MOLECULAR REPRODUCTION AND DEVELOPMENT 28:249-254 (1991)
The Effects of &-Amanitinand Cycloheximide on Nuclear Progression, Protein Synthesis, and Phosphorylation During Bovine Oocyte Maturation In Vitro P.M.M. KASTROP,~S.C.J. HULSHOF,~M.M. BEVERS,~O.H.J. DESTR~E: AND TH.A.M. KRUIP~ 'Department of Herd Health and Reproduction, University of Utrecht, and 2Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The Netherlands
ABSTRACT Bovine cumulus oocyte complexes were cultured for various periods and either denuded and orcein stained or radiolabeled with 35S-methionine or 32P-orthophosphate. Specific inhibitors were added to the culture medium to investigate mRNA and protein synthesis requirements for both nuclear and cytoplasmic changes during maturation in vitro. Inhibition of mRNA synthesis by a-amanitin during the first 2 h of culture prevented the phosphorylation of some specific proteins preceding GVBD and decreased the occurrence of GVBD from 97%to 27%. In addition, in oocytes that had undergone GVBD, only part of the changes in protein synthesis after GVBD were observed. Addition of a-amanitin after 3 h of culture had no effect on meiotic maturation. When cumulus oocyte complexes were cultured in the presence of cycloheximide, the phosphorylation of specific proteins was also blocked and only 5% of the oocytes underwent GVBD. Addition of cycloheximide after 4, 6, or 8 h of culture resulted in an increasing percentage of GVBD, but the oocytes became arrested in metaphase I. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was increasingly restored. It is concluded that after the onset of culture, both mRNA and protein synthesis are necessary for the phosphorylation of specific proteins and for GVBD. Furthermore, transcription during the first hours of culture is needed for the synthesis of new proteins after GVBD. Key Words: Cow, In vitro maturation, Inhibitors INTRODUCTION In prenatal life, mammalian oocytes become arrested at the diplotene stage of prophase I. The resumption of meiosis is triggered by the preovulatory LH-surge (Masui and Clarke, 1979). In vitro, meiotic maturation is mediated by the release of oocytes from their ovarian follicles into a suitable culture medium (Pincus and Enzmann, 1935; Edwards, 1965). Concomitant with the nuclear events, cytoplasmic rearrangements occur during meiotic maturation (Szollosi, 1972; Golbus and Stein, 1976; Schultz and Wassarman, 1977a; McGaughey and Van Blerkom, 1977; Warnes et al., 1977;
0 1991 WILEY-LISS, INC.
Van Blerkom and McGaughey, 1978). The regulatory mechanisms involved in meiotic maturation are quite different among species and are not fully understood. In cattle (Hunter and Moor, 1987; Sirard et al., 1989), in contrast to the mouse (Schultz and Wassarman, 1977b; Clarke and Masui, 1983), both protein synthesis and RNA synthesis are required for the germinal vesicle breakdown (GVBD). In addition, inhibition of protein synthesis does not prevent the extrusion of the first polar body in bovine oocytes that can proceed beyond metaphase I (Sirard et al., 1989). The aim of the present study was to extend the investigations of the role of mRNA and protein synthesis in bovine oocyte maturation. The specific inhibitors a-amanitin and cycloheximide were used to block mRNA or protein synthesis respectively. Especially the effect of these inhibitors on protein phosphorylation, preceding GVBD, and the reprogramming of protein synthesis, subsequent t o GVBD was examined.
MATERIALS AND METHODS Oocyte Recovery Ovaries were collected aseptically, kept in modified Dulbecco's phosphate buffered saline at 30°C, and transported to the laboratory within 1 h after slaughter. Cumulus oocyte complexes were obtained by aspirating antral follicles ( 6 8 mm) of bovine ovaries. The follicular contents were pooled in a conical centrifuge tube and allowed t o sediment for 15 minutes. The settled cumulus oocyte complexes were recovered from the follicular fluid, washed in HEPES-buffered medium M199 with Earle's salts (Flow Laboratories, U.K.) supplemented with 10% heat-treated fetal calf serum (FCS) and 10 kg/ml kanacylin, and classified under a stereomicroscope according to De Loos et al. (1989). Only oocytes surrounded by a compact and multilayered cumulus investment, i.e., categories 1-3, were used in this study, since these oocytes showed no Received July 16, 1990; accepted October 17, 1990. Address reprint requests to Dr. P.M.M. Kastrop, Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 7, 3584 CL Utrecht, The Netherlands.
