Morulae at compaction and the pattern of protein synthesis in mouse embryos

Differentiation (1994) 55: 175-184 Ontogeny, Neoplasia and Dimerentistion Therapy

0 Sorineer-Verlae 1994

Morulae at compaction and the pattern of protein synthesis in mouse embryos Roberto Mayor, Luis Izquierdo * Departamento de Biologia, Universidad de Chile, Casilla 653, Santiago, Chile Accepted in revised form August 11, 1993

Abstract. Compaction of mouse embryos at the 8-cell stage causes a drastic change in cell form and in cell-tocell contacts in 3-4 h. We have studied the effect of inhibitors of transcription (a-amanitin), DNA replication (aphidicolin) and compaction (cytochalasin D, EGTA, a-lactalbumin and Con A) on the pattern of protein synthesis using gel electrophoresis. Our results show that the pattern of protein synthesis is regulated principally by passage through S phase during each early cell cycle rather than by de novo transcription, while changes induced in cell form or contacts do not alter the pattern significantly.

Introduction From fertilization to the 8-cell stage of the mouse embryo the rate of total protein synthesis increases 1.5 fold and increases a further sevenfold to the blastocyst [ l , 58, 591. There is a distinctive pattern of synthesis of certain polypeptides prior to the 8-cell stage [15, 26, 27,411. The first molecular changes depend on maternal templates. The expression of the zygotic genome begins at the 2-cell stage in the mouse and somewhat later in humans and sheep [ l l , 12, 14, 22, 681. Although most polypeptides that change during this period have not been identified, it is known that heat shock proteins appear early in the second cell cycle [5], ribosomal proteins increase 11 fold between fertilization and the 8-cell stage [38] and actin increases almost 90 fold by the blastocyst stage [l]. The qualitative pattern of protein synthesis of 4- to 8-cell mouse or hamster embryos does not change significantly during subsequent development until the inner cell mass and trophoblast differentiate [32, 43, 631.

* Deceased Correspondence to: R. Mayor, Departamento de Biologia, Universidad de Chile, Casilla 653, Santiago, Chile

This report concerns the relationship between protein synthesis and the process of compaction in &cell mouse embryos. Compaction is morphologically characterized by the loss of the lobulated contour of the embryo, the disappearance of cellular outlines and the regionalisation of cells revealed by an apical pole that is full of microvilli and a basolateral surface which is almost devoid of them [35, 42, 621. Inhibitors of protein synthesis applied to early 8-cell embryos d o not interfere with these morphological features of compaction nor with the establishment, during compaction, of gap junctions and cytoskeletal connections [45, 461. However, protein synthesis and compaction may be related by, as yet, unknown mechanisms, since changes in cell form and cell-to-cell contact are correlated with changes in gene expression in a variety of differentiated cell types, such as, cytoskeletal genes in hepatocytes [6], c-myc and other growthassociated genes in 3T3 fibroblasts [16, 181 and the synthesis of proteoglycan in chondrocytes [47]. These effects have generally been ascribed to a role of the cytoskeleton and/or cell adhesion on protein synthesis [3,48, 501. Many agents that interfere with the development of early mammalian embryos seem not to overtly affect gene expression [30]. Polyploidy, induced by cytochalasin, does not affect the pattern of protein synthesis [52]. Continuous treatment with the same drug from the 2-cell stage onwards, inhibits compaction, except for cell polarization, but has no distinct effect on protein synthesis [53]. Increased cell number produced by embryo aggregation causes no change in lactate dehydrogenase isozyme expression [60]. Modification of the nucleo-cytoplasmic ratio by injection or extraction of cytoplasm or by bisection of eggs has no effect on protein synthesis or the expression of a stage-specific antigen [51]. In this paper we have investigated whether the changes in cell shape and adhesion that occur during compaction can affect the pattern of protein synthesis, and because the data, summarized above, suggest the operation of a developmental programme that is not modulated by epigenetic inputs [ 571, we also consider whether these changes are dependent on transcription or DNA replication.

