Stimulatory effect of okadaic acid, an inhibitor of protein phosphatases, on nuclear envelope breakdown and protein phosphorylation in mouse oocytes and one-cell embryos

DEVELOPMENTAL

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(1991)

Stimulatory Effect of Okadaic Acid, an Inhibitor of Protein Phosphatases, on Nuclear Envelope Breakdown and Protein Phosphorylation in Mouse Oocytes and One-Cell Embryos DANIELA.SCHWARTZANDRICHARD

M.SCHULTZ

Treatment of one-cell mouse embryos with okadaic acid (OA), which is an inhibitor of protein phosphatases 1 and ZA, induces a concentration-dependent precocious nuclear envelope breakdown (NEBD) of the pronuclei; at 10 ~Mokadaic acid, NEBD starts to occur after 1 hr and the embryos become committed to NEBD after about 45 min. Correlated with NEBD is the conversion of a protein of M, 32,000 (~32) to more highly phosphorylated forms. One-cell embryos cultured continuously in OA-containing medium do not cleave, whereas one-cell embryos incubated for 15-60 min prior to transfer to OA-free medium reveal a time-dependent inhibition in their ability to cleave. OA treatment of oocytes that are arrested from resuming spontaneous maturation by either a phosphodiesterase inhibitor or biologically active phorbol diester results in germinal vesicle breakdown and the maturation-associated changes in the pattern of protein phosphorylation, which include the apparent phosphorylation of ~32. Results of these experiments implicate protein phosphatases in the G2 to M transition of the cell cycle in both meiotic and mitotic cells. ‘cm1991 Academic press, I~C. INTRODIJCTION

Protein phosphorylation and dephosphorylation are involved in meiotic maturation of mouse oocytes; oocyte maturation entails a G2 to M transition in the cell cycle (Schultz, 1986 and references therein). For example, activation of either a CAMP-dependent protein kinase PKA (Bornslaeger et al., 1986a) or calcium, phospholipid-dependent protein kinase, PKC, inhibits oocyte maturation (Bornslaeger et ab, 1986b; Urner and Schoderet-Slatkine, 1984), and the dephosphorylation and phosphorylation of specific phosphoproteins occurs during a period of time in which the oocytes become committed to resume meiosis (Bornslaeger et al., 1986a). Specific changes in the pattern of protein phosphorylation occur during the first cell cycle (Besterman and Schultz, 1990; Howlett and Bolton, 1985; Howlett, 1986), and in particular, a set of proteins of h!, 32,000 (~32) becomes phosphorylated as the one-cell embryos enter M phase (Howlett and Bolton, 1985; Howlett, 1986). These results from studies of oocyte maturation and the first cell cycle are consistent with the role of protein phosphorylation and dephosphorylation in regulating the cell cycle (Lewin, 1990, and references therein). Although the emphasis of these previous studies has been on protein phosphorylation and the kinases that may be involved, little attention has been paid to the role of protein phosphatases (PP) in these early developmental processes. This has been due in large part to the lack of specific inhibitors of PP. In contrast to the plethora of protein kinases, the number of PPs seems to be 119

much smaller. The serine/threonine PPs fall essentially into four types: PPl (ATP-Mg’+-dependent) and PPBA (polycation-stimulated), which are structurally related to each other; PPBB, which is a calcium/calmodulin-dependent PP; and PPBC, which is a Mg2+-dependent PP (Cohen, 1989, and references therein). In addition, there are PP that catalyze the removal of phosphate from phosphotyrosine (Tonks and Charbonneau, 1989, and references therein). Okadaic acid (OA)-a polyether monocarboxylic acid synthesized by several types of dinoflagellates, concentrated in marine sponges, and responsible for diarrhetic shellfish poisoning-has recently been shown to be a very specific inhibitor of PPl and PPZA; the IC, of OA for PPl and PPBA is -0.1 and 10 nM, respectively (Bialojan and Takai, 1988; Cohen et al, 1990, and references therein). The IC,, for PPBB is -5 PM, and PPBC is essentially not inhibited by OA (Bialojan and Takai, 1988; Cohen et al., 1990). OA has proved to be a very useful reagent to study the role of protein phosphatases in oocyte maturation in lower species. OA induces hormone-independent maturation of both Xenopus (Goris et al., 1989) and starfish oocytes (Picard et al., 1989; Pondaven et al., 1989), and correlated with germinal vesicle breakdown (GVBD) is the appearance of maturation promoting factor (MPF) activity (Goris et al., 1989; Picard et al., 1989; Pondaven et al, 1989; Rime et aL, 1990). The OA-induced increase in the activity of MPF, which is a protein kinase composed of one molecule of a homologue of the yeast cdc2 mitotic protein kinase and a molecule of a cyclin (Labbe 001%1606191 Copyright All rights

