MOLECULAR REPRODUCTION AND DEVELOPMENT 33:324-332 (1992)

Protein Synthesis Requirements During Resumption of Meiosis in the Hamster Oocyte: Early Nuclear and Microtubule Configurations CARLOS E. P L A N C H A ~AND , ~ DAVID F. ALBERTINP 'Department of Anatomy and Cellular Biology, Tufts Uniuersity Health Science Schools, Boston, Massachusetts; 'Institute de Histologia e Embriologia, Faculdade de Medicina de Lisboa, Lisbon, Portugal

Key Words: Cell cycle, Germinal vesicle breakdown, Oocyte maturation

mals remains unclear (for review, see Motlik and Kubelka, 1990). It has been known for more than 50 years that mammalian oocytes arrested a t prophase I of meiosis when released from antral follicles and put into culture spontaneously resume meiosis (Pincus and Enzmann, 1935; Edwards, 1965; for review, see Thibault et al., 1987). This particular behavior of mammalian oocytes has made possible the investigation of translational requirements for meiotic resumption (for review, see Motlik and Kubelka, 1990). Mammalian oocytes from different species have been divided into two groups based on their dependence on protein synthesis for resumption of meiosis as assessed by germinal vesicle breakdown (GVBD) screening (Motlik et al., 1990; Motlik and Kubelka, 1990). Oocytes from some rodent species (mouse, rat) undergo GVBD and meiotic resumption in the presence of protein synthesis inhibitors (Wassarman et al., 1976; Schultz and Wassarman, 1977; Ekholm and Magnusson, 1979; Hashimoto and Kishimoto, 19881, whereas oocytes from sheep, pigs, and cattle fail to undergo GVBD under similar conditions (Moor and Crosby, 1986; Fulka et al., 1986; Hunter and Moor, 1987; Motlik et al., 1990; Mattioli e t al., 1991). The evaluation of meiotic resumption in mammalian oocytes commonly relies on either the light microscopic observation of germinal vesicle (GV) disappearance in living oocytes or chromosome-stained fixed preparations (Wassarman et al., 1976; Racowsky, 1984, 1986, Racowsky and Satterlie, 1985; Motlik et al., 1990; Motlik and Rimkevicova, 1990; Downs, 1990; Mattioli e t al., 1991). Data obtained via these approaches is restricted to determination of the meiotic stage, based solely on nuclear criteria. It is known that organelle rearrangements (Szolosi, 1972; Van Blerkom and Runner, 1984; Albertini, 1987) and a dramatic remodelling

INTRODUCTION The formation of active M phase-promoting factor (MPF) a t the Gz/Mtransition of the cell cycle represents a key step in the control of mitotic and meiotic divisions (Hunt, 1989). However, the precise role of translational and posttranslational processes associated with MPF activation during the resumption of meiosis in mam-

Received March 24, 1992; accepted June 2,1992. Address reprint requests to Carlos E. Plancha, Inst. Histologia e Embriologia, Faculdade de Medicina de Lisboa, 1699 Lisbon Codex, Portugal.

The organization of chromatin and ABSTRACT cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 IJ-g/mlpuromycin or cycloheximide for 5 hr. Both control (untreated)and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 h r in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 h r in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation IS inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption. 0 1992 Wiley-Liss,Inc.

0 1992 WILEY-LISS, INC.