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Our strategy in analyzing the control at compaction of the pattern of protein synthesis is: ( 1 ) To compare changes in protein synthesis caused by drugs that inhibit transcription (a-amanitin) or DNA replication (aphidicolin). Treatment with a-amanitin of 8-cell mouse embryos suppresses mRNA synthesis [40] and also prevents an increase in protein synthesis which is characteristic of this stage [22]. While treatment with aphidicolin of early 8-cell mouse embryos, though suppressing transcription, has no effect on their compaction [2, 17, 64, 651. (2) To compare changes in protein synthesis caused by drugs that inhibit compaction by different routes, i.e. by either by interferance with cytoskeletal components (cytochalasin D) or cell adhesion (EGTA, a-lactalbumin, Con A). Cytochalasin D inhibits compaction and causes its reversion probably by interference with actin polymerization [19]. Inhibition of compaction and decompaction by EGTA, has been attributed to calcium chelation. a-Lactalbumin inhibits the activity of /?-galactosyl transferase, a cell surface component involved in cell adhesion [4] and Con A interferes with compaction by binding to N-acetyl glucosamine [54]. Our results show that the pattern of protein synthesis seen in early embryos is dependent on properly scheduled DNA synthesis but not de-novo transcription, while modulation of cell form or cell-to-cell contacts does not alter the pattern significantly. Methods Collection and culture qf embryos. Female CFl mice were superovulated with 4 IU pregnant mare's serum (PMS) (Sigma) followed 44 h later by 5 IU human chorionic gonadotropin (hCG) (Sigma). and mated with CF1 males. The day in which the vaginal plug was detected is considered to be day 1 of gestation. Hours of development refer to compaction; thus, 0 h corresponds to 50% compaction among control embryos run in parallel with treated embryos and scored every 30 min. Eight-cell embryos were flushed from the oviduct early in the third day of gestation with Biggers' mcdium [7] supplemented with 4 mg/ml bovine scrum albumin (BSA; Sigma). Embryos were cultured in 30 pl microdrops of the same medium in plastic dishes (Greiner) under mineral oil in an atmosphere of 5% C 0 2 in humid air at 37" C.

ed with the labelled precursors during the last 2 h of treatment with each inhibitor. For two-dimensional electrophoresis, 40 embryos were labelled for 4 h. After incubation the embryos were rinsed free of drugs and BSA by several changes of Biggers' medium and then lysed in lop1 of sodium dodecyl sulphate (SDS) sample buffer [49]. In some cases not all the BSA was washed( out and a small distortion of the polypeptide pattern was observed/ around 66 kDa; in these cases the analysis of that region of the gel is not included in our conclusions. Thirty to 40 embryos were lysed for 2-D gels and 5 to 7 for 1-D gels. One-dimensional gels were performed using 10% polyacrylamide according to Laemmli [37] and two-dimensional gels (12 x 10 cm) were made according to O'Farrell [49]. Gels were processed for fluorography [9] and exposed to X-ray film (Kodak X-Omat) at -70" C. The one-dimensional electrophoresis gels were scanned with a densitometer (Gelman ACD-15). Films of two-dimensional electrophoresis were superimposed, 141 polypeptides routinely detected were analyzed, and 30 spots not always present were disregarded.

I

Uptake and incorporation qf [35S]-methionine.and control of DNA replication. Embryos which had been incubated for 22 h with inhibitors were labelled for 2 h with [35S]-methionine (2 pCi/ml). After rinsing the embryos three times with culture medium containing 50 mM methionine, total [35S]-methionine incorporation or incorporation into the trichloroacetic acid (TCA) insoluble fraction was measured. The effect of drugs on DNA replication was monitored by radioautography. Early 8-cell embryos were incubated for 24 h in Biggers' medium containing the drug and 1 pCi/ml [3H]-thymidine (Amersham), rinsed, treated with Na citrate (0.50/, for 2-5 min), fixed with methanol-acetic acid, air-dried, washed with 0.1% TCA at 4" C. Slides were covered with photographic emulsion (Kodak NTB-2) and after exposure, stained with toluidine blue, dehydrated and grains over the nuclei observed by light microscopy [65]. During DNA replication a big increase in the numbers of grains over the nuclei relative to the background enabled us to classify each embryo according the percentage of nuclei undergoing S phase.

