MOLECULAR REPRODUCTION AND DEVELOPMENT 3 2 1 - 8 (1992)
Changes in Protein Synthesis Pattern During In Vitro Maturation of Goat Oocytes F. LE GAL, L. GALL, AND V. DE SMEDT Unit6 de Biologie de la Fkcondation, Station de Physiologie Animale, I N R A , Jouy-en-Josas, France
ABSTRACT The regulation of meiotic events of goat oocytes from prophase I to metaphase tI was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e.,chromosome condensation, took place even in a cycloheximide-containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine.These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis. 0 1992 Wiley-Liss, Inc. Key Words: Meiotic maturation, Germinal vesicle breakdown, Cycloheximide
INTRODUCTION Oocytes enter meiosis in early prenatal life and progress to the diplotene stage of prophase I [germinal vesicle (GV) stage], at which they remain arrested until just prior to ovulation. The resumption of meiosis is mediated in vivo by a hormonal stimulus (Ireland and Roche, 1982; Dieleman e t al., 1983; Callesen et al., 1986) and is obtained in vitro by releasing oocytes from their follicular environment and culturing them under suitable conditions (Pincus and Enzman, 1935; Edwards, 1965; Thibault and Gerard, 1973). In both cases, oocytes undergo nuclear progression from GV to the metaphase I1 stage, and they remain arrested at this stage until fertilization. During meiotic maturation in mammals, many intracellular changes occur related to the nuclear structure, membrane transport, cytoplasmic distribution of organelles (reviewed by Thibault e t
0 1992 WILEY-LISS, INC.
al., 1987), and pattern of protein synthesis (Golbus and Stein, 1976; Schultz and Wassarman, 1977; Warnes et al., 1977; McGaughey and Van Blerkhom, 1977; Moor and Crosby, 1986; Kastrop et al., 1990). The reprogramming of protein synthesis plays a n essential role in the control of meiotic division. Mouse and r a t oocytes cultured in the presence of a protein synthesis inhibitor undergo GV breakdown (GVBD) but cannot proceed beyond this stage (Schultz and Wassarman, 1977; Ekholm and Magnusson, 1979; Szollosi et al., 1991). In contrast, resumption of meiosis in large domestic animals requires a n active protein synthesis for GVBD to occur (pig, Fulka et al., 1986; sheep, Moor and Crosby, 1986; cattle, Hunter and Moor, 1987). After GVBD, sensitivity to protein synthesis inhibition differs among species. Inhibition of protein synthesisjust after GVBD prevented the first polar body extrusion both in mouse (Clarke and Masui, 1983; Szollosi et al., 1991) and bovine (Sirard et al., 1989) oocytes, while inhibition of protein synthesis near metaphase I prevented that extrusion in mouse (Clarke and Masui, 1983) but did not in cattle (Sirard et al., 1989). However, in all species so far studied, some protein synthesis is still needed for the transition from metaphase I to metaphase 11; otherwise, chromatin decondensation occurs and a pronucleus-like structure appears (mouse, Siracusa et al., 1978; Clarke and Masui, 1983; cattle, Sirard et al., 1989). The present studies were undertaken to establish the timing of nuclear changes characterizing the maturation of goat oocytes and the modifications in the protein synthesis pattern associated with these changes. Also studied was the interdependency of these two phenomena. To this end, experiments were carried out using a protein synthesis inhibitor (cycloheximide).
MATERIALS AND METHODS Oocyte Collection and Culture Conditions Adult goats (more than 30 kg body weight) of both Alpine and Saanen breeds were used for experiments. Follicular development was stimulated by intramuscular injections of follicle-stimulating hormone (pFSH;
Received October 3, 1991; accepted November 25,1991. Address reprint requests to F. Le Gal, INRA, Unite de Biologie de la Fecondation, Station de Physiologie Animale, 78352 Jouy-enJosas cedex, France.
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F. LE GAL ET AL. TABLE 1. Time Sequence of I n Vitro Nuclear Maturation of Goat Oocytes* Time (hr) 3 6 7
8 9
12 15
18 27
No.
GV
GVBD
20
100
-
21 19 21
81
19
52
21
14 9
14
33 36 39
50
37
-
-
4.5 47
Pro-MI
MI
Ana-Tel I
Pro-MI1
MI1
-
-
-
-
-
10 16
-
19 62 51.5 50
-
-
-
2.5
-
4.5
-
10 10 39.5 42
-
-
-
77
8 2.5
10
4
-
18
-
-
-
-
86
*GV,germinal vesicle; GVBD, GV breakdown; Pro-MI,prometaphase I; MI, metaphase I; Ana-TelI, anaphase-telophase I transition; Pro-MII,prometaphase 11;MII, metaphase 11. Burns-biotec, Sanofi) 3-2-2 mg, 48-36-26 h r before slaughter on day 15-16 of the estrus cycle (day 0 = day of estrus). Ovaries were dissected a t 30-35"C, and nonatretic antral follicles (Moor et al., 1978) between 2 and 6 mm in diameter were isolated in phosphate-buffered saline (PBS) containing sodium pyruvate (36 mglliter), glucose (1g/liter), penicillin (0.06 giliter), and streptomycin (0.05 giliter). Cumulus-oocyte complexes (COCs) were released from the follicles and washed from the follicular fluid in TC 199-20 mM Hepes medium (Flow Laboratories). Mural granulosa cells were collected, centrifuged a t 1,200g for 5 min, washed twice, resuspended in TC 199Hepes medium, and diluted to lo6 cells/ml for coculture with the COCs. The TC 199-Hepes medium was supplemented for culture with FSH and luteinizing hormone (LH; 10 pgiml), estradiol17p (1pg/ml),fetal calf serum (10%)a s previously described for sheep oocytes (Staigmiller and Moor, 1984). The culture dishes were maintained on a rotary shaker a t a low speed throughout the incubation period. Temperature was kept at 39°C as previously described for goats (De Smedt et al., submitted).
plemented TC 199-Hepes without methionine; Proscience) at the onset of culture. Radioactive concentration varied from 750 pCi/ml for short labelling times (3 and 8 hr) to 250 pCi/ml for longer incubations (15-27 hr). Labelled oocytes were denuded of cumulus cells by repeated pipetting and assessed for maturation stage by using fluorescent DNA-binding dye (Hoechst dye 33342; Calbiochem) (10 pliml, 15 mn, 38°C) before being lysed individually in 15 pl sodium dodecyl sulfate (SDS)-sample buffer (Laemmli, 1970). Gel Electrophoresis Radiolabelled proteins were separated and resolved by SDS polyacrylamide gel electrophoresis (SDSPAGE) according to Laemmli (19701, using 7.5-18% linear gradient slab gels of 0.75 mm thickness. Colored proteins of known molecular weight (range 14-200 kD; Amersham) were run simultaneously as standards. When dried, the gels were exposed to Fuji X-ray films at -80°C for 10 days.
