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/ . Emhryol. exp. Morph. Vol. 60, pp. 303-319, 1980
Printed in Great Britain © Company of Biologists Limited 1980
303
Membrane sterols and the development of the
preimplantation mouse embryo
HESTER P.M. PRATT,1 JO KEITH
AND
JYOTSNA CHAKRABORTY2
SUMMARY
The role of membrane sterols in the compaction and subsequent development of the
preimplantation mouse embryo was studied by incubating embryos in 7-ketocholesterol and
other oxygenated sterols. These sterols have been shown to inhibit sterol synthesis and
deplete membranes of cholesterol in a variety of other cell types.
Compaction and subsequent blastocyst formation were normal when embryos were
incubated in physiological sterols but were inhibited by oxygenated sterols to a degree
which depended upon the concentration of sterol, duration of incubation and developmental
age of the embryos. Precompaction 8-cell embryos were most susceptible to the action of
these sterols and failed to compact (as assessed by cell flattening and increased intercellular
adhesion) but continued to divide, whilst later stage embryos developed normally. 7-ketocholesterol had a specific effect on the ultrastructure of the smooth endoplasmic reticulum
of treated embryos. The developmental and ultrastructural effects induced by the oxygenated
sterols could be reversed or prevented by the use of products of the blocked reaction (i.e.
mevalonate, desmosterol or cholesterol).
These results substantiate the evidence that preimplantation mammalian embryos are
capable of synthesizing membrane sterols from the 8-cell stage onwards and emphasize the
importance of the sterol composition of membranes for normal cytokinesis and compaction
of the mouse embryo.
INTRODUCTION
The presence of sterols in the cell membranes of all eukaryotes and some
prokaryotes (Nes, 1974) implies a fundamental role in membrane physiology
for these molecules. Studies of artificial bilayers of defined lipid composition
have demonstrated that sterols interact with phospholipids to counteract phase
transition effects and thereby create an intermediate fluidity state which stabilizes the bilayer and regulates its permeability (Lee, 1975). The variation in
sterol content amongst different intracellular membranes, the enrichment of
cholesterol in the normal plasma membrane (Bretscher, 1973) and the well
documented examples of pathological conditions associated with cells of
abnormal sterol composition (Papahadjopoulos, 1974; Cooper, 1977) all suggest
1
Author's address: Department of Anatomy, University of Cambridge, Downing Street,
Cambridge, CB2 3DY, U.K.
8
Author's address: Department of Physiology, Medical College of Ohio, C.S. No. 10008,
Toledo, Ohio 43699, U.S.A.
304
H. P. M. PRATT, J. KEITH, J. CHAKRABORTY
that the properties of membranes can be influenced by their component sterols.
Experimental modifications of the cholesterol content of membranes in intact
cells have confirmed that cholesterol can regulate the fluidity and permeability
of the lipid bilayer in natural as well as artificial membranes (Papahadjopoulos,
1974; Demel & de Kruyff, 1976). Sterol modification experiments have also
demonstrated that the activity of membrane transport enzymes can be influenced
and may be regulated by the cholesterol composition of their immediate lipid
microenvironment (Kimelberg, 1977; Warren, Houslay, Metcalfe & Birdsall,
1975). Other functions of cell membranes, namely the ability to undergo
cytokinesis (Pratt, Fitzgerald & Saxon, 1977) and natural or experimentally
induced cell fusion (Hope, Bruckdorfer, Hart & Lucy, 1977; Horwitz, Wight,
Ludwig & Cornell, 1978) as well as the capacity to bind and transmit proliferative and differentiative stimuli (Alderson & Green, 1975; Pratt et al. 1977) are
all radically and specifically altered by modifications in sterol composition.
With these observations in mind, it is clear that many of the crucial reorganizations of membranes, particularly plasma membranes, that occur during the
differentiation and proliferation of the preimplantation mouse embryo could
be influenced by, or possibly even mediated by, changes in their sterol composition. For example, the redistribution of plasma membrane components
(both pre-existing and newly synthesized) and the membrane fusion that
accompanies each cleavage or mitotic division must require a degree of fluidity
in the lipid matrix. Furthermore, the sequence of cell flattening and intercellular
adhesion that occurs during compaction of the morula and is associated with
changes in membrane transport (Biggers, Borland & Powers, 1977) together
with the development of intercellular junctions and a polarized epithelial
configuration (Ducibella, Ukena, Karnovsky & Anderson, 1977; Johnson,
Pratt & Handy side, 1980) could also be critically dependent on the sterol
composition of embryo plasma membranes.
