Activation of protein kinase C triggers premature compaction in the four-cell stage mouse embryo

DEVELOPMENTAL

138, l-15 (1990)

BIOLOGY

Activation

of Protein Kinase C Triggers Premature Compaction in the Four-Cell Stage Mouse Embryo

GLEN K. WINKEL,’ Depwtment

JAMES E. FERGUSON, MASATOSHI

of Zoology, University of Califmin, of Science, Kyoto University,

Faculty

Davis,

TAKEICHI,*

Califmia

Kitashirakasa,

Accepted Nwwmber

AND RICHARD

NUCCITELLI”

95616; and *Department of Biophysics, Sakyeku, Kyoto 606, Japan

13, 1989

During mouse preimplantation development, the cells of the mouse embryo undergo a progressive subcellular reorganization at compaction, which eventually results in the formation of two distinct cell types. We have investigated the effect that activators of the Caz+-phospholipid-dependent protein kinase (PKC) have on mouse compaction. Phorbol ester activation of PKC caused premature compaction of four-cell embryos within a few minutes of addition followed by a prolonged decompaction phase after 1 hr. This response was dose-dependent to concentrations as low as 250 pg/ml. Diacylglycerides also caused compaction; however, it was more sustained than with phorbol esters and was not followed by a phase of decompaction. Inhibition of PKC with sphingosine blocks induced compaction in a dose-dependent manner and also blocks normal compaction of eight-cell embryos. A monoclonal antibody to the cell adhesion molecule, E-cadherin, which mediates mouse embryo compaction, completely blocks compaction induced by these activators of PKC. Indirect immunofluorescence with a monoclonal antibody to E-cadherin indicates that PKC activation causes a rapid shift in the localization of this cell adhesion molecule, which coincides with the observed compaction. These results suggest that PKC plays a role in the initiation of compaction through its effect either directly or indirectly on E-cadherin. o lasn Academic PEWS, IIK

INTRODUCTION

great deal of research has focused upon the timing and mechanisms underlying compaction (Ziomek and Johnson, 1980; Johnson and Ziomek, 1981; Pratt et cd., 1982; Levy et al, 1986; Fleming et al, 1986a,b). Recent work by Levy and others (1986) has shown that inhibitors of protein synthesis initiate compaction prematurely at the four-cell stage. These inhibitors have their greatest effect if applied early in the third cell cycle. Therefore they proposed that an inhibitor protein, which was responsible for preventing compaction, might be synthesized early in the four-cell stage. An initiation signal for compaction acting at the posttranslational level would allow compaction to proceed by removal of this inhibition. This idea coupled with the evidence that the surface polarization is stable (Ducibella, 1982; Pratt et al, 1982; Johnson and Maro, 1985; Fleming et al., 1986a,b) and may be instructive in governing the polarity of the cytoplasm (Wiley and Obasaju, 1989) has led to the hypothesis that the signal to induce compaction and polarization may be initiated and propagated at the level of the plasma membrane (Johnson and Maro, 1985). Recent evidence has shown that cell-surface-mediated signal transduction events often utilize protein kinases as part of the transmembrane signaling mechanism (Berridge, 1987). Protein kinase C (PKC), in particular, has been demonstrated to play a crucial role in the release, secretion, and exocytosis of cellular constit-

Compaction of the blastomeres in the mouse embryo marks the beginning of differentiation in the preimplantation embryo (Johnson et ah, 1986). During compaction, previously round, undifferentiated cells become adherent through activation of the Ca’+-dependent cell-cell adhesion system (CDS). This causes cellular flattening which obscures the individual cell outlines and brings these cells together into tight apposition. Compaction is accompanied by other developmental changes including polarization of various cytoplasmic components (Houliston et ah, 1987; Johnson and Maro, 1984; Sobel, 1983; Sobel and Alliegro, 1985), formation of both gap junctions (Lo and Gilula, 19’79; McLachlin et al, 1983; Goodall and Johnson, 1982,1984) and apical tight junctions (Ducibella and Anderson, 1975; Fleming et ah, 1989), and polarization of the outer surfaces marked by accumulation of microvilli over the apical region (Ziomek and Johnson, 1980; Johnson and Ziomek, 1981; Johnson and Maro, 1984; Maro et al., 1985). Cell diversity appears soon after this embryonic reorganization with the appearance of two cell types, trophectoderm and inner cell mass. Because these major changes precede overt cell differentiation, a i Present address: Cardiovascular Research Institute, University of California, San Francisco, CA 94143. ‘To whom all correspondence should be addressed.

Box 0532,

1

0012-1606/90 $3.00 Copyright All rights

(c: 1990 by Academic Press. Inc. of reproduction in any form reserved

2

DEVELOPMENTALBIOLOGYV0~~~~138.1990

uents from a variety of endocrine and exocrine tissues as well as in the activation of many other cellular functions (Nishizuka, 1986). Moreover a role for PKC has been shown in a number of embryonic events including oocyte maturation (Bornslaeger et a& 1986), fertilization (Endo et ah, 1987), and neural induction (Otte et al., 1988). The evidence that PKC is involved in embryonic events, coupled with the fact that activated PKC acts within the plane of the plasma membrane, suggested that PKC might play a role in the initiation of compaction in the mouse embryo. PKC is an integral part of the signal transduction cascade of the phosphatidylinositol (PI) cycle and the phosphatidylcholine (PC) cycle (Irving and Exton, 1987). Typically, external signals detected by surface receptors stimulate phospholipase C, via a specific Gprotein (G,). Activated phospholipase C cleaves the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and 1,2-diacylglycerol (DAG). Ins(1,4,5)P, acts upon internal Cazf stores to raise intracellular calcium, while DAG transiently activates PKC within the plane of the membrane (Berridge, 1987). PKC can be artificially activated by external application of phorbol esters, although the fact that phorbol esters are not metabolized by the cell leads to a prolonged activation and subsequent down-regulation of PKC, distinct from the characteristic activation by DAG or its lipid analogs (Nishizuka, 1986). We report here that the activation of PKC by phorbol 12-myristate 13-acetate (PMA) causes premature compaction of the four-cell stage mouse embryo followed by complete decompaction. Furthermore, synthetic analogs of DAG induce a more rapid and sustained level of compaction than with PMA. Sphingosine, a potent inhibitor of PKC which competes for the PKC active site, blocks compaction induced by PKC activators as well as normal compaction of the eight-cell embryo. A monoclonal antibody to the E-cadherin adhesion molecule (ECCD-1) (Yoshida-Noro et ab, 1984) completely blocks compaction induced by phorbol ester or diacylglyceride. Those reagents that activate PKC also cause major shifts in the localization of E-cadherin. Therefore these results demonstrate that PKC plays a role in the initiation of compaction and may modulate the extent of compaction through its effects upon the cell adhesion molecule, E-cadherin. MATERIALS AND METHODS

