ABSTRACT
High-resolution autoradiographic studies were used to determine whether t12/t12 and tw32/tw32 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 tw32/tw32 embryos also exhibit non-physiological 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 tn/tn embryos, and second, the type of lipid deposited.
MATERIALS AND METHODS
Standard crosses were used to obtain T+/Z12 (Smith, 1956) and T+/tw32 (Bennett & Dunn, 1964; Hillman & Hillman, 1975) mice. Eight-week-old T+/tn (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 mutants since T+/tn males transmit the t12 and tw32 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 twS2/tw32 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-labeIled precursors were diluted with Brinster’s medium to a concentration of 5 μCi/ml medium and [14C]pyruvate was diluted to 2 μCi/ml medium.
After removal from culture, the embryos were washed in non-radioactive 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+/tw32inter se matings and three control litters were used for each study.
Homozygous t12 and tw32 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 al. 1970; Hillman & Hillman, 1975). These morphological characteristics were used, in the present study, to separate the homozygous tn/tn embryos from their litter-mates.
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 t32/t32 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.
An autoradiograph of a portion of a late morula t12/t12 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 tw32/tw32 and control morula embryos, × 18000.
An autoradiograph of a portion of a late morula t12/t12 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 tw32/tw32 and control morula embryos, × 18000.
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 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.
An autoradiograph of a tw32/tw32 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, × 6500.
An autoradiograph of a tw32/tw32 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, × 6500.
A portion of 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 t32/tw32 embryos (Fig. 2). Silver grains are seldom found over the lipid droplets but are scattered over other cellular organelles and membranes, × 40000.
A portion of 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 t32/tw32 embryos (Fig. 2). Silver grains are seldom found over the lipid droplets but are scattered over other cellular organelles and membranes, × 40000.
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. 4d). Silver grains were also found randomly scattered over the cytoplasm, nucleoplasm, membranes and cellular organelles.
(a) An autoradiograph of a late morula t12/t12 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. × 3600. (b) Lipid droplets from a tw32/tw32 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, × 14000.
(a) An autoradiograph of a late morula t12/t12 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. × 3600. (b) Lipid droplets from a tw32/tw32 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, × 14000.
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 tn/tn 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.
ACKNOWLEDGEMENTS
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.