In order to study the possible influence of membrane sterol composition
on these processes, we have investigated the effects of incubating mouse morulae
with synthetic sterols which have been shown in a variety of other systems to
inhibit the rate-limiting enzyme in sterol synthesis, HMG Co A reductase
(hydroxymethylglutaryl Co A reductase), and thus to deplete cellular membranes
of cholesterol (Brown & Golstein, 1974; reviewed, Kandutsch, Chen & Heiniger,
1978). The morphological and developmental consequences of this treatment
reinforce the biosynthetic evidence for sterol synthesis in preimplantation
embryos (Pratt, 1978) and suggest a role for sterols in the reorganization of the
embryo that occurs during compaction.
Membrane sterols andpreimplantation mouse embryos
305
MATERIALS AND METHODS
Animals
CFLP female mice were obtained from Anglia Labs., Carworth, Essex, U.K.,
superovulated using 5 i.u. PMS (pregnant mare's serum) (Folligon, lntervet)
and 5 i.u. hCG (human Chorionic Gonadotrophin) (Chorulon, lntervet)
44-48 h later and were then mated with CFLP males. Detection of a vaginal
plug the following morning was taken to indicate successful mating and the
embryos were staged chronologically by defining the time of the hCG injection
as 0 h. Mice were killed by cervical dislocation and the embryos recovered by
flushing the oviducts and uteri with culture media. When zona-free embryos
were used, the zona was removed with acid Tyrode's solution + 0-4 % polyvinylpyrrolidone pH 2-5.
Embryo culture
The conventional embryological technique of incubating embryos in microdrops of culture medium covered with paraffin oil could not be used without
modification since the solubility of the sterols in the oil would lead to uncertainty about the effective concentration of sterol in the culture medium. Embryos
were therefore incubated in groups of five to ten embryos in the wells (0-1 ml
medium per well) of the microtitre plates (Gibco Bio-cult Ltd, Scotland)
which are conventionally used for immunological assays. The plates were
covered with adhesive plate covers (Gibco Bio-cult Ltd, Scotland) which
permit gas exchange, and incubated at 37 °C in 95 % air, 5 % CO2. Embryos
were scored morphologically either by direct observation in the wells of the
microtitre plates or else by removing them to Petri dishes and examining them
under an inverted phase microscope. Qualitatively similar results were obtained
by using the conventional micro-drop technique and pre-equilibrating the
paraffin oil with the sterol-containing media though in this case the minimal
effective dose was substantially higher due to partitioning of the sterol into the
oil. In some experiments 2 % foetal calf serum was added to the medium.
Chromatographically pure sterols were obtained from Steraloids Ltd,
Croyden, U.K. Stock solutions were made up in benzene at 50 mg/ml and
stored under N 2 at 4 °C for a maximum of 2 weeks before use. Samples of these
stock solutions were assessed periodically for their chromatographic purity
and the absence of autooxidation products by thin layer chromatography
on silica gel plates using the solvent petroleum ether:ether:acetic acid (40:60:
0-1 vol./vol.) (Bowyer & Davies, 1976). Immediately before use, these stock
solutions were diluted into 5 % bovine serum albumin (BSA) 0-14MNaCl pH
7-0, vortexed repeatedly and cleared by centrifugation as described by Kandutsch
& Chen (1973) to give a sterol solution of 500 ju,g/ml. This solution was then
diluted into culture medium containing 4 mg/ml BSA (Biggers, Whitten &
Whittingham, 1971) to give the appropriate sterol concentration for embryo
306
H. P. M. PRATT, J. KEITH, J. CHAKRABORTY
culture. Benzene from the original sterol stock solution had no effect on normal
embryo development at these dilutions. The type of BSA (and associated fatty
acids) used for the original suspension of sterols was not an important factor in
the developmental inhibition observed since sterols diluted into delipidated
BSA had the same effect. However, for the experiments described here one
single batch of BSA was used throughout. Autooxidation of sterols during
long incubation periods was a potential problem and a-tocopherol was included
in some culture media as suggested by Kandutsch & Chen (1977). However
since this did not result in enhanced embryonic growth or development,
autooxidation was not considered to be a major contribution to the results
obtained and a-tocopherol was not included in routine cultures.
