached the desired cleavage stage (4-cell, 8-cell, latemorula (LM), late blastocyst (LB)). The embryos developed normally up to thelate blastocyst stage, prior to the time of hatching from the zona pellucida.Other embryos were allowed to develop in vivo, removed at specific times andtheir stage of development determined. There appeared to be no difference inthe rate of cleavage, determined by cell count, between those embryos develop-ing in vitro and those developing in vivo.

Total ATP content

Fifty embryos were removed from culture at random times during eachcleavage stage and assayed for total ATP content using luciferin-luciferaseassay. The protocol followed for this assay was a modification of two previouslypublished methods (Epel, 1969; Stanley & Williams, 1969). Each group ofembryos was collected in 10 [A Brinster's medium, in 0-4 ml centrifuge tubes,and 20 /A cold 0-5M 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'scontents were neutralized by adding 10 /A of a solution of 4 M - K 2 C O 3 and 1 Mtriethanolamine (TEA) buffer pH 8-0 (1:2-5, v/v), which precipitated the PCAas a potassium salt. (The presence of PCA in the solution interferes with theassay for ATP (Stanley & Williams, 1969).) The tube was centrifuged at 20000#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 [A of luciferin-luciferase solution(Sigma) was added to the vial and counted immediately on a Packard Tricarb3380 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 ATPcontent (Stanley & Williams, 1969). To obtain a calibration curve, a series ofknown quantities (pmoles) of ATP were treated and counted concurrently withthe extracted embryonic ATP. In this way pmoles of embryonic ATP could bedetermined for each embryonic stage. A minimum of six determinations wasmade for each preimplantation stage. A luciferin-luciferase assay of Brinster'smedium showed no detectable ATP content.

ATPIADP ratio

Total ATP was determined by luciferin before and after the addition of phos-phoenolpyruvate and pyruvate kinase (PK) to staged embryos. Pyruvate kinasein the presence of Mg2+ converts phosphoenolpyruvate and ADP to pyruvateand ATP, the conversion of ADP to ATP occurring in a 1:1 ratio (Conn &Stumpf, 1966). Thirty embryos at each stage were homogenized in 0-5M PCA,and the homogenate neutralized as for total ATP content. Twenty /A of 0-045 Mphosphoenolpyruvate and 10 [A of a solution containing 300 i.u. of pyruvate


kinase (Calbiochem), 0-05M TEA buffer, pH 7-4, and 0-0lM-MgCl2 were addedto the centrifuge tube. The tube was allowed to stand at room temperature for15 min, then centrifuged at 20000 # for 10 min. The supernatant solution wasadded to a scintillation vial containing 1 ml of 0-05 M TEA buffer, pH 7-4, andheated to 80 °C for 10 min to stop the enzymic reaction. The vial was cooled toroom temperature and the contents assayed for total ATP by the luciferinmethod. The number of counts in each sample was compared to similarly pre-pared samples of staged embryos without pyruvate kinase. The difference in thenumber of counts between the two samples gave the pmoles of ADP per embryoconverted to ATP. The pyruvate kinase reaction favors the production ofpyruvate from phosphoenolpyruvate. The reverse reaction is extremely smallespecially when the phosphoenolpyruvate and PK is in excess, the case of thepresent procedure. The limiting factor becomes the amount of ADP in thesample.

Labelling of embryos with 32PO 4

Thirty to 50 staged embryos were incubated for varying lengths of time (seespecific procedures below) in 0*05 ml Brinster's medium containing approxi-mately 150000 counts 32P-labelled H2PO4 (HCl-free) per 10 /d of medium. The32P was obtained from ICN (specific activity, 285 Ci/mg) and was diluted withmedium to the appropriate concentration. The exact counts in the medium weredetermined by spotting a 10 /A aliquot of medium on a piece of filter paperwhich was placed into a scintillation vial and then dried. Ten ml of toluene-based Omnifluor scintillation fluid was added and the sample counted.

