mevalonate reverse ths e developmental arres otf ...hence, lac of dolichok l may partly be the cause...

J. Embryol. exp. Morph. 75, 205-223 (1983) 2 0 5Printed in Great Britain © The Company of Biologists Limited 1983

Mevalonate reverses the developmental arrest ofpreimplantation mouse embryos by Compactin, an

inhibitor of HMG Co A reductase

By M. AZIM H. SURANI1, SUSAN J. KIMBER2 ANDJEREMY C. OSBORN3

From the A.R.C. Institute of Animal Physiology, Cambridge

SUMMARYHydroxymethyl 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 andnon-sterol isoprenes involved in membrane biogenesis, DNA replication and proteinglycosylation. The influence of two inhibitors of HMG Co A reductase, Compactin (orML236B) and an oxygenated sterol, Diosgenin, were tested on preimplantation developmentof mouse embryos. Compactin arrested development at about the 32-cell stage, leaving theblastomeres decompacted. Ultrastructural examination of the embryos revealed reducedmembrane apposition but no major effects on cell organelles. There was however apredominance of nuclei with highly condensed chromatin. Glycosylation of proteins alsoappeared to be inhibited as shown by reduced incorporation of sugar precursors but not thatof amino acids. The influence of Compactin was judged to be highly specific since onlylOjUg/ml (0-08 mM) mevalonic acid abolished the effects of Compactin. Mevalonate in em-bryos may not be primarily utilized in the synthesis of sterols since a specific inhibitor ofcholesterol synthesis, DL-4,4,10-/3-trimethyl-trans-decal-3-j3-ol had no detectable effect ondevelopment. The non-sterol isoprenes of mevalonate such as dolichol and isopentenyladenine may play a more significant role during early development since the influence ofCompactin resembled that previously described using tunicamycin, a specific inhibitor ofdolichol mediated synthesis of N-glycosidically linked glycoproteins. Hence, lack of dolicholmay partly be the cause of arrest of embryonic development by Compactin. Diosgenin causedembryonic arrest at about the 16-cell stage and the influence was not reversible by mevalonicacid. Cholesterol was able to rescue 50 % of the embryos but the effect of Diosgenin could benon-specific and probably caused by its entry into the plasma membrane.

INTRODUCTION

During preimplantation development in the mouse, two distinct tissues, theinner cell mass and the trophectoderm are formed at the blastocyst stage aftersix cleavage divisions (Gardner, 1971). Prior to this, a major Ca2+-dependent

1 Author's address: A.R.C. Institute of Animal Physiology, Animal Research Station, 307Huntingdon Road, Cambridge CB3 0JQ.

2 Author's present address: MRC Laboratory, Woodmansterne Road, Carshalton, SurreySM5 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 closemembrane apposition and cell flattening are observed (Ducibella & Anderson,1975; Lehtonen, 1980; Kimber, Surani & Barton, 1982), together with theregionalization of cell surface constituents (Handyside, 1980). At the 16-cellstage, 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 whilstthe outer cells give rise to trophectoderm (Surani & Handyside, 1983). Cellsurface properties determine cell interactions and adhesiveness and influencecell 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 be-tween cells (see Surani, Kimber & Barton, 1981).