$3.00

oh 1991 by Academic Press, Inc. of reproduction in any form reserved.

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et al., 1989a) and whose activity rises as cells enter mitosis and then declines as cells exit from M phase, is likely to be indirect, since correlated with the increase in MPF activity is dephosphorylation of the cdc2 subunit (Gautier et ah, 1989; Labbi! et al., 1989b). We report here that incubation of one-cell mouse embryos in medium containing OA induces a precocious nuclear envelope breakdown (NEBD) of the pronuclei in one-cell mouse embryos. Correlated with NEBD is the increased phosphorylation of p32 prior to NEBD. A 45min incubation in medium containing OA prior to transfer to OA-free medium is sufficient to induce NEBD in a significant fraction of the embryos. Last, meiotic arrest maintained by either 3-isobutyl-l-methyl xanthine (IBMX) or 12-0-tetradecanoyl phorbol13-acetate (TPA) is overcome with the same kinetics of maturation by addition of OA, which also induces the set of maturation-associated changes in protein phosphorylation. MATERIALS

Collection

and Culture

AND

METHODS

of Mouse Oocytes and Embryos

Fully grown, germinal vesicle (GV)-intact oocytes from preovulatory antral follicles were obtained from pregnant mare’s serum gonadotropin-primed CF-1 female mice (Harlan) and the oocytes were completely freed of attached cumulus cells as previously described (Schultz et ah, 1983). The collection medium was bicarbonate-free minimal essential medium (Earle’s salts) supplemented with pyruvate (100 pg/ml), gentamicin (10 pg/ml), polyvinylpyrrolidone (3 mg/ml), and 25 mM Hepes, pH 7.2 (MEM/PVP). To prevent GVBD, 0.2 mM IBMX was included (Schultz et ah, 1983). Oocytes were allowed to resume meiosis by culturing them at 37°C in a humidified atmosphere of 5% CO, in air in MEM/PVP in which the Hepes was replaced with 25 mh4 sodium bicarbonate and the IBMX was omitted. One-cell embryos were obtained from superovulated CF-1 female mice (Harlan) mated to B6DZFl/J males (Jackson Laboratory) as previously described (Poueymirou and Schultz, 1989). Following a brief treatment with hyaluronidase (3 mg/ml; Sigma, St. Louis, MO) in MEM/PVP, the embryos were washed through three drops of MEM/PVP and then cultured in CZB medium at 37°C in a humidified atmosphere of 5% CO, in air. CZB was chosen for the culture medium because it supports development of one-cell embryos obtained from outbred strains of mice to the blastocyst stage (Chatot et al., 1989), as well as higher levels of [35S]methionine incorporation (Poueymirou et al., 1989). Both GVBD and NEBD were scored by examining oocytes and one-cell embryos with a Wild M5A microscope at loo-fold magnification. NEBD was sometimes scored

with an Olympus CK2 microscope equipped with Hoffmann optics at ZOO-fold magnification. [“5S]Methionine and [“PlOrthophosphate Radiolabeling and Incorporaticm by Mouse Oocytes and Embryos