RESULTS Timing of Nuclear Events During At various times after the onset of culture, oocytes In Vitro Maturation were denuded of cumulus cells by repeated pipetting, As is shown in Table 1, oocytes after having been rinsed, and fixed in 95% ethanol: 90% acetic acid (3:l) for 24 hr a t 4°C and stained with lacmoid for determina- removed from their follicles underwent GVBD (characterized by the disappearance of both the nuclear envetion of their nuclear status. lope and the nucleolus) by 7-8 h r after explantation. Inhibition of Protein Synthesis After 9 hr, the majority of oocytes were in prometaProtein synthesis was inhibited a t different times phase I. This meiotic stage could be very long in goat during the maturation process by adding 20 pg/ml cy- species, since 6 h r later one-half of the oocytes were still cloheximide (Sigma) to the culture medium. In some in prometaphase I. Nevertheless, 18 h r after the onset experiments, protein synthesis was inhibited from the of culture, all the oocytes reached metaphase I (characbeginning of culture and the oocytes were subsequently terized by a complete individualization of the chromowashed and cultured in a n inhibitor-free medium to somes, which were organized in a metaphase plate constudy the reversibility of action of this drug. Nuclear figuration) or prometaphase I1 stage. Thus two groups maturation status was determined after lacmoid stain- of oocytes could be distinguished: one group with a very long prometaphase I stage (from 9 to 15 hr) and a short ing. metaphase I and another group with a short proRadiolabelling of Oocyte Proteins metaphase I stage (from 9 to 12 hr) followed by a TOstudy protein synthesis, [35S]metliionine (>1,000 long metaphase I (from 12 to 18 hr). Establishment of Ci mmolpl; Amersham) was added to the medium (sup- metaphase I1 (evidenced by the extrusion of the first Assessment of M a t u r a t i o n Stages
PROTEIN SYNTHESIS DURING IN VITRO OOCYTE MATURATION
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Fig. 1. Changes in chromatin configuration from the initiation of maturation to its completion. Photomicrographs of fixed and stained whole mounts of goat oocytes after various times of culture representing the essential steps of maturation. a: GV-stage oocyte. b: GVBDstage oocyte (8 hr). The chromatin is condensed. c: Metaphase I-stage
oocyte (15hr). The chromosomes are individualized and organized on a metaphase plate. The spindle microtubules are clearly visible. d Metaphase I1 stage oocyte (27 hr). The first polar body (PB) is extruded. Bars = 5 km.
polar body) was almost completed (>85%)a t 27 h r after the initiation of the maturation process (Table 1, Fig. 1a-d) .
effect was obtained when the drug was added after 2 h r of culture (100%GV stage a t the end of the 27 h r maturation period). Two classes of oocytes were found when the cycloheximide was added 5 h r after the onset of culture: 1)GV oocytes with condensed chromosomes (35%) as previously observed and 2) abnormal GVBD-metaphase I oocytes with extremely closed and condensed chromosomes (65%)(Fig. 2b). These structures were similar to
Effects of Protein Synthesis Inhibition Incubation of oocytes from the beginning of culture in the cycloheximide-containing medium (20 pg/ml) prevented the rupture of the GV (Table 2). Nevertheless, chromosomes were very condensed (Fig. 2a). The same
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F. LE G A L ET AL. TABLE 2. Effect of Cycloheximide on In Vitro Maturation of Goat Oocytes* Presence of CHX (hr) No. GV GVBD MI Ana-Tel I MI1 Nucleus-like 0 8 0 0 0 13 92 0-27 0 0 0 0 0 12 100 2-27 0 0 0 65 20 35 0 5-27 10 5 0 85 0 0 0-22 then without until 27 20 0 0 0 100 0 0 0-22 then withoutuntil46 21 0 0 78" 17.5 4.5 23 0 15-27 *CHX, cycloheximide; GV, germinal vesicle; GVBD, GV breakdown; MI, metaphase I; Ana-Tel I, anaphase-telophase I transition; MII, metaphase 11. aFifty-eightpercent have extruded the first polar body; 42%have not. They present either one nucleus or several micronuclei (50-50).
the clusters originally described in mouse oocytes by Hashimoto and Kishimoto (1988) and Szollosi et al. (1991). To establish further whether the transition from metaphase I to metaphase I1 was protein synthesis dependent, cycloheximide was administered close to metaphase I (15 h r after the onset of culture; Table 1). After 12 h r of incubation with the drug, 78% of oocytes reformed a nucleus-like structure containing decondensed chromatin (Fig. 2d). Two subpopulations were distinguished, i.e., oocytes that extruded their first polar body (58%)and those that failed to extrude it (42%). In the latter case, they exhibited either one nucleus or several micronuclei (50-50). The meiotic arrest induced by the addition of cycloheximide a t the onset of culture was reversible. Oocytes exposed to the drug during the first 22 h r of culture were able to progress through the maturation process after extensive washing and culture in a drug-free medium. Thus 5 hr after drug removal all the oocytes reached a t least the metaphase I stage (Table 2). This progression was accompanied by a n acceleration of the nuclear maturation process. Nevertheless, when cultured for 22 h r in a cycloheximide-containing medium and for 24 h r more without the drug, no oocyte overpassed the metaphase I stage. These metaphase I oocytes exhibited very condensed chromosomes, but the metaphase plate was slackened and the spindle presented a n abnormal morphology (Fig. 2c). This could be due to the oocyte aging, because, when the observation was made only 5 hr after removal of the drug, metaphase I was normal (not shown).