For the synthesis of N-glycosidically linked glycoproteins, the polyisoprenoidlipid, dolichol, is the major saccharide carrier (Hemming, 1977). We havepreviously shown that tunicamycin, a specific inhibitor of N-acetylglucosaminylpyrophosphoryl dolichol phosphate (Takatsuki, Arima & Tamura, 1971) sub-stantially inhibits glycosylation, disrupts compaction and alters the cell surfaceproperties (Surani, 1979; Surani, Kimber & Handyside, 1981; Atienza-Samols,Pine & Sherman, 1981). The levels of dolichol compared to cholesterol aresubstantially 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 aprecursor of divergent biosynthetic pathways of both sterol and non-sterolisoprenes (Brown etal. 1978; James & Kandutsch, 1979). These products includecholesterol, dolichol, isopentenyl adenine and ubiquinone (see Fig. 6). In thisstudy we have examined the consequences of inhibition of HMG Co A reductaseby Compactin (or ML236B) (Brown etal. 1978) and an oxygenated sterol, Dios-genin (Mills & Adamany, 1978). This study demonstrates that Compactin dis-rupts early embryonic development and inhibits protein glycosylation and theseeffects 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 using5i.u. pregnant mare's serum followed 42-48h later by 5i.u. human chorionicgonadotrophin (HCG) (Intervet, Milton, UK). Each female was caged with anFi (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 lactoneform of Compactin was converted to the acid form by heating at 50 °C for 1 h in0-1 M-NaOH (Kaneko, Hazama-Shimada & Endo, 1978). The solution was thenadjusted to pH8-0 with 1N-HC1 and the concentration of the compound to1 mg/ml in 0-01 M-tris-HCl, pH 8-2. The sodium salt of Compactin thus obtainedwas divided into lOjul aliquots and stored at -20 °C. DL-4,4,10-jS-trimethyl-trans-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, isopentenyladenine and Diosgenin (5,20a,22a,25D-spirosten-3/3-ol) were all obtainedfrom Sigma. Mevalonic acid was dissolved in the embryo culture medium at aconcentration of lmg//il medium and stored at — 20 °C. Dolichol, dolicholmonophosphate and coenzyme Qio were dissolved in chloroform at a concentra-tion of 10 mg/ml, 2 mg/ml and lmg/lOOjul, respectively. Isopentenyl adeninewas 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 weeksat — 20 °C under nitrogen. The rest of the compounds were freshly made on theday of the experiment. Diosgenin was made in ethanol (2 mg/ml). Cholesterolwas first dissolved in chloroform (100 jUg/jul) and 5 /il of this stock solution werethen added to 1 -0 ml of culture medium with 4 mg/ml bovine serum albumin withvigorous 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 2of pregnancy at about 45-48 h post HCG. Embryos were cultured in Brinster'smedium (Brinster, 1970) supplemented with 4 mg/ml bovine serum albumin(BMOC-3).

Two-cell embryos were used immediately in experiments or cultured over-night 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 thendivided into groups of 20-40 embryos. Subsequent cultures were carried out inmicrotitre plate wells containing 0-1-0-3 ml of each test medium and with ad-hesive 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 thetest mediums.


Observations and fixation of embryos for microscopyEmbryos 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 andviewed and photographed using a Zeiss microscope. Thin sections were stainedwith a saturated solution of uranyl acetate in 50 % ethanol followed by leadcitrate (Reynolds, 1963) and examined in an AEI801 electron microscope.

Determination of cell number in embryosThe number of cells was determined by counting nuclei in air-dried prepara-

tions of embryos (Tarkowski, 1966).

Incorporation of radioactive precursors into embryos and polyacrylamide gelelectrophoretic analysis

For the estimation of incorporation of [3H]leucine and [3H]sugar precursorsinto embryos, they were cultured in the presence of Compactin for approximate-ly 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 inglucose-free medium as described previously (Surani, 1979). For labelling ofembryos with sugar precursors, 50 / i of a 2x concentrated glucose-free mediumwas 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 labelledfor approximately 6 h in groups of between 10-30 embryos with the amino acidprecursors or in groups of 50-100 with the sugar precursors. At the end of thelabelling period, embryos were washed and processed to determine incorpora-tion of the precursors in embryos in the control group and compared with thosecultured in the presence of Compactin essentially as described previously(Surani, 1979).

Embryos labelled with [35S]methionine were also analysed on 8-15 % SDS-polyacrylamide gradient gels and the radiolabelled proteins visualized byfluorography exactly as described previously (Moor, Osborn, Cran & Walters,1981). In some cases, galactosyl glycopeptides were immunoprecipitated afterthe addition of peanut lectin followed by an addition of antibody against peanutlectin (PL Biochemicals) and the immune complexes were extracted usingProtein A-agarose gels. The method used was essentially as described elsewhere(Magnuson & Epstein, 1981). The extracted glycopeptides were also analysed bypolyacrylamide gel electrophoresis as described above.


Fluorescence microscopy

Control embryos and those cultured in Compactin were examined byfluorescence microscopy after treatment with FITC-Concanavalin A asdescribed (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 underwentcompaction and formed advanced morulae after 48 h in culture. When embryoswere cultured in 0-5-1-OjUg/ml Compactin, a small proportion (10-20%) for-med into poorly developed blastocysts. Embryos cultured in 2-0jUg/ml Compac-tin failed to develop into blastocysts and this was considered as the minimumconcentration of Compactin necessary to completely disrupt development.Similar preliminary experiments with Diosgenin (1-0-5-0/ig/ml) establishedthat 5-0 jUg/ml Diosgenin was necessary to disrupt development to the blastocyststage.