GV-intact oocytes were incubated in MEM/PVP plus bicarbonate containing 1 mCi/ml [35S]methionine (sp act > 1000 Ci/mmole, Amersham) or phosphate-free CZB medium containing 1 mCi/ml [32P]orthophosphate (ICN) for 2 hr as previously described (Bornslaeger et ab, 1986a). One-cell embryos were incubated in CZB containing 1 mCi/ml [35S]methionine or phosphate-free CZB medium containing 5 mCi/ml [32P]orthophosphate (ICN) for 2 hr at 37°C as previously described (Poueymirou and Schultz, 1989). Based on a ZO-hr cell cycle for the l-cell embryo (Howlett, 1986), one-cell embryos were radiolabeled in late S-phase. Following the period of radiolabeling, the oocytes or embryos were washed through six ZOO-p1 drops of bicarbonate-free MEM/PVP. The samples were then prepared for gel electrophoresis as described below. Acidsoluble and acid-insoluble radioactivity were determined as previously described (Poueymirou and Schultz, 1987). One-Dimensional

Gel Electrophwesis

One-dimensional gel electrophoresis was performed with a 4% stacking gel and a 10% separating gel in the presence of sodium dodecyl sulfate according to the method of Laemmli (1970). 35S-radiolabeled proteins were detected by fluorography (Bonner and Laskey, 1974) at -80°C using Kodak X AR5 X-ray film. 32P-labeled phosphoproteins were detected by autoradiography at -80°C using Kodak X AR5 X-ray film. Typically, 20-25 oocytes or embryos were analyzed, and except where indicated, equal amounts of acid-insoluble radioactivity were loaded onto the gels. Exposure times were usually about l-2 days. Materials

The initial source of OA used in these experiments was the generous gift of Dr. Hirota Fujiki, National Cancer Center Research Institute, Tokyo, Japan. When this source was consumed OA became commercially available from Moana Bioproducts, Inc. (Honolulu, HI). A 10 mMstock solution was made in dimethyl sulfoxide; control experiments revealed that the highest final concentration of dimethyl sulfoxide (0.1%) present in the medium following dilution of OA had no inhibitory effect on either oocyte maturation or development of onecell embryos (data not shown).

the same time that control embryos did and no pronuclei were observed in any of the OA-treated groups (data not shown). When one-cell embryos were incubated in medium containing 0.25 piV OA, 29% cleaved, 28% were fragmented, and 43% remained as one-cells that had no visible pronuclei (n = 109). In subsequent experiments, 10 PM OA was used, since this concentration resulted in a very rapid induction of NEBD. Efect of OA on Phosphorylation of p32

u0

1

2

3 Time

4

(hr)

FIG. 1. Time course and concentration dependence of OA-induced NEBD. One-cell embryos were transferred to medium containing OA and examined for NEBD as described under Materials and Methods. The experiment was performed four times and about 50 one-cell embryos were used in each case. Similar results were obtained in each experiment and the data have been pooled. (0) 0.5 j&fOA; (0) 1.0 PM OA; (A) 10 PM OA. Control one-cell embryos did not undergo NEBD during this time.

RESlJLTS

EJiect of OA on NEBD, Protein of One-Cell Embryos

Synthesis,

and Cleavage

The observation that OA can induce MPF activity, which is correlated with NEBD, in oocytes of lower species led us to examine if OA could induce precocious NEBD of pronuclei present in one-cell mouse embryos. Incubation of one-cell embryos, which were about 24 hr post hCG and thus likely to be in S phase, in increasing concentrations of OA resulted in a concentration-dependent increase in the incidence of precocious NEBD (Fig. 1). A very rapid response of the embryos to OA was that they became “crinkled,” and this change in morphology, which may be a manifestation of cytoskeletal changes induced by OA (Goris et al., 1989; Picard et al., 1989; Rime et nl., 1990; Rime and Ozon, 1990), reverted to the normal smooth appearance of the cells within an hour (data not shown). OA-treatment of one-cell embryos resulted in a rapid inhibition in protein synthesis (Fig. 2). Within 2 hr of culture in medium containing OA, the extent of incorporation of r5S]methionine into acid-insoluble radioactive material was inhibited by 90%, and this inhibition was almost 100% within a 4-hr culture period. The extent of incorporation of [35S]methionine into acid-insoluble radioactive material for control embryos was essentially constant during this period (Fig. 2). It was also observed that one-cell embryos incubated continuously in OA (0.5, 1, and 10 &V) did not cleave in

