J. Embryol. exp. Morph. 75, 205-223 (1983)
205
Printed in Great Britain © The Company of Biologists Limited 1983
Mevalonate reverses the developmental arrest of
preimplantation mouse embryos by Compactin, an
inhibitor of HMG Co A reductase
By M. AZIM H. SURANI 1 , SUSAN J. KIMBER 2 AND
JEREMY C. OSBORN3
From the A.R.C. Institute of Animal Physiology, Cambridge
SUMMARY
Hydroxymethyl glutaryl Co A reductase (HMG Co A reductase) is the key regulatory
enzyme in the conversion of acetate to mevalonate. Mevalonate is the precursor for sterol and
non-sterol isoprenes involved in membrane biogenesis, DNA replication and protein
glycosylation. The influence of two inhibitors of HMG Co A reductase, Compactin (or
ML236B) and an oxygenated sterol, Diosgenin, were tested on preimplantation development
of mouse embryos. Compactin arrested development at about the 32-cell stage, leaving the
blastomeres decompacted. Ultrastructural examination of the embryos revealed reduced
membrane apposition but no major effects on cell organelles. There was however a
predominance of nuclei with highly condensed chromatin. Glycosylation of proteins also
appeared to be inhibited as shown by reduced incorporation of sugar precursors but not that
of amino acids. The influence of Compactin was judged to be highly specific since only
lOjUg/ml (0-08 mM) mevalonic acid abolished the effects of Compactin. Mevalonate in embryos may not be primarily utilized in the synthesis of sterols since a specific inhibitor of
cholesterol synthesis, DL-4,4,10-/3-trimethyl-trans-decal-3-j3-ol had no detectable effect on
development. The non-sterol isoprenes of mevalonate such as dolichol and isopentenyl
adenine may play a more significant role during early development since the influence of
Compactin resembled that previously described using tunicamycin, a specific inhibitor of
dolichol mediated synthesis of N-glycosidically linked glycoproteins. Hence, lack of dolichol
may partly be the cause of arrest of embryonic development by Compactin. Diosgenin caused
embryonic arrest at about the 16-cell stage and the influence was not reversible by mevalonic
acid. Cholesterol was able to rescue 50 % of the embryos but the effect of Diosgenin could be
non-specific and probably caused by its entry into the plasma membrane.
INTRODUCTION
During preimplantation development in the mouse, two distinct tissues, the
inner cell mass and the trophectoderm are formed at the blastocyst stage after
six cleavage divisions (Gardner, 1971). Prior to this, a major Ca 2+ -dependent
1
Author's address: A.R.C. Institute of Animal Physiology, Animal Research Station, 307
Huntingdon Road, Cambridge CB3 0JQ.
2
Author's present address: MRC Laboratory, Woodmansterne Road, Carshalton, Surrey
SM5 4EF.
3
Author's present address: Department of Obstetrics & Gynaecology, St. Mary's Hospital,
Whitworth Park, Manchester M13 0JH.
206
M. A. H. SURANI, S. J. KIMBER AND J. C. OSBORN
morphogenetic event called compaction occurs at the 8-cell stage when close
membrane apposition and cell flattening are observed (Ducibella & Anderson,
1975; Lehtonen, 1980; Kimber, Surani & Barton, 1982), together with the
regionalization of cell surface constituents (Handyside, 1980). At the 16-cell
stage, a group of inner cells is established for the first time (Handyside, 1981;
Johnson & Ziomek, 1981) which presumably go to form the inner cell mass whilst
the outer cells give rise to trophectoderm (Surani & Handyside, 1983). Cell
surface properties determine cell interactions and adhesiveness and influence
cell position within the embryo (Kimber et al. 1982; Surani & Handyside, 1983)
in which cell surface changes (Hyafil, Morello, Babinet & Jacob, 1980; Johnson
& Calarco, 1980; Magnuson & Epstein, 1981; Kapadia, Feizi & Evans, 1981;
Kimber & Surani, 1982) probably have a major influence on interactions between cells (see Surani, Kimber & Barton, 1981).