Protein Synthesis During Maturation At the end of the labelling period, a nuclear examination of each oocyte was performed with Hoechst dye to correlate exactly the meiotic stage and the protein synthesis pattern. Figure 3 shows four main differences in the protein synthesis pattern between immature and mature oocytes. First, a polypeptide of 25 kD molecular weight was replaced by a new labelled one of 27 kD. Another shift resulted in a 34 kD appearing at the 33 kD region. A t the GV stage, a polypeptide of 43 kD was the major synthesized polypeptide, whereas its labelling dramatically decreased after 27 h r of culture. Twodimensional gel electrophoresis identified this polypep-
tide a s actin (not shown). The last marked change was the large increase in the synthesis of a 67 kD protein. The synthesis of this protein was maximal at 27 hr. Some other slight differences were observed, i.e., two polypeptides of 44 and 47 kD, respectively, were not synthesized by the immature oocyte. The time-dependent change of this protein synthesis pattern during the time course of maturation is shown in Figure 4. Thus, within individual oocytes, the shift from 25 to 27 kD was closely associated with GVBD. This change was not observed in GV-arrested oocytes even after 27 h r of culture (not shown). Polypeptides of 44 and 47 kD also appeared at GVBD. At the same time, a new polypeptide of 33 kD appeared, whereas concomitantly a polypeptide of 34 kD became barely detectable and finally disappeared during the second half of the maturation process. At the metaphase I stage (15 hr), the synthesis of a 67 kD polypeptide began to increase strongly and became predominant after 18 h r of culture. At the same time, the synthesis of actin greatly decreased. This drop was not observed in oocytes t h a t had not overpassed metaphase I at the end of culture (not shown). However, it is noteworthy that the incorporation rate of radioactive methionine was progressively reduced, particularly after the establishment of metaphase I. It has already been reported that oocyte permeability dropped as maturation progressed (Wassarmann, 1988).
DISCUSSION The results presented here show the timing of nuclear events in goat oocytes during in vitro maturation. Oocytes remained at the GV stage from the onset to 6 8 h r of culture. GVBD occurred between 7 and 9 hr. Metaphase I became established within 12-18 hr. Finally, most oocytes reached the metaphase I1 stage after 27 hr. These data are comparable to those reported for sheep (Moor and Crosby, 1985) and cattle (Sirard et al., 1989). Experiments on protein synthesis during in vitro maturation enabled us to identify some polypeptides whose synthesis was correlated with the meiotic nuclear status of the oocyte. Thus the shift from a polypeptide of 25 kD to one of 27 kD was due to the occurrence of GVBD, since it was never observed in a n oocyte arrested a t the GV stage even after 27 h r of culture (i.e.,
PROTEIN SYNTHESIS DURING IN VITRO OOCYTE MATURATION
5
Fig. 2. Effects of cycloheximide on the meiotic maturation of goat oocytes. Immature oocytes after isolation were incubated with cycloheximide (20 pgiml) for various periods of time. They were fixed and stained for evaluation of their meiotic status after 27 (a, b, d) or 46 (c, c') hr of culture. a: Cycloheximide was added a t the onset of culture. Oocyte is still at the GV stage, but the chromatin is very condensed. Many nucleoles are visible (arrowheads). b The drug was added 5 hr after the onset of culture. The chromosomes are closed and very condensed and form a cluster. c , c': The drug was added a t the onset of
culture and removed after 22 hr. Culture without inhibitor (after extensive washing) was prolonged for 24 hr. The chromosomes are very condensed and are organized on a very large metaphase plate. This structure is abnormal in terms of spindle morphology. d: The drug was added near the metaphase I stage (15 hr after the onset of culture). The oocyte has emitted the first polar body (PB). A nuclear membrane has been reformed, which surrounds the fully decondensed chromatin. This nucleus ( N ) has a pronucleus-like structure. Bars = 5 pm.
nonmeiosis-resuming oocyte; data not shown). However, we cannot determine whether this change was a combination of a n arrest of 25 kD polypeptide synthesis and the synthesis of a new 27 kD polypeptide or was a result of posttranslational modifications of the 25 kD polypeptide. The presence of the 34 kD polypeptide was correlated to the GV stage. At GVBD, there was a transient period
during which 34 and 33 kD polypeptides were both present. The presence of only the 33 kD polypeptide was associated with the metaphase I stage. This change in the electrophoretic motility between 34 and 33 kD has already been described in many species during mitosis and meiosis in Xenopus (Doree et al., 1989), in the starfish (Labbe et al., 19891, and in the mouse (Howlett, 1986; Morla e t al., 1989). It is now clear that the entry
6
F. LE GAL ET AL.
Fig. 3. Fluorogram of ["Slmethionine-labelled polypeptides from goat oocytes a t the GV stage and after maturation for 27 hr in vitro. Polypeptides were separated on a 7.5-18% linear gradient SDS-polyacrylamide gel. Each lane represents the pattern of one oocyte. Lane 1: Pattern of synthesis in GV-stage oocytes incubated with [%]methionine for 3 hr. Lane 2 Pattern of a metaphase 11-stage oocyte incubated during the 27 hr of maturation. The main differences observed were that 27, 33, 44, 47, and 67 kD polypeptides appeared, while 25 and 34 kD polypeptides disappeared. Actin synthesis has dropped.
of cells into the M phase is induced by a maturationpromoting factor (MPF) originally described by Masui and Markert (1971). One of the subunits of mammalian MPF is homologous to the gene product of the yeast gene cdc2. The protein coded for by the gene is a protein kinase with a relative molecular mass of 34 kD (Lee and Nurse, 1988; Lohka et al., 1988; Draetta and Beach, 1988). The 34 kD polypeptide observed in the goat may be related to the cdc2 gene product. Further studies are in progress to determine more precisely the behavior of these two polypeptides. The ultimate part of the maturation process was characterized by the increase in 67 kD polypeptide synthesis and the drop in actin synthesis. Actin was the major synthetic product of immature oocyte. Moor et al. (1983) reported that it accounted for 8-10% of the total synthesized proteins in the ovine oocyte. According to our results, the decrease in actin synthesis seemed to be a n essential characteristic of a normal maturation process, since a high rate of actin synthesis after 27 h r of
Fig. 4. Timing of protein synthesis pattern modifications during maturation of goat oocytes. L3"Slmethioninewas added at the onset of culture. Each lane represents the pattern of one oocyte. Lane 1: GV stage oocyte (same as lane 1in Fig. 3). Lane 2 After 8 hr of culture. GVBD-stage oocyte. Two shifts are detectable: 25-27 kD and 34-33 kD (34 kD polypeptide barely detectable); 44 and 47 kD polypeptides have also appeared. Lane 3: After 15 hr of culture. Metaphase I stage. The 34 kD polypeptide has totally disappeared. Lane 4 After 18 hr of culture. No observable difference could be observed compared with the 15 hr situation. Lane 5 After 27 hr of culture. Metaphase I1 stage. Actin synthesis has dropped, whereas the 67 kD polypeptide synthesis has become very extensive.