Preliminary experiments were also carried out using Compactin and Dios-genin in medium containing normal or fatty-acid-free albumin (from Sigma).There was no marked difference between the effect of the compounds in mediumwith fatty-acid-free albumin and in medium containing fatty acids. Fatty-acid-free albumin also did not hinder development to the blastocyst stage in theabsence of the inhibitors. In all cases except where indicated normal bovineserum albumin was present in the culture medium.

We first examined the influence of Compactin and Diosgenin on cell prolifera-tion on 8-cell precompacted embryos. After 24 h in culture (89 h post HCG), thenumber of cells in the control group and in the presence of Compactin was similarbut those cultured in Diosgenin had only about half the number of cells and thesehad not increased considerably after 31 h (96 h post HCG) in culture. Afterfurther 7h in culture, embryos in Compactin showed a slight increase in thenumber of cells to 32 cells compared with about 42 cells in the embryos in thecontrol group (Table 1). Both of the inhibitors have been previously shown tospecifically block HMG Co A reductase. Therefore the influence of the twomajor products of this pathway, mevalonic acid and cholesterol, on embryonicdevelopment in the presence of either Diosgenin or Compactin was examined.

The influence of mevalonic acid and cholesterol in the presence of Diosgeninis shown in Table 2. Mevalonic acid upto lmg/ml was unable to reverse theinfluence of Diosgenin. However, cholesterol at 50-100^g/ml was able torescue approximately 50 % of the embryos (see Fig. 1). Many of these embryoswhich were apparently rescued formed blastocysts that appeared to be poorlydeveloped. Similar experiments were carried out with Compactin (Table 3). As


Table 1. Influence of Compactin and Diosgenin on the number of cells in embryos

Group

Control

Compactin(2 Kg/ml)

Diosgenin(5pg/ml)

Time in culture (h)*(h post HCG)

24 (89)31 (96)

24 (89)31 (96)

24 (89)31 (96)

Mean cell no.±S.D.

29-2 ± 4-941-8±ll-9

28-8 ± 5-432-9 ± 8-2

12-1 ± 2-515-2 ± 2-7

No. of embryos

2127

3228

119

* Embryos were at the precompacted 8-cell stage (65 h post HCG) at the start of the experi-ment.

Table 2. Development of 8-cell embryos in Diosgenin and mevalonic acid orcholesterol

Group

Control

Diosgenin (5 jug/ml)

Diosgenin (5|Ug/ml)+ cholesterol (jug/ml)

2550

100

Diosgenin (5 jUg/ml)+ mevalonic acid (/ig/ml)

10100

1000

NumberExp.

7

3

373

234

Embryos

80

69

357835

273259

Mean cell no.*± S.D. (n)

44-8 ±11-9 (10)

13-9 ± 3-3(12)

12-4 ± 5-3(15)27-8 ± 4-8 (17)t29-9 ± 5-2 (16)t

14-9 ± 3-5(11)12-3 ± 7-2(12)14-9 ± 3-5(15)

No. blastocysts(%)t

67 (83-8)

3 ( 4-3)

1 ( 2-8)34 (43-6)19 (54-3)

000

* Determined at approximately 96 h post HCG (31 h in culture) in separate concurrentgroups 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 enablednearly 90% of the embryos to develop to the blastocyst stage. Even l^g/mlmevalonic acid was partially effective and rescued over 30 % of the embryos. Bycontrast, cholesterol at up to 100/ig/ml was virtually ineffective in reversing theinfluence of Compactin. The embryos that were rescued by mevalonic acidappeared to be normal when examined under an inverted microscope (see Fig.1). Furthermore, the number of cells in the embryos rescued by cholesterol in thepresence of Diosgenin was substantially less than the number of cells in the


Table 3. Development of 8-cell embryos in Compactin with mevalonic acid orcholesterol

Group

Control

Compactin (2 /zg/ml)

Compactin+ cholesterol (/^g/ml)

2550

100

Compactin+ mevalonic acid (jug/ml)

110

1001000

NumberExp.

11

11

3119

6565

Embryos

154

139

30166119

72608163

Mean cell no.*± S . D . (n)

42-2 ±12-1 (21)

33-3 ± 9-3(27)

32-2 ± 7-2(25)30-9 ± 6-8(29)31-3 ± 5-4(20)

38-8 ± 4-8(32)f42-2 ± 7-9(35)41-9 ±11-2 (30)39-7 ± 9-9(31)

No. blastocysts(%)t

141 (91-6)

3 ( 2-2)

003 ( 2-5)

24 (33-3)53 (88-3)73 (901)45 (71-4)

* 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 acidin the presence of Compactin had the normal number of cells.