For the synthesis of N-glycosidically linked glycoproteins, the polyisoprenoid
lipid, dolichol, is the major saccharide carrier (Hemming, 1977). We have
previously shown that tunicamycin, a specific inhibitor of N-acetylglucosaminyl
pyrophosphoryl dolichol phosphate (Takatsuki, Arima & Tamura, 1971) substantially inhibits glycosylation, disrupts compaction and alters the cell surface
properties (Surani, 1979; Surani, Kimber & Handyside, 1981; Atienza-Samols,
Pine & Sherman, 1981). The levels of dolichol compared to cholesterol are
substantially higher in many developing systems examined (Potter, Millet,
James & Kandutsch, 1981; Harford & Waechter, 1981; Carson & Lennarz,
1981). Hydroxymethyl-glutaryl Coenzyme A reductase (HMG Co A reductase)
is the key regulatory enzyme in the formation of mevalonic acid which is a
precursor of divergent biosynthetic pathways of both sterol and non-sterol
isoprenes (Brown etal. 1978; James & Kandutsch, 1979). These products include
cholesterol, dolichol, isopentenyl adenine and ubiquinone (see Fig. 6). In this
study we have examined the consequences of inhibition of HMG Co A reductase
by Compactin (or ML236B) (Brown etal. 1978) and an oxygenated sterol, Diosgenin (Mills & Adamany, 1978). This study demonstrates that Compactin disrupts early embryonic development and inhibits protein glycosylation and these
effects can be completely reversed by exogenous mevalonic acid.
MATERIALS AND METHODS
Animals
Embryos were obtained from 3 to 5-weeks old outbred strain of MF1 mice
(ARC colony established from OLAC stock) which were superovulated using
5i.u. pregnant mare's serum followed 42-48h later by 5i.u. human chorionic
gonadotrophin (HCG) (Intervet, Milton, UK). Each female was caged with an
Fi (C57BL/CBA) male and checked the following morning for the vaginal plug;
this was counted as day 1 of pregnancy.
Mevalonate reverses arrest of mouse embryos by Compactin 207
Chemicals
Compactin (ML-236B) was a gift from Dr Akira Endo (Sankyo, Tokyo,
Japan) and Dr R. Fears (Beecham Pharmaceuticals, Epsom, UK). The lactone
form of Compactin was converted to the acid form by heating at 50 °C for 1 h in
0-1 M-NaOH (Kaneko, Hazama-Shimada & Endo, 1978). The solution was then
adjusted to pH8-0 with 1 N - H C 1 and the concentration of the compound to
1 mg/ml in 0-01 M-tris-HCl, pH 8-2. The sodium salt of Compactin thus obtained
was divided into lOjul aliquots and stored at - 2 0 °C. DL-4,4,10-jS-trimethyltrans-decal-3-j3-ol (TMD) was a gift from Drs J. A. Nelson and T. A. Spencer
(Dartmouth College, NH, USA). DL-mevalonic acid lactone, cholesterol,
dolichol, dolichol monophosphate, retinoic acid, coenzyme Qio, isopentenyl
adenine and Diosgenin (5,20a,22a,25D-spirosten-3/3-ol) were all obtained
from Sigma. Mevalonic acid was dissolved in the embryo culture medium at a
concentration of lmg//il medium and stored at — 20 °C. Dolichol, dolichol
monophosphate and coenzyme Qio were dissolved in chloroform at a concentration of 10 mg/ml, 2 mg/ml and lmg/lOOjul, respectively. Isopentenyl adenine
was dissolved at 0-5 mg/ml ethanol. Retinoic acid was first dissolved in ethanol
(3 /^g/1 jUl ethanol) and then made up to 500/il in the embryo culture medium.
All these compounds were usually stored for 1 week and a maximum of 2 weeks
at — 20 °C under nitrogen. The rest of the compounds were freshly made on the
day of the experiment. Diosgenin was made in ethanol (2 mg/ml). Cholesterol
was first dissolved in chloroform (100 jUg/jul) and 5 /il of this stock solution were
then added to 1 -0 ml of culture medium with 4 mg/ml bovine serum albumin with
vigorous vortexing. Lectins and their antibodies were obtained from Vector
(California, USA), and protein A-Sepharose from Pharmacia (Uppsala,
Sweden). All radiochemicals were from Amersham (UK).
Recovery and culture of embryos
Two-cell embryos were flushed from oviducts between 2 and 5p.m. on day 2
of pregnancy at about 45-48 h post HCG. Embryos were cultured in Brinster's
medium (Brinster, 1970) supplemented with 4 mg/ml bovine serum albumin
(BMOC-3).