Recovery of Embryos Female CF-1 mice (Charles River) were induced to ovulate by intraperitoneal injection of 7.5 IU each of pregnant mare’s serum gonadotropin (Sigma, St. Louis,

MO) followed 48 hr later by 7.5 IU of human chorionic gonadotropin (hCG, Sigma). Females were mated overnight and checked for vaginal plugs the following morning. Two- and four-cell embryos were flushed from oviducts with flushing medium 1 (FM-l) (Spindle, 1980) 54 to 56 hr after injection of hCG. Four-cell embryos were incubated in 10 ~1 drops of T6 media under paraffin oil (Whittingham, 1971; Wiley et al., 1986) in an atmosphere of 9% (v/v) CO2 for 1 hr prior to addition of reagents. This relatively high COZ level appears to be required for PMA-stimulated compaction. Reagents and Culture Conditions Phorbol 12-myristate 13-acetate (PMA, Sigma) was dissolved in dry dimethyl sulfoxide (DMSO, Sigma) to a stock concentration of 0.1 mg/ml, pipetted into 20-~1 aliquots, and frozen until diluted appropriately to the final concentration with T6 media. DMSO had no effect on normal development or compaction during these experiments and was included in all control media at 0.01% . Four-cell embryos were divided into groups of 20 to 30 and placed into microdrops of T6 media containing increasing concentrations of PMA or 4a-phorbol (100 rig/ml) as a control. The protein kinase inhibitor, Dsphingosine (trans-D-erythro-2-amino-4-octadecene1,3-diol, Sigma), and DAG analogs, diCs (1,2-dioctanoyl-sn-glycerol) and OAG (1-oleoyl-2-acetyl-sn-glycerol, both from Sigma), were dissolved to 100 mM stock concentration in DMSO and frozen in 20-~1 aliquots until ready for use. Sphingosine was also made up in ethanol by the method of Lambeth et ah (1988) to control for cytotoxic effects reported when sphingosine was dissolved in DMSO. However, we found that the effects of sphingosine were identical with either solvent; therefore, we used DMSO as our primary solvent. Incubation conditions were modified for these lipophilic reagents because they were ineffective when embryos were cultured in microdrops under oil (Miller and Pursel, 1987). Eventually all experiments were conducted without paraffin oil since our results indicated that the apparent concentration of PMA and sphingosine in the aqueous phase was reduced by the presence of paraffin oil overlying our microdrops of media. Therefore, 50 ~1 of lipid-containing T6 was pipetted into each 200-~1well of a 3 X 8 Titertek microtiter plate (Flow). The microtiter plate was placed within a 150 X 25-mm petri dish containing 1 ml of sterile distilled water to maintain humidity and placed in the incubator. This was necessary to reduce the evaporation of media from the uncovered microwell plates during in vitro culture. When either sphingosine or the monoclonal antibody ECCD-1 were used in conjunction with PKC activators, they were added 15 min prior to Time 0.

WINKEL

ET

AL.

Activation

Immunoj5brescence

ECCD-2 antibody was diluted 1:200 in T6 media and incubated for 1 hr prior to the addition of embryos. Embryos were treated with PKC activators for varying periods of time and either placed directly into antibody-containing media or briefly fixed in phosphatebuffered 1% paraformaldehyde. Embryos were washed several times in FM-l and treated with goat anti-rat FITC antibody (1:200) for 15 min at room temp. They were washed with FM-l and then placed in a viewing chamber for epifluorescence. The pattern of immunofluorescence was the same whether cells were fixed or unfixed; therefore, we eliminated the fixation step as unfixed cells displayed less background staining. The fluorescent image was stored on an optical laser disk recorder and the digitized image was enhanced with a video image processing system. Scoring of Compactimz.

Beginning at 15-min intervals for the first hour, 30min intervals for the next 2 hr, and hourly for 5 hr, embryos were scored for degree of compaction, separated by extent of compaction into adjacent microdrops, counted, and returned to the incubator. Treated embryos were categorized into three groups on the basis of their degree of compaction when examined on a Wild dissecting microscope fitted with a heated stage. Embryos were classified as noncompacted if their cell outlines were clearly visible (Fig. la); partially compacted if they were beginning to compact, as evidenced by flattening of the cells and partial obscuring of the cell outlines (Fig. Id); and fully compact when none of the cell outlines were visible (Fig. lc). To quantify the extent of compaction for the population of cells a scoring system derived from Levy et al. (1986) was utilized: noncompacted cells were assigned a value of 0, partially compacted cells a value of 1, and fully compacted cells a value of 2. The population score at a particular time was then expressed as a percentage of the maximum possible score if all the cells were to have been fully compacted. Cell Counts and Nuclear

Staining

The cell number of fused embryos was determined by a modification of the air-drying technique of Tarkowski (1966). After staining with 2% acetoorcein (GIBCO), the number of nuclei were counted under the microscope with the aid of an ocular grid and hand counter. The number of nuclei were taken to be equivalent to the number of cells in the embryo. Any cell spreads which dispersed either poorly or excessively were not included. Treated and control embryos were incubated

of Protein

Kinase

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with (10 mg/ml) Hoechst dye and observed under epifluorescent illumination. The number of nuclei per blastomere was scored to determine if TPA-treated cells in our experiments were binucleate as suggested by Mystkowska and Sawicki (1987). RESULTS