The role of cholesterol in early embryonic development was examined furtherwith a specific inhibitor of its synthesis. TMD inhibits cyclization of squalene andhence prevents synthesis of lanosterol and cholesterol (Chang et al. 1979). Thisinhibitor was also of interest since its site of action is distal to the formation ofdolichol and therefore TMD would not affect the concentration of dolichol.TMD at a concentration of up to 25 /ig/ml was virtually ineffective in blockingdevelopment 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 stageonwards in the presence of TMD. Culture of embryos in the presence of TMDand fatty-acid-free albumin also did not influence development. The results weretherefore combined (Table 4).

The influence of Compactin on embryos was examined further. When 2- or8-cell embryos were cultured in the presence of Compactin, they developednormally at first, undergoing normal cleavage divisions and compaction at the8-cell stage and proceeded to the 32-cell stage. However the embryos started todecompact at the 16-cell stage and the majority of the embryos were decompac-ted after 30-35h in culture (Fig. 1). As shown in Fig. 1A, the embryos in thecontrol group were fully compacted or at the blastocyst stage. Embryos in thepresence of Compactin (Fig. IB) had rounded and distinct blastomeres.


Table 4. Develoment of2-cell and 8-cell embryos in DL-4,4,10-$-trimethyl-trans-decal-3-$-ol (TMD)*

Group

Control

TMD (jug/ml)1-252-55-0

10-012-525-050-0

Exp.

7

4467645

NumberEmbryos

75

645986928060

120

Mean cell no.±S .D . (n)t

41-7 ± 9-3(18)

39-5 ± 8-9(26)38-6 ± 7-6(22)37-6 ±10-1 (27)40-9 ±11-2 (29)37-7 ± 9-9(18)36-5 ± 9-3(12)All died after 24 h

No. blastocysts(%)t

66 (88-0)

51 (79-7)44 (74-6)70 (81-4)72 (78-3)69 (86-3)42 (70-0)

in culture§with almost no increase in thenumbers of cells

* Combined results of embryos cultured from 2-cell or 8-cell stage and in normal or fatty acidfree albumin.

t At 96 h post HCG in separate concurrent groups of embryos.J A t m h p o s t H C G .§ 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). Someof these embryos were sectioned and the semithin sections examined by lightmicroscopy. Figures 2A and 2B show that the embryos in the control group wereat the late morulae-early blastocyst stage. The outer blastomeres were charac-teristically flattened. Embryos cultured in Compactin (Fig. 2C and D) however,failed to show the flattened morphology of outer cells: the blastomeres generallytended to be rounder. Examination of the embryos by electron microscopy (Fig.3) revealed much-reduced areas of membrane apposition when embryos werecultured in the presence of Compactin. However, the unapposed peripheralsurfaces of the blastomeres had a high density of microvilli similar to the densityon the outer surface of control morulae. Large intercellular spaces were presentinside the morulae between the rounded blastomeres in contrast to the closelypacked arrangement of cells in control morulae. Although microvilli werepresent on their inner membranes, they did not interdigitate closely with thoseof adjacent blastomeres over the inner membrane area as in the control compac-ted morulae. However, in some instances adherens junctions were observed atthe apical regions of membrane contact between cells. Cytoplasmic blebs andlarge processes sometimes extended from the surface of Compactin-treated em-bryos (Fig. 3D) and there were regions of microvillar interdigitation. Themitochondrial population of both control late morulae and Compactin-treatedembryos consisted partly of the vacuolate type found in early preimplantationembryos and partly of the non-vacuole form with clearly defined cristae found


Fig. 1. Examination of embryos with inverted microscope under bright field at92-96h post HCG (27-31 h culture). (A) Control group with blastocysts and latemorulae, (B) 2 jug Compactin/ml; many of the embryos have undergone decompac-tion (C) 2jUg Compactin +10/ig mevalonic acid/ml; the majority of embryosdeveloped into normal blastocysts (D) 5-0 jug Diosgenin/ml; decompacted embryoswith fewer cells than in (B). (E) 5 peg Diosgenin + 100 pig cholesterol/ml; compactedmorulae and poor blastocysts (F) 5pig Diosgenin 4-100/ig mevalonic acid/ml; nomarked difference compared with (D). (Scale bar = 30jum.)


2A

v<.