Calculation of pmoles ofAT32P and 3 2 P 0 4

(1) AT32P. The conversion of counts in a sample to pmoles of AT3 2P wascalculated according to the formula of Francis, Mulligan & Wormall (1959):

moles of inorganic P O 4 .n . . / iX——: —:—T^—j j?— = new specific activity (1)total counts in 10 /A medium

new specific activity x corrected counts/emb./h = pmoles product/emb./h (2)

The molar concentration of inorganic phosphate in radioactive Brinster'smedium was determined by the assay method of Lowry & Lopez (1946). Thecorrected counts (per emb./h) of the sample were found by subtracting back-ground from the raw counts and dividing by the number of embryos per sample.The background for all studies of AT3 2P was obtained by counting only scintil-lation fluid.

(2) 3 2 P 0 4 . A time-course study was done to determine when 3 2PO4 reached

equilibrium within the embryos. For these studies, staged embryos were incu-bated in 3 2PO4 medium for 60 min. Samples of 50 embryos were removed, at 15min intervals, from the radioactive medium. The embryos were then quickly

ATP metabolism in mouse embryos 271washed four times, collected in 10 [A of the final wash, placed into an 80 °Coven and dried. The pmoles of embryonic phosphate were calculated from thesample counts minus background using equations (1) and (2).

Gross synthesis of ATP

Embryos were incubated in 32PO4-containing medium for 90 min, removingapproximately 50 embryos at 15 min intervals. Following the desired labellingperiod, each group of embryos was quickly washed, collected in 10 /A of non-radioactive medium and placed into a 0-4 ml centrifuge tube. Ten /A cold PCAwas added to each tube, the contents mixed on a Vortex, and then allowed tostand at 4°C for 20 min. Five /i\ of 0-01 M carrier ATP was added, the contentstwice 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 solutionswere spotted on PEI-cellulose TLC plates and chromatographed two-dimension-ally (Randerath & Randerath, 1967). The spots, identified by the fluorescenceof the carrier ATP, were cut out, placed into a scintillation vial containing 10 mlof scintillation fluid, and counted. Background was obtained by counting a vialcontaining only scintillation fluid. To serve as controls, either 10 [A of the lastwash or 10 /d of the radioactive medium was added to 5 fA of ATP carrier andchromatographed. In these controls the numbers of counts in the ATP carrierspot were not significantly above background.

ATP turnover

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

Net synthesis of ATP

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

ADP + 32PO4 «± AT32P. (3)

k


The rates of synthesis (kx) and turnover (k2) of ATP can be calculated from theequations

= ^ p = kx- k2 (ATP luciferin), (4)

&2/6O min = - 6 In x, (5)

(6)

Equations (4), (5) and (6) (Francis, Mulligan & Wormall, 1959) were modified foruse in the present studies. In (5), x represents the gross amount of ATP remainingafter a 10 min chase. The natural log of this number is multiplied by 6 to convertthe 10 min reading to 1 h. The k2 from (5) is used to calculate the rate of synthesis(k±) in (6), where y is the rate of gross synthesis of ATP in one hour. To comparek2 with kx, k2 has to be multiplied by the total ATP determined by luciferinassay. These equations are valid provided that the total embryonic ATP andphosphate remain relatively constant during the 1 h treatment, and that there isno great lag in the transport of 32PO4 in or out of the cell. These requirementsare 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-radioactivemedium containing 10~4M cyanide for 10 min or in radioactive medium contain-ing 10~4M cyanide for 60 min. This concentration of cyanide has been shown tobe 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 deter-mined by using the luciferin-luciferase method, while the effect of cyanide ongross ATP synthesis in the labelled sample was determined using the techniquedescribed above.

Statistical studies

Standard errors of the mean are included in the text where applicable.Significant differences between means (P

ATP metabolism in mouse embryos 273

1-6

f-

0-8

0-4

LM LB

Stage

Fig. 1. Changes in the total ATP level during early development. At each cleavagestage total ATP was assayed by luciferin (O O). The effect of cyanide on totalATP was determined after incubating embryos in 10"4M cyanide for 10 min beforebeing assayed by luciferin (x x) . 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 lossof total ATP, at each older cleavage stage up to but not including the lateblastocyst stage when the loss appears to level off (Fig. 1). There is a slightreduction in total ATP at the 2-cell stage (from 1-47 ± 0-07 to 1-37 ± 0-06pmoles), 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 % decreaseat late morula (from 0-65 ± 0-01 to 0-32 ± 0-02 pmoles), and a 26 % loss at thelate blastocyst stage (from 0-43 ± 0-01 to 0-32 ± 0-02 pmoles).