In experiments involving the addition of mevalonic acid a stock solution of
100 mM mevalonic acid was made up in culture medium and then diluted
appropriately for embryo culture.
Cell counting
Assessments of cell numbers were made by counting cell nuclei prepared
using the technique of Tarkowski (1966).
Electron microscopy
Embryos were collected in culture medium number 16 containing 4mg/ml
BSA and fixed for 1 h at room temperature in 2-5 % glutaraldehyde, 0-1 Mcacodylate buffer pH 7-2, 10% sucrose. Embryos were washed in 0-1 Mcacodylate buffer, post fixed in 1 % osmium tetroxide 0-1 M cacodylate buffer,
dehydrated in ethyl alcohol and embedded in Epon. Thin sections were stained
with lead citrate and viewed with a Phillips EM300.
RESULTS
Table 1 demonstrates that precompacted 8-cell embryos compacted (as
assessed by cell flattening at the light microscope level) normally and formed
blastocysts when cultured continuously in natural precursors of cholesterol
and in cholesterol itself at concentrations up to 100yMg/ml (the highest concentration tested). However when embryos were treated with a variety of sterol
synthesis inhibitors (Brown & Goldstein, 1974) a dose-related inhibition of
compaction and blastocyst formation was observed. 7-ketocholesterol and
6-ketocholestanol were the most potent inhibitors but 20-a-hydroxycholesterol
and 25-hydroxycholesterol were also effective. Embryos treated with 5 /xg/ml
of 7-ketocholesterol continued to divide through one to two cycles of cell
division during 48 h of culture in a simple medium containing BSA (Table 2)
before eventually lysing. The rate of proliferation was significantly reduced
compared to either control or cholesterol-treated embryos which did not differ
Membrane sterols and preimplantation mouse embryos
307
Table 1. Growth of 8-cell embryos (60-64 h post hCG) in sterols and sterol
synthesis inhibitors
Desmosterol
Lanosterol
7-dehydrocholesterol
Squalene
Mevalonic acid (5 m.M)
(10 mM)
Cholesterol synthesis inhibitors
7-ketocholesterol
6-ketocholestanol
25-hydroxycholesterol
20-a-hydroxycholesterol
—
ooo
Control
Cholesterol precursors
Cholesterol
10
10
10
10
—
1
5
10
20
50
1
5
10
50
5
10
25
5
10
25
% compacting;* % forming blastocysts
No. of
embryos
98
82
(49)
70
96
89
100
93
80
100
100
100
65
81
89
80
60
60
100
100
100
(57)
(81)
(19)
(10)
(15)
(15)
(15)
(9)
(9)
80
19
0
3
6
80
.12
0
0
80
6
0
0
0
80
0
0
0
(5)
(69)
(57)
(75)
(72)
19
0
0
47
0
0
13
0
0
ooo
/tg/ml
(5)
(17)
(14)
(4)
(12)
(24)
(16)
(15)
(15)
(11)
* Compaction was assessed by cell flattening using an inverted phase microscope and an
embryo was considered to be compacted if it was indistinguishable from an untreated control.
significantly from one another (Table 2). The viability and morphology of
7-ketocholesterol treated embryos was substantially improved by inclusion of
2 % foetal calf serum (FCS) in the medium though the rate of cell division still
remained reduced compared to untreated controls. In the presence of 2 %
FCS the minimum dose of 7-ketocholesterol sufficient to inhibit compaction
was increased to 20-50 /*g/ml. The inclusion of 2 % FCS thus provided a
means of analysing the ultrastructural consequences of long term (24-48 h)
sub-lethal inhibition.
Compacting embryos treated with 20-50/*g/ml 7-ketocholesterol (in the
absence of FCS) were demonstrably less compact than controls within 6 h of
308
H. P. M. PRATT, J. KEITH, J. CHAKRABORTY
Table 2. Cell numbers of control and sterol-treated 8-cell embryos after 24-48 h
in culture*
Sterol
None
Cholesterol
7-ketocholesterol
Concentration
Otg/ml)
Hrs in culture Mean cell numbert No. of embryos
—
—
50
50
5
5
24
48
24
48
24
48
14-2 ± 2-8
42-6 ±160 2
19-7+ 60
49-6±15-52
10 3± 20
23-4 ± 4-81
(39)
(27)
(14)
(10)
(17)
(10)
* Embryos placed in culture medium+4 mg/ml BSA at 60-64 h post hCG. Cell number
8±1.
f Mean ± standard deviation of the mean (4-10 embryos) at end of incubation period.
