MOLECULAR REPRODUCTION AND DEVELOPMENT 29:379-384 (1991)
Effect of 6-Dimethylaminopurine on Germinal Vesicle Breakdown of Bovine Oocytes J. FULKA, J R . ,M.L. ~ LEIBFRIED-RUTLEDGE,’ AND N.L. FIRST2 ‘Institute of Animal Production, Prague, Uhrineves, Czechoslovakia; ‘Department of Meat and Animal Science, University of Wisconsin, Madison, Wisconsin
ABSTRACT The effect of 6-dimethylaminopuline (6-DMAP)on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%;120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3%GV, 349/ 355) or absence (97.1%GV, 1651170) of cumulus cells. When lower concentrations of 6-DMAP were used (100500 pM), GVBD was observed in 87.9%of oocytes, but their maturation was arrested at late diakinesismetaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%).To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximidesupplemented medium approximately 80%of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased.These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes. Key Words: Oocyte maturation, Phosphorylation, 6DMAP, Cattle oocytes
INTRODUCTION Phosphorylation has been shown t o play an important role in the G2/M transition of mitosis (Adlakha and Rao, 1987; Cross et al., 1989) and meiosis (Lohka and Maller, 1987; Maller et al., 1989). In mammalian oocytes too, phosphorylation was observed to occur during meiotic resumption in sheep, mice, and cattle (Crosby et al., 1984; Moor and Crosby, 1986; Schultz, 1988;Kastrop et al., 1990).According to Schultz (19881, characteristic changes in protein phosphorylation precede germinal vesicle breakdown (GVBD), and these changes are probably directly or indirectly involved in the process of meiotic commitment. As meiosis I proceeds towards GVBD, a large increase in phosphorylation of cellular protein occurs in conjunction with the appearance of maturation-promoting factor (MPF) ac0 1991 WILEY-LISS, INC.
tivity (Lohka and Maller, 1987; Ozon et al., 1987). This large burst of phosphorylation may involve elements necessary for nuclear dissolution (e.g., lamins, histones, nucleoplasm, nucleolar proteins, intermediate filaments, etc.; reviewed by Adlakha and Rao, 1987). It has been shown recently that 6-dimethylaminopurine (6-DMAP) inhibits GVBD of starfish and sea urchin (Neant et al., 1989; Neant and Guerrier, 1988a), mollusc (Neant and Guerrier, 1988b), and mouse (Rime et al., 1989) oocytes. These investigators show that 6DMAP inhibits the burst of phosphorylation that normally occurs shortly before GVBD, without affecting the overall rate of protein synthesis. Although mice and cattle are both mammals, we cannot extrapolate results directly from one species to another. For example, it is possible to block GVBD in large domestic animals with protein synthesis inhibitors, but mouse GVBD was shown to be insensitive to this drug (Fulka et al., 1986b; Moor, 1988). For this reason, we tested 6-DMAP in cattle oocytes. Our results show that 6-DMAP inhibits GVBD in cattle as it does in other species, but the results also indicate some differences between species.
MATERIALS AND METHODS Bovine oocytes were aspirated from antral follicles 3-5 mm in diameter. The oocytes were then carefully selected under a stereomicroscope, and only those surrounded by several layers of cumulus cells were used (Leibfried and First, 1979). These oocytes were washed in culture medium containing different concentrations of 6-DMAP (0, 0.1, 0.25, 0.5, 1.0, 2.0 mM) and cultured in 0.1 ml of medium in a humid atmosphere of 5% C 0 2 in air at a temperature of 37°C for 24 hr. Ten oocytes were incubated in each droplet. The composition of medium was TC 199 with Earle’s salts (9 ml; Cellgro; Mediatech, Washington, DC), isotonic glucose solution (1 ml), bovine serum albumin (BSA) (4 mg/ml), gentamicin (50 pgiml), and pyruvic acid (0.2 mM). The 6-DMAP and cycloheximide (both from Sigma, St. Louis, MO) were dissolved in the glucose solution. When cumulus-free oocytes were used, the surrounding cells were removed by pipetting through a narrow-bore Received September 25, 1990; accepted April 10, 1991. Address reprint requests to Dr. N.L. First, Department of Meat and Animal Science, University of Wisconsin, 1675 Observatory Drive, Madison, WI 53706.
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pipette. These oocytes were cultured in medium containing 2 mM of 6-DMAP. The same concentration of 6-DMAP was used when reversibility of inhibition and the effect of this drug on metaphase I1 oocytes were tested. When reversibility was tested, the oocytes were cultured for 22-24 hr in 6-DMAP-supplemented medium; they then were washed in inhibitor-free medium and cultured in it for 22-24 hr. Some oocytes were fixed after the first incubation (controls). To assess the sensitive period in which it is possible to block GVBD, the oocytes were incubated in inhibitor-free medium for 2, 4, 6, and 8 hr, respectively, and then some oocytes were fixed as a control. The other oocytes were washed in 6-DMAP-supplemented medium and cultured in it up to 24 hr (22, 20, 18, 16 hr). To assess if the activity of 6-DMAP is different from that of protein synthesis inhibitors, the metaphase I1 oocytes (after 26 hr of culture) were exposed to 6-DMAP (2 mM) or cycloheximide (25 pg/ml) for 16 hr. In the last part of the experiment, we tested the level of maturation-promoting factor (MPF) in oocytes cultured for 24 hr in medium containing 500 pM of 6-DMAP. In these oocytes GVBD was observed nearly 90%of the time, but maturation was arrested in late diakinesis metaphase I-like stage. These oocytes were fused to germinal vesicle (GV) oocytes freshly isolated from ovaries and cultured in medium containing 500 FM of the inhibitor for 3 hr. As a control, metaphase I1 oocytes were fused with GV oocytes and cultured under the same conditions. The fusion procedure was exactly the same as that described by Fulka et al. (1986a), except a polyethylene glycol of molecular weight 1,000 (Sigma) was used. In each experiment, some oocytes were used after initial incubations as a control. At the end of all culture intervals, the cumulus cells were removed and the oocytes were fixed in acid-alcohol and evaluated at x40 with Normarski or phase-contrast optics. Each experiment was repeated at least three times.
