/. Embryol. exp. Morph. Vol. 33, 3, pp. 715-723, 1975
715
Printed in Great Britain
ATP metabolism in tn/tn mouse embryos
By LEONARD GINSBERG 1 AND NINA HILLMAN 1
Department of Biology, Temple University, Philadelphia
SUMMARY
Total ATP, ATP/ADP ratios, and the rates of synthesis and turnover of ATP were determined for t12-, tw3i- and r+/!T+-litters at the 2-cell, 4-cell, 8-cell, early morula, late morula
and late blastocyst stages. The results show that /"-litters have excessive rates of ATP metabolism up to the cleavage stage immediately before the stage of lethality for most tw32/tK32
embryos (early morula) and most t12/t12 embryos (late morula); mutant ATP metabolism
then falls below control levels. After the death of the tn homozygotes, ATP metabolism in the
/"-litters (composed of only T+/T+ and T+/tn embryos) returns to control levels. These data
are discussed in relation to the phenotypes of the homozygous tn embryos.
INTRODUCTION
The recessive lethal alleles (tn) at the complex T locus in the mouse can be
distributed into five complementation groups (Bennett & Dunn, 1964; Dunn &
Bennett, 1971). The alleles are assigned to these groups on the basis of similar
syndromes of phenotypic expressions and similar times of embryonic lethality.
Those homozygous tn alleles (t12 and tw32) which are lethal at the earliest
embryonic stages belong to the t12 complementation group. The t12/t12 genotype
is lethal to most embryos at the late morula stage, but these homozygous
embryos can be distinguished from their normal litter-mates at the 2-cell stage
by the presence of nuclear lipid and by the accumulation of excessive cytoplasmic lipid droplets (Hillman, Hillman & Wileman, 1970). The r 3 2 / ^ 3 2
embryos, like t12/t12 embryos, can be identified by the presence of excessive
cytoplasmic and nuclear lipid droplets at the 2-cell stage. However, unlike
t12lt12 embryos the lethal period for most twZ2jtwZ2 embryos is the early morula
stage. In addition, many tw32 homozygotes contain mitochondria with crystalline
depositions (Hillman & Hillman, 1975).
Both the accumulation of excessive lipid and the deposition of mitochondrial
crystals can occur as a result of non-physiological levels of energy metabolism
(Atkinson, 1965; Lehninger, Carafoli & Rossi, 1967; Newsholme & Start, 1973).
Therefore, an investigation of the aerobic metabolism of t12/t12 and twZ2jtwn
cleavage-stage mouse embryos was initiated in order to determine (1) if there
are differences in energy metabolism between correspondingly staged tn\tn and
1
Authors' address: Department of Biology, Temple University, Philadelphia, Pennsylvania
19122, U.S.A.
45
EMB
33
716
L. GINSBERG AND N. HILLMAN
wild-type embryos and (2) if these differences occur at the same developmental
stages in both t12/t12 and twZ2(twZ2 embryos.
MATERIALS AND METHODS
w32
Homozygous t
and t12 embryos were obtained by mating heterozygous
T lt males with superovulated (Edwards & Gates, 1959) heterozygous females
of the same genotype. Two-cell embryos were flushed from the oviducts with
Brinster's medium (Brinster, 1963) and were either assayed immediately or were
placed into culture (Brinster, 1963) until they reached the desired cleavage stage
(4-cell, 10-14 h; 8-cell, 24 h; early morula (EM), 36 h; late morula (LM), 45 h;
late blastocyst (LB), 60 h). Two-cell control embryos were obtained from a
closed colony of randomly breeding Swiss albino mice. The P-litters containing
mutants (subsequently referred to as P-litters) consisted of approximately 40 %
homozygous lethals as a result of the increased frequency of transmission of the
t12- and f^-bearing spermatozoa (Smith, 1956; Bennett & Dunn, 1964). The
data obtained in the tn experiments, except for the late blastocyst experiments,
were from total litters which included three genotypes (T+/T+, T+/tn, tn/tn). The
differences cited below in ATP metabolism between control and ^"-litters have
not been corrected to show the effect of the tn\tn genotype alone. Some experiments were done on P-litters at the late blastocyst stage; these contained only
T+/T+ and T+ltn embryos.
