/ . Embryol. exp. Morph. Vol. 30, J, pp. 267-282, 1973
Printed in Great Britain
267
ATP metabolism in cleavage-staged
mouse embryos
By L. GINSBERG 1 AND N. HILLMAN 1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
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.
INTRODUCTION
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 denned 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).
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 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.
MATERIALS AND METHODS
Supply and culture of embryos
The mice used in the present experiments were from a randomly breeding
closed colony of Swiss Albino mice. Eight- to 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
ATP metabolism in mouse embryos
269
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 [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's
contents were neutralized by adding 10 /A of a solution of 4 M - K 2 C O 3 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 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 Tricarb
3380 liquid scintillation counter preset for 3 H. 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.
ATPIADP 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 Mg 2+ 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 0-5M PCA,
and the homogenate neutralized as for total ATP content. Twenty /A of 0-045 M
phosphoenolpyruvate and 10 [A of a solution containing 300 i.u. of pyruvate
270
L. GINSBERG AND N. HILLMAN
kinase (Calbiochem), 0-05M TEA buffer, pH 7-4, and 0-0lM-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 with
32
PO 4
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 H 2 PO 4 (HCl-free) per 10 /d of medium. The
32
P 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 /A 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 ofAT32P
and 3 2 P 0 4
(1) AT32P. The conversion of counts in a sample to pmoles of AT 3 2 P was
calculated 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's
medium was determined by the assay method of Lowry & Lopez (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 AT 3 2 P was obtained by counting only scintillation fluid.
(2) 3 2 P 0 4 . A time-course study was done to determine when 3 2 PO 4 reached
equilibrium within the embryos. F o r these studies, staged embryos were incubated in 3 2 PO 4 medium for 60 min. Samples of 50 embryos were removed, at 15
min intervals, from the radioactive medium. The embryos were then quickly
ATP metabolism in mouse embryos
271
washed four times, collected in 10 [A 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 32PO4-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 /A of nonradioactive medium and placed into a 0-4 ml centrifuge tube. Ten /A 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 /i\ of 0-01 M carrier ATP was added, the contents
twice frozen at - 7 0 ° 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 ml
of scintillation fluid, and counted. Background was obtained by counting a vial
containing only scintillation fluid. To serve as controls, either 10 [A of the last
wash or 10 /d of the radioactive medium was added to 5 fA of ATP carrier and
chromatographed. In these controls the numbers of counts in the ATP carrier
spot were not significantly above background.
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 /A
medium and were immediately added to 10/^1 0-5M cold PCA. The other half
of the sample was reincubated in non-radioactive medium for an additional 10
min, collected in 10 /d of medium and added to 10 [A 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
ADP +
32
PO4 «± AT32P.
k
(3)
272
L. GINSBERG AND N. HILLMAN
The rates of synthesis (kx) and turnover (k2) of ATP can be calculated from the
equations
= ^ p = kx-
k2
(ATP luciferin),
&2/6O min = - 6 In x,
(4)
(5)
(6)
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 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 compare
k2 with kx, k2 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~ 4 M cyanide for 10 min or in radioactive medium containing 10~ 4 M cyanide for 60 min. This concentration of cyanide has been shown to
be the LD 50 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 Mest.
RESULTS
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 4- and 8-cell stages.
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 cleavage
stage total ATP was assayed by luciferin (O
O). The effect of cyanide on total
ATP was determined after incubating embryos in 10" 4 M cyanide for 10 min before
being assayed by luciferin (x
x ) . LM = late morula; LB = late blastocyst.
Effect of cyanide on total ATP
A 10 min treatment with 10~ 4 M 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 1-47 ± 0-07 to 1-37 ± 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).
ATPjADP 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.
Equilibrium of32PO^
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 32 PO 4 at each stage.
Embryonic 32PO4 reaches equilibrium after 30 min incubation in 32 PO 4
18
EHB
30
274
L. GINSBERG AND N. HILLMAN
LM
LB
Stage
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.
10 -
LM
LB
Stage
Fig. 3. Equilibrium of 32PO4 in cleavage-staged embryos.
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 32 PO 4 in the
8-cell and morula stages respectively (Fig. 3). By the late blastocyst stage the
equilibrium level drops to 2-0 pmoles 32PO4.
ATP metabolism in mouse embryos
^
275
0-6
E
oT 0-4
.« 0-2
o
a
15
30
45
60
Time of incubation (min)
75
90
Fig. 4. Time course study of 32 PO 4 into ATP. Fifty staged embryos were incubated
for varying lengths of time in 32 PO 4 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. O
O,
2-cell stage; x
x , 4-cell stage; A
A, late morula stage; •
• , late
blastocyst stage.
Gross synthesis of ATP
Embryos at each preimplantation stage were incubated for varying time
periods in 32 PO 4 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 32 PO 4 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.
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
18-2
276
L. G I N S B E R G AND N. H I L L M A N
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 32PO4
medium, samples of embryos were removed, ATP extracted, and separated by
two-dimensional chromatography (O
O). Additional samples were reincubated 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 by
the late blastocyst stage, an increase of 21 %.
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 (kj) and rates of
turnover (k2) (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 (1-11 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
ATP metabolism in mouse embryos
277
20
1-2
8 0-8
o
E
c
0-4
Stage
Fig. 6. Changes in net synthesis (kx) and net turnover (k2) of ATP during early
development. 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 ATP
synthesis (kx).
turnover of ATP in 60 min. At the 4- and 8-cell stages the respective kx and k2
values are equivalent. Later stages, late morula and late blastocyst, have a
higher 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 development. Incubation of embryos for 60 min in radioactive medium containing
10~ 4 M 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 0-34
pmoles) at 8-cell and 73 % (0-70 to 019 pmoles) at the morula stage, the oldest
stage studied (Fig. 6).
DISCUSSION
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 kx
and k2 values at these latter stages most likely reflect the increasing usage of
AT32P in macromolecular synthesis. The amount of AT32P used in these
278
L. GINSBERG AND N. HILLMAN
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. The
turnover rates at the later stages are therefore a better measure of synthesis than
are the kx values. The k2 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 as, 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 PO 4 (Chance & Hagihara, 1961) and that the cytoplasmic ATP,
ADP and PO 4 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
ATP metabolism
in mouse embryos
279
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 IE is like State I except it has a high substrate level; State III again has an
O 2 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
280
L. GINSBERG AND N. HILLMAN
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 (kj) 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
ATP metabolism
in mouse embryos
281
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.
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(Received 15 December 1972, revised 27 March 1973)