ATPjADP ratioThere 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 has10-5 ± 0-54 times more ATP than ADP, whereas the 4-cell embryo contains only5-8 ± 0-29 times more ATP than ADP. Although there is only a small decreasein total ATP between the 4- and 8-cell stages (Fig. 1), the ATP/ADP ratio dropsfrom 5-8 ± 0-29 to 2-5 ± 0-14 reflecting a twofold increase in ADP betweenthese 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.

Equilibrium of32PO^

Because of the size of the cleavage-staged mouse embryos it is extremelydifficult to directly measure inorganic phosphate pool size by any establishedmethod. The pool size can, however, be estimated by determining the equilibriumlevel of 32PO4 at each stage.

Embryonic 32PO4 reaches equilibrium after 30 min incubation in 32PO4

18 EHB 30


LM LBStage

Fig. 2. ATP/ADP ratios in preimplantation mouse embryos. Total ATP wasmeasured with luciferin in comparable samples before and after pyruvate kinasetreatment. The increase in ATP after enzyme action reflects the amount of ADPconverted to ATP.

10 -

LM LBStage

Fig. 3. Equilibrium of 32PO4 in cleavage-staged embryos.

containing medium. The time needed to reach equilibrium is the same for all pre-implantation stages. This equilibrium level is maintained for at least 30 additionalminutes under culture conditions. The 2-cell stage reaches equilibrium at 1-22pmoles 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 the8-cell and morula stages respectively (Fig. 3). By the late blastocyst stage theequilibrium level drops to 2-0 pmoles 32PO4.


^ 0-6

E

oT 0-4

.« 0-2o

a

15 30 45 60 75 90

Time of incubation (min)

Fig. 4. Time course study of 32PO4 into ATP. Fifty staged embryos were incubatedfor varying lengths of time in 32PO4 medium. The ATP was extracted and chro-matographed two-dimensionally on PEI-cellulose TLC. The ATP carrier spot wascut out and counted. The counts were converted to pmoles ATP/emb./h. O O,2-cell stage; x x , 4-cell stage; A A, late morula stage; • • , lateblastocyst stage.

Gross synthesis of ATP

Embryos at each preimplantation stage were incubated for varying timeperiods in 32PO4 containing medium and were then assayed for the incorpora-tion of radioactivity into AT32P (Fig. 4). The data from this time course studyshow that there is no significant incorporation into ATP for the first 15 min ofincubation at any cleavage stage. Equilibrium of 32PO4 incorporation into ATPis reached after 60 min of incubation at every stage examined. The concentrationreached at 60 min is constant for at least an additional 30 min. In order todetermine whether the long labelling periods necessary for AT32P accumulationto reach equilibrium were detrimental to development, embryos at each stagewere incubated for 90 min in radioactive medium and reincubated in non-radioactive medium. There was no effect of the radioactivity on the furtherdevelopment of these embryos up to hatching from the zona pellucida regardlessof the stage at which the embryos were treated.

Because a plateau of the accumulation of the isotope into ATP is reached at60 min, this length of incubation time in radioactive medium was used for allcleavage-staged embryos in determining the gross synthesis of ATP. Each stagewas 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 ATPbetween the 2-cell, 4-cell and 8-cell embryos, as well as between the late morulaand late blastocyst stages. A significant decrease in gross synthesis is foundbetween the 8-cell and late morula stage. There is a 55 % increase from the2-cell (0-22 ± 0-01 pmoles) to the 4-cell (0-49 ± 0-02 pmoles) and a 22 % increasebetween the latter and the 8-cell (0-63 ± 0-06 pmoles) stage in ATP accumulatedafter 1 h. Between the 8-cell and the late morula, the gross synthesis of ATPdecreases from 0-63 ± 0-06 to 0-27 ± 0-01 pmoles per embryo, a decrease of

18-2


0-8

0-6

0-4

0-2

LM LB

Stage

Fig. 5. The gross synthesis and turnover of AT32P. After 1 h incubation in 32PO4medium, samples of embryos were removed, ATP extracted, and separated bytwo-dimensional chromatography (O O). Additional samples were reincu-bated in cold medium for 10 min to determine the rate of gross turnover ( x x ).