12
P < 0001 Student's t test.
22
- P > 005 Student's t test.
treatment. Embryos incubated for longer periods in 20-50 /xg/ml + 2 % FCS
remained as a cluster of poorly compacted cells with their plasma membranes
deformed and aligned in regions of incipient intercellular contact but without
the generalized intercellular adhesion that occurs during normal compaction
(Figs. 1, 2). At the electron microscope level, embryos cultured for 2 4 h i n
20-50 /tg/ml 7-ketocholesterol + 2 % FCS formed fewer cell contacts and
junctional complexes than controls and had the general appearance of poorly
compacted morulae with occasional, small blastocoelic cavities (Fig. 3).
Numerous whorls were present in the cells of these embryos (Figs. 3, 6c). The
control and cholesterol-treated embryos on the other hand developed many
intercellular contacts with normal junctional complexes and compacted and
formed normal blastocysts (Fig. 4). Microvilli were localized to the basal and
apical regions of cells of 7-ketocholesterol-treated embryos and there was no
apparent effect of the inhibitor on the membranes of microvilli, mitochondria,
Golgi and membranous lamellae (Fig. 5). However, smooth endoplasmic
reticulum (ER) was always disrupted by the inhibitor although rough ER
remained morphologically undamaged even in regions where the two membrane
Fig. 1. (a) Phase-contrast micrograph of mouse embryos recovered at the 8-cell stage
(60-64 h post hCG) and cultured for 12 h in medium 16 + 2% FCS containing 20
/ig/ml cholesterol. The embryos are fully compacted as judged by cell flattening,
x 250. (b) As (a) but the medium contained 20 /tg/ml 7-ketocholesterol. Note that
the degree of cell flattening is substantially reduced compared with (a), x 250.
Fig. 2. (a) Phase-contrast micrograph of embryos shown in Fig. (1 a) after 36 h of
culture in 20/ig/ml cholesterol. The embryos have formed normal blastocysts.
x 250. (b) As (a) but after 36 h culture in 20 /ig/ml 7-ketocholesterol. Note the
absence of fluid accumulation and persistent reduction in cell flattening, x 650.
Membrane sterols and preimplantation mouse embryos
£7i
1a
309
310
H. P. M. PRATT, J. KEITH, J. CHAKRABORTY
1
).
Fig. 3. 8-cell embryo treated with 50/fg/ml of 7-ketocholesterol + 2 % FCS for
24 h. Small blastocoele with portions of extruded cells is identifiable ( *)• Two
cells (Ci and Q) are dividing. Numerous membranous whorls (arrows) are clearly
seen in several cells of this embryo, x 2000.
systems were continuous (Fig. 6 a). Rough and smooth ER were frequently
found in close contact with membranous whorls (Fig. 6 c) and membranebound dense bodies which were morphologically similar to lipid vesicles
(Fig. 6b, c). Partial disruption of localized areas of nuclear membrane was also
observed in 7-ketocholesterol-treated embryos. The damaging effects of the
inhibitor were more marked at higher concentrations. The smooth ER and
nuclear membranes of cholesterol-treated embryos were unaffected and indistinguishable from untreated controls (Fig. 4 b). Membrane whorls were
absent though cytoplasmic membrane-bound vesicles apparently containing
lipid were abundant (Fig. 4a).
The timing of 7-ketocholesterol action was examined by culturing 8-cell
Membrane sterols and preimplantation mouse embryos
i
f
Fig. 4. (a) 8-cell embryo treated with 50/ig/ml of cholesterol + 2 % FCS for 24 h.
Note the normal blastocyst morphology, x 2000. (b) Higher magnification of the
portions of two cells (Q and C2) from the embryo shown in Fig. 4 (a), showing
unaffected rough (arrow) and smooth (arrow heads) endoplasmic reticulum.
Cellular contact areas are normal (double arrows), x 54378.
311
312
H. P. M. PRATT, J. KEITH, J. CHAKRABORTY
>K
Fig. 5. 8-cell embryo treated with 50 /*g/ml of 7-ketocholesterol + 2 % FCS for 24 h.