B DFig. 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 groupshowing well compacted morula and early blastocyst. (C & D) 2 fig Compactin/ml;the peripheral blastomeres are rounder compared with the flattened cells in thecontrol group. Condensed chromatin (arrows) also observed in the majority of cells.(Scale bar= 10/im.)


Fig. 3. Electronmicrographs of (A) control late morula, 92-96 h post HCG. Closeassociation between membranes of adjacent cells is observed with blastomeresspread over one another. (Scale bar = 5 jum.) (B) Embryo cultured in 2 /ig Compac-tin/ml for 27-31 h (92-96 h post HCG) from the 8-cell stage. Note loose associationof 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 junction-al complexes (jc) including gap junction, cl, crystalline lamellae; mv, microvilli inwhich micro filaments were evident. (Scale bar = 1 fjm.) (D) Outer region of embryofrom the experimental group. Mitochondria (m) are both of the 'immature'vacuolate form and the form containing cristae. mv, microvilli (with micron"la-ments); b, blebs; er, endoplasmic reticulum; g, golgi apparatus. (Scale bar = 1 jum.)


in the blastocyst:' No differences could be found in the rough endoplasmicreticulum or in the distribution of microfilaments and microtubules betweencontrol and Compactin-treated embryos. One noticeable difference was foundin the nucleus. In the control embryos the majority of the nuclei could be detec-ted with distinct nuclear membranes, whereas in the experimental group thenuclear membranes were barely detectable and the chromatin was highly con-densed.

The influence of Compactin on the synthesis of macromolecules was alsoexamined. Eight-cell embryos were first cultured in the presence of Compactinfor 24 h and then labelled with amino acids and sugar precursors for 6 h after thistime. Figure 4 shows that the incorporation of amino acid precursors was notaffected 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 wassubstantially 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 polypeptidesor glycopeptides of embryos cultured in the presence of Compactin (Fig. 5).

The binding of fluorescein-conjugated Concanavalin A to the embryonic cellsurface was examined. Although occasionally there appeared to be a slight

r35S]Methionine (6)

3H]Leucine (5)

3H]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 deter-minations are in parenthesis. Compactin had no detectable effect on the acid-solublepool of the precursors.


reduction in the binding of Concanavalin A to embryos cultured in Compactin,there was no marked quantitative difference compared with the controls (datanot shown). Further detailed studies are needed to establish if Compactin has aneffect on the cell surface constituents of embryos.

Finally, since the influence of Compactin appears to be due to specific in-hibition of HMG Co A reductase, several key products of this pathway were usedto determine whether the effect of Compactin could be overcome by thesecompounds (Table 5). As mentioned previously, mevalonic acid was highly

Mrx10

200

92

69

46

30

- 3

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) glycopeptidesextracted from embryos in the control groups and (D) glycopeptides from embryosin the experimental group.


Table 5. Compounds tested for the reversal of the effects ofCompactin on embryos

ConcentrationCompound (jug/ml)

Mevalonic AcidCholesterolDolicholDolichol MonophosphateRetinoic AcidCoenzyme Q10

Isopentenyl adenine

1-100025-100

0-1-5-00-5-20

0-03-3-01-0

0-5-1-0

All the above compounds when tested in the absence ofCompactin had no detectable effectson development.

effective in reversing the influence of Compactin. Cholesterol however wasineffective. Other compounds tested were dolichol, dolichol monophosphate,retinoic acid, coenzyme Qio and isopentenyl adenine. All of these were ineffec-tive in reversing the influence of Compactin. The compounds were also testedin various concentrations and in a large variety of combinations. So far none ofthe combinations employed have been successful in reversing the influence ofCompactin (data not shown).

DISCUSSION

Compactin, a competitive inhibitor of HMG Co A reductase (Brown et al.1978) interrupts preimplantation development of mouse embryos after the16-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, Diosgeninacts relatively quickly and prevents compaction at the 8-cell stage but the effectis not reversible by mevalonic acid, although Diosgenin is also suggested to in-hibit HMG Co A reductase (Mills & Adamany, 1978). However, Diosgenin andother oxygenated sterols can sometimes act non-specifically when they becomeinserted in the plasma membrane (Gordon, Bass & Yachnin, 1980). Such non-specific effect is partly reversible by exogenous cholesterol which presumablydisplaces the compound from the plasma membrane. Similar results werepreviously obtained with other oxygenated sterols (Pratt, Keith & Chakraborty,1980), but such influence is not necessarily a reflection of de novo synthesis ofsterols during early development in the mouse. Indeed there is little synthesis ofsterols up to the blastocyst stage (Pratt, 1982; Carson, Hsu & Lennarz, 1982).Furthermore, cholesterol, the major product of mevalonic acid did not reversethe effects of Compactin on embryos; these embryos had approximately thesame number of cells (30) as in the blastocysts formed when cholesterol was