Two-cell embryos were used immediately in experiments or cultured overnight in microdrops of BMOC-3+BSA in Sterilin Petri dishes under paraffin oil.
The following morning, precompacted 8-cell embryos (approx. 65 h post HCG)
were removed and washed 6x through fresh medium. The embryos were then
divided into groups of 20-40 embryos. Subsequent cultures were carried out in
microtitre plate wells containing 0-1-0-3 ml of each test medium and with adhesive plate sealers. The drops were allowed to reach equilibrium at 37 °C in 5 %
CO2 in air for 30 min before groups of embryos were transferred to each of the
test mediums.
208
M. A. H. SURANI, S. J. KIMBER AND J. C. OSBORN
Observations and fixation of embryos for microscopy
Embryos were examined periodically and photographed using a Leitz Diavert
inverted microscope under phase contrast and bright field.
Embryos were fixed in 2-5 % glutaraldehyde, 1 % paraformaldehyde in
0-075 M-sodium cacodylate buffer (pH7-5) containing 2 mM-calcium, and 0-1 %
potassium ferricyanide. Embryos were post fixed in 1 % osmium tetroxide,
stained overnight in uranyl acetate, dehydrated and embedded in Epon. Thick
(0-5 fjm) and thin (50-100 nm) sections were stained with 1 % toluidine blue and
viewed and photographed using a Zeiss microscope. Thin sections were stained
with a saturated solution of uranyl acetate in 50 % ethanol followed by lead
citrate (Reynolds, 1963) and examined in an AEI801 electron microscope.
Determination of cell number in embryos
The number of cells was determined by counting nuclei in air-dried preparations of embryos (Tarkowski, 1966).
Incorporation of radioactive precursors into embryos and polyacrylamide gel
electrophoretic analysis
For the estimation of incorporation of [3H]leucine and [3H]sugar precursors
into embryos, they were cultured in the presence of Compactin for approximately 24 h commencing at the 8-cell stage. [3H]leucine (sp. act. 105Ci/mmol) and
[35S]methionine (sp. act. 900-1200 Ci/mmol) were added at 200/iCi/ml in
glucose-free medium as described previously (Surani, 1979). For labelling of
embryos with sugar precursors, 50 /i of a 2x concentrated glucose-free medium
was used to which the following radioactive precursors were added; 25/iCi
[3H]glucosamine (sp. act. 38Ci/mmol), 25/iCi [3H]mannose (sp. act. 2Ci/
mmol) and 25/iCi [3H]galactose (sp. act. 18Ci/mmol). Embryos were labelled
for approximately 6 h in groups of between 10-30 embryos with the amino acid
precursors or in groups of 50-100 with the sugar precursors. At the end of the
labelling period, embryos were washed and processed to determine incorporation of the precursors in embryos in the control group and compared with those
cultured in the presence of Compactin essentially as described previously
(Surani, 1979).
Embryos labelled with [35S]methionine were also analysed on 8-15 % SDSpolyacrylamide gradient gels and the radiolabelled proteins visualized by
fluorography exactly as described previously (Moor, Osborn, Cran & Walters,
1981). In some cases, galactosyl glycopeptides were immunoprecipitated after
the addition of peanut lectin followed by an addition of antibody against peanut
lectin (PL Biochemicals) and the immune complexes were extracted using
Protein A-agarose gels. The method used was essentially as described elsewhere
(Magnuson & Epstein, 1981). The extracted glycopeptides were also analysed by
polyacrylamide gel electrophoresis as described above.
Mevalonate reverses arrest of mouse embryos by Compactin 209
Fluorescence microscopy
Control embryos and those cultured in Compactin were examined by
fluorescence microscopy after treatment with FITC-Concanavalin A as
described (Kimber et al. 1982) using a Zeiss epifluorescence microscope.
RESULTS
In preliminary experiments Compactin was used in the range of 0-5-8-0 fig/
ml. Two-cell embryos developed to the 8-cell stage in all cases and underwent
compaction and formed advanced morulae after 48 h in culture. When embryos
were cultured in 0-5-1-OjUg/ml Compactin, a small proportion (10-20%) formed into poorly developed blastocysts. Embryos cultured in 2-0jUg/ml Compactin failed to develop into blastocysts and this was considered as the minimum
concentration of Compactin necessary to completely disrupt development.
Similar preliminary experiments with Diosgenin (1-0-5-0/ig/ml) established
that 5-0 jUg/ml Diosgenin was necessary to disrupt development to the blastocyst
stage.