PMA Triggers the Compaction of Four-Cell Embryos

Stage

The phorbol ester, PMA, had dramatic effects upon compaction of the four-cell stage embryo (Fig. 2). Within the first 15-min period after the addition of PMA (10 rig/ml), 43% of the population were compacted according to our scoring method. This percentage increased to 98% during the following 30 min, after which the number of compacted embryos began to decrease as these embryos began to decompact. This decompaction phase typically lasted for 3 to 4 hr, after which virtually all of the embryos became decompacted beyond their normal level of endogenous adhesion (Fig. lh). The inactive phorbol ester, 4a-phorbol, was completely ineffective in inducing compaction at concentrations up to lo-fold higher than PMA (100 rig/ml, data not shown). Likewise both protein synthesis inhibitors, puromycin and anisomycin (Sigma), although they caused a mild effect upon compaction (as Levy et al. (1986) observed), had no effect upon compaction induced by phorbol esters. This early compaction response to PMA exposure displayed a clear concentration dependence with concentrations as low as 1 rig/ml having the smallest detectable effect (Fig. 2a). Since phorbol esters applied for long periods of time are known to cause down-regulation of PKC (Nishizuka, 1984), we tested the possibility that the decompaction which we observed might have been due to this down-regulation. Our results indicated that after 1 hr of exposure to PMA cells began to decompact; therefore, we also measured the compaction of four-cell stage embryos in response to a 2-min pulse of PMA (10 rig/ml). After a 1-hr interval (to allow time for the decompaction phase to begin), we placed these embryos back into 10 rig/ml PMA. If exposure to PMA could activate PKC, then after an hour the PKC might have begun to down-regulate and reexposure to PKC should have a reduced effect. These embryos were still able to compact at the same rate as those exposed initially to the PMA, although they never reached the same level of overall compaction as embryos exposed to PMA continuously (Fig. 2a). This suggests that the PMA pulse may have caused only a fraction of the PKC to down-regulate so that the remaining PKC was then available to respond to the subsequent addition of PMA. We were concerned that some of the PMA would dissolve into the paraffin oil overlying the microdrops of

4

DEVELOPMENTALBIOLOGY

Controls

15 min

30 min

VOLUMF,138,1990

WINKEL

ET AL.

Activation

media, thereby reducing the aqueous concentration of PMA. Therefore we repeated our dose-response experiments in humidified chambers without paraffin oil. Under these conditions PMA appeared to cause maximum compaction at concentrations near 1 rig/ml. However, at concentrations above 5 rig/ml the overall level of compaction was reduced (Figs. 2b and 2~). This appeared to be due to decompaction of cells overriding the effect of compaction. At concentrations below 1 rig/ml, cells still responded to PMA; however, compaction was delayed and the overall level of compaction was reduced. However, in spite of the reduced effect upon compaction, these cells eventually began to decompact like embryos treated at the higher doses. Sphingosine

Inhibits

Compaction Induced by PMA

These results suggested that PKC activation can stimulate premature compaction. Therefore, we examined whether a specific antagonist of PKC would block this effect. Of the currently known inhibitors of PKC, sphingosine is one of the more specific, competes for the phorbol ester binding site on PKC, and may be used by the cell to modulate PKC (Hannun et ah, 1986). Therefore if phorbol ester were causing its effect upon compaction through activation of PKC, an antagonist such as sphingosine should block this effect. Our results show that at concentrations as low as 2 PCLM (under paraffin oil), sphingosine significantly decreased both the overall level of compaction and the rate of compaction induced by PMA (10 rig/ml; Fig. 3a). At 20 PLMsphingosine, compaction was completely inhibited, suggesting that the phorbol ester was unable to compete effectively with this high dose of sphingosine for the PKC active site. When these experiments were repeated without paraffin oil, the effect varied depending upon the initial concentration of PMA. When the initial concentration of PMA was 1 rig/ml, increasing concentrations of sphingosine decreased compaction in a dose-dependent manner (Fig. 3b). This is the expected result if sphingosine were competing with PMA for the active site on PKC. However at the higher dose of 10 rig/ml PMA, increasing concentrations of sphingosine had the opposite effect, causing increasing levels of compaction, also in a dose-dependent manner (Fig. 3~). At 30 min, 18% of

5

of Protein Kinase C

the PMA-treated embryos were compacted, compared to 26, 38, and 40% of the embryos with 1, 2, and 5 piIf sphingosine, respectively. Since PMA at this higher dose causes only a brief period of compaction followed by decompaction (Figs. 2c and 3c), sphingosine, by inhibiting the activation of PKC, extended the compaction phase causing increased levels of compaction. Therefore sphingosine probably acts to lower the effective concentration of PMA, reducing the decompaction effect such that an increased level of compaction was observed. Sphingosine Eight-Cell

Inhibits

Compaction of Normal

Embryos

Our results indicated that PKC activation could induce premature compaction, so we investigated whether PKC might also play a role in mediating normal compaction of eight-cell mouse embryos. To test this possibility, we allowed four-cell embryos to develop in the presence of increasing concentrations of sphingosine. Under our culture conditions, most of our control embryos compacted between 16 and 21 hr after being flushed from oviducts. Prior to this time the majority of the embryos, both control and sphingosine-treated, were not compacted. As can be seen from Table 1 and Fig. 4, sphingosine was effective in inhibiting normal compaction at very low concentrations. After 16 hr of culture at 2.5 $If, only 3% of these embryos were compacted compared to 63% of controls. Five hours later (21 hr of culture) just 10% were compacted while control embryos were 78% compacted. The cell number of sphingosine-treated embryos was also less than that of controls after 21 hr of culture (See Fig. 4d). At the higher concentrations of 5 and 10 &I, sphingosine was toxic to most of the embryos. We observed a general failure of development with unequal and fewer cellular divisions (Figs. 4c and 4d). After 21 hr many of the blastomeres of embryos treated with 5 and 10 PLM sphingosine had lysed (Table 1). However, at 2.5 p*Mand when cultured with paraffin oil (which diluted the effective concentration of sphingosine), embryos continued to develop, although they failed to compact at the same time as controls. Sphingosine at 2.5 PMalso inhibited blastulation of embryos after 41 hr of culture with