p32 resolves itself as three bands (upper, middle, and lower) following radiolabeling with [35S]methionine (Howlett and Bolton, 1985; Howlett, 1986; Besterman and Schultz, 1990), and phosphatase treatment results in detection of the species of highest electrophoretic mobility, i.e., the lower band (Howlett, 1986). p32 undergoes cell cycle changes in its state of phosphorylation during the first cell cycle, as evidenced by its conversion to the middle and upper species as the embryos enter M phase (Howlett, 1986). p32 is not the mouse cdc2 homolog, however, since its electrophoretic mobility differs from that of the mouse homolog, as detected by Western blotting using an antiserum directed towards a conserved peptide sequence of cdc2 (McConnell and Lee, 1989), and p32 present in interphase one-cell embryos does not contain phosphotyrosine (Besterman and Schultz, 1990). Thus, although p32 is not the cdc2 homolog, it may be involved in mitosis. Accordingly, one-cell embryos were treated with OA in order to induce precocious NEBD and the effect of OA on the phosphorylation state of p32 was assessed.

4000 t

”

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3-4

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FIG. 2. Effect of OA on incorporation of [%]methionine into acid-insoluble material. After 0, 1, 2, or 3 hr in CZB medium i- 10 pM OA, embryos were transferred to the appropriate medium containing [%]methionine and radiolabeled for 1 hr. The embryos were then processed for acid-insoluble radioactivity. Solid bar, control embryos; stippled bar, OA-treated embryos.

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jected to gel electrophoresis. Examination of the fluorograms indicated that during a 4-hr “chase” period, little of the more phosphorylated forms of p32 that were induced by the initial incubation in medium containing OA were converted to the less phosphorylated form, i.e., the lower band (Fig. 5). The slow extent of this reversibility may reflect the retention of OA in the embryo and thus its continued ability to maintain p32 in its more highly phosphorylated states.

1168458-

Length of Time Required

w 27-

FIG. 3. Fluorogram of 35S-radiolabeled proteins synthesized in control embryos and embryos treated with OA. Embryos were incubated in [%]methionine-containing medium (lanes 1-4) or [%]methioninecontaining medium supplemented with 10 &f OA (lanes 1’4’). After 15 min (lanes 1, l’), 30 min (lanes 2,2’), 45 min (lanes 3,3’), and 60 min (lanes 4,4’), embryos were removed and prepared for and subjected to electrophoresis; equal numbers of acid-insoluble radioactivity were applied. The experiment was performed twice and similar results were obtained; shown are the results of one experiment. The chevron points to the region where p32 migrates.

Incubation of one-cell embryos in medium containing OA resulted in a rapid conversion of p32 to the more phosphorylated species (Fig. 3); a discernable increase was observed within 15 min. It should be noted that this increase in phosphorylation of p32 occurred prior to NEBD, which started to occur between 45 and 60 min in these experiments. Incubation of one-cell embryos in medium containing OA also resulted in the apparent hyperphosphorylation of several proteins in addition to p32 (Fig. 4). In particular, OA induced the apparent hyperphosphorylation of phosphoproteins of M, -42,000, 44,000, 46,000, 70,000, 95,000, 105,000, 114,000, 140,000, 160,000, and 180,000. Reversibility

of OA-Induced

Phosphorylation

for OA to Induce NEBD

In order to determine how long of an exposure of onecell embryos to OA was required to induce NEBD, onecell embryos were incubated for increasing periods of time in medium containing 10 PM OA, then washed free of OA, cultured in OA-free medium, and scored for NEBD as a function of time (Fig. 6). A 30-min incubation had very little effect on inducing NEBD, whereas a 45-min incubation resulted in a significant fraction of the embryos that underwent NEBD; it should be noted that in these experiments at 45 min essentially none of the embryos had undergone NEBD. As previously observed, essentially all of the embryos incubated in OA for 2 hr prior to transfer to OA-free medium underwent NEBD. Thus, the commitment to undergo NEBD occurs