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differences in protein synthesis (Kastrop et al., 1990a). Kodak XAR5 films. Mixtures of proteins, with known The whole selection procedure was carried out at molecular weights (Mr) between 14.2 and 205 kDa approximately 30°C and within 2-3 h after slaughter. (Sigma Chemicals Co., St. Louis, MO), were run simultaneously as standards and visualized after electroCulture and Staining Procedure phoresis by Coomassie staining. Cumulus oocyte complexes were cultured in 200 pl drops of bicarbonate-buffered M199 with Earle’s salts RESULTS (Flow Laboratories, U.K.) supplemented with 10%FCS Effect of Cycloheximide and a-Amanitin on and 10 pg/ml bFSH under paraffin oil. Culture was Nuclear Development carried out a t 39°C in a humidified atmosphere of 5% When cumulus oocyte complexes were cultured for COz in air. Either immediately or at various times after onset of culture, cumulus oocyte complexes in the 24 h in the presence of cycloheximide, GVBD occurred inhibition groups were transferred t o 200 pl drops of in only 5% of the oocytes, whereas 97% of the oocytes culture medium containing either 10 pg/ml a-amanitin cultured in inhibitor-free medium underwent GVBD (Boehringer-Mannheim, West Germany) or 10 pg/ml (Fig. 1). Addition of cycloheximide at 4, 6, or 8 h after cycloheximide (BDH Chemicals Ltd, U.K.), based on the onset of culture resulted in 12, 38, and 81% of the protocols used by Hunter and Moor (1987) and Sirard oocytes having undergone GVBD, respectively. Howet al. (1989). When RNA or protein synthesis had to be ever, none of these oocytes in which GVBD took place inhibited immediately from the onset of culture, these reached metaphase 11. Only oocytes cultured for at least groups were selected and washed in medium containing 12 h in inhibitor-free medium were able to reach MIL corresponding concentrations of the inhibitor. At the A further delay in the addition of cycloheximide reend of incubation, cumulus oocyte complexes were sulted in an increasing proportion of oocytes with an either denuded and scored for their nuclear stage (pilot extruded first polar body. Addition of the inhibitor after 16 h of culture did not affect the percentage of oocytes experiments), or subsequently radiolabelled for 2 h. To assess the nuclear stage, a group of cumulus reaching MII. The presence of a-amanitin throughout 24 h of oocyte complexes were denuded mechanically, using a finely drawn Pasteur pipette, and mounted on slides. culture reduced the occurrence of GVBD from 97% The oocytes were fixed for 24 h in acetic acidlethano1 (control) to 27% (Fig. 1). In contrast, the inhibitory (1:3), stained with 1%aceto-orcein, and examined effect of a-amanitin was greatly decreased by delaying the addition of the inhibitor. When a-amanitin was under a phase-contrast microscope (400x1. added 1 or 2 h after the onset of culture, 66 and 79%, Radiolabelling and Electrophoresis respectively, of the oocytes underwent GVBD. No effect of Oocyte Proteins on nuclear maturation was observed when a-amanitin After various periods of culture, cumulus oocyte was added from 3 h of culture onwards. In spite of the complexes were either radiolabelled with [35S]-methio- strong inhibitory effect of a-amanitin on GVBD during nine or [32Pl-orthophosphate for 2 h. Radiolabeling the first 2 h of culture, substantial numbers of oocytes with [35Sl-methionine was performed in Dulbecco’s in which GVBD had occurred were still able to proceed phosphate-buffered saline, supplemented with 4 mg/ml to metaphase 11. bovine serum albumin, 0.36 mM pyruvate, 23.8 mM Effect of a-Amanitin on Protein lactate, 5.5 mM glucose, and 1 mCi/ml L-[35S]methioSynthesis Patterns nine (specific activity >1,000 Ci/mmol; Amersham, The results in Figure 1 showed that the inhibitory U.K.). Phosphorylation of proteins was achieved by labeling cumulus oocyte complexes with 0.5 mCi/ml action of a-amanitin on nuclear maturation was re[32P]-orthophosphate (carrier free; Amersham, U.K.), stricted to the first 2 h after the onset of culture. To using bicarbonate buffered saline as described by investigate whether the inhibition of mRNA synthesis Crosby et al. (1984). Labeled oocytes were denuded during this period was reflected in changes in the mechanically and assessed for morphological appear- protein synthesis patterns, cumulus oocyte complexes ance and the presence of a germinal vesicle or polar were radiolabelled with 35S-methionine after various body. The denuded oocytes were lysed individually by culture periods. After 2 and 6 h of culture, the protein adding 20 pl SDS-sample buffer (Laemmli, 19701, synthesis patterns exhibited no differences between followed by heating a t 100°C for 2 minutes. Aliquots of control oocytes (Fig. 2, lanes C) and oocytes cultured in 2 r~.lwere used to determine the amount of TCA the continuous presence of a-amanitin (Fig. 2, Lanes 1). precipitable counts. The remainder of each sample was Once GVBD had occurred in the control oocytes after stored at -20°C. 10 h of culture (Fig. 2, 10 h, lane C), the previously Electrophoresis was performed on 8-15% linear gra- described changes in protein synthesis patterns (Kasdient SDS-polyacrylamide gels, according to Laemmli trop et al., 1990b), especially at 130, 110, 82, and 54 (1970).Fluorography was carried out by treating [35Sl- kDa were observed. The protein patterns of oocytes methionine containing gels with Amplify@ (Amer- inhibited throughout the 10 h culture (Fig. 2, 10 h, sham, U.K.) for 30 minutes. All the gels were dried lane 1)showed no such changes and were identical with under vacuum at 80°C and subsequently exposed to the patterns after 2 and 6 h of culture. When a-
INHIBITORS AND BOVINE OOCYTE MATURATION
Fig. 1. The effect of cycloheximide and a-amanitin on the occurrence of GVBD. Cumulus oocyte complexes were cultured with cycloheximide (10 pg/ml) from 0,4,6,8, 12,14, 16, and 20 h after the onset of culture or with a-amanitin (10 pgiml) from 0, 1,2, 3, and 16 h after the onset of culture. After a 24-h culture period, oocytes were
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denuded and scored for their nuclear stage by orcein staining. The illustrated percentage GVBD is divided in the percentage of oocytes which proceeded to the MI and MI1 stage. The total number of oocytes examined is indicated in parentheses above bars.