FIG. 1. Effect of PMA and diacylglycerides on compaction. (a) Typical four-cell embryo prior to treatment. After 15 min in either diCx (c) or PMA (d) the majority of embryos are compacted, although those in diCa are more compacted than embryos in PMA. After 30 min, diC,-treated embryos (e) are fully compacted with completely indistinct cell outlines, while PMA-treated embryos (f), although still compacted, are not as compacted as those embryos treated with diCs. DiCa-treated embryos (g) are still compact after 1.5 hr, while those treated with phorbol ester (h) are already completely decompacted even beyond the endogenous level of compaction in controls (a). By 18 hr on the following day, control embryos (b) are at the morula stage. By contrast, PMA-treated embryos (j) are still at the four-cell stage, while diC,-treated embryos (i) resemble morula although cell number is reduced and there are several decompacted blastomeres. Bar (f) = 50 PM.

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DEVELOPMENTAL BIOLOGY

a

PMA

in microdrops

under

VOLUME 138,199O

pnrnffln

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PMA 10 W&al (n=20) PMA 5 @ml (23) PM‘4 lnghnl(22) PMA (2 mia Puke) 10 @ml added@ 1 hr (24)

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FIG. 2. Phorbol ester causes an increase in compaction followed by decompaction when used (a) in microdrops under paraffin oil at 1 to 10 rig/ml, (b) in a humidified chamber without paraffin oil at 1 rig/ml to 250 pg/ml, and (c) in a humidified chamber without paraffin oil at 2 to 10 rig/ml. In (a) (small triangles) PMA was applied for a 2-min interval, PMA was washed out and these embryos were cultured for 1 hr in T6 media. They were then reincubated in 10 rig/ml PMA to study the effects of PKC down-regulation on compaction. Note that at the highest concentration of PMA (10 rig/ml, c) PMA had the least effect on compaction as decompaction of cells was overiding the effect of compaction (see text). At the lowest concentration of PMA, 250 to 500 pg/ml, compaction was greatly retarded; however, decompaction still followed after the peak of compaction. These graphs are from one individual experiment. Each experimental run was repeated three times.

WINKEL

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Activation

ET AL.

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(10 rig/ml, ~22) + Sph 1 /A4 (23) + Sph 2pM (2d) + 5uM Sph (34)

20% 10% 0% 0

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FIG. 3. Effect of sphingosine on compaction induced by PMA. (a) Embryos were cultured in microdrops under paraffin oil which diluted the effects of both PMA and sphingosine (see Fig. 2a). Increasing concentrations of sphingosine decreased compaction in a dose-dependent manner. (b) When PMA was used in low concentration (1 rig/ml) in a humidified chamber without paraffin oil, sphingosine decreased compaction in a dose-dependent manner as above. (c) However, when PMA was used at high concentration (10 rig/ml) without paraffin oil, sphingosine increased compaction in a dose-dependent manner (see text). These graphs represent an individual experiment. Each run was repeated twice.

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only 19% of embryos forming blastocysts compared with controls (81%). Finally we examined whether sphingosine or PMA would decompact embryos which were already compacted. If PKC were instrumental in maintaining compaction, then inhibition of PKC might cause compacted morula to decompact. We placed compacted morula (n = 40) in 5 PLMsphingosine or 10 rig/ml PMA (n = 41) and scored the level of compaction for 3 hr. Compacted morula were 72% compacted at the outset using our scoring system. Within the first 30 min, PMA-treated embryos decompacted to 23% and by 3 hr these embryos were almost fully decompacted. In contrast, treatment with the PKC inhibitor, sphingosine, had no effect until 2 hr after treatment, when compaction was reduced to 55% and fell to 38% by 3 hr. This result indicates that inhibition of PMA with sphingosine or down-regulation induced by high doses of PKC can cause compacted morula to decompact, although PMA is more effective than sphingosine at these dosages. Diacylglyceride

(hr)

in humidified

of Protein

Treatment

Prolongs Compaction

PKC activators such as phorbol esters often cause nonphysiological effects on cells because they intercalate into the cell membrane and are metabolized very slowly (Nishizuka, 1984). PKC is physiologically activated via DAG, which is then rapidly metabolized causing only a transient activation of PKC. Therefore, we examined the effect of more natural activators of PKC on compaction. DiCs and OAG are two of the most effective lipid activators of PKC. When embryos were incubated in either reagent, the effects were strikingly different from PMA (Fig. 5). The whole population of diCs- and OAG-treated embryos became almost 100% compacted with every trial, whereas PMA-treated embryos never reached 100% compaction since some embryos had already begun to decompact within the first 30 min. The decompaction phase observed with PMAtreated embryos did not occur with diacylglyceride treatment. All of the embryos appeared to become completely compacted presumably because diacylglyceride treatment activated PKC without the down-regulation observed with phorbol ester. Compaction induced by these reagents was also qualitatively different from compaction induced with PMA. Although PMA-treated embryos became “fully compacted,” the location of the cell margins could still be readily determined (Fig. Id). However, diacylglyceride-treated embryos became so fully compacted that their cell margins were completely obscured, such that each four-cell embryo resembled a single cell (Figs. lc and le). This suggests that the diacylglycerides, which resemble the endogenous activators