27-

of p32

A pulse-chase experiment was performed to ascertain the extent of conversion of p32 to the lower band following transfer of the one-cell embryos to OA-free medium. One-cell embryos were radiolabeled in medium containing 10 PM OA for 1 hr, washed free of OA, and then cultured in OA-free medium. Immediately after the period of radiolabeling and at specified periods thereafter, the same number of embryos were removed and sub-

FIG. 4. Autoradiogram of “P-labeled phosphoproteins synthesized in control embryos and embryos treated with OA. The embryos were radiolabeled for 1 hr in the presence or absence of 10 FM OA. Equal numbers of embryos were then subjected to electrophoresis. Lane 1, control embryos; lane 2, OA-treated embryos. The experiment was performed three times and similar results were obtained in each case; shown is a representative example. The chevrons point to phosphoproteins that are apparently hyperphosphorylated in the presence of OA.

123 1 MI x i 0-3

2

3

4

5

6

7

8

9

10

11

12

100

r

80

8458-

27-

Time

--v---FIG. 5. Fluorogram of %-radiolabeled proteins synthesized in control embryos and embryos treated with OA and then cultured in the absence of OA and [%]methionine. Lanes 1, 3, 5, 7, 9, and 11 correspond to untreated embryos chased for 0, 1, 2, 3,4, and 18 hr, respectively, and lanes 2,4,6,8,10, and 12 correspond to OA-treated embryos chased for 0, 1, 2, 3, 4, and 18 hr, respectively. The experiment was performed twice and similar results were obtained in each case; shown are the results of one experiment. The chevron points to the region where 1132 migrates.

sometime between 30 and 45 min and is very close to the time when NEBD was actually observed.

ReL)ersibility of OA Trea,tment on Ability Embryos to Cleave

(hr)

FIG. 6. Time course of NEBD in one-cell embryos treated for increasing periods of time with OA. One-cell embryos were incubated in medium containing 10 FMOA for 30 min (0), 45 min (a), or 60 min (A) prior to transfer to OA-free medium and scored for NEBD at the indicated times. The experiment was performed four times and about 50 embryos were analyzed in each group for each experiment. Similar results were obtained in each case and the data were pooled and expressed as the mean.

Effect of OA on Induction of Oocyte Maturation Maturation-Associated Ch,anges in Protein Phosphorylation

and the

Activators of PKA, e.g., membrane-permeable CAMP analogs and phosphodiesterase inhibitors, inhibit spontaneous oocyte maturation in vitro (Schultz, 1986). The

of One-Cell

To examine the reversibility of OA treatment to inhibit cleavage of one-cell embryos, one-cell embryos were incubated in medium containing 10 PLMOA for increasing periods of time, washed free of OA, and then incubated in OA-free medium. The fraction of treated embryos that cleaved to the two-cell stage decreased as the length of the initial incubation in OA increased (Fig. 7). The decreased extent of cleavage, however, was unlikely due to an increased inhibition of protein synthesis that resulted from the increased length of the initial incubation in OA. When the treated embryos that were cultured overnight were radiolabeled for 2 hr in medium containing [35S]methionine, the extent of incorporation of radiolabel/embryo was relatively the same. For embryos initially treated with OA for 15,30,45, and 60 min, the extent of incorporation/embryo relative to control embryos was 68, 84, 76, and 70%, respectively (average of four experiments).

100 t

t 80 t

ia 2

-

60

b s 40

0 2-Cell

Fragmented

1 -Cell

FIG. 7. Effect of an initial incubation in OA-containing medium on cleavage to the two-cell stage. Embryos were incubated in 10 HM OA for 15 min (black bar), 30 min (open bar), 45 min (stippled bar), or 60 min (hatched bar), and then transferred to OA-free medium and scored for cleavage after an overnight incubation. The experiment was performed four times and about 50 embryos were analyzed in each group for each experiment. Similar results were obtained in each case and the data were pooled and expressed as the mean.