Preliminary experiments were also carried out using Compactin and Diosgenin in medium containing normal or fatty-acid-free albumin (from Sigma).
There was no marked difference between the effect of the compounds in medium
with fatty-acid-free albumin and in medium containing fatty acids. Fatty-acidfree albumin also did not hinder development to the blastocyst stage in the
absence of the inhibitors. In all cases except where indicated normal bovine
serum albumin was present in the culture medium.
We first examined the influence of Compactin and Diosgenin on cell proliferation on 8-cell precompacted embryos. After 24 h in culture (89 h post HCG), the
number of cells in the control group and in the presence of Compactin was similar
but those cultured in Diosgenin had only about half the number of cells and these
had not increased considerably after 31 h (96 h post HCG) in culture. After
further 7h in culture, embryos in Compactin showed a slight increase in the
number of cells to 32 cells compared with about 42 cells in the embryos in the
control group (Table 1). Both of the inhibitors have been previously shown to
specifically block HMG Co A reductase. Therefore the influence of the two
major products of this pathway, mevalonic acid and cholesterol, on embryonic
development in the presence of either Diosgenin or Compactin was examined.
The influence of mevalonic acid and cholesterol in the presence of Diosgenin
is shown in Table 2. Mevalonic acid upto lmg/ml was unable to reverse the
influence of Diosgenin. However, cholesterol at 50-100^g/ml was able to
rescue approximately 50 % of the embryos (see Fig. 1). Many of these embryos
which were apparently rescued formed blastocysts that appeared to be poorly
developed. Similar experiments were carried out with Compactin (Table 3). As
210
M. A. H. SURANI, S. J. KIMBER AND J. C. OSBORN
Table 1. Influence of Compactin and Diosgenin on the number of cells in embryos
Time in culture (h)*
(h post HCG)
Mean cell no.
±S.D.
No. of embryos
Control
24 (89)
31 (96)
29-2 ± 4-9
41-8±ll-9
Compactin
(2 Kg/ml)
Diosgenin
(5pg/ml)
24 (89)
31 (96)
24 (89)
31 (96)
28-8 ±
32-9 ±
12-1 ±
15-2 ±
21
27
32
28
11
9
Group
5-4
8-2
2-5
2-7
* Embryos were at the precompacted 8-cell stage (65 h post HCG) at the start of the experiment.
Table 2. Development of 8-cell embryos in Diosgenin and mevalonic acid or
cholesterol
Group
Number
Exp. Embryos
Mean cell no.*
± S.D. (n)
No. blastocysts
(%)t
Control
7
80
44-8 ±11-9 (10)
67 (83-8)
Diosgenin (5 jug/ml)
3
69
13-9 ± 3-3(12)
3 ( 4-3)
Diosgenin (5|Ug/ml)
+ cholesterol (jug/ml)
25
50
100
3
7
3
35
78
35
12-4 ± 5-3(15)
27-8 ± 4-8 (17)t
29-9 ± 5-2 (16)t
1 ( 2-8)
34 (43-6)
19 (54-3)
Diosgenin (5 jUg/ml)
+ mevalonic acid (/ig/ml)
10
100
1000
2
3
4
27
32
59
14-9 ± 3-5(11)
12-3 ± 7-2(12)
14-9 ± 3-5(15)
0
0
0
* Determined at approximately 96 h post HCG (31 h in culture) in separate concurrent
groups of embryos.
t Only includes well-compacted advanced morulae and a few early blastocysts.
t At 113 h post HCG (48h in culture).
little as 10 /ig/ml (0-08 HIM) mevalonic acid in the presence of Compactin enabled
nearly 90% of the embryos to develop to the blastocyst stage. Even l^g/ml
mevalonic acid was partially effective and rescued over 30 % of the embryos. By
contrast, cholesterol at up to 100/ig/ml was virtually ineffective in reversing the
influence of Compactin. The embryos that were rescued by mevalonic acid
appeared to be normal when examined under an inverted microscope (see Fig.