Results Inhibition of trunscription and D N A replication : effects on development

Addition of a-amanitin (15 pg/ml) to early 8-cell embryos arrested transcription but did not interfere with compaction (Fig. 1) but after a 24 h treatment embryos had decompacted, no blastulation had occurred and in-

Inhibition qf transcripticin and DNA replication. Transcription was inhibited with z-amanitin (Sigma) at concentration of 1 or 15 pg/ml in Biggers' medium [22. 401. DNA replication was inhibited with aphidicolin (Sigma) at a concentration of 2.5 pg/ml in the same medium [2. 17. 64. 651. Inhibition qf compaction. Cytochalasin D (CCD), a-lactalbumin and Con A was used at concentrations of 0.5 pg/ml [31], 2 mg/ml [4] and 100 pg/ml [54] respectively, in Biggers' medium. EGTA at 0.5 mM was the highest concentration that could be used without affecting the viability of embryos in long culture times. Calactate was replaced by Na-lactate in the culture medium used to dissolve EGTA. EGTA was purchased from Merck; CCD, a-lac and Con A from Sigma. One- and two-dimensional gel electroiphoresis of embryos incubated with [35S]-methionine.In the culture medium containing the inhibitor we added [35S]-methionine(Amersham, specific activity 10001400 Ci/mmol) to a final concentration of 2 pCi/pl. For one-dimensional electrophoresis, 20 embryos in each microdrop were incubat-

1

I

0

I

I

2

1

I

4

,

,6

24h

Fig. 1. Effect on compaction of drugs that inhibit transcription (1 5 pg/ml a-amanitin) or DNA replication (2.5 pg/ml aphidicolin). Ordinate: percentage of compacted embryos. Abscissa: time of development. 0 h corresponds to 50% compaction among control embryos. Vertical bars represent standard deviations. ( 0 =control; o = z-amanitin ; A = aphidicolin)

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but after 8 h, which partially coincides with the normal S phase, changes were observed. Three proteins decreased and at least nine proteins appeared (Fig. 3). After 24 h, two more proteins had decreased and at least six more proteins had appeared (Fig. 3). The densitogram traces indicate that proteins which appear during cc-amanitin or aphidicolin treatments are unlikely to be derived from the proteolytic degradation of other proteins. Eflect of a-amanitin and aphidicolin on uptake and incorporation of [35S]-methionineand on DNA replication

Fii.2. Effect on development of inhibitors of transcription (a, b: 15 pg/ml r-amanitin) or DNA replication (c, d : 2.5 pg/ml aphidicolin). Treatment began at -4 h and continued for 8 h (a, c) or 24 h (b,d). The effect of z-amanitin was studied on 42 embryos divided in two series and the effect of aphidicolin was observed on 32 embryos divided in two series

tracellular drops of blastocoelic fluid were not observed, although cell and nuclear divisions were not arrested (Fig. 2). Addition of aphidicolin (2.5 pg/ml) to early 8-cell embryos at -4 h onwards had no effect in the timing of the start of compaction but after 24 h the embryos had decompacted (Fig. 1). Cell and nuclear divisions were arrested and a blastocoel did not form, but accumulation of intracellular drops of fluid could be observed, in a process that mimics normal blastulation (Fig. 2). Inhibition of transcription and DNA replication: effecfs on the pattern of protein synthesis

The pattern of protein synthesis was studied by onedimensional gel electrophoresis of embryos that had been treated with a-amanitin (1 or 15 pg/ml) or with aphidicolin (2.5 pg/ml). Treatments of 2, 8 and 24 h were begun at - 2 h and [35S]-methionine was included for the last 2 h. Treatments of 2 or 8 h with both doses of a-amanitin had no appreciable effects on the pattern, by comparison with that of controls (Fig. 3b, note that the changes observed in the region around 70 kDa, 2 to 8 h after r-amanitin treatment, are due to the presence of BSA in the embryos; see Methods). However, treatments of 24 h reduced protein synthesis generally and also produced specific effects. Thus, three proteins appeared with both doses while one protein disappeared with the lower dose and another two with the higher dose (Fig. 3). A two hour treatment with aphidicolin produced no appreciable effects on the pattern of protein synthesis

