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/ . Embryo/, exp. Morph. Vol. 33, 3, pp. 725-730, 1975
Printed in Great Britain
725
Autoradiographic studies of tn/tn
mouse embryos
By MARY NADIJCKA 1 AND NINA HILLMAN 1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
High-resolution autoradiographic studies were used to determine whether t12/t12 and
Z"'32//"32 mouse embryos synthesize the excessive lipid which distinguishes these embryos
prior to their death. The studies show that the tn homozygotes synthesize neutral lipid which
is stored in intracellular lipid droplets. Cholesterol and phospholipid precursors are not
incorporated into these droplets.
INTRODUCTION
Three mutant recessive alleles (t12, tw32, t6), when homozygous, display similar
phenotypic characteristics (Hillman, Hillman & Wileman, 1970; Hillman &
Hillman, 1975; Nadijcka & Hillman, 1975). One of these characteristics is the
presence of excessive cytoplasmic lipid prior to the lethal periods of the homozygous mutant embryos. Both t12/t12 and twS2/tu'32 embryos also exhibit nonphysiological levels of ATP metabolism (Ginsberg & Hillman, 1975). Since it
has been reported that increased ATP synthetic rates can increase lipogenesis
by shunting acetyl CoA into fatty acid synthesis (Atkinson, 1965; Newsholme &
Start, 1973), it has been suggested that the aberrant ATP synthesis could result
in the characteristic excessive lipid. The present high-resolution autoradiographic study was undertaken to determine first, if the excessive lipid was synthesized by the tnjtn embryos, and second, the type of lipid deposited.
MATERIALS AND METHODS
Standard crosses were used to obtain T+lt12 (Smith, 1956) and T+/tw32
(Bennett & Dunn, 1964; Hillman & Hillman, 1975) mice. Eight-week-old
T+ltn (t12 or tw32) females were superovulated (Edwards & Gates, 1959) and
mated to heterozygous males of the same genotype (Hillman et al. 1970;
Hillman & Hillman, 1975). Pregnant females were sacrificed on gestation day 1
(day 0 = day of plug) and the 2-cell embryos were flushed from the excised
oviducts with Brinster's medium and placed into culture (Brinster, 1963). Each
litter contained more than the expected Mendelian ratio of homozygous
1
Authors'1 address: Department of Biology, Temple University, Philadelphia, Pennsylvania
19122, U.S.A.
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M. N A D I J C K A AND N. H1LLMAN
Fig. 1. An autoradiograph of a portion of a late morula t12jt12 embryo labelled from
the 8-cell to late morula stage with [14C]pyruvate. Note the presence of tracks over
the lipid droplets (L), myelin bodies (MB) and degradation bodies (DB). No tracks
are associated with mitochondria (M). Similar labelling patterns are found in
twS2/tu-32 a n d c o n t r o i morula embryos, x 18000.
mutants since T+/tn males transmit the t12 and ?u32 alleles in frequencies higher
than normal (Smith, 1956; Bennett & Dunn, 1964).
Individual litters developed in the standard medium until they reached the
early 8-cell stage when they were separately placed into radioactive medium.
Litters containing tw3*ltw*2 embryos remained in the radioactive medium until
they had reached the early morula stage of development (12 h of incubation)
and litters containing t12 homozygotes were kept in radioactive medium until the
late morula stage (24 h of incubation). The radioactive compounds used were
[14C]pyruvate (Amersham/Searle, 12-4mCi/mM), [3H]ethanolamine(Amersham/
Searle, 320 mCi/mM), [3H]choline chloride (New England Nuclear, 1 Ci/mM),
[3H]mevalonic acid (Amersham/Searle, 82 mCi/mM) and [3H]palmitic acid
(Schwarz/Mann, 27 Ci/mM). Prior to use, the 3H-labelled precursors were diluted
with Brinster's medium to a concentration of 5 /*Ci/ml medium and [14C]pyruvate
was diluted to 2/*Ci/ml medium.
After removal from culture, the embryos were washed in non-radioactive
Autoradiographic studies o/t n /t n embryos
727
m
Fig. 2. An autoradiograph of a tu'32jtwSZ embryo incubated from the 8-cell stage
until the early morula stage in [3H]ethanolamine-supplemented medium. Label is
scattered over the cytoplasm but is rarely found over the lipid droplets (L) of either
the homozygous tn embryos or the control embryos, x 6500.
medium and fixed immediately for electron microscopy. The fixation and embedding protocols of Stein & Stein (1971) for lipid high-resolution autoradiography were used in the present study. Thin sections were collected on copper
grids and coated with Ilford L-4 nuclear emulsion. After a suitable exposure
period (3H-precursors, 2 weeks; 14C-compound, 3 weeks to 3 months) the autoradiographs were developed with Dektol, acid-fixed and rinsed with distilled
water. The sections were stained with lead citrate and viewed with a Zeiss 9 A
electron microscope. Correspondingly staged litters of random-bred embryos
were incubated in the radioactive medium for similar lengths of time and processed in the same manner. At least three litters from both T+/t12 and T+jtwZ2
inter se matings and three control litters were used for each study.