57 %. The gross synthesis increases from 0-27 ± 0-01 to 0-34 ± 0-01 pmoles bythe late blastocyst stage, an increase of 21 %.

Turnover of ATP

Fig. 5 also shows the amount of accumulated AT32P remaining in embryoswhich were incubated in radioactive medium for 1 h and then reincubated incold medium for an additional 10 min. At each successive cleavage stage thereis a progressive increase in the percentage of ATP turnover, i.e. the amount ofAT32P present in embryos following reincubation when compared with the grossamount of AT32P in similarly staged embryos prior to reincubation. During the10 min reincubation period there is no loss of accumulated AT32P at the 2-cellstage, 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 a47 % 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 turnovervalues (Fig. 5), were used to calculate the rates of net synthesis (kj) and rates ofturnover (k2) (see Materials and Methods). The net rates of synthesis increasesby threefold between the 2- and 4-cell stages (from 0-22 to 0-66 pmoles), and bytwofold between the 4- (0-66 pmoles) and 8-cell (1-11 pmoles) stages (Fig. 6).There is a reduction from the 8-cell (1-11 pmoles) to the late morula (0-70pmoles), followed by a large increase between the late morula and late blasto-cyst (1-38 pmoles) stages. The turnover rate, k2, of ATP increases significantlyat successive cleavage stages (Fig. 6). Only at the 2-cell is there no detectable


20

1-2

8 0-8oEc

0-4

Stage

Fig. 6. Changes in net synthesis (kx) and net turnover (k2) of ATP during earlydevelopment. Values were calculated from the levels of gross synthesis, turnover,and total ATP (O O, kx\ x x , k2). A A, Effect of cyanide on ATPsynthesis (kx).

turnover of ATP in 60 min. At the 4- and 8-cell stages the respective kx and k2values are equivalent. Later stages, late morula and late blastocyst, have ahigher rate of turnover (k2) than synthesis (kx).

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 develop-ment. Incubation of embryos for 60 min in radioactive medium containing10~4M cyanide caused a reduction in kx values 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 0-34pmoles) at 8-cell and 73 % (0-70 to 019 pmoles) at the morula stage, the oldeststage studied (Fig. 6).

DISCUSSION

The present studies show that 2-cell mouse embryos are characterized by ahigh total ATP content, a high ATP/ADP ratio and a low level of both netsynthesis and turnover of ATP when compared with embryos of later cleavagestages. Beginning at the 4-cell stage, there is a decrease in total ATP and theATP/ADP ratio, together with increased synthesis and turnover of ATP.Embryos at each successively later cleavage stage contain progressively lesstotal ATP and lower ATP/ADP ratios. At the 8-cell stage the net turnover andnet synthesis of ATP are equivalent, whereas at both the late morula and lateblastocyst, net turnover exceeds net synthesis. The differences between the kxand k2 values at these latter stages most likely reflect the increasing usage ofAT32P in macromolecular synthesis. The amount of AT32P used in these


syntheses cannot bs determined nor included in the kx value of AT32P using the

present techniques, but would, however, be reflected in the turnover (k2) rates. Theturnover rates at the later stages are therefore a better measure of synthesis thanare the kx values. The k2 values thus show increased amounts of ATP synthesisat each successive cleavage stage. Preliminary studies on gross ATP synthesisand turnover have recently been published (Ginsberg & Hillman, 1972).