The general reduction in protein labelling of embryos treated with a-amanitin and aphidicolin observed in electrophoresis, was confirmed by measurements of radioactivity incorporated into TCA-insoluble precipitates. Indeed, a-amanitin (1 0 pg/ml) reduces uptake and incorporation to one fifth while aphidicolin (2.5 pg/ml) reduces uptake to one half and incorporation to one third (Fig. 4). The stage of the embryos correspond to those treated for 24 h in Fig. 3. The effect on DNA replication of a-amanitin and aphidicolin (and inhibitors of compaction) was analyzed using [3H]-thymidine incorporation followed by radioautography. Only aphidicolin inhibited DNA replication (Table 1). Inhibition of compaction and effects on further development

The effect on cell form and cell-to-cell contacts at compaction of CCD (0.5 pg/ml), EGTA (0.5 mM), a-lac (0.2%) and Con A (100 pg/ml) has been investigated. If early 8-cell embryos were incubated with EGTA, CCD or Con A, beginning at - 2 h of development (see Methods), they remained uncompacted while those incubated with a-lactalbumin began compaction at the same time as the controls but became decompacted 6 h later (Fig. 5). In another series, early 8-cell embryos (- 2 h) were submitted to the four treatments that inhibit compaction for 8 or 24 h and were examined microscopically (Fig. 6). Control embryos were totally compacted at 8 h and had formed blastocysts at 24 h (Fig. 6a, b). EGTA-treated embryos were uncompacted at 8 h and remained so at 24 h with no sign of cell or nuclear division or signs of intracellular drops of blastocoelic fluid (Fig. 6c, d). CCD-treated embryos were also uncompacted at 8 and 24 h. Cells had not divided but some contained two or more nuclei and the blastomeres were deformed (Fig. 6e, 0. Con A-treated embryos were also uncompacted at 8 h, although blastomeres appeared somewhat flattened against each other. By 24 h, some blastomeres had divided and most were deformed ; compaction and blastulation were not observed (Fig. 6g, h). Embryos treated with a-lactalbumin compacted at the same time as controls but had partially decompacted at 8 h, and then

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a

2h 24 h

--

b

v)

2h

8h

Fig. 3. Effect on the pattern of protein synthesis of inhibitors of transcription or DNA replication. a Densitograms of fluorographs of experiments in which the treatments began at - 2 h and incubations continued for 2, 8 or 24 h and during the last 2 h included ["S]-methionine. C, controls; a-am(l51, 15 Fg/ml a-amanitin; aam(1). 1 pg/ml a-amanitin; Aph, 2.5 pg/ml aphidicolin. Densitogram profiles are partially superimposed to facilitate comparisons, though adjustment is limited because gels have slightly different lengths. Proteins that change with treatments due to a-amanitin

24 h are indicated by white circles and those due to aphidicolin by black circles. Arrowheads pointing downwards indicate reduction and those pointing laterally indicate increase in synthesis. b SDSPAGE. Fluorographs corrrespond to densitograms in a. Scale of molecular weights: 180 kDa, 116 kDa, 84 kDa, 58 kDa, 48.5 kDa, 36.5 kDa, 26.6 kDa. Treatments were tested on 60 embryos divided in two series for a-amanitin and on 30 embryos divided in two series for aphidicolin

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C

a-am

Aph

Fig. 4. Effect of a-amanitin (r-am: 10 pg/ml) and aphidicolin (apph: 2.5 pg/ml) on uptake (U) into acid-soluble material and incorporation (I) into TCA-precipitable material of [35S]-methionine, as a percentage of the controls. Treatment began at - 2 h and continued for 24 h. During the last 2 h embryos were incubated with the labelled precursor. Counts/embryo per hour: controls U 25 757 and I 6389; aphidicolin U 13347 and I 2013; r-amanitin U 4859 and I 1260. Vertical bars represent the standard deviations

100%

60

20 0 I

I

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6 h

Fig. 5. Effect on compaction of cytochalasin D (CCD; 0.5 lg/ml), EGTA (0.5 mM), a-lac (0.2%), Con A (100 pg/ml). Ordinate: percentage of compacted embryos; abscisa: time of development. Results shown were obtained on 83 control embryos subdivided in 7 series, 90 embryos treated with EGTA in 9 series, 54 embryos treated with a-lactalbumin in 5 series, 60 embryos treated with CCD in 5 series and 60 embryos treated with Con A in 4 series. Vertical bars are the standard deviations. ( 0 =control; A = CCD; A = EGTA ; o = a-lac; =Con A) Table 1. Effect on DNA replication of a-amanitin, aphidicolin and inhibitors of compaction

