/. Embryol. exp. Morph. Vol. 56, pp. 157-168, 1980
Printed in Great Britain © Company of Biologists Limited 1980
\ 57
The metabolism of exogenous fatty acids by
preimplantation mouse embryos developing in vitro
By THOMAS J. FLYNN 1 AND NINA HILLMAN 1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
The utilization of fatty acids from the culture medium has been examined in preimplantation mouse embryos developing in vitro.
Incorporation of exogenous fatty acid into embryo lipids was examined by culturing 8-cell
mouse embryos for 2 h in a medium containing 01 mM [9, 10-3H]palmitic acid (900 mCi/
mmol). Lipids were extracted from the embryos, and the total lipid extract was fractionated
into various neutral lipid and polar lipid classes by thin-layer chromatography. Most of the
radioactivity, over 93 %, was recovered in neutral glycerides (mono- di-, and triacylglycerols).
About 2% of the total radioactivity was recovered in other neutral lipid species including
fatty acids, fatty alcohols, and sterol esters. The remainder of the radioactivity was recovered
in polar lipids. Seventy-four per cent of the polar lipid radioactivity was present in the
choline phosphatides. Other labelled phospholipid and glycolipid species included ethanolamine phosphatides, inositol and/or serine phosphatides, sphingomyelin, choline lysophosphatides, sulfatides, cerebrosides, and monoglycosylglycerides. Chemical degradation studies
of labelled embryo lipids indicated that the tritium label was entering into embryo lipids as
the fatty acid and not via metabolic recycling.
The oxidation of exogenous fatty acids by mouse embryos was assessed by incubating
variously staged embryos for 4h in medium containing 01 mM [U-14C]palmitic acid (50
mCi/mmol) and quantitating the production of 14CO2. The rate of fatty acid oxidation was
found to be relatively constant from the unfertilized egg up to the 8-cell stage and then increase
significantly between the 8-cell and late blastocyst stages. The results suggest that preimplantation mouse embryos developing in vitro can utilize fatty acids from the medium both for
incorporation into embryo lipids and for energy production via oxidation.
INTRODUCTION
Several reports have indicated that preimplantation mouse embryos in culture
can incorporate label from various radioactive exogenous substrates into lipids
(Wales, Quinn & Murdoch 1969; Wales & Whittingham, 1970; Wales, 1975).
However, no attempts were made to characterize the lipid species being synthesized.
We have recently carried out studies on the distribution of label from [U-14C]
glucose into various lipid classes and individual lipid species of mouse embryos
maintained in culture from the 2-cell to the morula stages (Flynn & Hillman,
1978). In these studies we demonstrated that the major class of labelled lipids
1
Authors' address: Department of Biology, Temple University, Philadelphia, PA 19122,
U.S.A.
II
EMB
56
158
T. J. FLYNN AND N. HILLMAN
was triacylglycerols. We also showed, by using specific chemical degradative
procedures, that most of the label in glycerolipids was present in the glycerol
portion of these compounds and not in the fatty acids. Since fatty acids are
necessary components for lipid synthesis, and because commercially obtained
serum albumins can contain significant quantities of fatty acids (Chen, 1967),
the possibility arose that preimplantation mouse embryos were utilizing fatty
acids from the culture medium.
Earlier autoradiographic studies on preimplantation embryos from mutant
mice (Nadijcka & Hillman, 1975) have demonstrated that these embryos can
take up labelled fatty acids from the culture medium. However, it was not
determined whether preimplantation embryos are capable of metabolizing
these exogenous fatty acids. The present investigation was undertaken, therefore, in order to assess the ability of preimplantation staged embryos to metabolize exogenous fatty acids. Both the incorporation of exogenous palmitic acid
into embryo lipids and the oxidation of palmitic acid to carbon dioxide were
examined.