Note the undamaged mitochondria (M), golgi (G), membranous lamellae (arrow
head) and plasma membrane (arrows) of two cells ( Q and C2). Damaged smooth
ER (double arrow heads) and membranous whorls (double arrows) are scattered
in the cytoplasm, x 27000.
Membrane sterols and preimplantation mouse embryos
313
precompaction embryos with 7-ketocholesterol for varying periods, removing
them to control media and observing their subsequent development (Table 3).
Developmental inhibition became irreversible within 6-12 h of exposure to
5 /xg/ml 7-ketocholesterol, the period during which untreated embryos became
fully compacted. Higher concentrations had irreversible effects within the first
6 h of culture (Table 3). Ultrastructural damage to the SER was detectable
within 4 h of treatment (Fig. 6) but could be overcome if cholesterol or serum
cholesterol (as FCS) was added to the culture within this period.
The influence of the age of the embryo on its susceptibility to treatment with
7-ketocholesterol was then assessed by taking embryos at 8-cell precompaction
stage (60-65 h post hCG), as well as compacted morulae (74-76 h post hCG)
and fluid-accumulating morulae (85-88 h post hCG) and culturing them
continuously in 5 /<g/ml 7-ketocholesterol. The precompaction 8-cell embryo
is more sensitive to the inhibitor than either of the later stage embryos (Table 4)
which proliferated at the same rate in the presence or absence of the inhibitor.
The average cell numbers of morulae cultured from 88 h post hCG for 48 h
in the presence of absence of 7-ketocholesterol were 39-8 ±4-9 and 35-5 ±6-8
for control and treated embryos respectively.
An attempt was made to investigate the possible mode of action of 7-ketocholesterol by incubating inhibited precompaction 8-cell embryos with the
immediate (mevalonic acid) or end (demosterol and cholesterol) products of
the putative blocked reaction (i.e. the conversion of HMG Co A to mevalonic
acid). The ability of embryos to compact (assessed by cell flattening) and form
blastocysts was at least partially recovered in media containing any of these
compounds in addition to 7-ketocholesterol (Table 5), and their rate of cellular
proliferation increased though it did not reach control levels.
DISCUSSION
The results presented in this paper demonstrate that incubation of preimplantation mouse embryos with sterols can inhibit compaction and blastocyst
formation to varying degrees depending upon the nature and dose of the sterol
(Table 1), the length of exposure (Table 3) and the age of the embryo (Table 4).
7-ketocholesterol and other oxygenated sterols (e.g. 20-a-hydroxycholesterol,
25-hydroxycholesterol and 6-ketocholestanol) have been used to block sterol
synthesis in a variety of cell types by their inhibition of the rate limiting enzyme,
hydroxymethylglutaryl Coenzyme A (HMG Co A) reductase (Brown &
Goldstein, 1974; Kandutsch & Chen, 1977; Kandutsch et al 1978). Preimplantation mouse embryos treated with these agents undergo specific and
irreversible changes in ultrastructure within 4 h of treatment (Fig. 6) and
exhibit subsequent reductions in cell proliferation., intercellular apposition,
junction formation and fluid accumulation (Figs. Ib, 2b, 3). These ultrastructural and developmental consequences of 7-ketocholesterol treatment reinforce
314
H. P. M. PRATT, J. KEITH, J. CHAKRABORTY
Membrane sterols and preimplantation mouse embryos
315
Table 3. Effect of exposure time on development inhibition by
7-ketocholesterol
Hrs in inhibitors*
1-6
12
18-24
Concentration
(/ig/ml)
No. of
% compacting % forming blastocysts embryos
97 1
73
10
7
182
5
10
25
50
5
5
1 4
90 1
13
10
0
0
42
2
(76)
(15)
(10)
(30)
(ID
(28)
* 8-cell embryos (60-64 h post hCG) were cultured in media (+4 mg/ml BSA) containing
the indicated concentrations of 7-ketocholesterol for the stated duration, then washed and
replaced in control medium for the remainder of the 48 h incubation period.
^ P < 0001 x2 test.
2fi
P > 005 x2 test.