The parameters of ATP metabolism determined in the present studies were
total ATP, total ADP and ATP/ADP ratios, gross synthesis and gross turnover
of AT32P and net synthesis and turnover of ATP. The procedures and analyses
for these determinations have been reported previously (Ginsberg & Hillman,
1973). Each experiment for a specific group of P-litter embryos was performed
together with correspondingly timed control litters and was repeated at least
three times. The levels of ATP metabolism in cleavage-stage, wild-type embryos
were reported previously (Ginsberg & Hillman, 1973). These studies have been
extended and the results, which do not differ significantly from those reported
earlier, are given for comparative purposes. Standard errors of the mean are
included where applicable. Significant differences between means (P < 0-05)
were determined by Student's ?-test.
+ n
RESULTS
ATPjADP ratios
n
w32
Although both t - and f -2-cell litters contain higher levels of total ATP
than do wild-type litters of corresponding stage, the ATP/ADP ratios of the
P-litters are lower than those of control 2-cell embryos (Table 1). The lower
mutant ATP/ADP ratios are a function of the significantly higher levels of total
ADP present in the P-litters.
The ATP/ADP ratio in ?12-litters remains constant between the 2-cell and late
ATP metabolism
in mutant mouse embryos
111
Table 1. Total ATP, ADP and ATPjADP ratios of cleavage-stage wild-type
(T+/T+) and mutant (t 12 ; t w32 ) litters
p-moles ATP/embryo
T+IT+
2-cell
4-cell
8-cell
EM
LM
LB
2-45±005
1-59±0-08
1-26±001
0-77±003
0-66±0-07
0-46 ±002
1-74 + 0-22
1-43±0-15
1-55±003
1-16±0-24
O-77±OO5
0-66 ±002
1-47±007
0-99±0-01
0-91 ±001
0-73 ±002
0-65 ±0-01
0-43 ±001
p-moles ADP/embryo
t12
tw32
T+/T+
1-58±008 0-53±006 015±001
1-12±0-08 0-85±0-03 0-17±0-01
1-19±0-06 0-34±003 0-39±002
0-40±0-10 013±002 0-42±004
0-16±0-01 0-19±0-05 0-39±003
0-27 ±007 0-32 ±005 0-33 ±002
ATP/ADP ratio
t12 tw32 T+IT+
1 55 3-28 9-80
1-42 1-68 5-82
106 4-56 2-33
1-93 8-92 1-74
413 405 1-67
1-70 206 1-30
4-cell stages even though there is a reduction in both ATP and ADP in the 4-cell
tx% embryos. In 4-cell f™32-litters, the ratio is reduced below the 2-cell ratio as a
result of a decrease in total ATP and an increase in total ADP. The ratio in
control litters also drops between the 2- and 4-cell stages as a result of a decrease
in total ATP. The 4-cell control ratio is still, however, higher than that found in
either mutant litter.
Between the 4- and 8-cell stages, the ATP/ADP ratios in both t12- and control
litters are reduced while the ratio in fw32-litters is greatly increased. The sharp
increase observed in fM)32-litters is a result of an extreme reduction in total ADP
in these embryos while their ATP content remains constant. The lowered ratio
in t12- and control litters is a result of a decreased ATP content in ?12-litters and
an increased ADP content in control litters. The final result at the 8-cell stage is
that the ATP/ADP ratio of f12-litters is lower than that in controls while the
ratio in ^<j32-litters is greater than that in controls.
Between the 8-cell and early morula stages, there is an increase in the ATP/
ADP ratios in mutant litters and a decrease in control litters. Both t12- and twZ2litters show a small reduction in total ATP associated with a large reduction in
total ADP, resulting in the increased ratios. A similar small reduction in total
ATP is found in control litters but is not associated with a loss in ADP content
and consequently the wild-type ratio decreases.
The ATP/ADP ratios at the late morula stage are variable. While the ratio
in wild-type embryos remains constant between the early and late morula
stages, it continues to increase in f12-litters and begins to decrease in f™32-litters.