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60

-0

1

2

3 Time

4

5

(hr)

FIG. 8. Effect of OA on GVBD in oocyte incubated in medium containing either IBMX or TPA. GV-intact oocytes were incubated in medium containing 0.2 mMIBMX or 10 rig/ml TPA (X), 0.2 mMIBMX containing either 1 FM OA (A) or 10 PM OA (A), or 10 rig/ml TPA containing either 1 &f OA (0) or 10 @f OA (O), or in inhibitor-free medium (+). The experiment was performed three times with 50 oocytes in each treatment group. Similar results were obtained in each experiment and the data were pooled and expressed as the mean.

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The effect of OA on the maturation-associated changes in protein phosphorylation, however, was examined (Fig. 9). OA-induced GVBD in oocytes incubated in an inhibitory concentration of IBMX manifested essentially the same set of maturation-associated changes in protein phosphorylation as did control oocytes, e.g., proteins of M, -32,000,70,000, and 150,000. In addition, several proteins displayed an apparent hyperphosphorylation, e.g., proteins of M, -45,000, 46,000,000, 105,000, 115,000, and 170,000. PKC activators also inhibit oocyte maturation (Bornslaeger et al., 1986b; Urner and Schorderet-Slatkine, 1984) but at a step distal to that of the PKA activators, since the maturation-associated decrease in oocyte CAMP occurs in the presence of TPA, but the full complement of maturation-associated changes in protein phosphorylation does not (Bornslaeger et al., 198613). Thus the ability of OA to induce maturation in oocytes incubated in the presence of TPA was assessed in order to determine if the step affected by OA was subsequent to the CAMP decrease but prior to that perturbed by

M,xiO3

mechanism of action of the CAMP analogs and phosphodiesterase is to inhibit the maturation-associated drop in oocyte CAMP that occurs during the commitment period (Schultz et ah, 1983). Since OA can induce hormoneindependent oocyte maturation in lower species (Goris et al., 1989; Picard et al., 1989; Pondaven et al., 1989), the ability of OA to induce maturation of oocytes inhibited from undergoing spontaneous oocyte maturation by PKA activators was examined. OA induced a concentration-dependent resumption of maturation, as assessed by GVBD, in oocytes incubated in 0.2 mM IBMX, which totally inhibits GVBD; at the higher concentrations of OA the kinetics of GVBD were similar to control oocytes (Fig. 8). It should be noted that the initial response of these GV-intact oocytes was a similar “crinkling” effect that was also observed when one-cell embryos were treated with OA (data not shown). In addition, oocytes continuously cultured in the presence of OA did not emit polar bodies (data not shown). A set of changes in protein synthesis and phosphorylation occur during oocyte maturation (Bornslaeger et al., 1986a,b; Bornslaeger et ah, 1988; Endo et ab, 1986). We did not examine the effect of OA on the changes in 35S-radiolabeled proteins, since OA treatment markedly inhibited protein synthesis in the oocytes (data not shown). Although GVBD does not require protein synthesis, protein synthesis is required for polar body emission and this may account for the inhibitory effect of OA on polar body emission (Wassarman et al., 1978).

’

2

3

180-

1168458-

(

27-

FIG. 9. Autoradiogram of s2P-labeled phosphoproteins synthesized in GV-intact oocytes, GVBD oocytes, and OA-treated oocytes. Lane 1, control oocytes incubated in medium containing 0.2 mMIBMX for 3 hr and then radiolabeled for 2 hr; lane 2, oocytes incubated in medium for 3 hr to allow GVBD and then radiolabeled for 2 hr; lane 3, oocytes incubated in medium containing 0.2 mM IBMX and 10 PM OA for 3 hr and then radiolabeled in the same medium for 2 hr. The experiment was performed two times and similar results were obtained in each case; shown are the results of one experiment. The large chevrons point to phosphoproteins that are apparently hyperphosphorylated in the presence of OA and the small chevrons point to the maturationassociated changes in protein phosphorylation.