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FIG. 4. Effect of sphingosine on normal compaction. Four-cell-stage embryos were cultured in sphingosine and scored after 16 hr in (a) control media, (b) 2.5 pM, (c) 5 FM, and (d) 10 pM sphingosine. While control embryos are primarily compacted morula, at every concentration sphingosine-treated embryos show few signs of compaction. At the highest concentrations (5 and 10 p&f), the cell numbers are reduced and some blastomeres appear irregular. After 21 hr of exposure, controls (e) are still compacted while 2.5 pM embryos (f) remain uncompacted. At 41 hr of treatment, control embryos (g) have already cavitated and formed blastocysts, while 2.5 pM embryos (h) have irregularly sized cells and very few embryos have begun to cavitate. Bar (b) = 50 pM.

of PKC, are more effective than phorbol esters in stimulating compaction of the four-cell mouse embryo. Since diacylglyceride-treatment resulted in sustained compaction, we investigated whether continuous acti-

vation of PKC was necessary during this compaction phase. The PKC inhibitor, sphingosine, competes with the diacylglycerides for their PKC binding site, and if continuous activation of PKC is necessary, sphingosine

WINKEL ET AL.

Activatkm

TABLE 1 EFPECT OF SPHINGOSINE ON COMI~ACTION OF EIGHT-CELL STAGE MOIJSE EMBRYOS 7%Population compacted” (hr in culture) Treatment Control Sphingosine, Sphingosine, Sphingosine, Sphingosine,

2.5 &f 5 KM 10 PM 10 &”

No. embryos 30 40 40 40 19

0

16

21

41

0 0 0 0 0

63 3 0 0 23

78 10 1 0 30

19 35 15b Oh 62

‘6 Blastocysts 81 19 0 0 42

Note. Early four-cell embryos were cultured in Ts media containing the indicated dose of sphingosine and scored for percentage of cells compacted and percentage of blastocysts after 41 hr of culture. “Percentages were calculated as described under Materials and Methods. “Sphingosine at 5 and 10 pM appeared to be toxic to mouse embryos. After 21 hr of culture 527% of 5 PM and 1OO’X of 10 PM embryos had lysed. ’ This group of embryos was cultured in microdrops under paraffin oil which substantially diluted the effects of sphingosine.

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Diacylglyceride-treated embryos after 18 hr of culture in either OAG or diCs were reduced in cell number compared to controls; however, they did divide more consistently than PMA-treated embryos. The average cell number after 21 hr of culture in diCs was 7.8 L 0.3 (n = 16) compared to controls with an average cell number of 11.8 k 0.6 (n = 23). PMA-treated embryos (1 rig/ml) averaged only 4.5 -t 0.2 (n = 30) cells per embryo. Further development of diacylglyceride-treated embryos was dependent upon the concentration of either OAG or diCx. At concentrations above 100 p&I, cell division was slowed and embryos neither compacted nor cavitated further. However at lower concentrations (50 PM or less), cell division was delayed, although these embryos still compacted and eventually formed blastocysts. Antibody to E-Cadherin PKC Activators

Completely Blocks the Eflect of

Compaction in the mouse embryo appears to employ two separate cell adhesion mechanisms. The earliest events of cell adhesion are mediated via the Ca’+-dependent cell adhesion system or CDS (Takeichi, 1987, 1988). The CDS acts during the early eight-cell stage causing compaction of the embryo. Subsequently, durshould cause a decrease in the observed compaction ing the late eight-cell stage, cell adhesion involving the whenever it is applied. Therefore we added sphingosine P-1,4-galactosyltransferase receptor (GalTase) particiat hourly intervals to embryos induced to compact with pates in maintaining the compacted state (Bayna et al., 50 &If OAG. Within 15 min, a small decrease in the 1988). To differentiate between these two cell adhesion number of compacted cells was observed. After a 1-hr systems and to determine whether the compaction inexposure to sphingosine, each population of cells was 50 to 80% less compacted (Fig. 6). This suggests that con- duced by PKC activators might be mediated through tinuous activation of PKC is necessary to sustain com- another mechanism, we utilized a monoclonal antibody paction of four-cell-stage mouse embryos. L(mg-Term Eflect of PKC Activators

on Development

Treatment of four-cell stage embryos with PMA initially caused a rapid compaction followed by a decompaction phase. By the following day, a majority of embryos were still at the four-cell stage. Mystkowska and Sawicki (1987) have shown that treatment with PMA prevents cytokinesis, but not mitosis resulting in binucleate embryos. However PMA (10 rig/ml) used with paraffin oil yielded only a few binucleate embryos (3/18). After 41 hr of treatment, control embryos had an average cell number of 45.8 & 11 (n = 18) while PMAtreated had an average cell number of 22.3 ? 8 (n = 24). These values were significantly different at the 0.0001 level (t = 8.4, df = 40). By contrast almost all of those embryos cultured for 19 hr without oil in PMA (1 rig/ml), were binucleate and 22/22 had only four blastomeres. Although these embryos were deficient in cell number, they did eventually compact and cavitate forming false blastocysts (blastocysts without an inner cell mass, due to reduced cell number).

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FIG. 5. Effect of the diacylglyceride, 1-oleoyl-2-acetyl-sn-glycerol (OAG) on compaction. At 100 pM concentration, embryos were fully compacted within 1 hr and remained mostly compacted throughout the experiment. Decreasing concentrations of OAG caused a reduced effect on compaction. DiCx responded similarly to OAG (data not shown). Data shown represent one trial and this dose-response study was repeated twice with both OAG and diC,.

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FIG. 6. Effect of addition of sphingosine on compaction induced by OAG when applied at hourly intervals. Three groups of embryos were cultured in 50 KM OAG and scored for extent of compaction. After 1 hr of culture, one group of embryos was transferred to OAG containing 5 pM sphingosine. After another hour of culture the second group of embryos was placed in medium with sphingosine. Each group began to decompact within 15 min of exposure to sphingosine. After 1 hr of sphingosine treatment, the extent of compaction was markedly reduced by 50 to 80%, suggesting that continuous activation of PKC appears necessary to sustain compaction of four-cell embryos. These data represent a single trial of this experiment.