The higher ATP/ADP ratio of 112-litters is a function of a sharp decrease in total
ADP between the early and late morula stages and is associated with only a
slight decrease in total ATP. The change in ratio is similar to that found in both
t12- and J""32-litters between the 8-cell and early morula stages. At the late morula
stage, however, the ratio in /™32-litters, while still higher than that in control
litters, has begun to decline. This is due mainly to a stabilization of the total
ADP content in these embryos.
45-2
718
L. GINSBERG AND N. HILLMAN
Table 2. Gross synthesis and turnover of ATZ2P by wild-type (T+/T+) and mutant
(t 12 ; t w32 ) litters
Gross synthesis
Gross turnover
A
12
fto32
T+/T+
0-31 ±001
0-64 ±002
0-67 ± 003
0-27 ± 002
0-21 ±001
0-35 ±001
0-37 ±004
0-71 ±003
031 ±004
0-20 ±003
0-33 ±005
0-32 ±002
0-22 ±001
0-49 ±002
0-63 ±006
0-24 ±001
0-27 ±001
0-34 ±001
t
2-cell
4-cell
8-cell
EM
LM
LB
A
c
e
12
t
fw32
0-30 ±001 0-36 ±002
0-49 ±001 0-65 ±002
0-47 ±001 0-30 ±003
0-21 ±002 018 ±002
016±001 0-30 ±003
017 ±001 019 ±001
T+IT+
0-22 ± 001
0-44 ±001
0-51 ±001
017 ±001
019 ±001
018 ±001
By the late blastocyst stage all of the tn/tn embryos have died and the tnlitters are composed only of viable T+/T+ and T+/tn embryos. The ATP/ADP
ratios in both P-litters decrease between the late morula and late blastocyst
stages. Although ATP/ADP ratios of the late blastocyst P-litters remain higher
than the control ratio, they are closer to normal than in the preceding cleavage
stages. The lower ratios are caused by both decreases in the total ATP content
and increases in the total ADP content of mutant litters. The control litter ATP/
ADP ratios also decrease between the late morula and late blastocyst stages,
owing to a decrease in the total ATP.
Gross synthesis and turnover (Table 2)
At the 2-cell stage, P-litters have higher rates of gross synthesis and turnover
of AT32P than do control litters at the same stage. This same pattern of higher
synthetic and turnover rates continues in the ?w32-litters through the 4-cell stage.
Gross synthesis in f12-litters also proceeds at the higher rate at the 4-cell stage,
but their turnover rate is reduced to near control levels. At the 8-cell stage, t12litters and control litters have similar rates of synthesis and turnover, while the
^ 32 -litters have significantly lower rates. At the early morula stage, the gross
synthetic and turnover rates are equivalent for all litters. The control and P a late morula litters continue to show approximately equivalent synthetic rates
while the ?12-late morula litters show lower rates of ATP synthesis. At this same
stage the t12- and control litters are alike in their rates of turnover while the
?w32-litters have higher turnover rates. By the late blastocyst stage all litters are
synthesizing and turning over ATP at equivalent rates.
Net ATP synthesis and turnover
The data from the gross synthesis and turnover experiments (Table 2) have
been converted to rates of net synthesis (fcj) and turnover (k2), as previously
described (Ginsberg & Hillman, 1973). There is a low level of net ATP synthesis
in all embryos at the 2-cell stage, but the mutant litters have a 1-3-1-6 times
ATP metabolism in mutant mouse embryos
20
719
r
1-5
10
0-5
EM
LM
LB
Stage
Fig. 1. A comparison of net ATP synthetic (k±) rates among wild-type (x— x),
ti2- ( # — • ) and /wM- (O—O) litters.
greater rate than do control litters. The net turnover rate of 2-cell f Mitters is
approximately 1-5 times that of control litters. These data show that the 2-cell
mutant litters both synthesize and turnover ATP faster than do control litters
(Figs. 1,2).
The higher rate of /"-litter net synthesis and turnover continues through the
late 4-cell stage. At this stage each group of embryos shows a two- to threefold
increase in net synthetic rate with the mutant litters still having a synthetic rate
1 -5-2-0 times that of control embryos. The net turnover rates at the 4-cell stage
reflect this same relationship, with P-litters having the highest turnover rates.