Although there have been no reported studies on rates of ATP synthesis andturnover, or ATP/ADP ratios in mouse embryos, there are two earlier studiesreporting luciferin assayed values for total ATP content in cleavage-stagedembryos. Our observations agree with the data of Quinn & Wales (1971), whomeasured total ATP in 2-cell, 8-cell, morula, and both early and late blastocystmouse embryos. Total ATP was not determined for 4-cell embryos. Althoughthe values for total ATP in the present report are slightly higher than the valuesreported by Quinn & Wales for correspondingly staged embryos, both reportsagree that there are progressive decreases at each successive cleavage stage.Both of these reports disagree with that of Epstein & Daentl (1971), who foundno change in total ATP between the 2-cell and late morula stages. It is likelythat differences in protocols, such as the removal or non-removal of PCA priorto counting, could result in the discrepancies found among these three reports.

The present data show that the synthesis of ATP is slightly inhibited bycyanide as early as the 2-cell stage and is inhibited in increasingly greateramounts at the older cleavage stages. Since cyanide blocks electron transport atcytochrome as, these findings indicate that ATP is being synthesized by oxida-tive phosphorylation and they suggest that the activity of the cytochrome systemincreases at each cleavage stage. It has been shown that the degree of activity ofthe cytochromes is directly determined by the relative ratios of mitochondrialATP, ADP and PO4 (Chance & Hagihara, 1961) and that the cytoplasmic ATP,ADP and PO4 relative ratios regulate the activity of the cytochrome system bycontrolling the activity of specific regulatory enzymes (Atkinson, 1965). In thepresent study, the measured total ATP and ATP/ADP ratios reflect the totalnucleotide level in the cell, and it is assumed that the levels in the cytoplasm andmitochondria are directly proportional.

The observed increase in ATP synthesis and turnover as well as the observedincreased inhibition of ATP synthesis by cyanide and the decreased ATP/ADPratios were predictable, therefore, in light of the studies of Mills & Brinster(1967) and Brinster (1967), who found that O2 and

14CO2 production fromglucose consumption increased in mouse embryos at each successive cleavagestage. Brinster further found (1967) that most of this CO2 was produced in theTCA cycle. From these combined data, Brinster suggested that the TCA cyclewas functional and showed increased activity as the embryos advanced fromone cleavage stage to another. In support of this hypothesis were the findings ofboth Brinster (1966) and Epstein, Wegienka & Smith (1969), who reported thatmalic 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 TCAintermediates at the 4-cell stage, with increasing rates of uptake in the progres-sively older cleavage stages.

The results of the present studies, together with those presented above, areevidence for an increase in the activities of both the TCA cycle and the electron-transport mechanism during mouse preimplantation stages. It is probable thatthese increases can be correlated with the mitochondrial configurational changeswhich occur in mouse cleavage-staged embryos (Hillman and Tasca, 1969;Stern, Biggers & Anderson, 1971). Piko & Chase (1971) have found that chlor-amphenicol treatment of cleavage-staged mouse embryos does not stop thesemitochondrial transformations. In an earlier study Piko (1970) noted thatuniformly labelled [14C]thymidine is incorporated into nuclear DNA but notinto mitochondrial DNA, during cleavage. Thus, the normal sequential differ-entiation does not involve de novo formation of the different-appearingmitochondria but merely the reorganization of the cristae of existing mito-chondria.

Hackenbrock (1966) has described four basic types of mitochondrial ultra-structural configurations: condensed, intermediate, swollen and orthodox.Either of the extremes of these mitochondria - condensed or orthodox - arepresent in cells or in a population of isolated mitochondria, depending upon themetabolic steady state of these organelles. Chance & Williams (1955,1956) havedefined the criteria of five metabolic steady states. State I has low substrate andO2 level, slow respiratory rate but an O2 consumption level greater than zero;State IE is like State I except it has a high substrate level; State III again has anO2 level greater than zero, but has both high substrate and ADP levels and therespiratory rate is fast; State IV has a high substrate level, low ADP level, slowrespiratory rate and again an O2 level greater than zero; State V is present underanaerobiosis in which substrate and ATP levels are both high but O2 consump-tion and respiration rates are equal to zero.