RESULTS Oocytes isolated from follicles and immediately fixed contained GV in all cases (671100%). The nuclear membrane was clearly visible, finely granulated nucleoplasm contained several clumps of chromatin, and in some cases the nucleolus was visible. After culture for 24 hr in medium without inhibitor, GVBD occurred in nearly all cases (120/124; 96.8%). Most oocytes completed meiosis I and were arrested at metaphase I1 with the first polar body extruded 103/120; (85.8%). When 6-DMAP was present, GVBD was completely blocked at a concentration of 2 mM. Nearly all oocytes contained GV with distinct nuclear membrane and several clusters of chromatin in the nucleoplasm (349/355; 98.3%) (Fig. 1). When lower concentrations were used (100500 mM), GVBD occurred in nearly all cells (160/182; 87.9%),but maturation was arrested at late diakinesis metaphase I-like stage. This means that the nuclear membrane had disappeared and that chromosomes were condensed, but their organization and morphology
were abnormal. Usually one cluster or several clusters of chromatin were observed (Fig. 2). If individual chromosomes were present, they were not organized on a spindle. At 1 mM 6-DMAP, GVBD occurred in approximately one-half of the oocytes (82/160; 51.3%). The inhibition of GVBD was not influenced by the absence of cumulus cells (2 mM 6-DMAP). Here, too, nearly all cells were arrested at the GV stage (1651170; 97%).These results are summarized in Table 1. The inhibitory effect of 6-DMAP was fully reversible. When the oocytes were cultured in 6-DMAP (2 mM) for 22-24 hr, then washed in control medium and cultured in it for another 20-24 hr, GVBD occurred in nearly all cells (2981302; 98.6%). Maturation proceeded up to the second metaphase (257/302; 85.0%; Fig. 3). However, the morphology of metaphase I1 chromosomes was abnormal in nearly all cases. The chromosomes were thinner and usually randomly arranged (Table 2). To assess the sensitive period in which it is possible to block GVBD, the oocytes were incubated in inhibitorfree medium (control medium) for various time intervals, then washed in 6-DMAP medium and cultured with it up to 24 hr. In control medium, GVBD occurred in most cases (89.2%) between 6 and 8 hr from the beginning of culture. When the oocytes were incubated for 4 hr in control medium and then in 6-DMAP medium, the process of GVBD was blocked almost completely (86.2% GV). On the other hand, transfer of the oocytes from control to 6-DMAP medium at 6 or 8 hr did not block oocytes at the GV stage. In most, GVBD occurred and chromosome clusters were observed in the cytoplasm (6 hr, 81.9% GVBD; 8 hr, 89% GVBD; Table 3). To assess if the action of 6-DMAP is different from that of inhibitors of protein synthesis, metaphase I1 oocytes were exposed to either 6-DMAP or cycloheximide. Concomitantly, some oocytes were similarly cultured, but the medium did not contain any inhibitor (control). In this group, activation (completion of meiosis 11, pronuclear formation) was observed infrequently 6/67; 7.5%). A low activation rate was also observed after incubation in 6-DMAP medium. Here only 14.5%of the oocytes contained pronuclei (20/138), whereas in cycloheximide-supplemented medium 82.4% of the oocytes were activated (891108). These results clearly show that the activity of 6-DMAP is quite different from that of cycloheximide. In the last experiment, the levels of MPF were tested in those oocytes incubated for 24 hr in the medium containing 500 pM of 6-DMAP. As is shown in Table 1, GVBD occurred in nearly all cases in this group, but the chromosomes usually formed clumps of chromatin. These oocytes were fused to freshly isolated oocytes and cultured in 6-DMAP medium for 3 hr. Fused oocytes containing two GVs were discarded. As a control, metaphase I1 oocytes (cultured for 24 hr in control medium) were fused to GV-containing oocytes and also cultured for 3 hr in 6-DMAP containing medium. In this group, complete GVBD was observed in nearly all
GVBD OF BOVINE OOCYTES
Fig. 1. Bovine oocytes cultured for 24 hr in medium containing 2 mM 6-DMAP. Germinal vesicle (GV) is clearly visible. ~ 6 0 0 . Fig. 2. Oocytes cultured in medium with 0.5 mM 6-DMAP. GVBD has occurred, and the cluster of chromatin is visible in the cytoplasm (arrow). x600. Fig. 3. Oocytes cultured in medium with 2 mM DMAP, then washed and cultured in inhibitor-free medium. Metaphase I1 (MI with the first polar body (PB) is present in the cytoplasm. The metaphase chromosomes are thinner and randomly arranged. No spindle is present. x300. Fig. 4. Two clusters of chromatin (C) are visible in the giant cell
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developed after fusion of a freshly isolated immature oocyte to an oocyte cultured for 24 hr in medium with 0.5 mM 6-DMAP. In Figures 4-6, the giant cells were cultured postfusion in medium containing 2 mM 6-DMAP for 3 hr. x600. Fig. 5. In some cases only slight changes occurred inside the GV after fusing oocytes as in Figure 4. Inside the GV, intensive chromatin condensation (C) is observed. ~ 3 0 0 . Fig. 6. After oocyte fusion as in Figure 4, GV of the immature oocyte remains completely intact. Arrow indicates a cluster of chromatin.
cases (411, and two groups of chromosomes were seen in 6-DMAP-incubated oocytes to GV-containing oocytes the cytoplasm. GVBD was incomplete or did not occur did not induce GVBD at the same frequency. In only 22 in four cases. In these cases, metaphase I1 oocytes were fused cells (35%) had GVBD of immature oocytes activated during manipulation and anaphase-telo- occurred, and usually two clumps of chromatin were phase I1 were present together with GV. Fusion of visible (Fig. 4).In 15 fused cells, GVBD was incomplete.