(ECCD-1) specific to E-cadherin, the cell adhesion molecule which mediates CDS. In every experiment in which we added ECCD-1 (1:200), compaction induced by PMA, diCs, or OAG was completely blocked, provided embryos were added to media containing ECCD-1 at least 15 min before the addition of any PKC activators (data not shown). Embryos treated with ECCD-1 failed to compact and furthermore completely decompacted such that they resembled decompacted PMA-treated embryos after 4 hr in PMA (Fig. lh). This suggests that the compaction induced by activators of PKC is not a result of nonspecific effects on the cell or cell membrane and implies therefore that PKC either directly or indirectly activates the CDS.

ized to small punctate regions at the contact point between the cells (Fig. ‘7b). The distribution of the cell adhesion molecules was quite different when diCs was used to induce compaction. In these embryos, as with PMA-treated embryos, the fluorescence on the outermost surfaces was dramatically reduced; however, the fluorescence was localized at the outermost portion of the junction between blastomeres (Fig. 7~). When we examined the antibody distribution of PMA-treated embryos at 30 min, the time of maximum compaction, we always observed a punctate region of fluorescence localized at the point of contact between blastomeres; however, it was broader and more intense than at 2.5 hr. DISCUSSION

We have shown that PKC activation results in premature compaction of the four-cell-stage mouse emCompaction induced by PKC activators occurred so bryo. These results are in agreement with a preliminary rapidly that we sought to determine the localization of report which showed that phorbol esters stimulated E-cadherin after activation of PKC. Therefore we compaction of four- as well as two-cell-stage embryos treated embryos with PMA and scored the distribution (Yamamura et al., 1987). However, our results varied of E-cadherin with immunofluorescence both before depending upon the reagent used to activate PKC: and after activation of PKC. For immunofluorescent Phorbol ester caused a biphasic response, resulting in a localization we used ECCD-2, a monoclonal antibody phase of rapid compaction followed by a prolonged desimilar to ECCD-1, which has a greater affinity for the compaction. In contrast, the analogs of DAG, OAG, and E-cadherin molecule but does not induce blastomere diCs stimulated only the compaction phase. It is known decompaction. Prior to induction of compaction, E-cad- that activation of PKC by phorbol esters is enhanced herin was uniformly localized around the cell mem- compared to activation via diacylglycerides since phorbranes of the blastomeres of the four-cell embryo with bol esters are not metabolized by the cell, which causes some concentration between the blastomeres (Fig. 7a). a prolonged activation of PKC. This chronic activation After a 2.5-hr treatment of embryos with PMA (when of PKC effectively extends a limited phase of a cellular most of the treated embryos would be decompacted) response which may distort the normal sequence of there was a dramatic decrease in overall fluorescence. events (Nishizuka, 1986). Since diacylglycerides are rapE-cadherin was no longer present along the outer idly metabolized, the transient activation of PKC is membranes of the blastomeres but instead was local- preserved and a more physiological cellular response is Localization

of E-Cadherin

during Induced Compaction

11

FIG. 7. Localization of E-cadherin with monoclonal antibody ECCD-2 after treatment with phorbol ester (10 rig/ml) and diCs (100 FM). (a) Fluorescent-labeled E-cadherin is localized uniformly around the blastomeres of the untreated four-cell embryo. Control embryos were unstained when primary or secondary antibody was omitted. (b) PMA-treated embryo after 2.5 hr. Embryo is completely decompacted and E-cadherin is localized only in a discrete region at the point of blastomere contact. (c) DiCs-treated embryo at 30 min of exposure. Embryo was fully compacted; however, treatment with the monoclonal antibody ECCD-2 causes mild decompaction. E-cadherin is localized near the outer junction of blastomere contact. Bar = 50 PLM.

observed. Chronic activation of PKC with phorbol esters may lead to either rapid down-regulation of PKC (Grove and Mastro, 1988; Ito et ab, 1988) or possibly activation of a negative feedback loop (Connolly et al., 1986; Woods et al., 1987; Nishizuka, 1988) which inhibits PKC. Grove and Mastro (1988) have reported that in primary lymphocytes treated with PMA, there was a 45% decrease in PKC activity within 30 min followed by a 70 to 80% decrease after 1 hr. The beginning of the phase of decompaction reported here correlates well with this time course of PKC down-regulation. When PKC is activated it becomes a target for Ca’+-specific proteases, known as calpains (Kishimoto et ah, 1989), which cleave PKC so that it is no longer available for activation. This proteolysis of the active fragment may be the mechanism behind the observed down-regulation. Alternatively, chronic activation of PKC by phorbol esters may continuously activate a negative feedback loop thereby inhibiting further activation of PKC. Although our results do not specifically favor one hypothesis over the other, the fact that sphingosine, a potent inhibitor of PKC (which does not cause downregulation of PKC but only blocks activation), is unable to decompact morula within the first hour suggests that down-regulation of PKC may be responsible for the observed decompaction. It has been suggested that sphingosine and lysosphingolipids may function as endogenous regulators of PKC (Hannun et al., 1986; Hannun and Bell, 1989). Therefore we used this lipid as a PKC inhibitor in our experiments. We found that sphingosine at low concentrations caused apparent inhibition of compaction induced by diacylglycerides. However sphingosine had varying effects upon phorbol ester-induced compaction. When PMA was administered at a dose that maximized compaction (1 rig/ml), sphingosine caused an inhibition of compaction. However, when PMA was used at a higher dose (10 rig/ml), sphingosine caused an increase in compaction. Our results indicate that higher doses of PMA cause only a brief period of compaction (Fig. 2c),