culture was in each case associated to oocytes that exhibited abnormal nuclear and cytoplasmic morphologies (data not shown). The specific 47 kD polypeptide synthesized before GVBD as reported by Moor and Crosby (1986) for ovine oocytes was also detected in the goat species. The reprogramming of protein synthesis that occurred during maturation is essential for the differentiation events required during fertilization and early development (reviewed by Moor e t al., 1983; Thibault et al., 1987; Wassarmann, 1988). In this respect, it would be interesting to determine the exact functions of these different proteins. The present results revealed a high sensitivity of goat oocytes to cycloheximide. In goats, protein synthesis was required for the GV to undergo breakdown, a s in pigs (Fulka et al., 1986). sheer, (Moor and Crosbv. 19867, and cattle (Hunter and Moo;, 1987).When cyc6:
PROTEIN SYNTHESIS DURING IN VITRO OOCYTE MATURATION heximide was administered after 5 h r of culture, 65% of oocytes overpassed GVBD, indicating that the required synthesis occurred within the first 5-6 h r of culture and that these specific proteins necessary for GVBD were not present in the ooplasm of fully grown oocytes. By contrast, resumption of meiosis in the mouse is not dependent on active protein synthesis, suggesting that the proteins required for M phase transition were probably already present in the prophase oocyte. These data indicate the substantial species differences in the protein synthesis required for GVBD. However, even if nuclear membrane breakdown was inhibited by cycloheximide, some chromosome condensation was observed in the karyoplasm, suggesting that the induction of chromosome condensation does not require protein synthesis, a phenomenon also described for pig (Kubelka e t al., 1988) and cattle (Simon e t al., 1989) oocytes. The present results and previous results for pigs, cattle, and sheep imply that the activity that induces chromatin condensation is different from the activity that induces the breakdown of the nuclear envelope. Inhibition of GVBD in goats by cycloheximide was partly reversible. When cycloheximide was present in the culture medium during the first 22 hr, removal of the drug resulted in 85% of oocytes reaching the metaphase I stage after 5 h r of culture in the drug-free medium. More remarkably, removal of the oocytes from the cycloheximide-containing medium resulted in a n acceleration of the GVBD process, as previously described for sheep (Moor and Crosby, 1986) and pigs (Kubelka et al., 1988). It is noteworthy that cycloheximide does not prevent chromosome condensation, indicating that the GVBD process is partially engaged. This could explain the acceleration observed after removal of the drug. However, under these conditions, oocytes did not proceed beyond the metaphase I stage (Table 2). We can conclude from all these results that inhibition of protein synthesis might lead to a partial MPF activation, which in this case is competent only for chromosome condensation. Thus protein synthesis is not the only mechanism involved in meiosis resumption. Synthesis of proteins is also required for the establishment of the metaphase I1 stage. The administration of cycloheximide 15 h r after the beginning of culture induced the formation of a nuclear envelope (78%), which indicated that the oocytes had entered interphase. However, two types of response to the protein synthesis inhibitor could be distinguished according to the meiotic stage reached by oocytes a t the time of drug addition. Of these oocytes, 52% had extruded their first polar body and 42% did not. However, in the latter case, one nucleus or several micronuclei were observed in the same proportion. As was previously indicated, after 15 h r of culture, one-half of the oocytes were in prometaphase, i.e., before the formation of a well-organized metaphase spindle, while the second one-half of the oocytes had reached full metaphase-stage organization. We can speculate that the drug applied a t the
7
prometaphase stage affects the formation of the spindle, inhibiting polar body extrusion, and induces formation of nuclear envelope(s) around dispersed chromosomes, whereas the drug applied at metaphase I does not inhibit polar body extrusion, suggesting that these oocytes escape the effect of the drug until telophase, and also induces the formation of a nuclear envelope. This nucleus reformation suggests a progression of the cycle onto a n interphase state, which might correspond to a loss of MPF activity. Similar results have been obtained in cattle (Sirard et al., 1989) and mouse (Clarke and Masui, 1983; Szollosi et al., 1991). These results reveal a n oocyte sensitivity to protein synthesis inhibition between prometaphase and metaphase I, while the transition from metaphase I to telophase is not protein synthesis dependent, a result already described for the mouse (Szollosi e t al., 1991). Nevertheless, the protein synthesis patterns obtained did not reveal newly synthesized polypeptides between metaphase I and metaphase 11. The only visible difference was the high rate of increase in the synthesis of a 67 kD polypeptide, suggesting a role for this protein in the control of metaphase I-metaphase I1 transition. It can also be speculated that a continuous synthesis of certain proteins is necessary for the establishment and maintenance of the metaphase I1 stage. Another nonexclusive possibility was a posttranslational regulation (like phosphorylation) of this transition, a possibility that might explain the absence of observational differences in the protein synthesis patterns. Indeed it has now been well established that phosphorylation plays a major role in nuclear changes and cell events. The role of phosphorylations during goat oocyte maturation will be the subject of further studies.
ACKNOWLEDGMENTS We thank Dr N. Crozet for critical reading the manuscript and R. Scandolo and F. Fort for the photographic work. This work was supported in part by grant from Conseils Regionaux des Regions Centre et PoitouCharentes to F.L.G.