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J. FULKA ET AL. TABLE 1. Effect of 6-DMAP on GVBD of Bovine Oocytes* 6-DMAP (mM) 0 0.1-0.5b 1.0 2.0 c+c 2.0 c-
No. of oocytes
GV
124 182 160 355 170
4 22 78 349 165
Stage of maturation after 22-24 hr of culturea Percent Percent LD-MI AI-TI MI1 GV GVBD 7 2
10 155+ 80+ 2+ 4f
103 3 2 4 1
3.2 12.1 48.7 98.3 97.1
96.8 87.9 51.3 1.7 2.9
*The number of degenerated oocytes did not exceed 5% of the total in all experiments. Each experiment was repeated at least three times. aGV, germinal vesicle; GVBD, GV breakdown; LD, late diakinesis; MI, metaphase I; AI, anaphase I; TI, telophase I; MII, metaphase I1 one polar body; chromosomes formed one or more clusters. True metaphase spindles were not observed. bResults of individual groups were not different, so the data were pooled. “C+(-), oocytes with or without cumulus.
+
+/-
phorylation processes (Gautier et al., 1989). In mammalian oocytes also, the process of GVBD is most likely Stage of maturationa under the control of MPF (Fulka, 1983; Moor, 1988), so No. of oocytes GV LD-MI AI-TI MI1 + 1 PB (%) it is highly probable that similar processes regulate the 302 4 35 6 257 onset of maturation. Indeed some changes in protein 46 (18)AB phosphorylation preceding GVBD were shown in 211 (82) N mouse (Schultz, 19881,sheep (Crosby et al., 1984; Moor, *The oocytes were cultured for 22-24 h r in 6-DMAP- 1988), and cow (Kastrop et al., 1990) oocytes. The supplemented medium (2 mM). Some oocytes were then discovery that 6-DMAP blocks phosphorylation withused as a control, the others were cultured in inhibitor free medium for the next 22-24 hr. Abbreviations as for Table 1. out affecting the overall rate of protein synthesis brings aN, normal MI1 configuration; AB, abnormal MI1 con- new insight into the study of the regulatory processes figuration. (Neant and Guerrier, 198813;Neant et al., 1989;Rime et al., 1989). As is shown in this study, in cow oocytes also, GV breakdown is effectively blocked when the oocytes are exposed to 6-DMAP. The effective dose of this drug is The nuclear membrane usually disappeared, but the similar to that used in the mouse (Rime et al., 1989)and remnants of nucleoplasm were still visible. In 26 pig (Motlik, personal communication). Moreover, as in oocytes GVBD did not occur. Here nearly intact GVs the mouse, this inhibition is reversible, but most of the were present together with the clumps of chromatin second metaphases are abnormal (Rime et al., 1989).It (Figs. 5, 6). The results are summarized in Table 4. is possible that some maturational processes occur even in the presence of 6-DMAP. It leads to asynchrony of DISCUSSION the maturation after washing out the inhibitor and The transition of the meiotic or mitotic cells from G2 culture in control medium. Our results show that the phase to M phase is under control of MPF, and its sensitive period in which it is still possible to block activity is controlled by the phosphorylation-dephos- GVBD lasts for approximately the first 4 hr. After this, TABLE 2. Reversibility of 6-DMAP Inhibition*
TABLE 3. Effect of Preincubation of Bovine Oocytes in Inhibitor-Free Medium and Subsequent Culture in 6-DMAP Supplemented Medium on GVBD Time sequence of GVBD in control medium Culture Stage of maturation interval (hr) GV/GVBD (%) 2 4 6 8
44/0 71/9 48/57 8/73
(100/0) (88.75/11.25) (45.7/54.3) (9.9/90.1)
Preincubation of oocytes in control medium with subsequent culture in 6-DMAP Control medium Stage of maturation 6-DMAP (hr) GVIGVBD (%)
+
2 4 6 8
++ 20 22 + 18 + 16
88/5 (94H5.4) 81/13 (86.2A3.8) 17/77 (18.U81.9) 14/116 (10.8/89.2)
GVBD OF BOVINE OOCYTES
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TABLE 4. Fusion of Bovine Primary Oocytes Treated With 6-DMAP or Secondary Oocytes to GV-Stage Oocytes* Chromatin configurationa iGVBD PCC CL GV CL MI1 15 26
AII-TI1 Type of fusion 2 x CL GVBD 6-DMAP X GV 22 MI1 X GV 41 4 *All oocytes were incubated for 3 h r postfusion in medium containing 6-DMAP (500 pM). “CL, clusters of chromatin; iGVBD, incomplete GVBD (usually nuclear membrane disappeared, but ruminants of nucleoplasm with condensed chromatin were clearly visible; GV, intact germinal vesicle; PCC, prematurely condensed chromosomes; MII, second metaphase; AII, TII, anaphase 11, telophase 11.
