MOLECULAR REPRODUCTION AND DEVELOPMENT 27:230-234 (1990)

Combined Effects of Protein Synthesis and Phosphorylation Inhibitors on Maturation of Mouse Oocytes In Vitro JAN MOTLIKAND ZORA RIMKEVICOVA Department of Genetics, Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, Czechoslovakia

ABSTRACT In denuded mouse oocytes, neither 3 nor 5 hours of preincubation in dbcAMP (1 mM) and cycloheximide (10 kg/ml), followed by further 3 hours in cycloheximide only, lowered the rate of GVBD (93% and 92%, respectively). It means that 3 and 5 hours preincubation in cycloheximide did not impair the ability of mouse oocytes to resume meiosis in medium with the protein synthesis inhibitor. To test the combined effects of inhibition of protein phosphorylation and protein synthesis, oocytes were cultured for 3,4, or 5 hours in 2 m M of 6-DMAP and subsequently for 3 hours in 10 pg/ml cycloheximide. The incubation in 6DMAP for 4 or 5 hours diminished (63% or 35% of GVBD, respectively) the ability of mouse oocytes to resume meiosis when subsequent protein synthesis was blocked by cycloheximide. However, the highly condensed bivalents were always visible in GVs. Thus the above treatment did not prevent chromatin condensation although GVBD was blocked. Key Words: Preincubation, dbcAMP, Cycloheximide

INTRODUCTION Protein synthesis and protein phosphorylation play a substantial role in the control of mitotic and meiotic divisions (see Hunt, 1989). Recent studies on mammalian oocytes have revealed that their sensitivity to protein synthesis inhibitors divides them in two groups. Rodent oocytes, as represented by mouse and rat, cultured in medium with puromycin or cycloheximide undergo germinal vesicle breakdown (GVBD) but cannot proceed beyond the circularly arranged bivalents stage (for review see Mangia and Canipari, 1977; Schultz and Wassarman, 1977; Ekholm and Magnusson, 1979). Rabbit denuded oocytes behave similarly in a cycloheximide-supplemented medium (Motlik et al., 1989). In contrast, resumption of meiosis in pig, sheep, and cattle oocytes requires a n active protein synthesis (Fulka Jr. et al., 1986; Moor and Crosby, 1986; Hunter and Moor, 1987). It means that oocytes of large domestic animals,

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with a slow sequence of GVBD, do not mature in media with a protein synthesis inhibitor (Motlik, 1989). Chromatin condensation and nuclear membrane breakdown either in mitotic or meiotic cycles, are associated with a burst of phosphorylation (Morgan et al., 1989; Murray and Kirschner, 1989). It has been shown recently that 6-dimethylaminopurine(6-DMAP),a n inhibitor of protein phosphorylation (Neant and Guerrier, 1988a1, specifically and reversibly blocks meiosis reinitiation in starfish (Neant and Guerrier, 1988a) and mouse (Rime et al., 1989) oocytes, presumably by inhibiting a relevant protein kinase activity (NBant et al., 1989). In the present study, we have tested a hypothesis that resumption of meiosis in rodent oocytes is dependent upon short-lived proteins (Mangia and Canipari, 1977; Ekholm and Magnusson, 1979). In the first series of experiments, GVBD was inhibited by dibutyryl CAMP(dbcAMP) while protein synthesis was simultaneously blocked by cycloheximide. The inhibition of protein synthesis did not prevent resumption of meiosis in mouse oocytes when the dbcAMP was removed. By contrast, in the second series of experiments, oocytes were subsequently cultured in a n inhibitor of protein phosphorylation (6-DMAP) and protein synthesis (cycloheximide). The results suggest that mouse oocytes, preincubated in 6-DMAP, require an active protein synthesis for GVBD.

MATERIALS AND METHODS Source of Oocytes Mouse oocytes were isolated from large antral follicles of sexually mature females (strain A) primed 48 h before with 5 IU pregnant mare’s serum gonadotropin. For experiments only oocytes surrounded by compact cumulus were chosen. Cumuli oophori were removed

Received February 2, 1990; accepted April 11, 1990. Address reprint requests to J a n Motlik, Department of Genetics, Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, 277 21 Libgchov, Czechoslovakia.