Homozygous t12 and twZ2 embryos can be distinguished from their respective
phenotypically wild-type litter-mates prior to their lethal periods by the presence
of excessive cytoplasmic lipid. Nuclear fibrillo-granular bodies and binucleated
cells are found in t12 homozygotes while tw32 homozygotes are characterized by
abnormal mitochondria and binucleated cells (Hillman et ah 1970; Hillman &
Hillman, 1975). These morphological characteristics were used, in the present
study, to separate the homozygous tnjtn embryos from their litter-mates.
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M. N A D I J C K A AND N. HILLMAN
Fig. 3. A portion of a t12/t12 embryo incubated in [3H]choline chloride-supplemented
medium from the 8-cell stage until the late morula stage. The pattern of labelling
is the same as that found in tw32/tw32 embryos (Fig. 2). Silver grains are seldom
found over the lipid droplets but are scattered over other cellular organelles and
membranes, x 40000.
RESULTS AND DISCUSSION
Incubation in medium supplemented with [14C]pyruvate, which can serve as
the sole energy source for the in vitro development of mouse embryos during
cleavage (Brinster, 1965), was used to determine if the excessive lipid was a result
of synthesis by the mutant embryo. Autoradiographs of embryos grown in
[14C]pyruvate showed that most of the lipid was labelled in t12/t12 and tw*2ltw*2
embryos, in their respective phenotypically wild-type litter-mates and in
correspondingly staged control embryos. This indicates that all of the embryos
are synthesizing lipid during the later cleavage stages. The tn homozygotes contained more lipid than the control embryos and most of this excessive lipid was
labelled. Label was also found scattered over all other cellular organelles, with
the exception of mitochondria, as well as over the cytoplasm and nucleoplasm of
the embryos. Fig. 1 shows a typical labelling pattern.
Neither the excessive lipid in tn homozygotes nor the lipid droplets in control
embryos were labelled after incubation in medium containing the phospholipid
precursors, [3H]ethanolamine (Fig. 2) and [3H]choline chloride (Fig. 3). Label
Autoradiographic studies o/t n /t n embryos
729
Fig. 4. (a) An autoradiograph of a late morula f12/'12 embryo, incubated in [3H]palmitic acid-supplemented medium. Most of the lipid droplets are heavily labelled
(arrows). This pattern is typical for both the tn/tn embryos and the control embryos,
x 3600. (6) Lipid droplets from a tu32/tuS2 embryo. Some lipid droplets are not
labelled by [3H]palmitic acid. This unlabelled lipid either was synthesized prior to
the treatment period or is not a neutral lipid. x 14000.
was, however, associated with cellular organelles, including mitochondria, and
was found over cellular and nuclear membranes. Embryos incubated in [3H]mevalonic acid, a cholesterol precursor, were completely unlabelled except for
the normal background. There are two possible explanations for this lack of
labelling: either the embryos are not synthesizing cholesterol or they are impermeable to this lipid precursor.
The majority of lipid droplets in normal embryos and most of the excessive
lipid deposits of the mutants were densely labelled following incubation in
medium supplemented with the neutral lipid precursor, [3H]palmitic acid
(Fig. 4). Those droplets which were not labelled may be assumed to be either
composed of non-neutral lipid or synthesized during the earlier cleavage stages
prior to incubation in radioactive medium (Fig. Ad). Silver grains were also found
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M. NADIJCKA AND N. HILLMAN
randomly scattered over the cytoplasm, nucleoplasm, membranes and cellular
organelles.
From the present series of experiments, it is impossible to determine whether
the excessive lipid deposits, characterizing the homozygous tn embryos, result
from excessive lipogenesis or from a lack of utilization of the lipid which is
being synthesized at normal rates. To distinguish between these two possibilities embryos must be processed for autoradiography after a suitable chase
period following their incubation in radioactive medium. We have found, however, that it is necessary to incubate the embryos in radioactive lipid precursors
for extensive periods of time (from early 8-cell until early or late morula) for
these embryos to be sufficiently labelled for autoradiographic analysis. In addition, mutant embryos must be incubated with radioactive label during the later
cleavage stages since it is during these developmental periods that tnjtn embryos
exhibit the greatest lipid deposition (Hillman et al. 1970; Hillman & Hillman,
1975). Since most of the tn/tn embryos die during the early (tw32) and late (t12)
morula stages, it is not possible to use a chase period, and consequently it is not
possible to determine the cause of their excessive lipid deposition. Nevertheless,
the studies do show that both t12/t12 and tw32/tw32 embryos are synthesizing the
excessive lipid and that this lipid is labelled only by the neutral lipid precursor.
The research was supported by U.S. Public Health Research Grant HD-00827. The
authors would like to acknowledge the technical assistance of Marie Morris and Geraldine
Wileman.
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