+
the inhibitor is ineffective. Kastrop et al. (1990) observed extensive phosphorylation in bovine oocytes from 3 hr after the beginning of culture. At this time, the oocytes were still sensitive to 6-DMAP in our study. This may indicate that 6-DMAP indeed blocks phosphorylation. When matured oocytes were exposed to either cycloheximide or 6-DMAP, activation was frequently observed in the former group (82.6%)and less frequently in the latter (14.5%).Inhibitors of protein synthesis are potent parthenogenetic activators (Clarke and Masui, 1983).The common configuration observed in activated eggs was one pronucleus with the second polar body. On the other hand, we did not observe the decondensation of chromatin in maturing oocytes in those experiments in which precultured oocytes were transferred into the medium containing 6-DMAP. This decondensation is frequently observed when metaphase I mouse, starfish, mollusc, and echinoderm oocytes are exposed to 6DMAP (Rime et al., 1989; Neant and Guerrier, 1988a,b; Neant et al., 1989). In our experiments, condensed clusters of chromatin were present, but there was no chromatin decondensation. It may be that this decondensation is possible only at certain stages of maturation or that there are some species-specificdifferences. Motlik (unpublished) also did not observe this decondensation in pig metaphase I oocytes. The next possibility is that the dose of 6-DMAP was too low to induce decondensation. However, in our experiments, the higher doses negatively influenced oocyte morphology. In the last experiment, the oocytes matured in the presence of low doses of 6-DMAP (500 pM) in which GVBD is observed in nearly all cases were fused to immature oocytes. Whereas after fusion of metaphase I1 oocytes to GV containing oocytes, GVBD was rapidly induced and two sets of chromosomes were present in the cytoplasm in all oocytes (activated oocytes excluded), cytoplasm of 6-DMAP-treated oocytes does not induce similar changes. In only one-third of the fused cells were two sets of condensed chromosomes (clusters) present, whereas in the rest GVBD was incomplete, or intact GVs were present together with the clusters of chromatin. This indicates lower levels of MPF in 6-
+
+
+
DMAP oocytes than in those matured in normal medium. In the second case, GVBD is usually observed between 40 and 60 min after the initiation of fusion, and by 3 hr of culture two sets of perfectly condensed chromosomes are present (Fulka et al., 1986a,b). This indicates that the activity of MPF was most likely negatively influenced by 6-DMAP. Taken together, our data clearly show an inhibitory effect of 6-DMAP on GVBD in bovine oocytes. These results may indicate the necessity of phosphorylation for GVBD in this species, too, as has been shown for mouse oocytes. The exact mechanism and the role of phosphorylation are largely unknown in this species, but we may expect that this process is important for the transition from G2 to M, as is described for somatic cells and the oocytes of other species.
ACKNOWLEDGMENTS This research was supported by the Department of Meat and Animal Science, College of Agricultural and Life Sciences, University of Wisconsin-Madison, and USDA grant 88-372403740. We thank Brad Haley for obtaining bovine material and Julie Busby for secretarial assistance. REFERENCES Adlakha RC, Rao PN (1987): Regulation of mitosis by nonhistone protein factors in mammalian cells. In RA Schlegel, MS Halleck, PN Rao (eds):“Molecular Regulation of Nuclear Events in Mitosis and Meiosis.” New York: Academic Press, pp 179-226. 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 Zoo1 229:459-466. Cross F, Roberts J , Weintraub H (1989): Simple and complex cell cycles. Annu Rev Cell Biol 5:341-395. Fulka J J r . (1983): Nuclear maturation in pig and rabbit oocytes after interspecific fusion. Exp Cell Res 146:212-281. Fulka J Jr., Motlik J., Fulka J , Crozet N (1986a): Activity of maturation promoting factor in mammalian oocytes after its dilution by single and multiple fusioins. Dev Biol 118:176-181. Fulka J Jr., Motlik J., Fulka J., Jilek F (1986b3: Effect of cycloheximide on nuclear maturation of pig and mouse oocytes. J Reprod Fertil 77:281-285.
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Gautier J, Matsukawa T, Nurse P, Maller J (1989): Dephosphorylation and activation of Xenopus p34 cdc2 protein kinase during the cell cycle. Nature 339:626-669.Kastrop PMM, Bevers MM, Destree OHJ, Kruip TAM (1990): Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro. J Reprod Fertil90:305-310. Leibfried L, First NL (1979): Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci 48:76-86. Lohka MJ, Maller JL (1987): Regulation of nuclear formation and breakdown in cell-free extracts in amphibian eggs. In RA Schlegel, MS Halleck, PN Rao (eds):“Molecular Regulation of Nuclear Events in Mitosis and Meiosis.” New York: Academic Press, pp 67-109. Maller JL, Gautier J , Langan TA, Shenoy S, Shalloway D, Nurse P (1989) Maturation-promoting factor and the regulation of the cell cycle. J Cell Sci 12 [Suppll:53-63. Moor RM (1988):Regulation ofthe meiotic cycle in oocytes of domestic animals. Ann NY Acad Sci 541:248-258. Moor RM, Crosby IM (1986): Protein requirements for germinal vesicle breakdown in ovine oocytes. J Embryo1 Exp Morphol94:207220.
Neant I, Charbonneau M, Guerrier P (19891: A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms. Dev Biol 132:304-314. Neant I, Guerrier P (1988a): Meiosis reinitiation in the mollusc patella vulgata. Regulation of MPF, CSF and chromosomes condensation activity by intracellular pH, protein synthesis and phosphorylation. Development 102:505-516. Neant I, Guerrier P (1988b): 6-dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinse activity. Exp Cell Res 176:68-79. Ozon R, Mulner 0, Boyer J, Belle R (1987):Role of protein phosphorylation in Xenopus oocyte maturation. In RA Schlegel, MS Halleck, PN Rao (eds): “Molecular Regulation of Nuclear Events in Mitosis and Meiosis.” New York: Academic Press, pp 111-130. Rime H, Neant I, Guerrier P, Olon R (1989): 6-Dimethylaminopurine (6-DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte. Dev Biol 133:169-179. Schultz RM (1988): Role of protein phosphorylation in meiotic maturation of mouse oocytes in vitro. Ann NY Acad Sci 541:217-227.