Hackenbrock (1966, 1968, 1972) and Hackenbrock, Rehn, Weinbach &LeMasters (1971) have suggested that the ultrastructural configuration of themitochondria can be correlated with the metabolic steady state of either isolatedmitochondria or of mitochondria within intact cells. Condensed mitochondriaare described as being electron-dense, and the inner membranes are irregularlyfolded (similar to those found in 2-cell mouse embryos), whereas orthodoxmitochondria contain a much less dense matrix and regularly arranged cristae(similar to those found in later cleavage-staged embryos). In an experimentin which Hackenbrock (1966) placed isolated mitochondria under different butspecific sets of conditions for any one of the five metabolic steady states, hefound that condensed mitochondria were initially found under those sets ofconditions which produced States I, II, III, and IV metabolism. Upon theutilization of endogenous substrate or upon the addition of either substrate


and/or ADP, mitochondria underwent oxidative phosphorylation and theirconfiguration changed from condensed to orthodox with only one exception.Under State II conditions, mitochondria were condensed and remained con-densed or became intermediate between condensed and orthodox upon theaddition of ADP and completion of oxidative phosphorylation.

If one subscribes to the hypothesis that mitochondrial configuration reflectsthe steady state of metabolism, and specifically the occurrence of oxidativephosphorylation or electron transport within that state, then it follows that themitochondrial configurational changes which occur within or between cleavagestages can also be correlated with different steady states of metabolism of theembryo. 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 ATPsynthesis (kj) by cyanide, and O2 consumption (Mills & Brinster, 1967) and

14CO2production are low (Brinster, 1967), suggesting State I metabolism. Since ADPis low at this state, active phosphorylation would not occur and consequentlythe mitochondria would not change to the orthodox configuration. At the 4-cellstage, the majority of mitochondria are condensed but there are, at the late4-cell, some mitochondria with a more orthodox appearance. At this stage, theATP level as well as the ATP/ADP ratio is lower than at the 2-cell stage. Theincreased relative amount of ADP could shift the metabolism from State I toState II. In the latter metabolic state the mitochondria are condensed and onlypartially transform following oxidative phosphorylation. This incomplete trans-formation could be correlated with the appearance of the few more orthodoxappearing mitochondria which are present at the late 4-cell stage. At the late4-cell and 8-cell stages, the embryos show greater permeability to TCA inter-mediates (Kramen & Biggers, 1971), suggesting increased substrate levels, andat the 8-cell, ATP is lower, ADP higher and O2 consumption (Mills & Brinster,1967) greater than at the earlier developmental stages. These conditions arebasic for a shift from State II to State III metabolism. The condensed popula-tions of mitochondria in 8-cell and older embryos could indicate State IIImetabolism alone or could be present together with State IV metabolism,oscillating between the two states within each mitochondrion. State III wouldbe present in those mitochondria with a relatively low ATP/ADP ratio and,upon the phosphorylation of all available ADP, enter State IV. This oscillationbetween steady states would result in an oscillation between condensed andorthodox configurations and would account for the mixed population of mito-chondrial forms found in the older cleavage-staged embryos.

Although it is not possible to determine continuing configurational changesby examining fixed sections of embryos at the ultrastructural level, it is possibleto show whether or not the embryos do have different steady states of metabo-lism at different cleavage stages. Each steady state reduces pyridine nucleotidesand consumes O2 in specific relative patterns. It is necessary therefore to deter-mine the level of reduction of nucleotides at each cleavage stage, and relate


these to the amount of oxygen consumption at corresponding stages in order todetermine definitely whether the embryos do in fact demonstrate differentmetabolic steady states at the different preimplantation stages. These studiesare now in progress.

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

REFERENCES

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./ . 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: JohnWiley.

DAENTL, D. L. & EPSTEIN, C. J. (1971). Developmental interrelationships of uridine uptake,nucleotide formation and incorporation into RNA by early mammalian embryos. DeviBiol. 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 gonado-tropins. / . Endocr. 18, 292-304.

ELLEM, K. A. O. & GWATKIN, R. B. L. (1968). Patterns of nucleic acid synthesis in the earlymouse 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 preimplanta-tion mouse embryos. Devi Biol. 26, 517-524.