amanitin was added 2 h after the onset of culture, 50 and 60 kDa (Fig. 4, lanes 1).When cycloheximide oocytes that remained blocked in the germinal vesicle was added 4 h after the onset of culture (Fig. 4, lanes 21, stage at 10 h (Fig. 2, lane 2+) showed also no changes only a slight increase was seen in the phosphorylation in their protein synthesis patterns. However, oocytes in of the relevant proteins. When the inhibitor was added which no germinal vesicle was detected showed a 6 h after the onset of culture (Fig. 4, lane 31, the different protein synthesis pattern (Fig. 2, lane 2-). phosphorylation was almost restored to normal. However, this pattern only partially matched the patDISCUSSION tern of the control oocytes (Fig. 2, 10 h, lane C). The present study demonstrates that both transcripEffect of a-Amanitin on Protein tional and translational activity during defined periods Phosphorylation Patterns of culture are crucial for GVBD and for protein syntheExtensive phosphorylation of several proteins be- sis and phosphorylation in bovine oocytes. Inhibition of tween 3 and 8 h after the onset of culture preceding mRNA synthesis for 2 h and of protein synthesis for GVBD was previously reported (Kastrop et al., 1990b). 4 h after the onset of culture effectively blocks the At 4 and 6 h after the onset of culture, four phosphopro- phosphorylation of proteins with molecular weights of teins with apparent molecular weights of 19, 24, and 24 and of those between 50 and 60 kDa. This indicates two between 50 and 60 kDa were observed (Fig. 3, that these phosphoproteins and/or proteins involved in lanes C). The continuous presence of a-amanitin during their phosphorylation are newly synthesized. Tranculture (Fig. 3, lanes 1)prevented the extensive phos- scripts for the proteins involved are synthesized during phorylation of the 24 kDa protein and those between 50 the first 2 h of culture. In addition, the data suggest and 60 kDa. However, when the inhibitor was added 2 that transcription is required for specific protein phos(Fig. 3, lanes 2) or 4 h (Fig. 3, lanes 3) after the onset phorylation necessary for subsequent GVBD. of culture, phosphorylation patterns were similar to Based on recent models by Smith (1989) and Gautier et al. (1990), our results suggest that the heavily those of the control oocytes. phosphorylated proteins at 24 and between 50 and 60 Effect of Cycloheximide on Protein kDa might be associated with Maturation-Promoting Phosphorylation Patterns Factor (MPF) activity. To obtain kinase activity, one of In addition to mRNA synthesis, the importance of the subunits of MPF, cyclin, has to be newly syntheprotein synthesis for protein phosphorylation during sized and phosphorylated (Murray and Kirschner, the period preceding GVBD was examined (Fig. 4). 1989). The two B-type cyclins in Xenopus run anomaInhibition of the protein synthesis by cycloheximide for lously on SDS gels and their apparent molecular sizes 4-6 h after the onset of culture, prevented the phos- range from about 48 and 51 to 52 and 55 kDa, dependphorylation of the proteins located a t 24 and between ing on gel conditions (Gautier et al., 1990). Thus, the
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P.M.M. KASTROP ET AL. transcription involved in the resumption of meiosis takes place within the oocyte or within the cumulus cells is still uncertain, since Osborn and Moor (1983) have demonstrated that denuded oocytes are hardly affected by a-amanitin. In addition, Hunter and Moor (1987) reported that a-amanitin prevented cumulus expansion in the bovine. This suggests either an impaired permeation system of the oocyte for a-amanitin or the essential involvement of the cumulus cells in the resumption of meiosis. The latter possibility is based on the hypothesis of Eppig and Downs (1987;Downs et al., 1988) postulating a regulatory mechanism in which an inhibitor, activated in the cumulus cells, prevents meiotic maturation, whereas a positive stimulus, e.g., induced by hormones and produced in the cumulus cells, overrides the inhibitory influence and promotes resumption of meiosis. The present data show that for completion of maturation, i.e., reaching metaphase 11, protein synthesis is required during different phases throughout culture.
Fig. 2. Effect of a-amanitin on protein synthesis patterns of bovine oocytes after 2, 6, and 10 h of culture and radiolabeling with 35S-methioninefor 2 h. Cumulus oocyte complexes were cultured and radiolabeled in the complete absence of a-amanitin (C),the continuous presence of 10 kg/ml a-amanitin (I), or in the media to which warnanitin was added 2 h after the onset of culture (2- and 2+). The oocytes cultured for 10 h and designated as C and 2- underwent GVBD, whereas all other oocytes possessed an intact germinal vesicle after radiolabeling. Differences in the protein synthesis patterns a t 130, 110, 82, and 54 kDa are marked by arrows.