Effect of 6-Dimethylaminopurine on Germinal Vesicle Breakdown of Bovine Oocytes J. FULKA, J R . ,M.L. ~ LEIBFRIED-RUTLEDGE,’ AND N.L. FIRST2 ‘Institute of Animal Production, Prague, Uhrineves, Czechoslovakia; ‘Department of Meat and Animal Science, University of Wisconsin, Madison, Wisconsin
ABSTRACT The effect of 6-dimethylaminopuline (6-DMAP)on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%;120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3%GV, 349/ 355) or absence (97.1%GV, 1651170) of cumulus cells. When lower concentrations of 6-DMAP were used (100500 pM), GVBD was observed in 87.9%of oocytes, but their maturation was arrested at late diakinesismetaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%).To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximidesupplemented medium approximately 80%of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased.These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes. Key Words: Oocyte maturation, Phosphorylation, 6DMAP, Cattle oocytes
INTRODUCTION Phosphorylation has been shown t o play an important role in the G2/M transition of mitosis (Adlakha and Rao, 1987; Cross et al., 1989) and meiosis (Lohka and Maller, 1987; Maller et al., 1989). In mammalian oocytes too, phosphorylation was observed to occur during meiotic resumption in sheep, mice, and cattle (Crosby et al., 1984; Moor and Crosby, 1986; Schultz, 1988;Kastrop et al., 1990).According to Schultz (19881, characteristic changes in protein phosphorylation precede germinal vesicle breakdown (GVBD), and these changes are probably directly or indirectly involved in the process of meiotic commitment. As meiosis I proceeds towards GVBD, a large increase in phosphorylation of cellular protein occurs in conjunction with the appearance of maturation-promoting factor (MPF) ac0 1991 WILEY-LISS, INC.
tivity (Lohka and Maller, 1987; Ozon et al., 1987). This large burst of phosphorylation may involve elements necessary for nuclear dissolution (e.g., lamins, histones, nucleoplasm, nucleolar proteins, intermediate filaments, etc.; reviewed by Adlakha and Rao, 1987). It has been shown recently that 6-dimethylaminopurine (6-DMAP) inhibits GVBD of starfish and sea urchin (Neant et al., 1989; Neant and Guerrier, 1988a), mollusc (Neant and Guerrier, 1988b), and mouse (Rime et al., 1989) oocytes. These investigators show that 6DMAP inhibits the burst of phosphorylation that normally occurs shortly before GVBD, without affecting the overall rate of protein synthesis. Although mice and cattle are both mammals, we cannot extrapolate results directly from one species to another. For example, it is possible to block GVBD in large domestic animals with protein synthesis inhibitors, but mouse GVBD was shown to be insensitive to this drug (Fulka et al., 1986b; Moor, 1988). For this reason, we tested 6-DMAP in cattle oocytes. Our results show that 6-DMAP inhibits GVBD in cattle as it does in other species, but the results also indicate some differences between species.
MATERIALS AND METHODS Bovine oocytes were aspirated from antral follicles 3-5 mm in diameter. The oocytes were then carefully selected under a stereomicroscope, and only those surrounded by several layers of cumulus cells were used (Leibfried and First, 1979). These oocytes were washed in culture medium containing different concentrations of 6-DMAP (0, 0.1, 0.25, 0.5, 1.0, 2.0 mM) and cultured in 0.1 ml of medium in a humid atmosphere of 5% C 0 2 in air at a temperature of 37°C for 24 hr. Ten oocytes were incubated in each droplet. The composition of medium was TC 199 with Earle’s salts (9 ml; Cellgro; Mediatech, Washington, DC), isotonic glucose solution (1 ml), bovine serum albumin (BSA) (4 mg/ml), gentamicin (50 pgiml), and pyruvic acid (0.2 mM). The 6-DMAP and cycloheximide (both from Sigma, St. Louis, MO) were dissolved in the glucose solution. When cumulus-free oocytes were used, the surrounding cells were removed by pipetting through a narrow-bore Received September 25, 1990; accepted April 10, 1991. Address reprint requests to Dr. N.L. First, Department of Meat and Animal Science, University of Wisconsin, 1675 Observatory Drive, Madison, WI 53706.
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pipette. These oocytes were cultured in medium containing 2 mM of 6-DMAP. The same concentration of 6-DMAP was used when reversibility of inhibition and the effect of this drug on metaphase I1 oocytes were tested. When reversibility was tested, the oocytes were cultured for 22-24 hr in 6-DMAP-supplemented medium; they then were washed in inhibitor-free medium and cultured in it for 22-24 hr. Some oocytes were fixed after the first incubation (controls). To assess the sensitive period in which it is possible to block GVBD, the oocytes were incubated in inhibitor-free medium for 2, 4, 6, and 8 hr, respectively, and then some oocytes were fixed as a control. The other oocytes were washed in 6-DMAP-supplemented medium and cultured in it up to 24 hr (22, 20, 18, 16 hr). To assess if the activity of 6-DMAP is different from that of protein synthesis inhibitors, the metaphase I1 oocytes (after 26 hr of culture) were exposed to 6-DMAP (2 mM) or cycloheximide (25 pg/ml) for 16 hr. In the last part of the experiment, we tested the level of maturation-promoting factor (MPF) in oocytes cultured for 24 hr in medium containing 500 pM of 6-DMAP. In these oocytes GVBD was observed nearly 90%of the time, but maturation was arrested in late diakinesis metaphase I-like stage. These oocytes were fused to germinal vesicle (GV) oocytes freshly isolated from ovaries and cultured in medium containing 500 FM of the inhibitor for 3 hr. As a control, metaphase I1 oocytes were fused with GV oocytes and cultured under the same conditions. The fusion procedure was exactly the same as that described by Fulka et al. (1986a), except a polyethylene glycol of molecular weight 1,000 (Sigma) was used. In each experiment, some oocytes were used after initial incubations as a control. At the end of all culture intervals, the cumulus cells were removed and the oocytes were fixed in acid-alcohol and evaluated at x40 with Normarski or phase-contrast optics. Each experiment was repeated at least three times.