MATERIALS AND METHODS
Incorporation of palmitic acid into embryo lipids
Two-cell embryos were collected from superovulated female mice (Edwards &
Gates, 1959). The embryos were washed and then cultured in ovum culture
medium (Brinster, 1972) up to the 8-cell stage (approximately 24 h). The 8-cell
embryos were collected and transferred to radioactive medium which was prepared as follows: 0-2 ml of a 1 mM solution of palmitic acid in benzene and
approximately 0-25 mCi of [9,10-3H(n)]palmitic acid (New England Nuclear)
were added to a sterile glass tube. The benzene was evaporated with a gentle
stream of nitrogen gas. Two ml of the ovum culture medium described by Brinster
(1972), which had been prepared with fatty acid-free bovine serum albumin (Miles
Research), was added. The tube was then placed in a Sonitron-80 sonic bath and
sonicated for 0-5 h. An aliquot of the labelled medium was chromatographed on
a Sephadex G-25 column, which was eluted with 0-1 M sodium phosphate buffer,
pH 7-4. Over 99 % of the radioactivity was recovered in the void volume with the
albumin fraction, indicating that the palmitic acid was complexed with the
albumin. The concentration of palmitate in this medium is 0-1 mM, and the
molar ratio of palmitic acid to albumin is 1-2. These values are within the
physiological range (Spector, 1967). The specific radioactivity is approximately
900 mCi/mmol. From 60 to 180 8-cell embryos were cultured for 2 h in 0-1 ml
drops of this medium overlaid with silicone oil (Dow-Corning 200). Paraffin oil,
which is often used for embryo culture, was found to be unsuitable because it
extracted fatty acids from the medium.
Mouse embryo fatty acid metabolism
159
Extraction and fractionation of labelled lip ids
After the 2 h incubation in labelled medium, embryos were collected, washed
four times in non-radioactive medium, and then extracted for 2 h at 0 °C with
1-9 ml chloroform-rnethanol-water (1:2:0-8, v/v/v) according to the procedure
of Bligh & Dyer (1959). After partition by the addition of 0-5 ml each of chloroform and water, the aqueous layers were removed and discarded. The chloroform layers were evaporated to dryness with a stream of nitrogen, and the lipid
extract was then taken up in 1-0 ml chloroform. Aliquots of this chloroform
extract were used for further analysis.
Thin-layer chromatographic procedures
All thin-layer chromatography was carried out on mylar-backed silica gel
60 thin-layer plates (E. Merck). The thin-layer plates were cut to a size of 2-5 x
7-5 cm for one-dimensional chromatography and 5-0 x 5-0 cm for two-dimensional chromatography. The following solvent systems were used for onedimensional development of the plates: (1) hexane-diethylether-glacial acetic
acid (75:25:1, v/v/v) for the characterization of neutral lipids; (2) diisopropylether-glacial acetic acid (96:4, v/v) followed by hexane-diethylether-glacial
acetic acid (80:20:1, v/v/v) for the characterization of chloroform-soluble
products from alkaline methanolysis. The following two-dimensional system
was used for the characterization of polar lipids: (3) chloroform-methanolwater (62:25:4, v/v/v) in the first direction followed by chloroform-methanolglacial acetic acid-water (25:15:4:2, v/v/v/v) in the second direction. Appropriate non-labelled lipid standards, which were obtained from either Applied
Science Laboratories or Supelco, Inc., were added to embryo lipids as carriers
and to provide enough material for detection on the thin-layer plates. Spots were
visualized by spraying the plates with 0-6 % K2Cr2O7 in 55 % H2SO4 and then
heating to 120 °C for 1 h in order to char the lipids. For the determination of
radioactivity, spots were scraped from the plates into scintillation vials containing 1 ml water and 10 ml Hydromix (Yorktown Research).
Mild alkaline methanolysis of lipids
Alkaline methanolysis of lipids was carried out essentially as described by
Ambron & Pieringer (1971). Lipid samples were dried in a test tube with a
stream of nitrogen and then redissolved in 0-3 ml toluene-methanol (1:1, v/v).