Acetate

IHMG Co A

(HMG Co A)(Reductase) COMPACTIN

MEVALONIC ACID

IFarnesyl-P-P

Squalene Dolichol

TMDI

Dol-P

Lanosterol

Cholesterol

UDP GlcNAc

TUNICAMYCIN

YDol-PP-GlcNAc

Polypeptide

Polypeptide-N-oligosaccharide

Fig. 6. The biosynthetic pathway in the formation of mevalonic acid and the diver-gent pathways of two major isoprenes, dolichol and cholesterol. The site of inhibi-tory action of Compactin, TMD and tunicamycin are indicated.

present along with Diosgenin. In addition, TMD, a specific inhibitor ofsqualence cyclization (Chang et al. 1979) had no effect on development to theblastocyst stage. The combined observations indicate that products ofmevalonate other than cholesterol, such as the non-sterol isoprenes dolichol andisopentenyl adenine, may be equally important if not crucial for the rescue ofembryos by mevalonic acid in the presence of Compactin after the 16-cell stage.


Only 0-03-0-11 % of acetate is generally incorporated into dolichol comparedwith sterols, but their levels can increase by 10- to 100-fold in developing systemswhere cell differentiation is underway (James & Kandutsch, 1980; Potter et al.1981; Harford & Waechter, 1980; Carson & Lennarz, 1981). This increase indolichol, the key lipid saccharide carrier in the glycosylation of N-glycosidically-linked glycoproteins (Hemming, 1977), and mannosylphosphoryl dolichol (Har-ford & Waechter, 1980; Carson & Lennarz, 1981) is probably essential for rapidchanges in the cell surface glycoproteins during cell interactions and morpho-genesis. In this respect it was interesting to observe the effect of Compactin onthe incorporation of sugar precursors into embryos which was markedly reducedcompared with the incorporation of amino acids. Since overall proteinsynthesis, both quantitative and qualitative remains unaffected, this suggests aninhibition of protein glycosylation probably resulting from the lack of dolichol.We cannot entirely rule out the effect of Compactin on polypeptides sincequalitative differences may be detected if they are analysed by the two-dimensional gel-electrophoretic system. The decrease in the incorporation ofthe sugars was not caused by the influence of Compactin on intracellular mem-branes since the endoplasmic reticulum and other organelles such as mitochon-dria developed normally. The influence of Compactin first becomes detectableat the 16-cell stage when some of the interstitial glycoproteins such as lamininare detected between cells (Leivo, Vaheri, Timpl & Wartiovaara, 1980). How-ever, further work is necessary to demonstrate that the decompaction of em-bryos caused by Compactin is as a result of changes in the cell surfaceglycoproteins.

There are similarities between the effects of Compactin and tunicamycin onmouse embryos. Tunicamycin, a specific inhibitor of synthesis of dolichol-linkedsaccharides (Takatsuki et al. 1981) also caused decompaction of embryos andinhibited protein glycosylation (Surani, 1979; Ateinza-Samols et al. 1981) andcaused changes in the cell surface properties of blastomeres (Surani et al. 1981).In sea urchin embryos, dolichol alone can overcome the inhibitory effects ofCompactin on protein glycosylation as well as development (Carson & Lennarz,1979,1981). Although dolichol alone (or in combination with other products ofmevalonic 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 thecompounds only enter the plasma membrane and not the cytoplasm.

Isopentenyl adenine, another product of mevalonic acid may be crucial duringdevelopment for DNA replication and cell growth. It appears that in embryoscultured in Compactin, the cells ceased division at a specific point in the cellcycle, since the majority of them had highly condensed chromatin and lackedclear nuclear membrane. This finding may be significant since isopentenyladenine 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 221Recent 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 viadolichol-mediated protein glycosylation and suggests that mevalonate itself maybe required during morphogenesis and embryonic development when alterationsin cell shape occur.

We thank Mrs S. C. Barton for expert assistance and the research workers cited in the paperfor their generous gifts of various compounds.

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(Accepted 16 January 1983)

mevalonate reverse ths e developmental arres otf ...hence, lac of dolichok l may partly be the cause...

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