probably by chronic activation and down-regulation of PKC. Therefore, sphingosine, by antagonizing the effect of PMA, probably reduced the initial activation and subsequent down-regulation of PKC such that compaction was enhaneed over decompaction. Sphingosine was also able to prevent compaction of four-cell embryos cultured overnight. Although we observed some toxic effects at higher concentrations (10 PM), lower concentrations (2.5 p&f) caused no observable toxicity yet still prevented compaction. Likewise, sphingosine also caused compacted eight-cell stage embryos to decompact; however, this effect was not apparent until after 2 hr. These results suggest that sphingosine is acting specifically as an inhibitor of PKC. However, sphingosine has also been reported to inhibit calcium- and calmodulin-dependent myosin light chain kinases (Hannun et ah, 1986; Jefferson and Schulman, 1988) when used at high dosages. Recent evidence that calmodulin antagonists also block compaction (Yamamura and Spindle, 1988) suggests that sphingosine may act by inhibition of calmodulin-dependent kinases. However, if the observed effects were due to inhibition of these kinases, then we would not expect sphingosine to enhance compaction when used to inhibit PMA-induced compaction (Fig. 3~). Furthermore sphingosine has been shown to inhibit calmodulin-dependent kinases and myosin light chain kinases at doses (lo-100 pM) significantly higher than those we used (Jefferson and Schulman, 1988). Therefore, we interpret this to indicate that inhibition of these kinases is a minor contribution, which suggests that sphingosine specifically inhibits PKC. Compaction in the mouse embryo is mediated by the cell adhesion molecule E-cadherin (also known as uvomorulin). This adhesion molecule is part of a family of related cell adhesion molecules which are expressed differentially in tissues during development (Takeichi, 1987) and therefore may play a role in tissue sorting (Nose et al., 1988) and axonal guidance (Matsunaga et al., 1988). During mouse preimplantation development,

12

DEVELOPMENTALBIOLOGY

E-cadherin is synthesized at the two-cell stage coincident with activation of the embryonic genome (Vestweber et al., 1987). It is distributed uniformly over the blastomeres of the two- and four-cell embryos with increased concentration between the blastomeres of the four-cell embryo. During compaction of the eight-cell embryo, E-cadherin becomes restricted to the cell membranes between adjacent cells. Blockage of E-cadherin with a monoclonal antibody has been shown to alter the timing and orientation of polarization of mouse blastomeres (Shirayoshi et al., 1983; Johnson et aZ.,1986). We have shown that activators of PKC cause compaction to occur prematurely at the four-cell stage and that the localization of E-cadherin is identical to that observed during normal compaction. This suggests that we have activated a component of the normal compaction sequence. However, in several cases (especially with PMA treatment), we observed embryos that were fully decompacted while the E-cadherin was still localized within the cell junction (Fig. 7b). This suggests that the process of cell adhesion may involve both localization of the E-cadherin and a change in the relative adhesiveness of the molecule. Activation of PKC apparently causes both events to occur since we observed simultaneous localization and compaction. However, the mechanism through which this is accomplished is not clear. The localization of E-cadherin could be mediated either directly or indirectly via actin microfilaments. Since the distribution of E-cadherin closely coincides with the localization of actin (Hirano et al, 1987), it is possible that activation of PKC also affects the distribution of actin. A recent report indicates that activation of PKC causes stage-specific effects upon actin microfilaments (Bloom, 1989). Furthermore the observation that the activation of PKC has been shown to increase the interaction of integral transmembrane proteins (integrins) with the cytoplasmic actin-linking protein talin (Burn et al., 1988) indicates a potential mechanism for the localization of integrins. Similar mechanisms which incorporate these or other actinlinking proteins may function in the localization of Ecadherin and other cell adhesion molecules. The relative binding properties of E-cadherin might also be altered through activation of PKC. Danilov and Juliano (1989) showed that PKC activation increased the adhesion of tissue culture cells to fibronectin. Furthermore, recent evidence by Mackie et al. (1989) indicates that the neural cell adhesion molecule, N-CAM, is phosphorylated by two “independent” kinases which may alter the binding properties of N-CAM. Similarly, PKC might directly phosphorylate E-cadherin thereby either increasing its binding to actin-binding proteins or altering its adhesiveness to adjacent cadherins. Recent evidence indicating that PKC activation increases

V0~~~~138,1990

phosphorylation of the c-erbA encoded thyroid hormone receptor (Goldberg et aZ., 1988) and that phosphorylation of other transmembrane receptors modify their function (Sibley et al, 1987) suggests that a similar form of regulation of E-cadherin may be possible. Therefore the phosphorylation of specific proteins may be part of the underlying mechanism which results in increased cell adhesion. Our results complement the hypothesis suggested by Levy et al. (1986) that compaction is triggered by a post-translation initiation signal. We are proposing that activation of PKC is the initiation signal or an integral part of the signal that leads to inhibition of a “putative compaction-restraining factor” (Levy et al, 1986). However, the mechanism underlying PKC activation in the eight-cell embryo is not clearly understood. PKC could be activated via three potential mechanisms: A receptor-ligand interaction on the cell surface which activates PI metabolism; activation of the PI cycle via cyclic events during early cleavage divisions; or through modulation of other lipid components in the cell membrane. PKC is frequently activated by receptor-mediated activation of the phosphatidylinositol cycle. Generally this begins with the activation of a G-protein which stimulates phopholipase C to cleave PIP2 into Ins(1,4,5)P, and DAG. Ins(1,4,5)P, (and some of its metabolites) raise intracellular calcium levels, while DAG transiently activates PKC (Berridge, 1987). However there is currently no evidence that a receptor-ligand interaction in the mouse preimplantation embryo may stimulate the PI cycle and mouse compaction. A recent report by Schuch et al. (1989) suggests that neural cell adhesion molecules may be involved in activation of the PI cycle. Whether E-cadherin may participate in a similar interaction remains to be determined, although this may be a mechanism which stimulates the PI cycle and would lead to activation of PKC. Alternatively, the PI cycle might be triggered by cyclic events as is the regulation of cell division. During cell division, the levels of cyclins and maturation-promoting factor (MPF) rise and fall at each cell cycle (Murray and Kirschner, 1989; Murray et al., 1989) without apparent involvement of a receptor-ligand interaction. Recent evidence has shown that in organisms as diverse as sea urchins (Forer and Sillers, 1987; Twigg et al., 1988), Xenopus Zaevis(Han et al., 1988), yeast (Uno et al, 1988), and mouse (Izquierdo and Becker, 1982) inhibition of the PI cycle suspends or delays cell division. This evidence suggests that the PI cycle may be activated in a cyclical fashion during cell division, perhaps stimulated by the alternating levels of cyclins or MPF. Since compaction begins soon after the third cleavage division, then activation of the PI cycle due to changes in the levels of cyclins during mitosis may be part of the

WINKEL ET AL.