two bovine phosphoproteins between 50 and 60 kDa might be cyclins, whereas the phosphoprotein of 24 kDa might act as a MPF substrate. Messenger RNA synthesis during the first hours of culture is not only necessary for phosphorylation and GVBD, but also for the synthesis of new proteins after GVBD since in oocytes exposed to a-amanitin from 2 h after the onset of culture, only part of the changes in protein synthesis occur. Since the addition of a-amanitin 2 h after the onset of culture results in prevention Of GVBD in a number Of o°CyteS, it is suggested that the population of Oocyte is less homogeneous than supposed on the basis Of the morphological assessment. This is supported by the observation that in about a quarter of the oocyteGVBD Occurs in the presence from the Onset Of culture. Apparently, in these oocytes the transcripts necessary for GVBD are already present. Whether the
Fig. 3. Effect of a-amanitin on protein phosphorylation patterns of oocytes cultured for 4 (left panel) and 6 h (right panel) and radiolabeled with 32P-orthophosphate for 2 h. Cumulus oocyte complexes were cultured and radiolabeled in a-amanitin free media (C), media containing 10 pg/ml a-amanitin (1)or media with a-amanitin added 2 h (2) or 4 h (3) after the onset of culture. Phosphoprotein bands at 19, 24, and between 50 and 60 kDa were marked by an arrow. The different intensities of the phosphoprotein bands in the 4 and 6 h panels were caused by various exposure times.
INHIBITORS AND BOVINE OOCYTE MATURATION
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species, virtually all changes observed in protein synthesis patterns occur after GVBD (McGaughey and Van Blerkom, 1977; Schultz and Wassarman, 1977a; Van Blerkom and McGaughey, 1978; Moor et al., 1981; Moor and Crosby, 1986). In conclusion, transcription especially during the first 3 h and translation throughout culture are prerequisites for both nuclear and cytoplasmic changes during bovine oocyte maturation. Evaluation of the models by Smith (1989) and Gautier et al. (1990), with regard to bovine oocyte maturation, will involve further analyses of the specific phosphoproteins.
ACKNOWLEDGMENTS This study was supported by grants of the Dutch Program Committee for Agriculture Biotechnology. We thank Mr. Th. van Beneden for his skilled technical assistance.
REFERENCES
Fig. 4. Effect of cycloheximide on protein phosphorylation patterns of oocytes cultured for 4 (left panel) and 6 h (right panel) and radiolabeled with 32P-orthophosphate for 2 h. Cumulus oocyte complexes were cultured and radiolabelled in cycloheximide free media (C), in the continuous presence of 10 pg/ml cycloheximide (l), or in media to which cycloheximide was added 4 h (2) and 6 h (3) after the onset of culture. The different intensities of the phosphoprotein bands in the 4 and 6 h panels were caused by various exposure times. Phosphoprotein bands at 19, 24, and between 50 and 60 kDa are marked by a n arrow.
To achieve GVBD, de novo protein synthesis is necessary particularly beyond 4 h of culture. Once GVBD has occurred, protein synthesis is still needed to proceed to metaphase 11, as oocytes become arrested in metaphase I when the inhibitor is present up to 12 h of culture. In a previous report, changes in the protein synthesis patterns were exclusively observed after GVBD, 8 and 16 h after the onset of culture (Kastrop et al., 1990b). The striking changes observed in the protein synthesis patterns after 8 h of culture, i.e., shortly after GVBD, apparently are caused by the newly synthesized proteins required to reach metaphase 11. Although no changes in protein synthesis patterns are observed before GVBD, the effect of cycloheximide on protein phosphorylation clearly demonstrates that essential proteins are additionally synthesized during this period. Probably, the amounts of these proteins form only a small proportion of the total de novo synthesized proteins and therefore escape detection. Also in other
Clarke HJ, Masui Y (1983): The induction of reversible and irreversible chromosome decondensation by protein inhibition during meiotic maturation of mouse oocytes. Dev Biol 97:291-301. Crosby IM, Osborn JC, Moor RM (1984):Changes in protein phosphorylation during the maturation of mammalian oocytes in vitro. J Exp Zool 229:459-466. De Loos F, Van Vliet C, Van Maurik P, Kruip TAM (1989): Morphology of immature bovine oocytes. Gamete Res 24:1-8. Downs SM, Daniel SAJ, Eppig JJ (1988): Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: Evidence for a positive stimulus of somatic cell origin. J Exp Zool 245:86-96. Edwards RG (1965): Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 208:349-351. Eppig J J , Downs SM (1987): The effect of hypoxanthine on mouse oocyte growth and development in vitro: Maintenance of meiotic arrest and gonadotropin-induced oocyte maturation. Dev Biol 119:313321. Gautier J , Minshull J , Lohka M, Glotzer M, Hunt T, Maller J L (1990): Cyclin is a component of Maturation Promoting Factor from Xenopus. Cell 60:487-494. Golbus MS, Stein MP (1976): Qualitative patterns of protein synthesis in the mouse oocyte. J. Exp. Zool. 198:337342. Hunter AG, Moor RM (1987): Stage-dependent effects of inhibiting ribonucleic acids and protein synthesis on meiotic maturation of bovine oocytes in vitro. J Dairy Sci 70:1646-1651. Kastrop PMM, Bevers MM, Destree OHJ, Kruip ThAM (1990a): Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro. Molec Reprod Dev 261222-226. Kastrop PMM, Bevers MM, Destrke OHJ, Kruip ThAM (1990b): Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro. J Reprod Fertil, in press. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Masui Y, Clarke H J (1979): Oocyte maturation. Int Rev Cytol 57:186-282. McGaughey RW, Van Blerkom J (1977): Patterns of polypeptide synthesis of porcine oocytes during maturation in vitro. Dev Biol 56:241-254. Moor RM, Crosby IM (1986): Protein requirements for germinal vesicle breakdown in ovine oocytes. J Embryol Exp Morphol94:207220. Moor RM, Osborn JC, Cran DG, Walters DE (1981):Selective effect of gonadotropins on cell coupling, nuclear maturation and protein synthesis in mammalian oocytes. J Embryol Exp Morphol61:347365.