GVBD INHIBITION IN MOUSE OOCYTES

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TABLE 1. Combined Effects of dbcAMP and Cycloheximide on Resumption of Meiosis in Mouse Denuded Oocytes Time of

culture in dbcAMP (h)" 3

Time of culture in dbcAMP + CYCLO (h)'

Time of

culture in CYCLO (hY 3 3

5 3 3 5 5

3

3

Nuclear configuration Number of oocytes 94 105 124 107 112 95 102

Late GV diakinesis 91 3 9 8 116 105 2 8 104 93 2 8 94

Percent of GVBDd 3.1 91.5 93.6 1.8+ 92.8' 2.1* 92.1"

"The culture medium was supplemented with 1 mM dbcAMP. 'The culture medium was supplemented with 1 mM dbcAMP and 10 pg/ml cycloheximide. 'The culture medium was supplemented with 10 kg/ml cycloheximide. d + , *: These values are highly significantly different (a= 0.001).

ing both 1 mM dbcAMP and 10 kg/ml of cycloheximide for 3 or 5 h. After thorough washing, the oocytes were cultured in medium supplemented with cycloheximide only. In these oocytes, neither 3 nor 5 h of preincubaCulture of Oocytes in Media With Inhibitors tion in dbcAMP and cycloheximide, followed by a furMouse denuded oocytes were cultured in 0.1 ml of the ther 3 h in cycloheximide supplemented medium only, modified Parker's medium as droplets under paraffin oil lowered the GVBD rate (93% and 92%, respectively) at 38°C under 5% COz in air. The culture medium con- (Table 1, 1.5 and 7). These data indicate that protein tained 72 ml isotonic TC 199 medium (USOL, Prague, synthesis was not required for mouse oocytes to resume Czechoslovakia), 18 mll.45% NaHCO, + 0.002 phenol meiosis when the dbcAMP was removed. In the second series of experiments, the sequential red, 10 ml 5.5% (v/v) glucose solution, 4 mg sodium pyruvate, 10 mg freeze-dried calf serum growth proteins culture in the protein phosphorylation (6-DMAP) and (USOL)/ml, 50 IU penicillin, and 5 mg streptomycidml. protein synthesis (cycloheximide) inhibitor were done. For the experiments, the control medium was supple- Pilot experiments showed th a t only concentrations of mented with dbcAMP (SERVA, Heidelberg, FRG) at 6-DMAP above 1mM fully inhibited GVBD in denuded concentration 1 mM alone, and/or cycloheximide (10 mouse oocytes. For this reason, 2 mM 6-DMAP, which kg/ml) (SERVA). 6-DMAP (Sigma Chemical Co., St. inhibited GVBD and chromatin condensation in 97% of oocytes (Table 2), was used in all further experiments. Louis, MO) was used a t 2.0 mM concentration. The reversibility test of the 3-h 6-DMAP block to GVBD revealed that a n additional 3 h incubation withEvaluation of Oocytes out 6-DMAP resulted in full reversibility (93% of At the end of culture all oocytes were mounted on GVBD). However, if the oocytes were exposed to 6slides, fixed in a n acetic acid:alcohol (1:3) for 24 h, DMAP for 5 h before incubating 3 h in the control stained with orcein, and examined under a phase-con- medium, only 85% of oocytes underwent GVBD. The trast microscope. The frequency of GVBD given in Ta- remaining oocytes (15%) possessed GV with the highly ble 1 and Table 2 was compared on the basis of chi- condensed bivalents. square analysis. All data were pooled together from After 3 h of preincubation in 6-DMAP, the subsefive replicate experiments. quent culture in cycloheximide (3 h) allowed GVBD in 82% of oocytes (see Table 2). Then culture in 6-DMAP RESULTS was prolonged to 4 h, however, only 63% of oocytes GVBD occurred in 96% of mouse denuded oocytes subsequently underwent GVBD. Moreover, the reafter 3 h of culture in the control medium (data not maining oocytes possessed GV filled with the highly shown). The medium supplemented with 1 mM condensing bivalents (Fig. 1). This phenomenon was dbcAMP very effectively blocked (3.1% GVBD) re- even more pronounced after 5 h of preincubation in sumption of meiosis (see Table 1). In contrast, the me- 6-DMAP, when nearly 65% of oocytes showed GV with dium with cycloheximide (10 kg/ml) allowed to 92% of the condensing or highly condensed bivalents (Fig. 2). oocytes to undergo GVBD. The dbcAMP block was fully Although 35% of oocytes had undergone GVBD, remareversible since the subsequent culture in cyclohexi- nents of nuclear membrane were frequently visible mide (3 h) led to GVBD in 94% of oocytes (Table 1,1.3). around the late diakinesis bivalents (Fig. 3a,b). These These data led to culture oocytes in medium contain- data demonstrate that 6-DMAP diminished the ability

mechanically and denuded oocytes, with germinal vesicle (GV) containing a large compact nucleolus, were used in the following experimental schema.