1). Furthermore, the number of cells in the embryos rescued by cholesterol in the
presence of Diosgenin was substantially less than the number of cells in the
Mevalonate reverses arrest of mouse embryos by Compactin 211
Table 3. Development of 8-cell embryos in Compactin with mevalonic acid or
cholesterol
Group
Control
Compactin (2 /zg/ml)
Compactin
+ cholesterol (/^g/ml)
25
50
100
Compactin
+ mevalonic acid (jug/ml)
1
10
100
1000
Number
Exp. Embryos
Mean cell no.*
± S . D . (n)
No. blastocysts
(%)t
154
42-2 ±12-1 (21)
11
139
33-3 ± 9-3(27)
141 (91-6)
3 ( 2-2)
3
11
9
30
166
119
32-2 ± 7-2(25)
30-9 ± 6-8(29)
31-3 ± 5-4(20)
0
0
3 ( 2-5)
6
5
6
5
72
60
81
63
38-8 ± 4-8(32)f
42-2 ± 7-9(35)
41-9 ±11-2 (30)
39-7 ± 9-9(31)
24 (33-3)
53 (88-3)
73 (901)
45 (71-4)
11
* After 31 h in culture (96 h post HCG) in separate groups of concurrent embryos.
$ At 113 h post HCG (48 h in culture).
t Only well-compacted morulae and early blastocysts were counted.
embryos in the control group. However, the embryos rescued by mevalonic acid
in the presence of Compactin had the normal number of cells.
The role of cholesterol in early embryonic development was examined further
with a specific inhibitor of its synthesis. TMD inhibits cyclization of squalene and
hence prevents synthesis of lanosterol and cholesterol (Chang et al. 1979). This
inhibitor was also of interest since its site of action is distal to the formation of
dolichol and therefore TMD would not affect the concentration of dolichol.
TMD at a concentration of up to 25 /ig/ml was virtually ineffective in blocking
development to the blastocyst stage. At 50/ig/ml TMD seemed highly toxic.
Similar results were obtained with embryos cultured from 2-cell or 8-cell stage
onwards in the presence of TMD. Culture of embryos in the presence of TMD
and fatty-acid-free albumin also did not influence development. The results were
therefore combined (Table 4).
The influence of Compactin on embryos was examined further. When 2- or
8-cell embryos were cultured in the presence of Compactin, they developed
normally at first, undergoing normal cleavage divisions and compaction at the
8-cell stage and proceeded to the 32-cell stage. However the embryos started to
decompact at the 16-cell stage and the majority of the embryos were decompacted after 30-35h in culture (Fig. 1). As shown in Fig. 1A, the embryos in the
control group were fully compacted or at the blastocyst stage. Embryos in the
presence of Compactin (Fig. IB) had rounded and distinct blastomeres.
212
M. A. H. SURANI, S. J. KIMBER AND J. C. OSBORN
Table 4. Develoment of2-cell and 8-cell embryos in DL-4,4,10-$-trimethyl-transdecal-3-$-ol (TMD)*
Group
Control
TMD (jug/ml)
1-25
2-5
5-0
10-0
12-5
25-0
50-0
Exp.
Number
Embryos
7
75
4
4
6
7
6
4
5
64
59
86
92
80
60
120
Mean cell no.
± S . D . (n)t
No. blastocysts
41-7 ± 9-3(18)
66 (88-0)
(%)t
39-5 ± 8-9(26)
51 (79-7)
38-6 ± 7-6(22)
44 (74-6)
37-6 ±10-1 (27)
70 (81-4)
40-9 ±11-2 (29)
72 (78-3)
37-7 ± 9-9(18)
69 (86-3)
42 (70-0)
36-5 ± 9-3(12)
All died after 24 h in culture§
with almost no increase in the
numbers of cells
* Combined results of embryos cultured from 2-cell or 8-cell stage and in normal or fatty acid
free albumin.
t At 96 h post HCG in separate concurrent groups of embryos.
JAtmhpostHCG.
§ Presence of 100 ;Ug cholesterol/ml did not prevent the toxic effects of TMD.
Mevalonic acid prevented this effect of Compactin on embryos (Fig. 1C). Some
of these embryos were sectioned and the semithin sections examined by light
microscopy. Figures 2A and 2B show that the embryos in the control group were
at the late morulae-early blastocyst stage. The outer blastomeres were characteristically flattened. Embryos cultured in Compactin (Fig. 2C and D) however,
failed to show the flattened morphology of outer cells: the blastomeres generally
tended to be rounder. Examination of the embryos by electron microscopy (Fig.