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Murray AW, Kirschner MW (1989):Cyclin synthesis drives the early embryonic cell cycle. Nature 339:275-280. Osborn JC, Moor RM (1983):Time-dependent effects of cu-amanitin on nuclear maturation and protein synthesis in mammalian oocytes. J Embryo1 Exp Morphol 73:317-338. Pincus G, Enzmann EV (1935): The comparative behavior of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J Exp Med 62:665-675. Schultz RM, Wassarman PM (1977a):Specific changes in the pattern of protein synthesis during meiotic maturation of mammalian oocytes in vitro. Proc Natl Acad Sci USA 74538-541. Schultz RM, Wassarman PM (1977b): Biochemical studies of mammalian oogenesis: Protein synthesis during oocyte growth and meiotic maturation in the mouse. J Cell Sci 24:167-194. Sirard MA, Florman HM, Leibfried-Rutledge ML, Barnes FL, Sims
ML, First NL (1989): Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes. Biol Reprod 40:1257-1263. Smith LD (1989): The induction of oocyte maturation: Transmembrane signaling events and regulation of the cell cycle. Development 107:685-699. Szollosi D (1972):Changes of some cell organelles during oogenesis in mammals. In J D Biggers, AW Schuetz (eds): “Oogenesis.” Baltimore: University Park Press, pp 47-64. Van Blerkom J , McGaughey RW (1978):Molecular differentiation of the rabbit ovum. 1. During oocyte maturation in vivo and in vitro. Dev Biol 63:139-150. Warnes GM, Moor RM, Johnson MH (1977): Changes in protein synthesis during maturation of sheep oocytes in vivo and in vitro. J Reprod Fertil 49:331-335.
The Effects of &-Amanitinand Cycloheximide on Nuclear Progression, Protein Synthesis, and Phosphorylation During Bovine Oocyte Maturation In Vitro P.M.M. KASTROP,~S.C.J. HULSHOF,~M.M. BEVERS,~O.H.J. DESTR~E: AND TH.A.M. KRUIP~ 'Department of Herd Health and Reproduction, University of Utrecht, and 2Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The Netherlands
ABSTRACT Bovine cumulus oocyte complexes were cultured for various periods and either denuded and orcein stained or radiolabeled with 35S-methionine or 32P-orthophosphate. Specific inhibitors were added to the culture medium to investigate mRNA and protein synthesis requirements for both nuclear and cytoplasmic changes during maturation in vitro. Inhibition of mRNA synthesis by a-amanitin during the first 2 h of culture prevented the phosphorylation of some specific proteins preceding GVBD and decreased the occurrence of GVBD from 97%to 27%. In addition, in oocytes that had undergone GVBD, only part of the changes in protein synthesis after GVBD were observed. Addition of a-amanitin after 3 h of culture had no effect on meiotic maturation. When cumulus oocyte complexes were cultured in the presence of cycloheximide, the phosphorylation of specific proteins was also blocked and only 5% of the oocytes underwent GVBD. Addition of cycloheximide after 4, 6, or 8 h of culture resulted in an increasing percentage of GVBD, but the oocytes became arrested in metaphase I. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was increasingly restored. It is concluded that after the onset of culture, both mRNA and protein synthesis are necessary for the phosphorylation of specific proteins and for GVBD. Furthermore, transcription during the first hours of culture is needed for the synthesis of new proteins after GVBD. Key Words: Cow, In vitro maturation, Inhibitors INTRODUCTION In prenatal life, mammalian oocytes become arrested at the diplotene stage of prophase I. The resumption of meiosis is triggered by the preovulatory LH-surge (Masui and Clarke, 1979). In vitro, meiotic maturation is mediated by the release of oocytes from their ovarian follicles into a suitable culture medium (Pincus and Enzmann, 1935; Edwards, 1965). Concomitant with the nuclear events, cytoplasmic rearrangements occur during meiotic maturation (Szollosi, 1972; Golbus and Stein, 1976; Schultz and Wassarman, 1977a; McGaughey and Van Blerkom, 1977; Warnes et al., 1977;
0 1991 WILEY-LISS, INC.
Van Blerkom and McGaughey, 1978). The regulatory mechanisms involved in meiotic maturation are quite different among species and are not fully understood. In cattle (Hunter and Moor, 1987; Sirard et al., 1989), in contrast to the mouse (Schultz and Wassarman, 1977b; Clarke and Masui, 1983), both protein synthesis and RNA synthesis are required for the germinal vesicle breakdown (GVBD). In addition, inhibition of protein synthesis does not prevent the extrusion of the first polar body in bovine oocytes that can proceed beyond metaphase I (Sirard et al., 1989). The aim of the present study was to extend the investigations of the role of mRNA and protein synthesis in bovine oocyte maturation. The specific inhibitors a-amanitin and cycloheximide were used to block mRNA or protein synthesis respectively. Especially the effect of these inhibitors on protein phosphorylation, preceding GVBD, and the reprogramming of protein synthesis, subsequent t o GVBD was examined.