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J. MOTLfK AND Z. RIMKEVICOVA TABLE 2. Combined Effects of 6-Dimethylaminopurine and Cycloheximide on Resumption of Meiosis in Mouse Denuded Oocytes First Second culture (h) culture (h) 6-DMAP" 3 3 5 3 4 5

Control 3 3

Germinal vesicle with Late Percent Number of Filamentous Condensed CYCLOb oocytes bivalents bivalents diakinesis of GVBD" 96 93 3 3.11+* 117 8 109 93.1t 127 19 108 85.1*" 3 141 25 116 82.3' 148 3 55 93 62.8 3 142 92 50 35.2'-

"The control medium was supplemented with 2 mM of 6-DMAP. bThe control medium was supplemented with 10 pg/ml cycloheximide. + , *: These values are highly significantly different (a = 0.001); t, ': these values are significantly different !a = 0.1).

Fig. 3a,b. Mouse oocytes cultured in 6-DMAP for 5 h and subsequently in cycloheximide-supplemented medium for 3 h. These oocytes passed GVBD but the remanents of nuclear membrane (arrowed) are visible around the late diakinesis bivalents. X 700.

Fig. 1. Mouse oocyte cultured in 6-DMAP (2 mM) for 4 h and subsequently in cycloheximide (10 pgiml) for 3 h. Germinal vesicle (GV) is filled with the condensing and highly condensed bivalents. Position of nucleolus (n) is indicated. x 900. Fig. 2. Mouse oocyte cultured in 6-DMAP for 5 h and subsequently in cycloheximide-supplemented medium for 3 h. Germinal vesicle (GV) is filled with the highly condensed bivalents. x 1100.

of mouse oocytes to resume meiosis in medium supplemented with the protein synthesis inhibitor in a timedependent manner.

DISCUSSION Resumption of meiosis in mouse oocytes isolated from Graafian follicles is not dependent on a n active protein synthesis. Neither of the protein synthesis inhibitors cycloheximide and puromycin block GVBD (Schultz and Wassarman, 1977). Rat oocytes also resume meiosis in the presence of puromycin, but when they were cultured in dbcAMP and puromycin (2 or 4 h) and subsequently in medium with puromycin alone (4 h), GVBD was prevented (Ekholm and Magnusson, 1979). The authors suggested the existence of shortlived proteins necessary for initiation of meiosis. In our experiments with mouse oocytes, the same experimental schema, except using cycloheximide instead of puromycin, did not support this hypothesis. Neither 3 nor 5 h preincubation with dbcAMP and cycloheximide impaired the ability of mouse oocytes to resume meiosis in medium with protein synthesis inhibitor. Our data suggest that the protein synthesis inhibition by cycloheximide does not deplete mouse oocytes of protein(s)