0-3 ml 0-2 N KOH in methanol was added, and the sample was incubated at
37 °C for 20 min. The reaction was stopped by the addition of three drops of
ethyl formate, and the reaction products were partitioned by the addition of 0-5 ml
chloroform and 0-3 ml water.
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T. J. FLYNN AND N. HILLMAN
Fatty acid oxidation
The procedure used for the measurement of carbon dioxide production from
fatty acid was adapted from that described by Brinster (1967 a, 19676) for the
measurement of carbon dioxide production from glycolytic substrates.
Medium containing no energy substrates was prepared as described by Brinster
(1967a). Fatty acid-free bovine serum albumin was used in the preparation of
this medium. Medium labelled with [14C]palmitic acid was prepared as follows.
About 20 /tCi of [U-14C]palmitic acid (New England Nuclear) and 0-4 ml of
1 mM solution of unlabelled palmitic acid in benzene were added to a sterile
glass tube. The palmitic acid was dried as a thin film on the sides of the tube by
evaporating the benzene with a gentle stream of nitrogen gas. Four ml of the
substrate-free medium was added, and the tube was sealed and placed in a
Sonitron-80 sonic bath for 0-5 h. The concentration of palmitate in this medium
is 0-1 mM and the specific radioactivity is approximately 50 mCi/mmol.
One- and 2-cell embryos were obtained from superovulated females and
utilized directly. One-cell embryos were washed for 5 min at room temperature
in medium containing bull sperm hyaluronidase (Sigma), 300 U/ml, in order to
remove the cumulus cells (Biggers Whitten & Whittingham, 1971). Later-staged
embryos were obtained by placing 2-cell embryos into culture medium (Brinster,
1972) and allowing them to develop to the desired stage. Embryos were collected
and washed through four drops of energy-substrate-free medium. The embryos
were then counted and transferred into 50 /A of [14C]palmitate-labelled medium
under three drops of silicone oil, all in a sterile 6 mm x 40 mm glass tube. The
culture tube and an empty 2 ml shell vial were sealed in a scintillation vial with a
rubber serum bottle stopper. The vial was flushed with 5 % CO2 in air, and then
incubated at 37 °C for 4 h. At the end of the incubation period, 0-1 ml of 1 NH2SO4 was injected into the culture tube, and 1 ml of 1 M hyamine hydroxide
(Amersham/Searle) was injected into the shell vial. The scintillation vials were
left sealed at room temperature for 48 h. After 48 h, the rubber stoppers and the
culture tubes were removed. Ten ml Hydromix was added and mixed well with
the Hyamine. Radioactivity was then measured in a liquid scintillation counter.
The percent recovery of 14CO2 by this procedure was quantitated by carrying out
incubations as above with a medium containing known amounts of NaH14CO3.
Blank values were established from control incubations which contained no
embryos.
At the specific activity of labelled palmitate used, approximately 2 c.p.m.
detected was equivalent to one fmol CO2 produced. The average number of
embryos used per determination varied from 35 for late blastocysts to 225 for
unfertilized eggs. In all experiments, the number of embryos used was sufficient
to give at least 150 c.p.m. above the blank values.
Mouse embryo fatty acid metabolism
161
Determination of cell number of blastocysts.
The number of cells per blastocyst was determined by counting nucleii prepared by the air-drying technique of Tarkowski (1966). Blastocysts were collected and subjected to hypotonic treatment in 0-25 % sodium citrate for 10-15
min. The blastocysts were then transferred to microscope slides, fixed with
ethanol-glacial acetic acid (3:1, v/v), and stained with Giemsa Stain (Harleco).
The number of normal nucleii was determined under a light microscope.
Measurement of radioactivity
Radioactivity was measured in a Packard Tricarb Model 3380 liquid scintillation spectrometer. Counting efficiency was determined by the channels ratio
method.
Statistical methods
For the lipid labelling studies, data were transformed into angles ('arcsin
transformation') in order to compute standard deviations (Zar, 1974). Statistical
significance was determined using Student's ?-test.