MATERIALS AND METHODS Oocyte Recovery Ovaries were collected aseptically, kept in modified Dulbecco's phosphate buffered saline at 30°C, and transported to the laboratory within 1 h after slaughter. Cumulus oocyte complexes were obtained by aspirating antral follicles ( 6 8 mm) of bovine ovaries. The follicular contents were pooled in a conical centrifuge tube and allowed t o sediment for 15 minutes. The settled cumulus oocyte complexes were recovered from the follicular fluid, washed in HEPES-buffered medium M199 with Earle's salts (Flow Laboratories, U.K.) supplemented with 10% heat-treated fetal calf serum (FCS) and 10 kg/ml kanacylin, and classified under a stereomicroscope according to De Loos et al. (1989). Only oocytes surrounded by a compact and multilayered cumulus investment, i.e., categories 1-3, were used in this study, since these oocytes showed no Received July 16, 1990; accepted October 17, 1990. Address reprint requests to Dr. P.M.M. Kastrop, Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 7, 3584 CL Utrecht, The Netherlands.
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differences in protein synthesis (Kastrop et al., 1990a). Kodak XAR5 films. Mixtures of proteins, with known The whole selection procedure was carried out at molecular weights (Mr) between 14.2 and 205 kDa approximately 30°C and within 2-3 h after slaughter. (Sigma Chemicals Co., St. Louis, MO), were run simultaneously as standards and visualized after electroCulture and Staining Procedure phoresis by Coomassie staining. Cumulus oocyte complexes were cultured in 200 pl drops of bicarbonate-buffered M199 with Earle’s salts RESULTS (Flow Laboratories, U.K.) supplemented with 10%FCS Effect of Cycloheximide and a-Amanitin on and 10 pg/ml bFSH under paraffin oil. Culture was Nuclear Development carried out a t 39°C in a humidified atmosphere of 5% When cumulus oocyte complexes were cultured for COz in air. Either immediately or at various times after onset of culture, cumulus oocyte complexes in the 24 h in the presence of cycloheximide, GVBD occurred inhibition groups were transferred t o 200 pl drops of in only 5% of the oocytes, whereas 97% of the oocytes culture medium containing either 10 pg/ml a-amanitin cultured in inhibitor-free medium underwent GVBD (Boehringer-Mannheim, West Germany) or 10 pg/ml (Fig. 1). Addition of cycloheximide at 4, 6, or 8 h after cycloheximide (BDH Chemicals Ltd, U.K.), based on the onset of culture resulted in 12, 38, and 81% of the protocols used by Hunter and Moor (1987) and Sirard oocytes having undergone GVBD, respectively. Howet al. (1989). When RNA or protein synthesis had to be ever, none of these oocytes in which GVBD took place inhibited immediately from the onset of culture, these reached metaphase 11. Only oocytes cultured for at least groups were selected and washed in medium containing 12 h in inhibitor-free medium were able to reach MIL corresponding concentrations of the inhibitor. At the A further delay in the addition of cycloheximide reend of incubation, cumulus oocyte complexes were sulted in an increasing proportion of oocytes with an either denuded and scored for their nuclear stage (pilot extruded first polar body. Addition of the inhibitor after 16 h of culture did not affect the percentage of oocytes experiments), or subsequently radiolabelled for 2 h. To assess the nuclear stage, a group of cumulus reaching MII. The presence of a-amanitin throughout 24 h of oocyte complexes were denuded mechanically, using a finely drawn Pasteur pipette, and mounted on slides. culture reduced the occurrence of GVBD from 97% The oocytes were fixed for 24 h in acetic acidlethano1 (control) to 27% (Fig. 1). In contrast, the inhibitory (1:3), stained with 1%aceto-orcein, and examined effect of a-amanitin was greatly decreased by delaying the addition of the inhibitor. When a-amanitin was under a phase-contrast microscope (400x1. added 1 or 2 h after the onset of culture, 66 and 79%, Radiolabelling and Electrophoresis respectively, of the oocytes underwent GVBD. No effect of Oocyte Proteins on nuclear maturation was observed when a-amanitin After various periods of culture, cumulus oocyte was added from 3 h of culture onwards. In spite of the complexes were either radiolabelled with [35S]-methio- strong inhibitory effect of a-amanitin on GVBD during nine or [32Pl-orthophosphate for 2 h. Radiolabeling the first 2 h of culture, substantial numbers of oocytes with [35Sl-methionine was performed in Dulbecco’s in which GVBD had occurred were still able to proceed phosphate-buffered saline, supplemented with 4 mg/ml to metaphase 11. bovine serum albumin, 0.36 mM pyruvate, 23.8 mM Effect of a-Amanitin on Protein lactate, 5.5 mM glucose, and 1 mCi/ml L-[35S]methioSynthesis Patterns nine (specific activity >1,000 Ci/mmol; Amersham, The results in Figure 1 showed that the inhibitory U.K.). Phosphorylation of proteins was achieved by labeling cumulus oocyte complexes with 0.5 mCi/ml action of a-amanitin on nuclear maturation was re[32P]-orthophosphate (carrier free; Amersham, U.K.), stricted to the first 2 h after the onset of culture. To using bicarbonate buffered saline as described by investigate whether the inhibition of mRNA synthesis Crosby et al. (1984). Labeled oocytes were denuded during this period was reflected in changes in the mechanically and assessed for morphological appear- protein synthesis patterns, cumulus oocyte complexes ance and the presence of a germinal vesicle or polar were radiolabelled with 35S-methionine after various body. The denuded oocytes were lysed individually by culture periods. After 2 and 6 h of culture, the protein adding 20 pl SDS-sample buffer (Laemmli, 19701, synthesis patterns exhibited no differences between followed by heating a t 100°C for 2 minutes. Aliquots of control oocytes (Fig. 2, lanes C) and oocytes