At the 8-cell stage, the /12-litters continue to have the highest ATP synthetic and
turnover rates, but the relationship between ^ 32 -litters and wild-type litters has
now reversed. At this developmental stage, both the synthesis and turnover of
ATP by the ?""32-litters are significantly lower than the control levels.
At the early morula stage, {w32-litters have only one-half the synthetic rate
and one-fourth the turnover rate of control litters. At this same stage the
synthetic and turnover rates of ?12-litters are also reduced to below those in
control litters. This reduction in the rates of ATP synthesis and turnover in tnlitters continues into the late morula stage. At late morula, the /12-litters have the
lowest rates of synthesis and turnover.
720
L. GINSBERG AND N. HILLMAN
30
20
EM
LM
LB
Stage
Fig. 2. The rates of net ATP turnover (A:2 (ATP luciferin)) from T+/T+ (x — x),
t12- ( • — • ) and tw32- (O—O) litters.
At the late blastocyst stage, when the P-litters are composed of only T+(T+
and T+ltn embryos, there is no significant difference between mutant and control
litters in either their rates of net synthesis or net turnover.
DISCUSSION
The present studies show that the homozygous tn genotypes cause nonphysiological rates of ATP synthesis prior to embryonic death. Two-cell tvlitters have lower ATP/ADP ratios, higher levels of total ATP, and higher
rates of net synthesis and turnover of ATP than do wild-type embryos at the
same stage. These increased levels of ATP metabolism continue until the 8-cell
stage in /w32-litters and until the early morula stage in ?12-litters. At these respective stages, the levels of ATP metabolism in mutant embryos fall below control
levels and remain low until the death of the homozygous tn embryos. Following
the death of the tn\tn embryos, the ATP metabolism of the ^-litters returns to
control levels. Since the P-litters are then composed ofT+/T+ and T+/tn embryos,
the data indicate that a single dose of the tn allele, in the presence of the T+
allele, does not affect embryonic metabolism.
The decline in the rates of ATP metabolism at different cleavage stages in
t12- and ?ty32-litters can be correlated with studies which show that these two
tn homozygotes have different lethal stages (Hillman et ah 1970; Hillman &
ATP metabolism in mutant mouse embryos
wS2
721
Hillman, 1975). The lethal period of t
homozygotes ranges from the 8-cell
stage to the early morula stage, with most dying at the early morula stage,
whereas the lethal period of t12 homozygotes ranges from the 8-cell to early
blastocyst stages with most dying at the late morula stage. The observed decreases in ATP metabolism occur, therefore, one stage prior to the respective
lethal periods of most twZ2ltwZ2 and t12/t12 embryos.
Prior to death, the tw32/tw32 and t12lt12 embryos, as well as t&/te embryos, can
be distinguished from their litter-mates by excess cytoplasmic lipid droplets
(Hillman et al. 1970; Hillman & Hillman, 1975; Nadijcka & Hillman, 1975a). A
high-resolution autoradiographic study of tx2jt12 and tw32/tw32 embryos has
shown that excessive neutral lipid is synthesized and stored by the mutant
embryos (Nadijcka & Hillman, 19756). Although the preimplantation lethals
(t12 and /u'32) contain excess lipid droplets as early as the 2-cell stage, these
droplets become more numerous in the later cleavage stages. The excessive lipid
deposition occurs both during the periods of excessive ATP metabolism (2- to
4-cell stages) and after the mutants' ATP metabolism falls below control levels.
It has been noted in other systems that acetyl-CoA, in the presence of excessive total ATP, is diverted to fatty acid and lipid synthesis (Atkinson, 1965;
Newsholme & Start, 1973). In mutant embryos the temporal coincidence of
excessive mutant ATP metabolism and the presence of excessive lipid suggests
the occurrence of a similar divergence of acetyl-CoA. This hypothesis is supported by two recent studies on ?6//6 embryos. In the first, Nadijcka & Hillman
(1975#) have shown that tG/te embryos cannot be distinguished from their littermates before the late blastocyst stage. At this time, the homozygotes can be
identified by the presence of excessive cytoplasmic lipid. In the second, Ginsberg
& Hillman (unpublished data) have shown that litters containing f6/?6 embryos do
not differ from control litters in ATP metabolism before the late blastocyst
stage. At this time, however, ^-litters exhibit excessive ATP metabolism. Therefore, the first distinctive phenotypic characteristic (excessive lipid) of the t6/t6
genome occurs at a time coincident with excessive ATP metabolism. In the
preimplantation lethal genotypes (t12/t12 and twZ2jtwZ2), the increase in the
numbers of lipid droplets following the decrease in mutant ATP metabolism
could be a result of a continued diversion into lipid metabolism of the acetylCoA which is no longer being used for ATP synthesis. It is apparent, however,
that more extensive studies will have to be undertaken to determine the relationship of the aberrant ATP metabolism and the excessive lipid in these embryos.