Treatmen t

Grains over the nuclei

Control a-amanitin aphidicolin CCD EGTA a-lactalbumin

+ ++ + +

+, More than 50% of nuclei with grains; -, less than 20% of nuclei with grains; CCD, cytochalasin D * The grains over the nuclei was analyzed as described in Methods

Fig. 6. Effect on development of inhibitors of compaction. Controls (a, b), 0.5 mM EGTA (c, d), 0.5 pg/ml CCD (e, f), 100 pg/ml Con A (g, h), 0.2% a-lac (i,j). Continuous treatment began at -2 h (legend to Fig. 1). k f r column: embryos after 8 h incubation. Righr column: embryos after 24 h incubation. 287 embryos were examined in this series

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24 h

b

2h

8h

24 h

Fig. 7. Effect on the pattern of protein synthesis of inhibitors of compaction. a Densitograms of fluorographs of experiments in which the treatments began at -2 h and continued for 2, 8 or 24 h. During the last 2 h of treatment the embryos were incubated with 2 pCi/pl [35S]-methionine. C: controls; 0.5 m M EGTA; 0.5 pg/ml CCD; 0.7 5% a-lac; 100 pg/ml Con A. Densitogram

profiles are partially superimposed to facilitate comparisons but adjustment is an approximation because gels differ in length. b SDS-PAGE. Fluorographs correspond to densitograms in a. Scale of molecular weights: 180 kDa, 116 kDd, 84 kDa, 58 kDa, 48.5 kDa, 36.5 kDa and 26.6 kDa. In this series 110 embryos were used.

continued to compact. Blastomeres divided and by 24 h most embryos had blastulated (Fig. 6i, j).

(Fig. 7 a and b). The same number of embryos (usually five) were run in each lane except for EGTA- and Con A-treated embryos, whose number was increased to ten because these drugs reduced uptake and incorporation of [35S]-methionine (see below). Relative abundance of each protein was estimated on densitograms by measuring the area covered by distinct peaks relative to bands that d o not change appreciably. Notable changes in electrophoretic pattern occur during incubation of control and treated embryos while differences between the treatments at any one time are small.

Effect on the pattern of protein synthesis of treatments that inhibit compaction :one-dimensional gel electrophoresis

The effect of the four inhibitors of compaction on the pattern ofprotein synthesis was examined by 1D electrophoresis using embryos labelled with [35S]-methionine

181 a U+

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Fig. 8. Effect of inhibitors of compaction on uptake (U)into acidsoluble material and incorporation (I)into TCA-precipitable material of ["Sl-methionine, relative to control embryos. Treatments were began at -2 h and continued for 24 h. During the last 2 h embryos were incubated with the labelled precursor. Counts/embryo per hour: controls U 25757 and I 6389; CCD U 26831 and 16752; I-lac U 25612 and I 6292; EGTA U 10702 and I 3519; ConA U 1364 and I 1065. Verticolhurscorrespond to the standard deviations

Controls were repeated four times and experiments with each inhibitor were repeated three times. The resolution of the fluorograms is however only acceptable for those proteins whose rate of synthesis is high. Effect of treatments that inhibit compaction on uptake and incorporation o f f 3sS]-methionine

Uptake of 20% [3sS]-methionine into control embryos, CCD- or a-lac-treated embryos were similar but was reduced to half in EGTA-treated embryos and to less than one fifth in Con A-treated embryos. Incorporation into TCA precipitable material was slighly less affected in both cases (Fig. 8). In view of these results we only used two-dimensional electrophoresis to compare control embryos with embryos incubated either with CCD or a-lac. Effect on the pattern of protein synthesis of CCD and a-lac treatments :two-dimensionul gel electrophoresis

The high resolving power of two-dimensional gel electrophoresis was used to give a more detailed analysis of the pattern of protein synthesis in early embryos. Control early 8-cell embryos and embryos cultured for 4 or 24 h in a medium containing a-lac or CCD were labelled for 4 h with [35S]-methionine starting at - 4 h. One hundred and forty-one proteins could be clearly detected (see Methods). In control embryos, 119 polypeptides did not change with time, 17 appeared from compaction onwards and 4 disappeared during the same interval (Fig. 9). One of the latter proteins (42.5 kDa)