RESULTS
Effects of 2 h exposure of 8-cell embryo to [9, 10-sH]paImitic acid on further
in vitro development
Tritium-labelled compounds in the culture medium have previously been
shown to adversely affect the development of preimplantation mouse embryos
in vitro (Snow, 1973). Therefore, an experiment was performed to assure that the
amounts of radioactivity used in the present experiments (approximately 100
/^Ci/ml) were not causing severe radiation damage to the embryos. After the 2 h
labelling period, some embryos were washed, transferred into unlabelled culture
medium, and allowed to develop for an additional 45 h. Control embryos were
manipulated in a similar manner except for exposure to the isotope. As shown
in Table 1, the percentage of 8-cell embryos developing to blastocysts was similar
for both groups. The number of cells per blastocyst in the 3H-treated group decreased slightly but not significantly (P > 0-05) from that in the controls. Both
longer labelling periods and higher medium specific radioactivity were observed
to significantly impair embryonic development (data not shown).
Incorporation of label from [9, 10-3H] palmitic acid into embryo lip ids
(a) Total lipid extract. An average of 620 d.p.m./embryo or 75000 d.p.m./
experiment was recovered in the total lipid extract. Based on the specific activity
of the palmitic acid in the medium, the net incorporation of palmitic acid into
total embryo lipids was 317-2 ±24-2 fmol/embryo/2 h + s.E.M.
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T. J. FLYNN AND N. HILLMAN
Table 1. Effects of 2 h exposure to [9, lO-3H]palmitic acid on the
in vitro development of 8-cell mouse embryos to blastocysts
Embryos
Control
[3H]palmitate
% developing
into blastocysts
from 8-cell
Mean cell no.
per blastocyst
97-0
94-6
65-8 ±3-8
57-3 ± 3 0
±S.E.M.
8-cell mouse embryos were incubated for 2 h in medium containing [9, 10-3H]palmitic
acid, and then transferred to unlabelled medium for further development. Control embryos
were manipulated similarly except for exposure to the isotope. Both percent development
into blastocysts and mean cell number per blastocyst were determined. Data were pooled from
two independent experiments.
Table 2. Distribution of label from [9, 10-sH]palmitic acid in
neutral lipids
Neutral lipid class
Triacylglycerols
Monoalkyldiacylglycerols
Diacylglycerols
Fatty acids
Fatty alcohols
Monoacylglycerols
Sterol esters
Polar lipids at origin
% of total
lipid radioactivity
83-4
8-9
10
10
0-5
0-4
0-3
41
angle ±S.E.M.
6615± 1-31
17-34±O-38
5-66 ±0-40
5-58 ±0-68
407 ±0-36
2-45 ±0-73
211 ±0-73
11 32± 1 10
Individual neutral lipid classes were obtained by thin-layer chromatography of the total
lipid extract in solvent system 1. Results are expressed as proportion of the total lipid radioactivity recovered in each neutral lipid class in terms of percentages and their angular transformations. Values were obtained from seven determinations made in two independent
experiments.
(b) Neutral lipids. The distribution of total lipid radioactivity among individual
neutral lipid species is shown in Table 2. The major labelled neutral lipids are the
triacylglycerols and their monoether analogs, the monoalkyldiacylglycerols.
Together, these two neutral lipid classes contain over 92 % of the total embryo
lipid radioactivity. An additional 1-4% of the radioactivity was recovered in the
diacylglycerol and monoacylglycerol fractions. Thus, over 93 % of the total
labelled embryo lipid was recovered in neutral glycerides. Radioactivity was also
found in the fatty acid, fatty alcohol, and sterol ester fractions. About 4 % of the
total lipid label remained at the origin with the polar lipid fraction.