Table 4. Effect of developmental age on susceptibility to inhibition
by 7-ketocholesterol (5 ftg/ml)
Hrs post hCG*
7-ketocholesterol
60-65
74-76
85-88
Control
60-65
Cellnumberf % compactingJ % forming blastocystsJ No. of embryos
7 ±0-7 (7)
18 ±3-2 (7)
28 ±5-9 (4)
191
662
792
51
51 2
632
(84)
(53)
(24)
7-5 ±1 (4)
962
892
(45)
* Developmental age of the embryos when placed in control media (containing 4 mg/ml
BSA) or media containing 5 /*g/ml 7-ketocholesterol.
t Mean number of cells in embryos at start of incubation ± standard deviation. Number
of embryos analysed in parentheses.
X % of embryos which compacted (or remained compacted) and formed blastocysts
during a 48 h incubation period.
**P < 0001 x2 test.
2 2
- P > 005 x2 test.
Fig. 6 (a) 8-cell embryo treated with 20/*g/ml of 7-ketocholesterol + 2 % FCS for
24 h. Continuity between the damaged smooth ER (arrows) and intact rough ER
(arrow heads) is clearly visible, x 31920. (b, c) 8-cell embryo treated with 50/*g/ml
of 7-ketocholesterol+ 4 mg/ml BSA for 4 h. Selective damage in the smooth ER
(arrows Fig. 6b) and membranous whorls (arrows Fig. 6 c) is clearly seen. Lipid-like
dense bodies surrounded by membrane remained undamaged (arrow heads) 6b
x 36700, 6c x 27100.
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H. P. M. PRATT, J. KEITH, J. CHAKRABORTY
Table 5. Development of8-cell embryos (60-64 h post hCG) in 7-ketocholesterol
and mevalonic acid or cholesterol*
% forming
% compacting blastocysts No. of embryos Cell numberf
Mevalonic acid (10 ITLM)
Cholesterol (50 /*g/ml)
7-ketocholesterol (5 /tg/ml)
7-ketocholesterol (5 /tg/ml)
+ Mevalonic acid (10 ITLM)
+ Cholesterol (50 /tg/ml)
+ Desmosterol (50/^g/ml)
1001
96 1
192
1001
81 1
62
(10)
(47)
(69)
53-7 ±13-3 (8)
580±13-5 (6)
21 -5 ± 6-4 (6)
733
23
683
703
(30)
(19)
(10)
270±3-8 (5)
3 5 0 ± 7 1 (18)
32-4 ±7-2 (5)
743
903
* Embryos were cultured for 60 h in media+ 4 mg/ml BSA.
t Blastocysts or the most advanced embryos were counted at the end of the culture period.
Mean ± S.D. Number of embryos counted in parentheses.
X
*P < 0001 x2 test.
2 3
- P < 005 x2 test.
l
»P > 005 x2 test.
the biosynthetic evidence that mouse embryos are capable of synthesizing
membrane sterols from the 8-cell stage onwards (Huff & Eik-Ness, 1966; Pratt,
1978). First, only those sterols known to inhibit HMG Co A reductase (Brown
& Goldstein, 1974) induced abnormal development (Table 1), and all of these
exerted their effects within short periods of time (Table 3) which are compatible
with the half life (3^ hours) of the enzyme (Brown & Goldstein, 1974). Secondly
the developmental abnormalities could be overcome by simultaneous exposure
of the embryos to both the inhibitor and natural precursors of cholesterol or
cholesterol itself (Table 5). This observation probably accounts for the improved
viability and morphology of embryos treated with 7-ketocholesterol in the
presence of FCS and its associated serum cholesterol, a fact which was exploited
when analyzing the ultrastructure of embryos exposed to long term (24-28 h)
sub-lethal treatment with 7-ketocholesterol. Finally the fact that the ultrastructural disruptions induced by 7-ketocholesterol were restricted to smooth
endoplasmic reticulum (SER) (Figs. 5, 6) argues for a primary action on these
membranes which are known to be the sites of sterol synthesis (Chesterton,
1968). We consider it unlikely that this SER is derived from rough ER as a
secondary consequence of 7-ketocholesterol treatment since rough ER remains
intact and two parameters of membrane-bound ribosome activity are unaffected,
namely total protein synthesis and the assembly of membrane glycoproteins as
assessed by the binding of concanavalin A (Pratt, unpublished). Furthermore,
the membrane whorls associated with these lesions (Fig. 6 c) have been observed
in other cell types where sterol synthesis has been inhibited (Dietert & Scallen,
1969) and are possible abnormal intermediates in membrane biosynthesis. The
Membrane sterols and preimplantation mouse embryos
317
proliferation of SER observed here and in other systems has been suggested
to be compensatory growth to replace damaged membranes (Yates, Arai &
Rappoport, 1967). HMG Co A reductase activity (Pratt, 1978) of inhibited
embryos is now being analyzed to provide a direct demonstration of the action
of 7-ketocholesterol on sterol synthesis.