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cle breakdown in ovine oocytes. J Embryol Exp Morphol 94207220. Moor RM, Crosby IM (1987):Cellular origin, hormonal regulation and biochemical characteristics of polypeptides secreted by Graafian follicles of sheep. J Reprod Fertil79:469483. Moor RM, Crosby IM, Osborn J C (1983): Growth and maturation of mammalian oocytes. In: In Vitro Fertilization and Embryo Transfer. London: Academic Press, pp 39-63. Moor RM, Hay MF, Dott DM, Cran DG (1978):Macroscopic identification and steroidogenic function of atretic follicles in sheep. J Endocrinol77:309-318. Morla AO, Draetta G, Beach D, Wang JYJ (1989): Reversible tyrosine phosphorylation of cdc2: Dephosphorylation accompanies activation during entry into mitosis. Cell 58:193-203. Pincus G, Enzman EV (1935): The comparative behavior of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J Exp Med 62665. Schultz RM, Wassarman PM (1977): Biochemical studies of mammalian oogenesis: Protein synthesis during oocyte growth and meiotic maturation in the mouse. J Cell Sci 24:167-194. Simon M, Jilek F, Fulka J J r (1989): Effect of cycloheximide upon maturation of bovine oocytes. Reprod Nutr Dev 29533-540. Siracusa G, Whittingham DG, Molinaro M, Vivarelli E (1978):Parthenogenetic activation of mouse oocytes induced by inhibitors of protein synthesis. J Embryol Exp Morphol43:157-166. 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. Staigmiller RB, Moor RM (1984): Effect of follicle cells on the maturation and developmental competence of ovine oocytes matured outside the follicle. Gamete Res 9:221-229. Szollosi MS, Debey P, Szollosi D, Rime H, Vautier D (1991):Chromatin behaviour under influence of puromycin and 6-DMAP a t different stages of mouse oocyte maturation. Chromosoma 100:339-354. Thibault C , Gerard M (1973): Cytoplasmic and nuclear maturation of rabbit oocyte in vitro. Ann Biol Anim Biochem Biophys 13:145-156. Thibault C, Szollosi D, Gerard M (1987): Mammalian oocyte maturation. Reprod Nutr Dev 27:865-896. Warnes GM, Moor RM, Johnson MH (1977): Changes in protein synthesis during maturation of sheep oocytes in vivo and in vitro. J Reprod Fertil49:331-335. Wassarman PM (1988):The mammalian ovum. In: Knobil E and Neil J (eds): “The Physiology of Reproduction.” New York: Raven Press pp 69-103.
Changes in Protein Synthesis Pattern During In Vitro Maturation of Goat Oocytes F. LE GAL, L. GALL, AND V. DE SMEDT Unit6 de Biologie de la Fkcondation, Station de Physiologie Animale, I N R A , Jouy-en-Josas, France
ABSTRACT The regulation of meiotic events of goat oocytes from prophase I to metaphase tI was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e.,chromosome condensation, took place even in a cycloheximide-containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine.These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis. 0 1992 Wiley-Liss, Inc. Key Words: Meiotic maturation, Germinal vesicle breakdown, Cycloheximide
INTRODUCTION Oocytes enter meiosis in early prenatal life and progress to the diplotene stage of prophase I [germinal vesicle (GV) stage], at which they remain arrested until just prior to ovulation. The resumption of meiosis is mediated in vivo by a hormonal stimulus (Ireland and Roche, 1982; Dieleman e t al., 1983; Callesen et al., 1986) and is obtained in vitro by releasing oocytes from their follicular environment and culturing them under suitable conditions (Pincus and Enzman, 1935; Edwards, 1965; Thibault and Gerard, 1973). In both cases, oocytes undergo nuclear progression from GV to the metaphase I1 stage, and they remain arrested at this stage until fertilization. During meiotic maturation in mammals, many intracellular changes occur related to the nuclear structure, membrane transport, cytoplasmic distribution of organelles (reviewed by Thibault e t
0 1992 WILEY-LISS, INC.
al., 1987), and pattern of protein synthesis (Golbus and Stein, 1976; Schultz and Wassarman, 1977; Warnes et al., 1977; McGaughey and Van Blerkhom, 1977; Moor and Crosby, 1986; Kastrop et al., 1990). The reprogramming of protein synthesis plays a n essential role in the control of meiotic division. Mouse and r a t oocytes cultured in the presence of a protein synthesis inhibitor undergo GV breakdown (GVBD) but cannot proceed beyond this stage (Schultz and Wassarman, 1977; Ekholm and Magnusson, 1979; Szollosi et al., 1991). In contrast, resumption of meiosis in large domestic animals requires a n active protein synthesis for GVBD to occur (pig, Fulka et al., 1986; sheep, Moor and Crosby, 1986; cattle, Hunter and Moor, 1987). After GVBD, sensitivity to protein synthesis inhibition differs among species. Inhibition of protein synthesisjust after GVBD prevented the first polar body extrusion both in mouse (Clarke and Masui, 1983; Szollosi et al., 1991) and bovine (Sirard et al., 1989) oocytes, while inhibition of protein synthesis near metaphase I prevented that extrusion in mouse (Clarke and Masui, 1983) but did not in cattle (Sirard et al., 1989). However, in all species so far studied, some protein synthesis is still needed for the transition from metaphase I to metaphase 11; otherwise, chromatin decondensation occurs and a pronucleus-like structure appears (mouse, Siracusa et al., 1978; Clarke and Masui, 1983; cattle, Sirard et al., 1989). The present studies were undertaken to establish the timing of nuclear changes characterizing the maturation of goat oocytes and the modifications in the protein synthesis pattern associated with these changes. Also studied was the interdependency of these two phenomena. To this end, experiments were carried out using a protein synthesis inhibitor (cycloheximide).
MATERIALS AND METHODS Oocyte Collection and Culture Conditions Adult goats (more than 30 kg body weight) of both Alpine and Saanen breeds were used for experiments. Follicular development was stimulated by intramuscular injections of follicle-stimulating hormone (pFSH;
Received October 3, 1991; accepted November 25,1991. Address reprint requests to F. Le Gal, INRA, Unite de Biologie de la Fecondation, Station de Physiologie Animale, 78352 Jouy-enJosas cedex, France.
2
F. LE GAL ET AL. TABLE 1. Time Sequence of I n Vitro Nuclear Maturation of Goat Oocytes* Time (hr) 3 6 7
8 9
12 15
18 27
No.
GV
GVBD
20
100
-
21 19 21
81
19
52
21
14 9
14
33 36 39
50
37
-
-
4.5 47
Pro-MI
MI
Ana-Tel I
Pro-MI1
MI1
-
-
-
-
-
10 16
-
19 62 51.5 50
-
-
-
2.5
-
4.5
-
10 10 39.5 42
-
-
-
77
8 2.5
10
4
-
18
-
-
-
-
86
*GV,germinal vesicle; GVBD, GV breakdown; Pro-MI,prometaphase I; MI, metaphase I; Ana-TelI, anaphase-telophase I transition; Pro-MII,prometaphase 11;MII, metaphase 11. Burns-biotec, Sanofi) 3-2-2 mg, 48-36-26 h r before slaughter on day 15-16 of the estrus cycle (day 0 = day of estrus). Ovaries were dissected a t 30-35"C, and nonatretic antral follicles (Moor et al., 1978) between 2 and 6 mm in diameter were isolated in phosphate-buffered saline (PBS) containing sodium pyruvate (36 mglliter), glucose (1g/liter), penicillin (0.06 giliter), and streptomycin (0.05 giliter). Cumulus-oocyte complexes (COCs) were released from the follicles and washed from the follicular fluid in TC 199-20 mM Hepes medium (Flow Laboratories). Mural granulosa cells were collected, centrifuged a t 1,200g for 5 min, washed twice, resuspended in TC 199Hepes medium, and diluted to lo6 cells/ml for coculture with the COCs. The TC 199-Hepes medium was supplemented for culture with FSH and luteinizing hormone (LH; 10 pgiml), estradiol17p (1pg/ml),fetal calf serum (10%)a s previously described for sheep oocytes (Staigmiller and Moor, 1984). The culture dishes were maintained on a rotary shaker a t a low speed throughout the incubation period. Temperature was kept at 39°C as previously described for goats (De Smedt et al., submitted).