RESULTS Oocytes isolated from follicles and immediately fixed contained GV in all cases (671100%). The nuclear membrane was clearly visible, finely granulated nucleoplasm contained several clumps of chromatin, and in some cases the nucleolus was visible. After culture for 24 hr in medium without inhibitor, GVBD occurred in nearly all cases (120/124; 96.8%). Most oocytes completed meiosis I and were arrested at metaphase I1 with the first polar body extruded 103/120; (85.8%). When 6-DMAP was present, GVBD was completely blocked at a concentration of 2 mM. Nearly all oocytes contained GV with distinct nuclear membrane and several clusters of chromatin in the nucleoplasm (349/355; 98.3%) (Fig. 1). When lower concentrations were used (100500 mM), GVBD occurred in nearly all cells (160/182; 87.9%),but maturation was arrested at late diakinesis metaphase I-like stage. This means that the nuclear membrane had disappeared and that chromosomes were condensed, but their organization and morphology
were abnormal. Usually one cluster or several clusters of chromatin were observed (Fig. 2). If individual chromosomes were present, they were not organized on a spindle. At 1 mM 6-DMAP, GVBD occurred in approximately one-half of the oocytes (82/160; 51.3%). The inhibition of GVBD was not influenced by the absence of cumulus cells (2 mM 6-DMAP). Here, too, nearly all cells were arrested at the GV stage (1651170; 97%).These results are summarized in Table 1. The inhibitory effect of 6-DMAP was fully reversible. When the oocytes were cultured in 6-DMAP (2 mM) for 22-24 hr, then washed in control medium and cultured in it for another 20-24 hr, GVBD occurred in nearly all cells (2981302; 98.6%). Maturation proceeded up to the second metaphase (257/302; 85.0%; Fig. 3). However, the morphology of metaphase I1 chromosomes was abnormal in nearly all cases. The chromosomes were thinner and usually randomly arranged (Table 2). To assess the sensitive period in which it is possible to block GVBD, the oocytes were incubated in inhibitorfree medium (control medium) for various time intervals, then washed in 6-DMAP medium and cultured with it up to 24 hr. In control medium, GVBD occurred in most cases (89.2%) between 6 and 8 hr from the beginning of culture. When the oocytes were incubated for 4 hr in control medium and then in 6-DMAP medium, the process of GVBD was blocked almost completely (86.2% GV). On the other hand, transfer of the oocytes from control to 6-DMAP medium at 6 or 8 hr did not block oocytes at the GV stage. In most, GVBD occurred and chromosome clusters were observed in the cytoplasm (6 hr, 81.9% GVBD; 8 hr, 89% GVBD; Table 3). To assess if the action of 6-DMAP is different from that of inhibitors of protein synthesis, metaphase I1 oocytes were exposed to either 6-DMAP or cycloheximide. Concomitantly, some oocytes were similarly cultured, but the medium did not contain any inhibitor (control). In this group, activation (completion of meiosis 11, pronuclear formation) was observed infrequently 6/67; 7.5%). A low activation rate was also observed after incubation in 6-DMAP medium. Here only 14.5%of the oocytes contained pronuclei (20/138), whereas in cycloheximide-supplemented medium 82.4% of the oocytes were activated (891108). These results clearly show that the activity of 6-DMAP is quite different from that of cycloheximide. In the last experiment, the levels of MPF were tested in those oocytes incubated for 24 hr in the medium containing 500 pM of 6-DMAP. As is shown in Table 1, GVBD occurred in nearly all cases in this group, but the chromosomes usually formed clumps of chromatin. These oocytes were fused to freshly isolated oocytes and cultured in 6-DMAP medium for 3 hr. Fused oocytes containing two GVs were discarded. As a control, metaphase I1 oocytes (cultured for 24 hr in control medium) were fused to GV-containing oocytes and also cultured for 3 hr in 6-DMAP containing medium. In this group, complete GVBD was observed in nearly all
GVBD OF BOVINE OOCYTES
Fig. 1. Bovine oocytes cultured for 24 hr in medium containing 2 mM 6-DMAP. Germinal vesicle (GV) is clearly visible. ~ 6 0 0 . Fig. 2. Oocytes cultured in medium with 0.5 mM 6-DMAP. GVBD has occurred, and the cluster of chromatin is visible in the cytoplasm (arrow). x600. Fig. 3. Oocytes cultured in medium with 2 mM DMAP, then washed and cultured in inhibitor-free medium. Metaphase I1 (MI with the first polar body (PB) is present in the cytoplasm. The metaphase chromosomes are thinner and randomly arranged. No spindle is present. x300. Fig. 4. Two clusters of chromatin (C) are visible in the giant cell
381
developed after fusion of a freshly isolated immature oocyte to an oocyte cultured for 24 hr in medium with 0.5 mM 6-DMAP. In Figures 4-6, the giant cells were cultured postfusion in medium containing 2 mM 6-DMAP for 3 hr. x600. Fig. 5. In some cases only slight changes occurred inside the GV after fusing oocytes as in Figure 4. Inside the GV, intensive chromatin condensation (C) is observed. ~ 3 0 0 . Fig. 6. After oocyte fusion as in Figure 4, GV of the immature oocyte remains completely intact. Arrow indicates a cluster of chromatin.
cases (411, and two groups of chromosomes were seen in 6-DMAP-incubated oocytes to GV-containing oocytes the cytoplasm. GVBD was incomplete or did not occur did not induce GVBD at the same frequency. In only 22 in four cases. In these cases, metaphase I1 oocytes were fused cells (35%) had GVBD of immature oocytes activated during manipulation and anaphase-telo- occurred, and usually two clumps of chromatin were phase I1 were present together with GV. Fusion of visible (Fig. 4).In 15 fused cells, GVBD was incomplete.