Fig. 9. Two dimensional gel electrophoresis of embryos in which compaction has been inhibited. a Diagram of a fluorogrdph that summarizes observations on two-dimensional electrophoresis of control embryos. White spots represent 119 polypeptides that do not change during or after compaction; arrow's point to 17 polypeptides that appear during or after compaction; black spors correspond to 4 polypeptides that disappear during or after compaction and the arrow head points to one of these polypeptides which does not disappear in embryos treated with CCD or a-lac. b Two-dimensional gel electrophoresis of control embryos and of embryos treated with CCD or a-lac. Treatments began at - 4 h and continued for 4 or 24 h. Fluorographs on the left side correspond to treatments for 4 h and those on the right side to 24 h. During the last 4 h of treatment embryos were incubated with the labelled precursor

did not disappear when embryos were treated with CCD or a-lac and thus might be regulated by changes in cell form or cell to cell contacts during compaction (Fig. 9a). However, further studies would be needed to be certain that this is not a common effect of both drugs that is unrelated to compaction. The comparison of fluorographs reveals that 25% of the proteins are affected differentially by CCD or a-lac (Fig. 9) and were therefore disregarded.

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Discussion Inhibition of DNA transcription and replication aflects the pattern of protein synthesis

%-Amanitininhibits eukaryotic mRNA synthesis specifically [ 131 and also blocks cleavage and blastulation of mouse embryos explanted at various stages [23,69]. The lack of effect of a-amanitin on early 2-cell mouse embryos shows that polypeptide synthesis at this stage is dependent on maternal transcripts and is largely regulated post-transcriptionally [22, 401. Early 8-cell embryos compact in the presence of aamanitin at a dose that prevents mRNA synthesis, but after 24 h treatment they have decompacted. Intracellular drops of blastocoelic fluid have not formed and blastulation has not occurred, but nuclear and cell divisions were not arrested (Fig. 2). During the last 2 h of this 24 h period, the incorporation of labelled methionine into TCA precipitable material was reduced to 20% (Fig. 4). Treatment with a-amanitin for 2 or 8 h had no appreciable effect on the pattern of protein synthesis, showing that the mRNA used during this time has a longer lifetime than the period of treatment. After 24 h treatment when compaction is complete, certain polypeptides appeared and disappeared (Fig. 3). Absence of polypeptides suggests that they are translated from mRNA that is normally transcribed during this period, but the appearance of polypeptides is not easily interpreted since the densitograms provide no evidence for proteolytic degradation. In any case, neither the pattern of protein synthesis nor the morphological changes are altered by the inhibition of transcription during compaction and the effects on synthesis or morphology are detected at a later stage, when embryos should begin blastulation. Continuous treatment with aphidicolin (a competitive inhibitor of alpha and delta DNA polymerases that does not interfere with beta and gamma polymerases [24,29]), starting from 4 h prior to compaction, did not inhibit compaction. However 24 h later, the embryos had decompacted, cell and nuclear division were arrested and the blastocoel had not formed though intracellular drops of fluid could be observed, suggesting that some of the steps in the blastulation - such as the pumping of liquid by the blastomeres - have not been affected by the treatment (Fig. 2). Others have shown that the continuous presence of aphidicolin (2 pg/ml) from GI of the 4-cell stage onwards prevents the third round of cell division but does not interfere with compaction [64] and when uncompacted 8-cell embryos were incubated with aphidiColin (2.5 pg/ml for 8 h) during the S phase, neither compaction nor blastulation were inhibited [17]. Judged by the criterion of inhibition of blastocyst formation, embryos are most sensitive to aphidicolin treatment (0.5 pg/ ml for 16 h) either at the late 4-cell stage, possibly by inhibiting the fourth DNA-replication cycle, or at the uncompacted 8-cell stage and then sensitivity decreases markedly during compaction [2, 651. The aphidicolin treatments used inhibit DNA synthesis more than 90% and would therefore be expected