(c) Polar lipids. The distribution of total polar lipid radioactivity among
individual polar lipid classes is shown in Table 3. Radioactivity was recovered in
both phospholipid and glycolipid species. The major labelled polar lipids are
the choline phosphatides, which account for almost three fourths of the total
Mouse embryo fatty acid metabolism
163
Table 3. Distribution of label from [9, 10-zH]palmitic acid in polar lipids
Polar lipid class
Phospholipids
Choline phosphatides
Ethanolamine phosphatides
Sphingomyelin
Inositol and/or serine phosphatides*
Choline lysophosphatides
Glycolipids
Sulfatides
Cerebrosides
Monoglycosylglycerides
% of total
polar lipid
radioactivity
angle ±S.E.M.
73-7
6-9
2-4
1-3
0-5
5919 + 0-75
15-10 ±1-06
7-65 ± 1 -42
5-78 ±1-03
3-68±O-85
4-9
3-5
2-2
12-78 ±0-66
9-69 ±2-39
7-89 ±2-20
Individual polar lipid classes were obtained by 2-dimensional chromatography of the total
lipid extract in solvent system 3. Results are expressed as proportion of the total polar lipid
radioactivity recovered in each polar lipid class in terms of percentages and their angular
transformations. Values were obtained from seven determinations made in two independent
experiments.
* These two compounds were not clearly separated by the chromatographic system used.
polar lipid radioactivity. Other labelled phospholipid classes include the ethanolamine phosphatides, sphingomyelin, inositol and/or serine phosphatides,
and choline lysophosphatides. About 10% of the total polar lipid radioactivity
migrated on thin layer plates with glycolipid standards. Almost half of the
glycolipid radioactivity was found in the sulfatide fraction, and the remainder
was recovered in cerebrosides and monoglycosylglycerides.
Identification of the labelled moiety
In order to verify that label from [9,10-3H]palmitic acid was entering into
mouse embryo lipids relatively intact and not via metabolic recycling of the
tritium label, the total extract was treated with mild alkali in methanol. Alkaline
methanolysis cleaves lipid-bound fatty acid esters from the parent lipid with a
resultant formation of the corresponding fatty acid methyl esters. The recovery
of radioactivity in chloroform soluble and aqueous soluble products after alkali
treatment of the total lipid extract is shown in Table 4. Over 98 % of the radioactivity is recovered in the chloroform-soluble material. This finding is consistent
with the radioactivity being present in fatty acids and not in the water soluble
glycerol backbones of the glycerides. As a further check on the identity of the
labelled moiety of mouse embryo lipids, the chloroform soluble products from
alkaline methanolysis were examined by thin-layer chromatography. The results
are shown in Table 5. About 95 % of the label is recovered in fatty acids and
fatty acid methyl esters. Almost 2 % of the label is found in the fatty alcohol
fraction, and less than 1 % of the radioactivity is recovered in monoalkylglyceryl-
164
T. J. FLYNN AND N. HILLMAN
Table 4. Distribution of radioactivity after alkaline methanolysis
of the total lipid extract
H 2O-soluble radioactivity
CHC13-soluble radioactivity
A
angle ±S.E.M.
°/
/o
981
r
%
1-9
82-77 ±1-38
angle ±S.E.M.
7-23 ±1-38
The total lipid extract was subjected to alkaline methanolysis as described in Materials and
Methods. Radioactivity was determined in both the chloroform-soluble and aqueous products
from methanolysis. Results are expressed as proportion of the starting radioactivity recovered
in the chloroform-soluble and aqueous products in terms of percentages and their angular
transformations. Values were obtained from seven determinations made in two independent
experiments.
Table 5. Distribution of radioactivity in chloroform soluble products
from alkaline methanolysis of the total lipid extract
Compound
Fatty acid methyl esters
Fatty acids
Fatty alcohols
Monoalkylglyceryl ethers
Polar compounds at origin
% of total
radioactivity
91-4
3-5
1-7
0-2
2-4
angle±s.E.M.