382
J. FULKA ET AL. TABLE 1. Effect of 6-DMAP on GVBD of Bovine Oocytes* 6-DMAP (mM) 0 0.1-0.5b 1.0 2.0 c+c 2.0 c-
No. of oocytes
GV
124 182 160 355 170
4 22 78 349 165
Stage of maturation after 22-24 hr of culturea Percent Percent LD-MI AI-TI MI1 GV GVBD 7 2
10 155+ 80+ 2+ 4f
103 3 2 4 1
3.2 12.1 48.7 98.3 97.1
96.8 87.9 51.3 1.7 2.9
*The number of degenerated oocytes did not exceed 5% of the total in all experiments. Each experiment was repeated at least three times. aGV, germinal vesicle; GVBD, GV breakdown; LD, late diakinesis; MI, metaphase I; AI, anaphase I; TI, telophase I; MII, metaphase I1 one polar body; chromosomes formed one or more clusters. True metaphase spindles were not observed. bResults of individual groups were not different, so the data were pooled. “C+(-), oocytes with or without cumulus.
+
+/-
phorylation processes (Gautier et al., 1989). In mammalian oocytes also, the process of GVBD is most likely Stage of maturationa under the control of MPF (Fulka, 1983; Moor, 1988), so No. of oocytes GV LD-MI AI-TI MI1 + 1 PB (%) it is highly probable that similar processes regulate the 302 4 35 6 257 onset of maturation. Indeed some changes in protein 46 (18)AB phosphorylation preceding GVBD were shown in 211 (82) N mouse (Schultz, 19881,sheep (Crosby et al., 1984; Moor, *The oocytes were cultured for 22-24 h r in 6-DMAP- 1988), and cow (Kastrop et al., 1990) oocytes. The supplemented medium (2 mM). Some oocytes were then discovery that 6-DMAP blocks phosphorylation withused as a control, the others were cultured in inhibitor free medium for the next 22-24 hr. Abbreviations as for Table 1. out affecting the overall rate of protein synthesis brings aN, normal MI1 configuration; AB, abnormal MI1 con- new insight into the study of the regulatory processes figuration. (Neant and Guerrier, 198813;Neant et al., 1989;Rime et al., 1989). As is shown in this study, in cow oocytes also, GV breakdown is effectively blocked when the oocytes are exposed to 6-DMAP. The effective dose of this drug is The nuclear membrane usually disappeared, but the similar to that used in the mouse (Rime et al., 1989)and remnants of nucleoplasm were still visible. In 26 pig (Motlik, personal communication). Moreover, as in oocytes GVBD did not occur. Here nearly intact GVs the mouse, this inhibition is reversible, but most of the were present together with the clumps of chromatin second metaphases are abnormal (Rime et al., 1989).It (Figs. 5, 6). The results are summarized in Table 4. is possible that some maturational processes occur even in the presence of 6-DMAP. It leads to asynchrony of DISCUSSION the maturation after washing out the inhibitor and The transition of the meiotic or mitotic cells from G2 culture in control medium. Our results show that the phase to M phase is under control of MPF, and its sensitive period in which it is still possible to block activity is controlled by the phosphorylation-dephos- GVBD lasts for approximately the first 4 hr. After this, TABLE 2. Reversibility of 6-DMAP Inhibition*
TABLE 3. Effect of Preincubation of Bovine Oocytes in Inhibitor-Free Medium and Subsequent Culture in 6-DMAP Supplemented Medium on GVBD Time sequence of GVBD in control medium Culture Stage of maturation interval (hr) GV/GVBD (%) 2 4 6 8
44/0 71/9 48/57 8/73
(100/0) (88.75/11.25) (45.7/54.3) (9.9/90.1)
Preincubation of oocytes in control medium with subsequent culture in 6-DMAP Control medium Stage of maturation 6-DMAP (hr) GVIGVBD (%)
+
2 4 6 8
++ 20 22 + 18 + 16
88/5 (94H5.4) 81/13 (86.2A3.8) 17/77 (18.U81.9) 14/116 (10.8/89.2)
GVBD OF BOVINE OOCYTES
383
TABLE 4. Fusion of Bovine Primary Oocytes Treated With 6-DMAP or Secondary Oocytes to GV-Stage Oocytes* Chromatin configurationa iGVBD PCC CL GV CL MI1 15 26
AII-TI1 Type of fusion 2 x CL GVBD 6-DMAP X GV 22 MI1 X GV 41 4 *All oocytes were incubated for 3 h r postfusion in medium containing 6-DMAP (500 pM). “CL, clusters of chromatin; iGVBD, incomplete GVBD (usually nuclear membrane disappeared, but ruminants of nucleoplasm with condensed chromatin were clearly visible; GV, intact germinal vesicle; PCC, prematurely condensed chromosomes; MII, second metaphase; AII, TII, anaphase 11, telophase 11.