The greater sensitivity of the precompaction 8-cell embryo to 7-ketocholesterol
as compared with the compacting or fully compacted embryo could be explained
by a reduced permeability to sterols at these later stages. However, this is not
borne out by experiments using labelled cholesterol (Tarver & Pratt, unpublished
observations), or by examination of the localization of sterols in embryos
before or during compaction since sterols appear to be taken up from the
medium to similar extents irrespective of embryonic age. On the other hand,
synthesis of sterols is not detectable until the 8- to 16-cell stage (Pratt, 1978 and
unpublished observations) and the increased susceptibility of the precompaction
8-cell embryos to 7-ketocholesterol may therefore be due to the immaturity of
the sterol-synthesizing machinery and its inability to compensate for damage.
In cells exposed to 7-ketocholesterol a secondary consequence of blocked
sterol synthesis is a depletion in the cholesterol content of cellular membranes,
particularly the plasma membrane (Kandutsch & Chen, 1977). Some properties of 7-ketocholesterol-treated embryos could be attributed to cholesteroldepleted plasma membranes. For example, intercellular adhesion and fluid
accumulation are reduced or absent, cell proliferation declines and methionine
uptake and N a + / K + ATP ase activity (assessed by ouabain sensitivity) are
reduced (Pratt, unpublished). Similar phenomena have been observed in cells
with cholesterol-depleted membranes (Papahadjopoulos, 1974; Kimelberg,
1977; Horwitz et al. 1978). However 7-ketocholesterol is not having an indiscriminate effect on all cellular processes since total protein synthesis, binding
of concanavalin A, presence of the glycolytic pathway and phospholipid synthesis (Pratt, 1980) are similar in sterol-treated and normal embryos.
Compaction of the 8-cell embryo involves the coordinated reorganisation
of the cytoskeleton which is vital for the subsequent differentiation of the
embryo (Johnson et al. 1980). Important features of the compaction process
are the deformation of the nearly spherical cleavage stage blastomeres to form
an epithelial-type tissue and the ability of the component cells to spread across
their neighbours and adhere to them (Ducibella et al. 1977). This process is
known to be Ca 2 + dependent, requires the participation of intact microfilaments (Ducibella & Anderson, 1975) and can be inhibited by antibodies
directed against cell surface determinants (Kemler, Babinet, Eisen & Jacob,
1977; Johnson et al. 1979). From the experiments described in this paper it
seems that sterol-mediated properties of the membrane may be additional
important features to be considered. These properties could include intercellular adhesion (possibly influenced by phase separation effects (Hope et al.
1977), activity of membrane-bound enzymes (Kimelberg, 1977) and cell motility
21
EMB 60
318
H. P. M. PRATT, J. KEITH, J. CHAKRABORTY
(mediated through interactions between sterols and proteins associated with the
cytoskeleton (discussed, Pratt, 1978)).
In conclusion the results described here are consistent with evidence that the
preimplantation mouse embryo is capable of synthesizing its own membrane
sterols (Pratt, 1978) and emphasize the role that sterol synthesis plays in the
normal metabolism of the embryo as well as its probable importance in facilitating the changes in surface properties that occur during development.
Future studies will be aimed at analyzing the sterol composition of 7-ketocholesterol-treated embryos and investigating any consequent modifications to
membrane properties by studying lateral mobility of membrane lipids and
proteins (Johnson & Edidin, 1978) and the activities of membrane transport
sites (Holmberg & Johnson, 1979).
We wish to thank Mr Dave Tarver and Ms Gin Flach for assistance with these experiments
and Dr David Bowyer and Dr Martin Johnson for helpful discussions. This work was supported by grants from the Ford Foundation and the Medical Research Council to H.P. M.P.
J.C. was in receipt of Grant No: BRSG-507-RR-05700-09.
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{Received 26 February 1980, revised 20 June 1980)