PKC activators or if the OA-sensitive step was distal to the step affected by PKC activators. OA induced a concentration-dependent resumption of maturation in oocytes incubated in 10 rig/ml TPA, which totally inhibits GVBD. Again, at the higher concentrations of OA the kinetics of GVBD were similar to control oocytes (Fig. 8), and moreover, the kinetics of maturation were similar at both concentrations of OA in the presence of either IBMX or TPA. Thus, the step in maturation affected by OA appeared distal to that affected by activation of PKC.

The ability of OA to induce GVBD in oocytes and NEBD in one-cell embryos suggests a role for either PPl or PPZA in cell cycle regulation in the germ cell and early embryo; GVBD/NEBD constitute one aspect of the G2 to M transition. Since dephosphorylation of specific proteins occurs during oocyte maturation and the first cell cycle (Bornslaeger et rx,l,, 1986a; Howlett, 1986), oocytes and one-cell embryos possess protein phosphatases. Although we do not know if PPl and PPZA are present in oocytes and early embryos, it is likely that they are, since these enzymes are widely distributed among different cell types. In addition, although OA is a more specific inhibitor of PPZA than PPl, our experimental protocol-incubating the cells in medium containing OA rather than microinjecting OA to a known final intracellular concentration-prevents assigning the effects induced by OA treatment to one of the phosphatases, since we do not know the final intracellular concentration reached during the incubation period. Inhibition of PPZB is unlikely to be the target of OA action to induce GVBD/NEBD, since calmodulin antagonists inhibit, rather than stimulate, GVBD in oocytes (Bornslaeger et ul., 1984) or NEBD in one-cell embryos (Poueymirou and Schultz, 1990). It should be noted that PPl is implicated in the G2 to M transition in both A.sl,er:qill~s &ultrns (Doonan and Morris, 1989) and fission yeast (Booher and Beach, 1989; Ohkura et ul., 1989). The results of our experiments on the ability of OA to induce GVBD in oocytes incubated in medium containing IBMX confirm those presented in a recent report that OA induces GVBD in oocytes incubated in medium containing a membrane-permeable CAMP analog, which inhibits spontaneous maturation (Rime and Ozon, 1990). We extend this observation by demonstrating that OA induces maturation in oocytes arrested from undergoing GVBD by the PKC activator, TPA, which inhibits oocytes maturation at a step distal to that effected by activation of PKA (Bornslaeger et uL., 198613). It should also be noted that the kinetics of GVBD incubated in medium containing 10 puM OA and either IBMX or TPA