to prevent chromosome condensation, centrosome duplication, spindle formation and cytokinesis; however, the control of embryonic cell cycles differs from somatic cycles [25]. Inhibition of DNA synthesis does not prevent morphogenesis of the sea urchin embryo [lo, 661; in Drosophila, nuclear divisions may occur and centrosomes continue dividing [55]; in Xenopus, cells divide at a normal rate and embryos differentiate for several hours [56] and in C. elegans, the expression of biochemical markers that appear at different times during gut development are not affected by inhibition of DNA synthesis [20]. In mammals, our results and those of others are also consistent with the idea that some of the developmental process may be uncoupled from strict cell cycle dependence. An effect of aphidicolin on protein synthesis is evident when embryos are treated from 4 h before compaction until 8 h, which includes most of the S phase (Fig. 3). A number of proteins appear even though uptake and incorporation of labelled methionine is reduced to half of the controls (Fig. 4). Some of the proteins that appear during this treatment could correspond to polypeptides present at an earlier stage (eg. proteins around 150, 75, 45 and 40 kDa, Fig. 3, 8 h treatment), suggesting that their degradation is dependent on DNA replication, but it is not possible to be sure that they are the same proteins that were present at earlier stages based only on their molecular weight. Since synthesis of proteins 8 h after compaction is not dependent on transcription, the modifications in the polypeptide pattern induced by inhibition of DNA synthesis during this time are probably post transcriptional changes as in the sea urchin embryos [33], but additional evidence is necessary to support this conclusion in mice. Inhibition of compaction does not affect the pattern of protein synthesis significantly

The inhibitors used to investigate the relation between compaction and the pattern of protein synthesis, operate by different mechanisms. These include interference with the cytoskeleton and disruption of cell to cell contacts. Cytochalasin causes inhibition of compaction by interfering with polymerization of actin, which at the 8-cell stage is a major component of the cytoskeleton [34,39]. CCD inhibit formation of microfilaments which are also found in the cytoskeletal connections established between adjoining cells during compaction [45]. CCD is thought to inhibit protein synthesis by releasing mRNA from the cytoskeletal framework [50] but we did not observe inhibition of protein synthesis by this drug under conditions where compaction is affected. EGTA is thought to cause decompaction as a result of chelation of calcium that is involved in Ca-dependent adhesion. In early mammalian embryos, as well as in many differentiated epithelia, this is mediated by the glycoprotein called uvomorulin that is protected from proteolytic degradation by calcium [28, 36, 671. Antibodies against uvomorulin inhibit early but not late stages of compaction [28, 441. EGTA which probably

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has many effects as a consequence of calcium chelation is thought to affect translation by altering the phosphorylation of proteins that control it [21] which may be also the case here. u-Lactalbumin interferes with compaction by inhibiting fl-galactosyltransferase [4], a cell surface component involved in cell adhesion whose activity increases a few hours after the beginning of compaction. Con A, which binds to N-acetyl glucosamine, also interferes with compaction [ 5 5 ] . Our data confirm the effect of drugs on early compaction and also show that in late stages these drugs have very different effects on development (Figs. 5, 6 ) . Observations reported in this paper d o not suggest that changes in cell form or contacts during compaction regulate protein synthesis. The four drugs used to inhibit compaction caused no clear-cut common changes in the pattern of protein synthesis revealed by one- or twodimensional electrophoresis (Figs. 7, 9). except for one polypeptide of 42.5 kDa (out of 141 analyzed) which did not disappear during CCD or a-lac treatment. Further study would be needed to show this was connected with compaction. Changes in phosphorylation have been found in preimplantation mouse embryos, in particular in the 3538 kDa spots in 8 cell embryos [43]. These proteins were no longer detectable if embryos were labelled in the presence of cycloheximide. Furthermore, a phorbol ester (PMA) in the culture medium increased the phosphorylation of proteins which are absent in the 4-cell stage at the 8-cell stage. Moreover, embryos cultured in Cafree medium had diminished phosphorylation of the same proteins [8]. In conclusion we suggest that the developmental program in early mouse embryogenesis that leads to embryo compaction is dependent on new rounds of DNA synthesis, which initiate programs of specific protein synthesis that are not affected by inhibitors of transcription or compaction. A ~ k n o i i , / . ~ ~ c . m ~ We n l . ~thank . Dr. M. Sargent for critical reading of the manuscript and M. Brennan for editing the manuscript. This work was partially financed by grants from the University of Chile and FONDECYT.

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