plemented TC 199-Hepes without methionine; Proscience) at the onset of culture. Radioactive concentration varied from 750 pCi/ml for short labelling times (3 and 8 hr) to 250 pCi/ml for longer incubations (15-27 hr). Labelled oocytes were denuded of cumulus cells by repeated pipetting and assessed for maturation stage by using fluorescent DNA-binding dye (Hoechst dye 33342; Calbiochem) (10 pliml, 15 mn, 38°C) before being lysed individually in 15 pl sodium dodecyl sulfate (SDS)-sample buffer (Laemmli, 1970). Gel Electrophoresis Radiolabelled proteins were separated and resolved by SDS polyacrylamide gel electrophoresis (SDSPAGE) according to Laemmli (19701, using 7.5-18% linear gradient slab gels of 0.75 mm thickness. Colored proteins of known molecular weight (range 14-200 kD; Amersham) were run simultaneously as standards. When dried, the gels were exposed to Fuji X-ray films at -80°C for 10 days.
RESULTS Timing of Nuclear Events During At various times after the onset of culture, oocytes In Vitro Maturation were denuded of cumulus cells by repeated pipetting, As is shown in Table 1, oocytes after having been rinsed, and fixed in 95% ethanol: 90% acetic acid (3:l) for 24 hr a t 4°C and stained with lacmoid for determina- removed from their follicles underwent GVBD (characterized by the disappearance of both the nuclear envetion of their nuclear status. lope and the nucleolus) by 7-8 h r after explantation. Inhibition of Protein Synthesis After 9 hr, the majority of oocytes were in prometaProtein synthesis was inhibited a t different times phase I. This meiotic stage could be very long in goat during the maturation process by adding 20 pg/ml cy- species, since 6 h r later one-half of the oocytes were still cloheximide (Sigma) to the culture medium. In some in prometaphase I. Nevertheless, 18 h r after the onset experiments, protein synthesis was inhibited from the of culture, all the oocytes reached metaphase I (characbeginning of culture and the oocytes were subsequently terized by a complete individualization of the chromowashed and cultured in a n inhibitor-free medium to somes, which were organized in a metaphase plate constudy the reversibility of action of this drug. Nuclear figuration) or prometaphase I1 stage. Thus two groups maturation status was determined after lacmoid stain- of oocytes could be distinguished: one group with a very long prometaphase I stage (from 9 to 15 hr) and a short ing. metaphase I and another group with a short proRadiolabelling of Oocyte Proteins metaphase I stage (from 9 to 12 hr) followed by a TOstudy protein synthesis, [35S]metliionine (>1,000 long metaphase I (from 12 to 18 hr). Establishment of Ci mmolpl; Amersham) was added to the medium (sup- metaphase I1 (evidenced by the extrusion of the first Assessment of M a t u r a t i o n Stages
PROTEIN SYNTHESIS DURING IN VITRO OOCYTE MATURATION
3
Fig. 1. Changes in chromatin configuration from the initiation of maturation to its completion. Photomicrographs of fixed and stained whole mounts of goat oocytes after various times of culture representing the essential steps of maturation. a: GV-stage oocyte. b: GVBDstage oocyte (8 hr). The chromatin is condensed. c: Metaphase I-stage
oocyte (15hr). The chromosomes are individualized and organized on a metaphase plate. The spindle microtubules are clearly visible. d Metaphase I1 stage oocyte (27 hr). The first polar body (PB) is extruded. Bars = 5 km.
polar body) was almost completed (>85%)a t 27 h r after the initiation of the maturation process (Table 1, Fig. 1a-d) .
effect was obtained when the drug was added after 2 h r of culture (100%GV stage a t the end of the 27 h r maturation period). Two classes of oocytes were found when the cycloheximide was added 5 h r after the onset of culture: 1)GV oocytes with condensed chromosomes (35%) as previously observed and 2) abnormal GVBD-metaphase I oocytes with extremely closed and condensed chromosomes (65%)(Fig. 2b). These structures were similar to
Effects of Protein Synthesis Inhibition Incubation of oocytes from the beginning of culture in the cycloheximide-containing medium (20 pg/ml) prevented the rupture of the GV (Table 2). Nevertheless, chromosomes were very condensed (Fig. 2a). The same
4
F. LE G A L ET AL. TABLE 2. Effect of Cycloheximide on In Vitro Maturation of Goat Oocytes* Presence of CHX (hr) No. GV GVBD MI Ana-Tel I MI1 Nucleus-like 0 8 0 0 0 13 92 0-27 0 0 0 0 0 12 100 2-27 0 0 0 65 20 35 0 5-27 10 5 0 85 0 0 0-22 then without until 27 20 0 0 0 100 0 0 0-22 then withoutuntil46 21 0 0 78" 17.5 4.5 23 0 15-27 *CHX, cycloheximide; GV, germinal vesicle; GVBD, GV breakdown; MI, metaphase I; Ana-Tel I, anaphase-telophase I transition; MII, metaphase 11. aFifty-eightpercent have extruded the first polar body; 42%have not. They present either one nucleus or several micronuclei (50-50).
the clusters originally described in mouse oocytes by Hashimoto and Kishimoto (1988) and Szollosi et al. (1991). To establish further whether the transition from metaphase I to metaphase I1 was protein synthesis dependent, cycloheximide was administered close to metaphase I (15 h r after the onset of culture; Table 1). After 12 h r of incubation with the drug, 78% of oocytes reformed a nucleus-like structure containing decondensed chromatin (Fig. 2d). Two subpopulations were distinguished, i.e., oocytes that extruded their first polar body (58%)and those that failed to extrude it (42%). In the latter case, they exhibited either one nucleus or several micronuclei (50-50). The meiotic arrest induced by the addition of cycloheximide a t the onset of culture was reversible. Oocytes exposed to the drug during the first 22 h r of culture were able to progress through the maturation process after extensive washing and culture in a drug-free medium. Thus 5 hr after drug removal all the oocytes reached a t least the metaphase I stage (Table 2). This progression was accompanied by a n acceleration of the nuclear maturation process. Nevertheless, when cultured for 22 h r in a cycloheximide-containing medium and for 24 h r more without the drug, no oocyte overpassed the metaphase I stage. These metaphase I oocytes exhibited very condensed chromosomes, but the metaphase plate was slackened and the spindle presented a n abnormal morphology (Fig. 2c). This could be due to the oocyte aging, because, when the observation was made only 5 hr after removal of the drug, metaphase I was normal (not shown).