A second characteristic which is common to both tw32ltw32 and ?6/?6 embryos
is the presence of crystal-containing mitochondria. The crystals are similar to
those which have been described both in treated mitochondria isolated from
adult tissues and in mitochondria observed in injured, neoplastic and treated
cells (Brierley & Slautterback, 1964; Trump, Croker & Mergner, 1971; Bonucci,
Derenzini & Marinozzi, 1973). These crystalline deposits are composed of
divalent cations and inorganic phosphate (Lehninger, Rossi & Greenawalt,
722
L. GINSBERG AND N. HILLMAN
1963; Greenawalt, Rossi & Lehninger, 1964). The cations which have been
found to be accumulated by mitochondria are Ca2+, Mn 2+ , Sr2+ and Ba2+. Of
these four, all except Ba2+ can be accumulated in sufficient quantities to result
in 'massive loading' of the mitochondria with consequent granular or crystalline
depositions. The uptake of the cations by mitochondria occurs only in the
presence of excessive ATP and ADP. The accumulation of the cations, and the
subsequent formation of crystals has been shown to be respiration-dependent
and stoichiometrically related to electron transport (Lehninger et al. 1967).
Their formation can be prevented by respiratory uncouplers and inhibitors such
as 2,4-dinitrophenol, antimycin A and cyanide. When Ca 2+ , Mn 2+ or Sr2+ is
accumulated and deposited as a phosphate granule or crystal, no oxidative
phosphorylation of ADP occurs. Oxidative phosphorylation of ADP and cation
accumulation are thus alternative processes and cannot occur simultaneously.
Because the formation of crystals prevents oxidative phosphorylation of ADP,
there is a net reduction of ATP synthesis and a decreased ATP/ADP ratio.
Excessive ATP synthesis, followed by the formation of crystalline deposits, a
reduction of ATP synthesis and a decreased ATP/ADP ratio is the sequence of
events which is found in the tw32/twS2 homozygotes. Studies are now in progress
to determine if this same sequence occurs in tejt6 embryos.
A possible explanation for the excessive levels of ATP metabolism in early
cleavage-stage tn/tn embryos can be drawn from recent studies on ^-bearing
spermatozoa. Ginsberg & Hillman (1974) have reported that te-, tw32- and t12bearing epididymal spermatozoa have greater oxygen uptake in denned media
supplemented with pyruvate, lactate or succinate than do J + -bearing spermatozoa. Pyruvate and lactate are the carbohydrates present in Brinster's medium
(Brinster, 1963) and are also found in mouse oviducts (Bishop, 1957; Hamner &
Williams, 1965; Holmdahl & Mastroianni, 1965; David, Brackett, Garcia &
Mastroianni, 1969). If tnjtn embryos, like P-bearing spermatozoa, utilize
pyruvate and lactate more efficiently than do correspondingly staged T+jT+
embryos, the increased utilization could result in the increased ATP metabolism
found in 2- and 4-cell t12lt12 and twS2ltwZ2 embryos. The metabolic imbalance
caused by this excessive carbohydrate oxidation would result both in excessive
levels of ATP synthesis and in excessive amounts of acetyl-CoA, the prerequisites for excessive lipid synthesis and storage and for the deposition of
mitochondrial crystals.
The research was supported by U.S. Public Health Research Grant HD-00827. The
authors would like to acknowledge the aid of Dr Ralph Hillman in the preparation of this
manuscript.
ATP metabolism in mutant mouse embryos
723
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