EPSTEIN, C. J., WEGIENKA, E. A. & SMITH, C. W. (1969). Biochemical development of pre-implantation 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. Universityof London: Athlone Press.

GINSBERG, L. & HILLMAN, N. (1972). ATP synthesis in normal and mutant cleavage-stagedmouse embryos. Genetics 71, Suppl. 3, S 20.

HACKENBROCK, C. R. (1966). Ultrastructural bases for metabolically linked mechanicalactivity in mitochondria. I. Reversible ultrastructural changes with change in metabolicsteady state in isolated liver mitochondria. / . Cell Biol. 30, 269-297.

HACKENBROCK, C. R. (1968). Ultrastructural bases for metabolically linked mechanicalactivity in mitochondria. II. Electron transport-linked ultrastructural transformations inmitochondria. / . Cell Biol. 37, 345-369.

HACKENBROCK, C. R. (1972). Energy-linked ultrastructural transformations in isolated livermitochondria and mitoplasts. / . Cell Biol. 53, 450-465.


HACKENBROCK, C. R., REHN, T. G., WEINBACH, E. C. & LEMASTERS, J. J. (1971). Oxidativephosphorylation and ultrastructural transformation in mitochondria in the intact ascitestumor cell. / . Cell Biol. 51, 123-137.

HILLMAN, N. & TASCA, R. (1969). Ultrastructural and autoradiographic studies of mousecleavage stages. Am. J. Anat. 126, 151-174.

KNOWLAND, J. & GRAHAM, C. (1972). RNA synthesis at the two-cell stage of mouse develop-ment. / . Embryol. exp. Morph. 11, \61-\16.

KRAMEN, M. A. & BIGGERS, J. D. (1971). Uptake of tricarboxylic acid cycle intermediates bypreimplantation mouse embryos in vitro. Proc. natn. Acad. Sci. U.S.A. 68, 2656-2659.

LOWRY, O. H. & LOPEZ, J. A. (1946). The determination of inorganic phosphate in thepresence of labile phosphate esters. / . biol. Chem. 162, 421-428.

MILLS, Jr., R. M. & BRINSTER, R. L. (1967). Oxygen consumption of preimplantation mouseembryos. Expl Cell Res. 47, 337-344.

MINTZ, B. (1964). Synthetic processes and early development in the mammalian egg. / . exp.Zool. 157, 85-100.

MONESI, V. & SALFI, V. (1967). Macromolecular synthesis during early development in themouse 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: theeffect of ethidium bromide and chloramphenicol. Cell Biol. 51, 443 (abstr.).

QUINN, P. & WALES, R. G. (1971). Adenosine triphosphate content of pre-implantationmouse embryos. J. Reprod. Fert. 25, 133-135.

RANDERATH, K. & RANDERATH, E. (1967). Thin-layer separation methods for nucleic acidderivatives. Meth. Enzym. 12, 323-347.

STANLEY, P. E. & WILLIAMS, S. G. (1969). Use of the liquid scintillation spectrometer fordetermining adenosine triphosphate by the luciferase enzyme. Analyt. Biochein. 29,381-392.

STERN, S., BIGGERS, J. & ANDERSON, E. (1971). Mitochondria and early development of themouse. / . 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 proteinsynthesis. Am. Zool. 7, 170 (Abstr.).

TASCA, R. J. & HILLMAN, N. (1970). Effects of actinomycin D and cycloheximide on RNAand protein synthesis in cleavage stage mouse embryos. Nature, Lond. 225, 1022-1025.

THOMSON, J. L. (1967). Effect of inhibitors of carbohydrate metabolism on the developmentof preimplantation mouse embryos. Expl Cell Res. 46, 252-262.

WOODLAND, H. R. & GRAHAM, C. F. (1969). RNA synthesis during early development of themouse. Nature, Lond. Ill, 327-331.

(Received 15 December 1972, revised 27 March 1973)

atp metabolism in cleavage-staged mouse embryos · the molar concentratio o inorganif nc phosphat...

Documents