Protein Synthesis During Maturation At the end of the labelling period, a nuclear examination of each oocyte was performed with Hoechst dye to correlate exactly the meiotic stage and the protein synthesis pattern. Figure 3 shows four main differences in the protein synthesis pattern between immature and mature oocytes. First, a polypeptide of 25 kD molecular weight was replaced by a new labelled one of 27 kD. Another shift resulted in a 34 kD appearing at the 33 kD region. A t the GV stage, a polypeptide of 43 kD was the major synthesized polypeptide, whereas its labelling dramatically decreased after 27 h r of culture. Twodimensional gel electrophoresis identified this polypep-
tide a s actin (not shown). The last marked change was the large increase in the synthesis of a 67 kD protein. The synthesis of this protein was maximal at 27 hr. Some other slight differences were observed, i.e., two polypeptides of 44 and 47 kD, respectively, were not synthesized by the immature oocyte. The time-dependent change of this protein synthesis pattern during the time course of maturation is shown in Figure 4. Thus, within individual oocytes, the shift from 25 to 27 kD was closely associated with GVBD. This change was not observed in GV-arrested oocytes even after 27 h r of culture (not shown). Polypeptides of 44 and 47 kD also appeared at GVBD. At the same time, a new polypeptide of 33 kD appeared, whereas concomitantly a polypeptide of 34 kD became barely detectable and finally disappeared during the second half of the maturation process. At the metaphase I stage (15 hr), the synthesis of a 67 kD polypeptide began to increase strongly and became predominant after 18 h r of culture. At the same time, the synthesis of actin greatly decreased. This drop was not observed in oocytes t h a t had not overpassed metaphase I at the end of culture (not shown). However, it is noteworthy that the incorporation rate of radioactive methionine was progressively reduced, particularly after the establishment of metaphase I. It has already been reported that oocyte permeability dropped as maturation progressed (Wassarmann, 1988).
DISCUSSION The results presented here show the timing of nuclear events in goat oocytes during in vitro maturation. Oocytes remained at the GV stage from the onset to 6 8 h r of culture. GVBD occurred between 7 and 9 hr. Metaphase I became established within 12-18 hr. Finally, most oocytes reached the metaphase I1 stage after 27 hr. These data are comparable to those reported for sheep (Moor and Crosby, 1985) and cattle (Sirard et al., 1989). Experiments on protein synthesis during in vitro maturation enabled us to identify some polypeptides whose synthesis was correlated with the meiotic nuclear status of the oocyte. Thus the shift from a polypeptide of 25 kD to one of 27 kD was due to the occurrence of GVBD, since it was never observed in a n oocyte arrested a t the GV stage even after 27 h r of culture (i.e.,
PROTEIN SYNTHESIS DURING IN VITRO OOCYTE MATURATION
5
Fig. 2. Effects of cycloheximide on the meiotic maturation of goat oocytes. Immature oocytes after isolation were incubated with cycloheximide (20 pgiml) for various periods of time. They were fixed and stained for evaluation of their meiotic status after 27 (a, b, d) or 46 (c, c') hr of culture. a: Cycloheximide was added a t the onset of culture. Oocyte is still at the GV stage, but the chromatin is very condensed. Many nucleoles are visible (arrowheads). b The drug was added 5 hr after the onset of culture. The chromosomes are closed and very condensed and form a cluster. c , c': The drug was added a t the onset of
culture and removed after 22 hr. Culture without inhibitor (after extensive washing) was prolonged for 24 hr. The chromosomes are very condensed and are organized on a very large metaphase plate. This structure is abnormal in terms of spindle morphology. d: The drug was added near the metaphase I stage (15 hr after the onset of culture). The oocyte has emitted the first polar body (PB). A nuclear membrane has been reformed, which surrounds the fully decondensed chromatin. This nucleus ( N ) has a pronucleus-like structure. Bars = 5 pm.
nonmeiosis-resuming oocyte; data not shown). However, we cannot determine whether this change was a combination of a n arrest of 25 kD polypeptide synthesis and the synthesis of a new 27 kD polypeptide or was a result of posttranslational modifications of the 25 kD polypeptide. The presence of the 34 kD polypeptide was correlated to the GV stage. At GVBD, there was a transient period
during which 34 and 33 kD polypeptides were both present. The presence of only the 33 kD polypeptide was associated with the metaphase I stage. This change in the electrophoretic motility between 34 and 33 kD has already been described in many species during mitosis and meiosis in Xenopus (Doree et al., 1989), in the starfish (Labbe et al., 19891, and in the mouse (Howlett, 1986; Morla e t al., 1989). It is now clear that the entry
6
F. LE GAL ET AL.
Fig. 3. Fluorogram of ["Slmethionine-labelled polypeptides from goat oocytes a t the GV stage and after maturation for 27 hr in vitro. Polypeptides were separated on a 7.5-18% linear gradient SDS-polyacrylamide gel. Each lane represents the pattern of one oocyte. Lane 1: Pattern of synthesis in GV-stage oocytes incubated with [%]methionine for 3 hr. Lane 2 Pattern of a metaphase 11-stage oocyte incubated during the 27 hr of maturation. The main differences observed were that 27, 33, 44, 47, and 67 kD polypeptides appeared, while 25 and 34 kD polypeptides disappeared. Actin synthesis has dropped.