3) revealed much-reduced areas of membrane apposition when embryos were
cultured in the presence of Compactin. However, the unapposed peripheral
surfaces of the blastomeres had a high density of microvilli similar to the density
on the outer surface of control morulae. Large intercellular spaces were present
inside the morulae between the rounded blastomeres in contrast to the closely
packed arrangement of cells in control morulae. Although microvilli were
present on their inner membranes, they did not interdigitate closely with those
of adjacent blastomeres over the inner membrane area as in the control compacted morulae. However, in some instances adherens junctions were observed at
the apical regions of membrane contact between cells. Cytoplasmic blebs and
large processes sometimes extended from the surface of Compactin-treated embryos (Fig. 3D) and there were regions of microvillar interdigitation. The
mitochondrial population of both control late morulae and Compactin-treated
embryos consisted partly of the vacuolate type found in early preimplantation
embryos and partly of the non-vacuole form with clearly defined cristae found
Mevalonate reverses arrest of mouse embryos by Compactin 213
Fig. 1. Examination of embryos with inverted microscope under bright field at
92-96h post HCG (27-31 h culture). (A) Control group with blastocysts and late
morulae, (B) 2 jug Compactin/ml; many of the embryos have undergone decompaction (C) 2jUg Compactin +10/ig mevalonic acid/ml; the majority of embryos
developed into normal blastocysts (D) 5-0 jug Diosgenin/ml; decompacted embryos
with fewer cells than in (B). (E) 5 peg Diosgenin + 100 pig cholesterol/ml; compacted
morulae and poor blastocysts (F) 5pig Diosgenin 4-100/ig mevalonic acid/ml; no
marked difference compared with (D). (Scale bar = 30jum.)
214
M. A. H. SURANI, S. J. KIMBER AND J. C. OSBORN
<.
v
2A
B
D
Fig. 2. Semi-thin sections of embryos at 92-96 h post HCG (27-31 h culture)
examined by Zeiss phase contrast microscope. (A & B) embryos from control group
showing well compacted morula and early blastocyst. (C & D) 2 fig Compactin/ml;
the peripheral blastomeres are rounder compared with the flattened cells in the
control group. Condensed chromatin (arrows) also observed in the majority of cells.
(Scale bar= 10/im.)
Mevalonate reverses arrest of mouse embryos by Compactin 215
Fig. 3. Electronmicrographs of (A) control late morula, 92-96 h post HCG. Close
association between membranes of adjacent cells is observed with blastomeres
spread over one another. (Scale bar = 5 jum.) (B) Embryo cultured in 2 /ig Compactin/ml for 27-31 h (92-96 h post HCG) from the 8-cell stage. Note loose association
of cells and nuclei of some cells with condensed chromatin (arrows). Vacuoles (V)
are present in some cases but only a few small lipid droplets (L). (Scale bar = 5 jum.)
(C) Control embryo showing outer region of contact between two cells with junctional complexes (jc) including gap junction, cl, crystalline lamellae; mv, microvilli in
which micro filaments were evident. (Scale bar = 1 fjm.) (D) Outer region of embryo
from the experimental group. Mitochondria (m) are both of the 'immature'
vacuolate form and the form containing cristae. mv, microvilli (with micron"laments); b, blebs; er, endoplasmic reticulum; g, golgi apparatus. (Scale bar = 1 jum.)
216
M. A. H. SURANI, S. J. KIMBER AND J. C. OSBORN
in the blastocyst:' No differences could be found in the rough endoplasmic
reticulum or in the distribution of microfilaments and microtubules between
control and Compactin-treated embryos. One noticeable difference was found
in the nucleus. In the control embryos the majority of the nuclei could be detected with distinct nuclear membranes, whereas in the experimental group the
nuclear membranes were barely detectable and the chromatin was highly condensed.
The influence of Compactin on the synthesis of macromolecules was also
examined. Eight-cell embryos were first cultured in the presence of Compactin
for 24 h and then labelled with amino acids and sugar precursors for 6 h after this
time. Figure 4 shows that the incorporation of amino acid precursors was not
affected by Compactin, rather there was an increase in the incorporation of both
[35S]methionine (117 %) and [3H]leucine (135 %) relative to the control values.
On the contrary, the incorporation of all of the sugar precursors tested was
substantially reduced to between 34-48 % relative to the control values.
Both the polypeptides and glycopeptides were also analysed on 8-15 %
polyacrylamide gels. No qualitative differences were detectable in polypeptides
or glycopeptides of embryos cultured in the presence of Compactin (Fig. 5).