73-69±2-19
10-44 ±1 06
4-98 ±2-36
1-51 ±0-98
8-79 ±0-74
The chloroform-soluble products from alkaline methanolysis of the total lipid extract were
chromatographed on thin-layer plates in solvent system 2. Results are expressed as proportion
of the starting radioactivity recovered in each of the indicated compounds in terms of percentages and their angular transformations. Values were obtained from seven determinations
made in two independent experiments.
ethers. About 2 % of the radioactivity remains at the origin with polar compounds. The radioactivity at the origin is probably contained in monetherlysophosphatides (e.g. lysoplasmalogens) and polar sphingolipids. The ether
bonds in the former compounds and the amide bonds in the latter would not be
degraded by mild alkali treatment.
Fatty acid oxidation
The oxidation of exogenous fatty acids by preimplantation embryos at various
stages of development is shown in Table 6. The rate of fatty acid oxidation
remains relatively constant from the unfertilized egg to the 2-cell stage. There
is a significant (P < 0-02) increase in fatty acid oxidation between the 2-cell
and 8-cell stages and a highly significant (P < 0-002) increase in the rate of
fatty acid oxidation between the 8-cell and morula stages. The rate of fatty acid
oxidation continues to increase between the morula and late blastocyst stages.
Mouse embryo fatty acid metabolism
165
Table 6. Carbon dioxide production from [U-uC]palmitic acid by
preimplantation mouse embryos*
Time after
ovulation (hr)
Stage of development
fmol CO2
produced /embryo/hr ±
S.E.M.
12
12
36
60
84
84
108
Unfertilized 1-cell
Fertilized 1-cell
2-cell
8-cell
Morula
Early Blastocyst
Late Blastocyst
79 ±3 (3)
75 ±3 (4)
75 ±4 (4)
100 + 6 (4)t
244 + 23 (3)J
375 ± 29 (3)§
409 ±63 (3)
* Embryos at the indicated stages were incubated for 4 h with [U-14C]palmitic acid as the
sole energy substrate. 14CO2 production was measured as described in Materials and Methods.
The number of determinations at each stage is given in parentheses.
t Significantly different (P < 002) from preceding stage of development.
X Significantly different (P < 0002) from preceding stage of development.
§ Significantly different (P < 005) from preceding stage of development.
DISCUSSION
The data clearly demonstrate that preimplantation mouse embryos can take
up and utilize exogenous fatty acids both for incorporation into embryo lipids,
and for oxidation to carbon dioxide.
The bulk of the label from palmitic acid, over 93 %, was recovered in neutral
glycerides (Table 2). This rinding agrees with our earlier study (Flynn & Hillman,
1978) which showed that triacylglycerols are the major class of lipids labelled
with [U-14C]glucose as the precursor. This apparently active metabolism of
triacylglycerols by these early embryos may have developmental significance. A
major biological function of triacylglycerols is to serve as a storage form of
metabolic energy. It is possible that preimplantation embryos synthesize and
store triacylglycerols in preparation for a specific developmental event such as
the hatching of the blastocyst.
This hypothesis is supported by the observations of Thomson & Brinster
(1966) and Ozias & Stern (1973). They reported that glycogen, which is also
a storage form of metabolic energy, accumulates between the 1-cell and blastocyst stages in mouse embryos, but is rapidly depleted during the expansion and
hatching of the blastocyst. It has also been reported (Cholewa & Whitten, 1970)
that mouse embryos can develop in vitro from the 2-cell to the blastocyst stage
with no albumin present in the culture medium. However, a larger number of
these blastocysts collapsed or failed to hatch relative to blastocysts obtained
from 2-cell embryos cultured in the presence of albumin. It is possible that the
fatty acids associated with albumin are necessary for optimal hatching in vitro.
Similarly, although 2-cell embryos can develop in vitro into blastocysts in the
166
T. J. FLYNN AND N. HILLMAN
absence of glucose (Brinster, 1965), hatching, attachment, and trophoblast
outgrowth of the blastocysts is markedly inhibited without glucose (Wordinger
& Brinster, 1976). Since glucose is a major source of glycerol-3-phosphate
for lipid synthesis, the observed requirement for glucose may be related to a
requirement for lipid synthesis in normal in vitro hatching.