Activation

signal that initiates compaction. Protein synthesis inhibitors will inhibit the reappearance of cyclins if applied immediately after mitosis (Evans et ah, 1983; Twigg et ah, 1988). Likewise protein synthesis inhibitors prevent formation of a putative compaction-inhibiting factor in mouse embryos if applied soon after the second cleavage division (Levy et al., 1986). Therefore activation of phosphatidylinositol metabolism through cyclic events may be responsible for activation of PKC which would then act to stimulate mouse embryo compaction. PKC might also be regulated by modulating lipid components of the membrane which are not created through PI metabolism. Our results indicate that sphingolipids exert an inhibitory effect upon PKC and therefore upon embryonic compaction. It has also been reported that sphingosine (Hannun et al., 1986) and lysosphingolipids (Hannun and Bell, 1989) inhibit PKC activity in other systems. This suggests that these membrane metabolites may function as negative effectors for cellular processes involving PKC. Thus if these sphingolipids were maintained at an elevated level, compaction would be inhibited. During preimplantation development, changes in the expression of gangliosides on the cell surface may be accompanied by corresponding intracellular changes in the levels of sphingolipids, hence providing a mechanism to regulate the onset of compaction. Conversely, unsaturated fatty acids (such as oleic and arachidonic acid) have also been shown to activate PKC either in the presence or absence of Ca2+ or phospholipid (McPhail et ah, 1984; Rando, 1988) and certain lysophospholipids can biphasically regulate PKC (Oishi et ab, 1988). Since the amount of phosphatidylcholine in the membrane is much greater than that of phosphatidylinositol, metabolism of PC via activation of phospholipase C could theoretically generate more DAG than from PI metabolism (Irving and Exton, 1987, Blackshear et al, 1988; Pelech and Vance, 1989). Therefore it is possible that PKC and, subsequently, compaction might be regulated by other membrane lipid metabolites. After this paper was submitted for publication, an apparently contradictory report concluded that the activation of PKC inhibits compaction of mouse blastomeres (Bloom, 1989). However this apparent contradiction with our results can be resolved by comparing the concentration of phorbol ester applied. When we utilized the phorbol ester, PMA, at a high concentration (10 PALM, without an oil overlay), we observed a brief phase of compaction followed by decompaction. Bloom typically used concentrations of PMA 25 PM or greater. Although we did not use concentrations of this magnitude, on the basis of our dose-response studies, we would expect that this concentration would further reduce the initial phase of compaction. However, even if

of Protein

Kinase

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C

compaction had occurred under these conditions, Bloom would not have observed the early compaction phase since she scored compaction at 1-hr intervals. We found it necessary to score compaction at more frequent intervals (15 min) due to the rapid biphasic effects of phorbol ester treatment. Therefore our results are consistent with those of Bloom, since our concentration of phorbol was considerably lower, thereby allowing us to observe the biphasic effect of PMA treatment. Furthermore, Bloom reported that PMA caused a depolymerization of actin in the cytocortex. Since Hirano et al. (1987) have shown that actin bundles colocalize with E-cadherin, this suggests that actin microfilaments are involved in the maintenance of compaction. Therefore the decompaction which Bloom observed is probably due to a rapid down-regulation of PKC induced by high doses of phorbol esters, which disrupt the cytoskeleton and lead to decompaction. We have shown that activation of PKC stimulates compaction of the four-cell mouse embryo. Likewise, inhibition of PKC prevents compaction induced by PKC activators as well as normal compaction of eight-cell embryos. This suggests that PKC plays an influential role in compaction probably through its effects upon the cytoskeleton and E-cadherin. This involvement of PKC in compaction indicates that compaction and probably polarization in the mouse embryo involve a complex cascade of post-translational events. In particular, the growing evidence for a multitude of protein kinases utilized in cell regulation (Hunter, 1987) suggests that these protein kinases play a major role during mammalian embryogenesis. The authors thank Jin-Kwan Han for technical and methodological assistance. Also, thanks to Carolyn Larabell for editorial comments. This work was supported by NIH Grant HD 22594 to R.N., G.W. was supported in part by NIH Reproductive Biology Training grants HD 07131 and HD 22594, and J.F. was supported in part by NIH Training Grant GM 07377. REFERENCES BAYNA, E. M., SIIAPER, J. II., and SHTJR, B. D. (1988). Temporally specific involvement of cell surface p-1,4-galactosyltransferase during mouse embryo morula compaction. Cell 53,145-157. BERRIDGE, M. J. (1987). Inositol trisphosphate and diacylglycerol: Two interacting second messengers. Annu. Rev. B&hem. 56,159-193. BLACKSHEAR, P. J., NAIRN, A. C., and Kuo, J. F. (1988). Protein kinases 1988: A current perspective. FASEB J. 2,2957-2969. BLOOM, T. L. (1989). The effects of phorbol ester on mouse blastomeres: A role for protein kinase C in compaction? Development 106, 159-171. BORNSLAEGER, E. A., POLJEYMIROU, W. T., MATTEI, P., and SCHIJLTZ, R. M. (1986). Effect of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes. Exp. Cell Res. 165, 507-517. BURN, P., KUPFER, A., and SINGER, S. J. (1988). Dynamic membranecytoskeletal interactions: Specific association of integrin and talin arises in viva after phorbol ester treatment of peripheral blood lymphocytes. Proc. Natl. Acad. Ski. USA 85,497-501.

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