+
the inhibitor is ineffective. Kastrop et al. (1990) observed extensive phosphorylation in bovine oocytes from 3 hr after the beginning of culture. At this time, the oocytes were still sensitive to 6-DMAP in our study. This may indicate that 6-DMAP indeed blocks phosphorylation. When matured oocytes were exposed to either cycloheximide or 6-DMAP, activation was frequently observed in the former group (82.6%)and less frequently in the latter (14.5%).Inhibitors of protein synthesis are potent parthenogenetic activators (Clarke and Masui, 1983).The common configuration observed in activated eggs was one pronucleus with the second polar body. On the other hand, we did not observe the decondensation of chromatin in maturing oocytes in those experiments in which precultured oocytes were transferred into the medium containing 6-DMAP. This decondensation is frequently observed when metaphase I mouse, starfish, mollusc, and echinoderm oocytes are exposed to 6DMAP (Rime et al., 1989; Neant and Guerrier, 1988a,b; Neant et al., 1989). In our experiments, condensed clusters of chromatin were present, but there was no chromatin decondensation. It may be that this decondensation is possible only at certain stages of maturation or that there are some species-specificdifferences. Motlik (unpublished) also did not observe this decondensation in pig metaphase I oocytes. The next possibility is that the dose of 6-DMAP was too low to induce decondensation. However, in our experiments, the higher doses negatively influenced oocyte morphology. In the last experiment, the oocytes matured in the presence of low doses of 6-DMAP (500 pM) in which GVBD is observed in nearly all cases were fused to immature oocytes. Whereas after fusion of metaphase I1 oocytes to GV containing oocytes, GVBD was rapidly induced and two sets of chromosomes were present in the cytoplasm in all oocytes (activated oocytes excluded), cytoplasm of 6-DMAP-treated oocytes does not induce similar changes. In only one-third of the fused cells were two sets of condensed chromosomes (clusters) present, whereas in the rest GVBD was incomplete, or intact GVs were present together with the clusters of chromatin. This indicates lower levels of MPF in 6-
+
+
+
DMAP oocytes than in those matured in normal medium. In the second case, GVBD is usually observed between 40 and 60 min after the initiation of fusion, and by 3 hr of culture two sets of perfectly condensed chromosomes are present (Fulka et al., 1986a,b). This indicates that the activity of MPF was most likely negatively influenced by 6-DMAP. Taken together, our data clearly show an inhibitory effect of 6-DMAP on GVBD in bovine oocytes. These results may indicate the necessity of phosphorylation for GVBD in this species, too, as has been shown for mouse oocytes. The exact mechanism and the role of phosphorylation are largely unknown in this species, but we may expect that this process is important for the transition from G2 to M, as is described for somatic cells and the oocytes of other species.
ACKNOWLEDGMENTS This research was supported by the Department of Meat and Animal Science, College of Agricultural and Life Sciences, University of Wisconsin-Madison, and USDA grant 88-372403740. We thank Brad Haley for obtaining bovine material and Julie Busby for secretarial assistance. REFERENCES Adlakha RC, Rao PN (1987): Regulation of mitosis by nonhistone protein factors in mammalian cells. In RA Schlegel, MS Halleck, PN Rao (eds):“Molecular Regulation of Nuclear Events in Mitosis and Meiosis.” New York: Academic Press, pp 179-226. 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 Zoo1 229:459-466. Cross F, Roberts J , Weintraub H (1989): Simple and complex cell cycles. Annu Rev Cell Biol 5:341-395. Fulka J J r . (1983): Nuclear maturation in pig and rabbit oocytes after interspecific fusion. Exp Cell Res 146:212-281. Fulka J Jr., Motlik J., Fulka J , Crozet N (1986a): Activity of maturation promoting factor in mammalian oocytes after its dilution by single and multiple fusioins. Dev Biol 118:176-181. Fulka J Jr., Motlik J., Fulka J., Jilek F (1986b3: Effect of cycloheximide on nuclear maturation of pig and mouse oocytes. J Reprod Fertil 77:281-285.
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Gautier J, Matsukawa T, Nurse P, Maller J (1989): Dephosphorylation and activation of Xenopus p34 cdc2 protein kinase during the cell cycle. Nature 339:626-669.Kastrop PMM, Bevers MM, Destree OHJ, Kruip TAM (1990): Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro. J Reprod Fertil90:305-310. Leibfried L, First NL (1979): Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci 48:76-86. Lohka MJ, Maller JL (1987): Regulation of nuclear formation and breakdown in cell-free extracts in amphibian eggs. In RA Schlegel, MS Halleck, PN Rao (eds):“Molecular Regulation of Nuclear Events in Mitosis and Meiosis.” New York: Academic Press, pp 67-109. Maller JL, Gautier J , Langan TA, Shenoy S, Shalloway D, Nurse P (1989) Maturation-promoting factor and the regulation of the cell cycle. J Cell Sci 12 [Suppll:53-63. Moor RM (1988):Regulation ofthe meiotic cycle in oocytes of domestic animals. Ann NY Acad Sci 541:248-258. Moor RM, Crosby IM (1986): Protein requirements for germinal vesicle breakdown in ovine oocytes. J Embryo1 Exp Morphol94:207220.
Neant I, Charbonneau M, Guerrier P (19891: A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms. Dev Biol 132:304-314. Neant I, Guerrier P (1988a): Meiosis reinitiation in the mollusc patella vulgata. Regulation of MPF, CSF and chromosomes condensation activity by intracellular pH, protein synthesis and phosphorylation. Development 102:505-516. Neant I, Guerrier P (1988b): 6-dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinse activity. Exp Cell Res 176:68-79. Ozon R, Mulner 0, Boyer J, Belle R (1987):Role of protein phosphorylation in Xenopus oocyte maturation. In RA Schlegel, MS Halleck, PN Rao (eds): “Molecular Regulation of Nuclear Events in Mitosis and Meiosis.” New York: Academic Press, pp 111-130. Rime H, Neant I, Guerrier P, Olon R (1989): 6-Dimethylaminopurine (6-DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte. Dev Biol 133:169-179. Schultz RM (1988): Role of protein phosphorylation in meiotic maturation of mouse oocytes in vitro. Ann NY Acad Sci 541:217-227.