of cells into the M phase is induced by a maturationpromoting factor (MPF) originally described by Masui and Markert (1971). One of the subunits of mammalian MPF is homologous to the gene product of the yeast gene cdc2. The protein coded for by the gene is a protein kinase with a relative molecular mass of 34 kD (Lee and Nurse, 1988; Lohka et al., 1988; Draetta and Beach, 1988). The 34 kD polypeptide observed in the goat may be related to the cdc2 gene product. Further studies are in progress to determine more precisely the behavior of these two polypeptides. The ultimate part of the maturation process was characterized by the increase in 67 kD polypeptide synthesis and the drop in actin synthesis. Actin was the major synthetic product of immature oocyte. Moor et al. (1983) reported that it accounted for 8-10% of the total synthesized proteins in the ovine oocyte. According to our results, the decrease in actin synthesis seemed to be a n essential characteristic of a normal maturation process, since a high rate of actin synthesis after 27 h r of
Fig. 4. Timing of protein synthesis pattern modifications during maturation of goat oocytes. L3"Slmethioninewas added at the onset of culture. Each lane represents the pattern of one oocyte. Lane 1: GV stage oocyte (same as lane 1in Fig. 3). Lane 2 After 8 hr of culture. GVBD-stage oocyte. Two shifts are detectable: 25-27 kD and 34-33 kD (34 kD polypeptide barely detectable); 44 and 47 kD polypeptides have also appeared. Lane 3: After 15 hr of culture. Metaphase I stage. The 34 kD polypeptide has totally disappeared. Lane 4 After 18 hr of culture. No observable difference could be observed compared with the 15 hr situation. Lane 5 After 27 hr of culture. Metaphase I1 stage. Actin synthesis has dropped, whereas the 67 kD polypeptide synthesis has become very extensive.
culture was in each case associated to oocytes that exhibited abnormal nuclear and cytoplasmic morphologies (data not shown). The specific 47 kD polypeptide synthesized before GVBD as reported by Moor and Crosby (1986) for ovine oocytes was also detected in the goat species. The reprogramming of protein synthesis that occurred during maturation is essential for the differentiation events required during fertilization and early development (reviewed by Moor e t al., 1983; Thibault et al., 1987; Wassarmann, 1988). In this respect, it would be interesting to determine the exact functions of these different proteins. The present results revealed a high sensitivity of goat oocytes to cycloheximide. In goats, protein synthesis was required for the GV to undergo breakdown, a s in pigs (Fulka et al., 1986). sheer, (Moor and Crosbv. 19867, and cattle (Hunter and Moo;, 1987).When cyc6:
PROTEIN SYNTHESIS DURING IN VITRO OOCYTE MATURATION heximide was administered after 5 h r of culture, 65% of oocytes overpassed GVBD, indicating that the required synthesis occurred within the first 5-6 h r of culture and that these specific proteins necessary for GVBD were not present in the ooplasm of fully grown oocytes. By contrast, resumption of meiosis in the mouse is not dependent on active protein synthesis, suggesting that the proteins required for M phase transition were probably already present in the prophase oocyte. These data indicate the substantial species differences in the protein synthesis required for GVBD. However, even if nuclear membrane breakdown was inhibited by cycloheximide, some chromosome condensation was observed in the karyoplasm, suggesting that the induction of chromosome condensation does not require protein synthesis, a phenomenon also described for pig (Kubelka e t al., 1988) and cattle (Simon e t al., 1989) oocytes. The present results and previous results for pigs, cattle, and sheep imply that the activity that induces chromatin condensation is different from the activity that induces the breakdown of the nuclear envelope. Inhibition of GVBD in goats by cycloheximide was partly reversible. When cycloheximide was present in the culture medium during the first 22 hr, removal of the drug resulted in 85% of oocytes reaching the metaphase I stage after 5 h r of culture in the drug-free medium. More remarkably, removal of the oocytes from the cycloheximide-containing medium resulted in a n acceleration of the GVBD process, as previously described for sheep (Moor and Crosby, 1986) and pigs (Kubelka et al., 1988). It is noteworthy that cycloheximide does not prevent chromosome condensation, indicating that the GVBD process is partially engaged. This could explain the acceleration observed after removal of the drug. However, under these conditions, oocytes did not proceed beyond the metaphase I stage (Table 2). We can conclude from all these results that inhibition of protein synthesis might lead to a partial MPF activation, which in this case is competent only for chromosome condensation. Thus protein synthesis is not the only mechanism involved in meiosis resumption. Synthesis of proteins is also required for the establishment of the metaphase I1 stage. The administration of cycloheximide 15 h r after the beginning of culture induced the formation of a nuclear envelope (78%), which indicated that the oocytes had entered interphase. However, two types of response to the protein synthesis inhibitor could be distinguished according to the meiotic stage reached by oocytes a t the time of drug addition. Of these oocytes, 52% had extruded their first polar body and 42% did not. However, in the latter case, one nucleus or several micronuclei were observed in the same proportion. As was previously indicated, after 15 h r of culture, one-half of the oocytes were in prometaphase, i.e., before the formation of a well-organized metaphase spindle, while the second one-half of the oocytes had reached full metaphase-stage organization. We can speculate that the drug applied a t the
7
prometaphase stage affects the formation of the spindle, inhibiting polar body extrusion, and induces formation of nuclear envelope(s) around dispersed chromosomes, whereas the drug applied at metaphase I does not inhibit polar body extrusion, suggesting that these oocytes escape the effect of the drug until telophase, and also induces the formation of a nuclear envelope. This nucleus reformation suggests a progression of the cycle onto a n interphase state, which might correspond to a loss of MPF activity. Similar results have been obtained in cattle (Sirard et al., 1989) and mouse (Clarke and Masui, 1983; Szollosi et al., 1991). These results reveal a n oocyte sensitivity to protein synthesis inhibition between prometaphase and metaphase I, while the transition from metaphase I to telophase is not protein synthesis dependent, a result already described for the mouse (Szollosi e t al., 1991). Nevertheless, the protein synthesis patterns obtained did not reveal newly synthesized polypeptides between metaphase I and metaphase 11. The only visible difference was the high rate of increase in the synthesis of a 67 kD polypeptide, suggesting a role for this protein in the control of metaphase I-metaphase I1 transition. It can also be speculated that a continuous synthesis of certain proteins is necessary for the establishment and maintenance of the metaphase I1 stage. Another nonexclusive possibility was a posttranslational regulation (like phosphorylation) of this transition, a possibility that might explain the absence of observational differences in the protein synthesis patterns. Indeed it has now been well established that phosphorylation plays a major role in nuclear changes and cell events. The role of phosphorylations during goat oocyte maturation will be the subject of further studies.
ACKNOWLEDGMENTS We thank Dr N. Crozet for critical reading the manuscript and R. Scandolo and F. Fort for the photographic work. This work was supported in part by grant from Conseils Regionaux des Regions Centre et PoitouCharentes to F.L.G.
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