The binding of fluorescein-conjugated Concanavalin A to the embryonic cell
surface was examined. Although occasionally there appeared to be a slight
r35
S]Methionine (6)
3
H]Leucine (5)
3
H]Galactost
}
H]Glucosamine
[3H]Mannose (4)
50
100
% Compactin/control
150
Fig. 4. Incorporation of amino acids and sugar precursors in embryos labelled for 6 h
(89-95 h post HCG) after 24 h culture of 8-cell embryos in 2jug Compactin/ml.
(Relative to the control values.) The values are mean ±S.D. The number of determinations are in parenthesis. Compactin had no detectable effect on the acid-soluble
pool of the precursors.
Mevalonate reverses arrest of mouse embryos by Compactin 217
reduction in the binding of Concanavalin A to embryos cultured in Compactin,
there was no marked quantitative difference compared with the controls (data
not shown). Further detailed studies are needed to establish if Compactin has an
effect on the cell surface constituents of embryos.
Finally, since the influence of Compactin appears to be due to specific inhibition of HMG Co A reductase, several key products of this pathway were used
to determine whether the effect of Compactin could be overcome by these
compounds (Table 5). As mentioned previously, mevalonic acid was highly
M r x10 - 3
200
92
69
46
30
14.9
A
B C
D
Fig. 5. Analysis of polypeptides and glycopeptides of embryos labelled with
[35S]methionine as described in the text. Labelled polypeptides in embryos from (A)
the control group (B) embryos cultured in 2/xg Compactin/ml (C) glycopeptides
extracted from embryos in the control groups and (D) glycopeptides from embryos
in the experimental group.
218
M. A. H. SURANI, S. J. KIMBER AND J. C. OSBORN
Table 5. Compounds tested for the reversal of the effects ofCompactin on embryos
Concentration
(jug/ml)
Compound
Mevalonic Acid
Cholesterol
Dolichol
Dolichol Monophosphate
Retinoic Acid
Coenzyme Q10
Isopentenyl adenine
1-1000
25-100
0-1-5-0
0-5-20
0-03-3-0
1-0
0-5-1-0
All the above compounds when tested in the absence ofCompactin had no detectable effects
on development.
effective in reversing the influence of Compactin. Cholesterol however was
ineffective. Other compounds tested were dolichol, dolichol monophosphate,
retinoic acid, coenzyme Qio and isopentenyl adenine. All of these were ineffective in reversing the influence of Compactin. The compounds were also tested
in various concentrations and in a large variety of combinations. So far none of
the combinations employed have been successful in reversing the influence of
Compactin (data not shown).
DISCUSSION
Compactin, a competitive inhibitor of HMG Co A reductase (Brown et al.
1978) interrupts preimplantation development of mouse embryos after the
16-cell stage. Mevalonic acid (1-10/ig/ml), the product of this enzyme activity
(see Fig. 6) can totally reverse the influence ofCompactin. In contrast, Diosgenin
acts relatively quickly and prevents compaction at the 8-cell stage but the effect
is not reversible by mevalonic acid, although Diosgenin is also suggested to inhibit HMG Co A reductase (Mills & Adamany, 1978). However, Diosgenin and
other oxygenated sterols can sometimes act non-specifically when they become
inserted in the plasma membrane (Gordon, Bass & Yachnin, 1980). Such nonspecific effect is partly reversible by exogenous cholesterol which presumably
displaces the compound from the plasma membrane. Similar results were
previously obtained with other oxygenated sterols (Pratt, Keith & Chakraborty,
1980), but such influence is not necessarily a reflection of de novo synthesis of
sterols during early development in the mouse. Indeed there is little synthesis of
sterols up to the blastocyst stage (Pratt, 1982; Carson, Hsu & Lennarz, 1982).