were essentially identical to those of control oocytes. Thus, the step effected by OA treatment may be close to that of MPF activation. OA, which induces Hl kinase activity in mouse oocytes (Rime and Ozon, 1990), also induces the set of maturation-associated changes in protein phosphorylation. The increased level of phosphorylation of the proteins of M, 70,000 that we observe is likely to represent hyperphosphorylation of nuclear lamins that occurs upon GVBD/NEBD, and consistent with this is the apparent decrease in electrophoretic mobility that is associated with phosphorylation. OA treatment also results in the apparent hyperphosphorylation of a set of proteins of ikfr 32,000; as discussed below, these proteins become phosphorylated as one-cell embryos enter M phase. OA treatment also results in hyperphosphorylation of several proteins whose level of phosphorylation does not change during maturation. Although the identity of these proteins is not known, the species at M, 105,000 could possibly be nucleoline (M,. 92,000), which is a major nucleolar protein. Maturation is associated with an increase in the apparent phosphorylation of this protein and OA stimulates an apparent hyperphosphorylation. Nucleoline is a substrate for the cdc2 kinase, and phosphorylation is associated with a decrease in electrophoretie mobility (Peter et tsl., 1990). It should also be noted that progesterone-independent maturation induced by OA in X~U~JJUS luet% oocytes results in the apparent hyperphosphorylation of a protein of M, 110,000, which is not present in enucleated oocytes (Rime et ul., 1990). Also similar to the situation in Xenop1.s (Rime et (xl., 1990), OA-induced maturation of mouse oocytes is associated with the phosphorylation of a protein of M, -46,000. This protein could be elongation factor EF-1-y (Belle et (]I., 1989), which is a substrate for cdc2 kinase (Mulner-Lorillon et (II., 1989). As one-cell embryos enter M phase, p32 is converted to the more phosphorylated forms (Howlett, 1986). In OA-treated one-cell embryos the phosphorylation of p32 is rapid and occurs prior to NEBD. If p32 is a substrate for ~34”~“‘, which is activated by OA. the increased level of p32 phosphorylation induced by OA may reflect the increase in the level of this protein kinase. Although these results indicate a correlation between p32 phosphorylation and NEBD, a causal relationship still remains to be determined. In addition, OA-induced NEBD results in the apparent hyperphosphorylation of two phosphoproteins of M, -45,000. The phosphorylation of two phosphoproteins of this molecular weight also occurs as one-cell embryos enter M phase (Howlett, 1986). OA also induces the apparent hyperphosphorylation in one-cell embryos of several proteins that may also be hyperphosphorylated in oocytes treated with OA. This

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observation is expected, since most of the proteins in the one-cell embryo are maternally derived. Continuous culture of one-cell embryos in medium containing OA inhibits cleavage to the two-cell stage. This inhibition of cleavage may be due to the inability of these embryos that had undergone precocious NEBD to complete DNA synthesis and/or the requirement for protein synthesis during G2 that is essential for cleavage of one-cell embryos (Howlett, 1986). A brief treatment of one-cell embryos with OA also results in a marked inhibition of cleavage. One explanation for this result is that the extent of inhibition of PPBA by OA depends on the concentration of the enzyme (Cohen et ul, 1989). This suggests that OA may form a tight inhibitory complex with PPBA. Thus, during the chase period, even though OA is not present in the medium, the protein phosphatase may still be inhibited. This may explain the very slow conversion of the more phosphorylated forms of p32 in the pulse-chase experiments. The ability of OA to induce both GVBD and precocious NEBD suggests that neither PPl or PPBA is directly involved in activation of the cdc2 kinase. During maturation of oocytes in other species, activation of the cdc2 kinase is associated with dephosphorylation (Gautier et ab, 1989; Labbe et al., 1989b). Thus, an OA-insensitive protein phosphatase whose activity is modulated either directly or indirectly by PPUPPBA, e.g., a cdc2 phosphatase whose activity is activated by phosphorylation, may be involved in the dephosphorylation of cdc2. It is also possible that the induction of MPF activity by OA is related to cyclin phosphorylation (Pondaven et al., 1990; Roy et ub, 1990). Last, the ability of OA to inhibit a biological phenomenon that requires protein synthesis should be interpreted with caution, since results presented here indicate that OA can inhibit protein synthesis. Although the basis for this inhibition is not known, both initiation factor-2 and elongation factor-2, which has an M, 100,000, are inhibited by phosphorylation (Rowlands et ul., 1988; Ryazanov and Davydova, 1989) and OA treatment results in the apparent hyperphosphorylation of proteins in this molecular weight range. This research was supported by grants from the NIH (HD 22681 and HD 21355) to R.M.S. lvofoteo&led ire proof: Recently, Gavin ef crl. (EJsp CeU Rex 192,75-81, 1991) reported that microinjection of okadaic acid results in GVBD of mouse oocytes cultured in the presence of cAMP analogs, phosphodiesterase inhibitors, or biologically phorbol diesters. REFERENCES BELL& R., DERANCOURT,J., POULHE, R., CAPONY, J. P., OZON, R., and MULNER-LORILLON, 0. (1989). A purified complex from Xenopus oocytes contains a ~47 protein, an i?/ 1+7:osubstrate of MPF, and a P30

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