cultured in 2 r~.lwere used to determine the amount of TCA the continuous presence of a-amanitin (Fig. 2, Lanes 1). precipitable counts. The remainder of each sample was Once GVBD had occurred in the control oocytes after stored at -20°C. 10 h of culture (Fig. 2, 10 h, lane C), the previously Electrophoresis was performed on 8-15% linear gra- described changes in protein synthesis patterns (Kasdient SDS-polyacrylamide gels, according to Laemmli trop et al., 1990b), especially at 130, 110, 82, and 54 (1970).Fluorography was carried out by treating [35Sl- kDa were observed. The protein patterns of oocytes methionine containing gels with Amplify@ (Amer- inhibited throughout the 10 h culture (Fig. 2, 10 h, sham, U.K.) for 30 minutes. All the gels were dried lane 1)showed no such changes and were identical with under vacuum at 80°C and subsequently exposed to the patterns after 2 and 6 h of culture. When a-
INHIBITORS AND BOVINE OOCYTE MATURATION
Fig. 1. The effect of cycloheximide and a-amanitin on the occurrence of GVBD. Cumulus oocyte complexes were cultured with cycloheximide (10 pg/ml) from 0,4,6,8, 12,14, 16, and 20 h after the onset of culture or with a-amanitin (10 pgiml) from 0, 1,2, 3, and 16 h after the onset of culture. After a 24-h culture period, oocytes were
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denuded and scored for their nuclear stage by orcein staining. The illustrated percentage GVBD is divided in the percentage of oocytes which proceeded to the MI and MI1 stage. The total number of oocytes examined is indicated in parentheses above bars.
amanitin was added 2 h after the onset of culture, 50 and 60 kDa (Fig. 4, lanes 1).When cycloheximide oocytes that remained blocked in the germinal vesicle was added 4 h after the onset of culture (Fig. 4, lanes 21, stage at 10 h (Fig. 2, lane 2+) showed also no changes only a slight increase was seen in the phosphorylation in their protein synthesis patterns. However, oocytes in of the relevant proteins. When the inhibitor was added which no germinal vesicle was detected showed a 6 h after the onset of culture (Fig. 4, lane 31, the different protein synthesis pattern (Fig. 2, lane 2-). phosphorylation was almost restored to normal. However, this pattern only partially matched the patDISCUSSION tern of the control oocytes (Fig. 2, 10 h, lane C). The present study demonstrates that both transcripEffect of a-Amanitin on Protein tional and translational activity during defined periods Phosphorylation Patterns of culture are crucial for GVBD and for protein syntheExtensive phosphorylation of several proteins be- sis and phosphorylation in bovine oocytes. Inhibition of tween 3 and 8 h after the onset of culture preceding mRNA synthesis for 2 h and of protein synthesis for GVBD was previously reported (Kastrop et al., 1990b). 4 h after the onset of culture effectively blocks the At 4 and 6 h after the onset of culture, four phosphopro- phosphorylation of proteins with molecular weights of teins with apparent molecular weights of 19, 24, and 24 and of those between 50 and 60 kDa. This indicates two between 50 and 60 kDa were observed (Fig. 3, that these phosphoproteins and/or proteins involved in lanes C). The continuous presence of a-amanitin during their phosphorylation are newly synthesized. Tranculture (Fig. 3, lanes 1)prevented the extensive phos- scripts for the proteins involved are synthesized during phorylation of the 24 kDa protein and those between 50 the first 2 h of culture. In addition, the data suggest and 60 kDa. However, when the inhibitor was added 2 that transcription is required for specific protein phos(Fig. 3, lanes 2) or 4 h (Fig. 3, lanes 3) after the onset phorylation necessary for subsequent GVBD. of culture, phosphorylation patterns were similar to Based on recent models by Smith (1989) and Gautier et al. (1990), our results suggest that the heavily those of the control oocytes. phosphorylated proteins at 24 and between 50 and 60 Effect of Cycloheximide on Protein kDa might be associated with Maturation-Promoting Phosphorylation Patterns Factor (MPF) activity. To obtain kinase activity, one of In addition to mRNA synthesis, the importance of the subunits of MPF, cyclin, has to be newly syntheprotein synthesis for protein phosphorylation during sized and phosphorylated (Murray and Kirschner, the period preceding GVBD was examined (Fig. 4). 1989). The two B-type cyclins in Xenopus run anomaInhibition of the protein synthesis by cycloheximide for lously on SDS gels and their apparent molecular sizes 4-6 h after the onset of culture, prevented the phos- range from about 48 and 51 to 52 and 55 kDa, dependphorylation of the proteins located a t 24 and between ing on gel conditions (Gautier et al., 1990). Thus, the
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P.M.M. KASTROP ET AL. transcription involved in the resumption of meiosis takes place within the oocyte or within the cumulus cells is still uncertain, since Osborn and Moor (1983) have demonstrated that denuded oocytes are hardly affected by a-amanitin. In addition, Hunter and Moor (1987) reported that a-amanitin prevented cumulus expansion in the bovine. This suggests either an impaired permeation system of the oocyte for a-amanitin or the essential involvement of the cumulus cells in the resumption of meiosis. The latter possibility is based on the hypothesis of Eppig and Downs (1987;Downs et al., 1988) postulating a regulatory mechanism in which an inhibitor, activated in the cumulus cells, prevents meiotic maturation, whereas a positive stimulus, e.g., induced by hormones and produced in the cumulus cells, overrides the inhibitory influence and promotes resumption of meiosis. The present data show that for completion of maturation, i.e., reaching metaphase 11, protein synthesis is required during different phases throughout culture.