Furthermore, cholesterol, the major product of mevalonic acid did not reverse
the effects of Compactin on embryos; these embryos had approximately the
same number of cells (30) as in the blastocysts formed when cholesterol was
Mevalonate reverses arrest of mouse embryos by Compactin 219
Acetate
I
HMG Co A
(HMG Co A)
(Reductase)
COMPACTIN
MEVALONIC ACID
I
Farnesyl-P-P
Squalene
TMD
Dolichol
I
Dol-P
Lanosterol
UDP GlcNAc
Cholesterol
TUNICAMYCIN
Y
Dol-PP-GlcNAc
Polypeptide
Polypeptide-N-oligosaccharide
Fig. 6. The biosynthetic pathway in the formation of mevalonic acid and the divergent pathways of two major isoprenes, dolichol and cholesterol. The site of inhibitory action of Compactin, TMD and tunicamycin are indicated.
present along with Diosgenin. In addition, TMD, a specific inhibitor of
squalence cyclization (Chang et al. 1979) had no effect on development to the
blastocyst stage. The combined observations indicate that products of
mevalonate other than cholesterol, such as the non-sterol isoprenes dolichol and
isopentenyl adenine, may be equally important if not crucial for the rescue of
embryos by mevalonic acid in the presence of Compactin after the 16-cell stage.
220
M. A. H. SURANI, S. J. KIMBER AND J. C. OSBORN
Only 0-03-0-11 % of acetate is generally incorporated into dolichol compared
with sterols, but their levels can increase by 10- to 100-fold in developing systems
where cell differentiation is underway (James & Kandutsch, 1980; Potter et al.
1981; Harford & Waechter, 1980; Carson & Lennarz, 1981). This increase in
dolichol, the key lipid saccharide carrier in the glycosylation of N-glycosidicallylinked glycoproteins (Hemming, 1977), and mannosylphosphoryl dolichol (Harford & Waechter, 1980; Carson & Lennarz, 1981) is probably essential for rapid
changes in the cell surface glycoproteins during cell interactions and morphogenesis. In this respect it was interesting to observe the effect of Compactin on
the incorporation of sugar precursors into embryos which was markedly reduced
compared with the incorporation of amino acids. Since overall protein
synthesis, both quantitative and qualitative remains unaffected, this suggests an
inhibition of protein glycosylation probably resulting from the lack of dolichol.
We cannot entirely rule out the effect of Compactin on polypeptides since
qualitative differences may be detected if they are analysed by the twodimensional gel-electrophoretic system. The decrease in the incorporation of
the sugars was not caused by the influence of Compactin on intracellular membranes since the endoplasmic reticulum and other organelles such as mitochondria developed normally. The influence of Compactin first becomes detectable
at the 16-cell stage when some of the interstitial glycoproteins such as laminin
are detected between cells (Leivo, Vaheri, Timpl & Wartiovaara, 1980). However, further work is necessary to demonstrate that the decompaction of embryos caused by Compactin is as a result of changes in the cell surface
glycoproteins.
There are similarities between the effects of Compactin and tunicamycin on
mouse embryos. Tunicamycin, a specific inhibitor of synthesis of dolichol-linked
saccharides (Takatsuki et al. 1981) also caused decompaction of embryos and
inhibited protein glycosylation (Surani, 1979; Ateinza-Samols et al. 1981) and
caused changes in the cell surface properties of blastomeres (Surani et al. 1981).
In sea urchin embryos, dolichol alone can overcome the inhibitory effects of
Compactin on protein glycosylation as well as development (Carson & Lennarz,
1979,1981). Although dolichol alone (or in combination with other products of
mevalonic acid) failed to reverse the influence of Compactin on mouse embryos,
we have not ruled out the possibility that in the mouse embryos many of the
compounds only enter the plasma membrane and not the cytoplasm.
Isopentenyl adenine, another product of mevalonic acid may be crucial during
development for DNA replication and cell growth. It appears that in embryos
cultured in Compactin, the cells ceased division at a specific point in the cell
cycle, since the majority of them had highly condensed chromatin and lacked
clear nuclear membrane. This finding may be significant since isopentenyl
adenine is implicated in DNA-polymerase-dependent DNA replication
(Habenicht, Glomset & Ross, 1980; Quesney-Huneeus, Wiley & Siperstein,
1979, 1980). This aspect requires further investigation.
Mevalonate reverses arrest of mouse embryos by Compactin 221
Recent studies indicate that mevalonate itself has an influence on cell shape
since in its absence, cells tend to round up (Schmidt etal. 1982; Cohen, Massoglia
& Gospodarowicz, 1982). This role of mevalonate is independent of its effect via
dolichol-mediated protein glycosylation and suggests that mevalonate itself may
be required during morphogenesis and embryonic development when alterations
in cell shape occur.
We thank Mrs S. C. Barton for expert assistance and the research workers cited in the paper
for their generous gifts of various compounds.
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(Accepted 16 January 1983)