GVBD INHIBITION IN MOUSE OOCYTES necessary for the maturation promoting factor (MPF) activation (Masui and Markert, 1971). However, the observed discrepancies between rat and mouse might be caused by the different drugs used. Cycloheximide acts reversibly on peptidyl transferase (see Vazquez, 1974). A formation of peptide bonds is blocked but the chain is not released from the polyribosome. In contrast, puromycin, a structural analogue of tRNA molecule, causes the premature release of a nascent polypeptide from the ribosome. This possibility is the focus of our present research. In all species studied (see Neant and Guerrier, 19891, the appearance of MPF activity during meiosis reinitiation is temporally associated with a high level of protein phosphorylation. During starfish oocyte maturation, two successive phosphorylation bursts are observed (Doree et al., 1983). MPF activity and the extent of protein phosphorylation oscillate in parallel. While the first increase in protein phosphorylation and MPF activity occurs in the absence of protein synthesis, the reappearance of MPF after first polar body extrusion requires the synthesis of new proteins in both starfish (Doree et al., 1983) and frog (Gerhart et al., 1984) oocytes. Similarly, MPF activity in mouse oocytes oscillate and the first appearance of MPF is insensitive to cycloheximide; however, the reappearance of MPF activity at the second metaphase requires a n active protein synthesis (Hashimoto and Kishimoto, 1989). A burst of phosphorylation occurs during GVBD of mouse oocytes as well (Rime et al., 1989). These data are fully supported by the finding that 6-DMAP, a n inhibitor of protein phosphorylation (Neant and Guerrier, 1988a), reversibly prevents resumption of meiosis in starfish oocytes (Neant and Guerrier, 1988a) and in oocytes from the mollusc Patella vulgata (Neant and Guerrier, 1988b). GVBD in mouse cumulus-free oocytes is fully but reversibly blocked by 1.2 mM of 6-DMAP (Rime et al., 1989). The present experiments with 2 mM concentration of 6-DMAP showed that the inhibition was nearly absolute (90%) and fully reversible after the 3 h incubation in the inhibitor. However, when reversibility was tested after the 5 h incubation, it was revealed for the first time that 15% of oocytes did not regain their ability to break down their nuclear membrane, although the chromatin in nucleoplasm was highly condensed. This phenomenon was clearly expressed in 38% and 65% of oocytes preincubated for 4 and 5 h in 6-DMAP, respectively, and subsequently cultured in cycloheximide. These data suggest that inhibition of protein phosphorylation reduced the ability of mouse oocytes to break down their nuclear envelope while chromatin condensation activity was fully kept. The mouse oocytes showing GV filled with highly condensed bivalents are very reminiscent of pig oocytes cultured in cycloheximide (Kubelka et al., 1988). In pig oocytes, cycloheximide inhibits nuclear membrane breakdown but does not prevent chromatin condensation. If we accept that MPF is responsible for induction

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of chromatin condensation and GVBD, then inhibition of protein synthesis in pig oocytes might allow only a partial MPF activation t h a t results in chromatin condensation only (Motlik, 1989). After 6-DMAP treatment mouse oocytes possess chromatin condensation activity similar to pig freshly isolated GV oocytes. This may imply t h a t inhibition of protein phosphorylation at the GV stage reduces the species specificity of mammalian oocytes, since a t this stage both pig and 6DMAP-treated mouse oocytes require ongoing active protein synthesis for nuclear membrane disintegration. The present experiments with mouse oocytes and the previous results with pig oocytes (Kubelka et al., 1988) imply that a n activity inducing chromatin condensation is separable from the activity that induces nuclear envelope breakdown. This conclusion is supported by results achieved in stable mitotic extracts (Newport and Spann, 1987) in which chromatin condensation is independent of lamin depolymerization and membrane breakdown. DNA from bacteriophage lambda can titrate chromosome condensation activity in mitotic extracts and the specific inhibitors of topoisomerase I1 block chromosome condensation. Since the above treatments do not influence lamin and membrane release, these events are thought to be independent of the chromatin condensation pathway (Newport and Spann, 1987). The complete subunit composition of MPF is not yet certain; however, nearly all MPF preparation contain a 32-34 kD subunit and the other subunit, varying in size according to the species origin, corresponds to Btype cyclins (see Murray and Kirschner, 1989). PreMPF molecules can be detected in interphase cells (Dunphy and Newport, 1988) and ~ 3 4 " in ~ "starfish ~ oocytes is stable during meiosis, while MPF level oscillates (Labbe et al., 1988). This implies that the protein synthesis requirement for mitosis and the second meiotic division (see above) could be explained by the synthesis of a n MPF activating protein (Hunt, 1989). We postulate that cyclin-like molecules are synthesized by pig oocytes between 8 and 14 h after meiosis resumption (Motlik, 1989; Moor et al., 1989) but fully grown mouse oocytes in the GV stage, which are not sensitive to protein synthesis inhibitors, already possess such molecules. One way to interpret the present results is to suppose that a n inhibition of protein phosphorylation depletes cyclin-like molecules from mouse oocytes and thereby causes their dependency on a n active protein synthesis for nuclear membrane breakdown.

ACKNOWLEDGMENTS The authors thank Dr. Tim Hunt, Dr. Josef Fulka, and Dr. Vaclav KopeEny for critical reading of the manuscript. We are indebted to Dr. Pierre Guerrier for providing 6-DMAP and for encouragement of this re-

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search. Thanks to Dr. Yasuo Shioya for hospitality of his laboratory during preparation of the manuscript.

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Combined effects of protein synthesis and phosphorylation inhibitors on maturation of mouse oocytes in vitro.

In denuded mouse oocytes, neither 3 nor 5 hours of preincubation in dbcAMP (1 mM) and cycloheximide (10 micrograms/ml), followed by further 3 hours in...
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