The pattern of incorporation of label from palmitic acid into mouse embryo
phospholipids (Table 3) is similar to that observed with [U-14C]glucose as the
precursor (Flynn & Hillman, 1978). The distribution of label among the various
phospholipid classes is roughly in accord with the known relative abundances of
these lipids in mammals (Bishop, 1971).
The major labelled glycolipid class was the sulfatides (Table 3). These compounds were not significantly labelled by [U-14C]glucose (Flynn & Hillman,
1978). However, the glucose-labelling studies were conducted on morulae, while
the present studies were carried out on 8-cell embryos. The observed differences
in labelling could be due to a differential utilization of the two labelled precursors. A more intriguing possibility is a developmental stage specificity in
glycolipid synthesis. We have previously discussed the possible relationship
between glycolipids and early embryonic antigen expression (Flynn & Hillman,
1978).
The data in Tables 4 and 5 indicate that most of the radioactivity from [9,
10-3H]palmitic acid entered into embryo lipids as the fatty acid and not via
metabolic recycling of the tritium label. Mouse embryos can oxidize palmitic
acid to carbon dioxide (Table 6). Presumably, any tritium lost from the labelled
palmitate by oxidation would be greatly diluted by the cellular water and in the
2 h incubation period, should not be significantly reincorporated into lipids via
recycling. However, minor metabolic alterations such as chain elongation or
desaturation of the labelled palmitic acid cannot be ruled out. The recovery
of label in fatty alcohols (Tables 2 and 5) and in monoalkylglyceryl ethers
(Table 5) suggests that preimplantation mouse embryos can reduce fatty acids
to the corresponding fatty alcohols and incorporate them into ether lipids.
From studies with mammalian cells in culture, it is known that exogenous
fatty acids can compete with other respiratory substrates for oxidative metabolism (Spector, 1972). The rates of carbon dioxide production from palmitic
acid shown in Table 6 are about an order of magnitude less than similar values
reported for glucose (Brinster, 1967 a) or lactate and pyruvate (Brinster, 19676).
However, these earlier studies by Brinster (1967a, 19676) were presumably not
carried out in fatty acid-free medium. Thus, further studies are needed to assess
the relative importance of exogenous fatty acids as oxidative substrates in preimplantation mouse embryos. A recent report (Kane, 1979) has shown that fatty
acids are an important energy source for preimplantation rabbit embryos.
The /?-oxidation of fatty acids to carbon dioxide takes place within the mitochondria (Lehninger, 1970). The observed significant increase in carbon dioxide
production from palmitic acid between the 8-cell and morula stages is indicative
Mouse embryo fatty acid metabolism
167
of a change in mitochondrial oxidative function at this stage of development.
Mills & Brinster (1967) have reported that oxygen consumption by preimplantation mouse embryos remains relatively constant up to the 8-cell stage, but then
undergoes a highly significant increase between the 8-cell and morula stages.
Thus, there appears to be a correlation between changes in the rates of oxygen
uptake and fatty acid oxidation in these embryos.
One question which remains to be answered is the contribution of endogenous
fatty acid synthesis to the overall metabolism of fatty acids by preimplantation
mouse embryos. Our previous study (Flynn & Hillman, 1978) showed that label
from [U-14C]glucose was going preferentially into the glycerol portion rather
than the fatty acid portion of embryonic lipids. Since this earlier study was
carried out with serum albumin which probably contained fatty acids, it is
presumed that the presence of exogenous fatty acids inhibited endogenous fatty
acid synthesis from glucose. The inhibition by exogenous lipids of endogenous
lipid synthesis in mammalian cells in culture is a well documented phenomenon
(Spector, 1972). We are currently investigating lipid biosynthesis in preimplantation mouse embryos in the absence of an exogenous source of fatty acids.
This study was supported by USPHS Grant HD 00827 from the National Institutes of
Health. The excellent technical assistance of Ms Marie Morris is greatly appreciated.
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{Received 25 January 1979, revised 12 October 1979)