Fig. 2. Effect of a-amanitin on protein synthesis patterns of bovine oocytes after 2, 6, and 10 h of culture and radiolabeling with 35S-methioninefor 2 h. Cumulus oocyte complexes were cultured and radiolabeled in the complete absence of a-amanitin (C),the continuous presence of 10 kg/ml a-amanitin (I), or in the media to which warnanitin was added 2 h after the onset of culture (2- and 2+). The oocytes cultured for 10 h and designated as C and 2- underwent GVBD, whereas all other oocytes possessed an intact germinal vesicle after radiolabeling. Differences in the protein synthesis patterns a t 130, 110, 82, and 54 kDa are marked by arrows.
two bovine phosphoproteins between 50 and 60 kDa might be cyclins, whereas the phosphoprotein of 24 kDa might act as a MPF substrate. Messenger RNA synthesis during the first hours of culture is not only necessary for phosphorylation and GVBD, but also for the synthesis of new proteins after GVBD since in oocytes exposed to a-amanitin from 2 h after the onset of culture, only part of the changes in protein synthesis occur. Since the addition of a-amanitin 2 h after the onset of culture results in prevention Of GVBD in a number Of o°CyteS, it is suggested that the population of Oocyte is less homogeneous than supposed on the basis Of the morphological assessment. This is supported by the observation that in about a quarter of the oocyteGVBD Occurs in the presence from the Onset Of culture. Apparently, in these oocytes the transcripts necessary for GVBD are already present. Whether the
Fig. 3. Effect of a-amanitin on protein phosphorylation patterns of oocytes cultured for 4 (left panel) and 6 h (right panel) and radiolabeled with 32P-orthophosphate for 2 h. Cumulus oocyte complexes were cultured and radiolabeled in a-amanitin free media (C), media containing 10 pg/ml a-amanitin (1)or media with a-amanitin added 2 h (2) or 4 h (3) after the onset of culture. Phosphoprotein bands at 19, 24, and between 50 and 60 kDa were marked by an arrow. The different intensities of the phosphoprotein bands in the 4 and 6 h panels were caused by various exposure times.
INHIBITORS AND BOVINE OOCYTE MATURATION
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species, virtually all changes observed in protein synthesis patterns occur after GVBD (McGaughey and Van Blerkom, 1977; Schultz and Wassarman, 1977a; Van Blerkom and McGaughey, 1978; Moor et al., 1981; Moor and Crosby, 1986). In conclusion, transcription especially during the first 3 h and translation throughout culture are prerequisites for both nuclear and cytoplasmic changes during bovine oocyte maturation. Evaluation of the models by Smith (1989) and Gautier et al. (1990), with regard to bovine oocyte maturation, will involve further analyses of the specific phosphoproteins.
ACKNOWLEDGMENTS This study was supported by grants of the Dutch Program Committee for Agriculture Biotechnology. We thank Mr. Th. van Beneden for his skilled technical assistance.
REFERENCES
Fig. 4. Effect of cycloheximide on protein phosphorylation patterns of oocytes cultured for 4 (left panel) and 6 h (right panel) and radiolabeled with 32P-orthophosphate for 2 h. Cumulus oocyte complexes were cultured and radiolabelled in cycloheximide free media (C), in the continuous presence of 10 pg/ml cycloheximide (l), or in media to which cycloheximide was added 4 h (2) and 6 h (3) after the onset of culture. The different intensities of the phosphoprotein bands in the 4 and 6 h panels were caused by various exposure times. Phosphoprotein bands at 19, 24, and between 50 and 60 kDa are marked by a n arrow.
To achieve GVBD, de novo protein synthesis is necessary particularly beyond 4 h of culture. Once GVBD has occurred, protein synthesis is still needed to proceed to metaphase 11, as oocytes become arrested in metaphase I when the inhibitor is present up to 12 h of culture. In a previous report, changes in the protein synthesis patterns were exclusively observed after GVBD, 8 and 16 h after the onset of culture (Kastrop et al., 1990b). The striking changes observed in the protein synthesis patterns after 8 h of culture, i.e., shortly after GVBD, apparently are caused by the newly synthesized proteins required to reach metaphase 11. Although no changes in protein synthesis patterns are observed before GVBD, the effect of cycloheximide on protein phosphorylation clearly demonstrates that essential proteins are additionally synthesized during this period. Probably, the amounts of these proteins form only a small proportion of the total de novo synthesized proteins and therefore escape detection. Also in other
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