PROTEIN SYNTHESIS REQUIREMENTS IN MEIOSIS of the microtubule (MT) cytoskeleton (Mattson and Albertini, 1990; Van Blerkom, 1991; Albertini et al., 1992) occur concomitant with nuclear events during oocyte maturation, but the dependence of these cytoplasmic events on protein synthesis has not yet been analyzed. Furthermore, it has been shown that alterations in MT organization at entry into M phase, such as loss of interphase microtubule arrays, centrosome activation, and spindle pole formation, are subject to regulation by MPF (Verde et al., 1990). Consequently, to evaluate fully oocyte maturation, both cytoplasmic and nuclear events must be assessed simultaneously by methods that afford a clearer image of these compartments. The present study was undertaken to determine the extent to which early nuclear and cytoplasmic microtubule rearrangements depend on protein synthesis during the resumption of meiosis in cultured cumulus-enclosed hamster oocytes. Double fluorescence analysis of oocytes treated with cycloheximide (CX) and puromycin (PUR) shows that meiotic spindle assembly requires protein synthesis, whereas key aspects of meiotic resumption, including chromatin condensation and cytoplasmic MT rearrangements, are less sensitive to protein synthesis inhibition. MATERIALS AND METHODS Collection and Culture of Oocytes Mature female golden Syrian hamsters (Mesocricetus aurutus) 45-60 days of age were kept in lighting conditions of 16 h r light-8 h r dark (lights on at 0500 hr), and were stimulated by intraperitoneal injection of 30 IU pregnant mare’s serum gonadotropin (PMSG; Sigma, St. Louis, MO). Cumulus-oocyte complexes (COC) were collected 68-78 hr post-PMSG administration by puncture of antral follicles. Animals were asphyxiated with carbon dioxide, and the ovaries were excised and briefly washed in sterile phosphate-buffered saline (PBS). Ovaries were then transferred to a sterile petri dish containing Eagle’s minimum essential medium (MEM) with Hanks’ salts plus 25 mM Hepes buffer (Gibco, Grand Island, NY), and the large antral follicles (2700 pm in diameter) were carefully punctured. COC were either immediately stripped of cumulus and fixed or cultured intact in Falcon 3037 organ culture dishes in 1 ml Medium 199 with Hanks’ salts and 25 mM Hepes buffer (Gibco) supplemented with 10% newborn calf serum (Gibco), 1mM L-glutamine, 2.5 mM Na lactate, 0.3 mM Na pyruvate, 1pl/ml insulin-transferrin-selenium solution (ITS; Collaborative Research, Lexington, MA), 100 U/ml penicillin, and 100 pg/ml streptomycin (modified from Racowsky, 1984,1986).Culture of COC was carried out a t 37-38°C in a humidified atmosphere of 5% CO, in air. Less than 15 min transpired between ovary removal and COC culture. In protein synthesis inhibition experiments, either PUR or CX was used at a concentration of 100 pg/ml in culture medium. Although no direct measurements of

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the protein synthesis inhibition efficiency were performed in this study, CX at this concentration has been shown to suppress protein synthesis completely in mouse oocytes (Siracusa et al., 1978; Hashimoto and Kishimoto, 1988; Downs, 1990). PUR was also used because it blocks protein synthesis by a different mechanism, although it has been reported to be less effective (Motlik and Rimkevicova, 1990; Downs, 1990). In some experiments, inhibitors were also added to the collection medium, with identical results. Treatment reversibility was tested by incubating inhibitor-treated oocytes in drug-free medium for 3.5 and 5 h r prior to fixation. Fixation and Fluorescence Microscopy At the time of collection (To) or after 1-1.5, 2-2.5, 3-3.5,44.5, and 5-5.5 h r of incubation (T1--T5),cumulus cells were removed mechanically by pipetting through a narrow-bore pipette, and oocytes were fixed and processed for fluorescence microscopy. Oocytes were simultaneously fixed and extracted in 2% paraformaldehyde in a microtubule-stabilizing buffer containing 0.1% or 0.5% Triton X-100, 1 pM axol, 10 unitdm1 aprotinin, and 50% deuterium oxide (Herman e t al., 1983) for 3 0 4 0 min a t 37°C and were either immediately labelled for tubulin and DNA or stored at 4°C in 0.05% Tween 20 in PBS containing 0.02% sodium azide prior to processing. For tubulin labelling, a mouse IgG monoclonal antibody to P-tubulin (Lot. No. N122020; Accurate Chemical Co., Westbury, NY) (1/50,60 min, 37°C) followed by a fluorescein isothiocyanate (F1TC)-conjugated goat anti mouse IgG (Lot. No. 29046, Organon Teknika-Cappel, Malvern, PA) (1/50, 45 min, 37°C) was used. An affinity-purified rabbit antiserum against tubulin ( a gift from Dr. J a n De Mey) followed by affinity-purified TRITC-conjugated goat antirabbit IgG (Sigma) was also used, and comparable results were obtained. No immunostaining was observed when primary antibodies were omitted. DNA was stained by Hoechst 33258 (Polysciences Inc., Warrington, PA) (1pg/ml). In some cells TRITC-conjugated phalloidin (Molecular Probes Inc., Eugene, OR) (1unitiml, 30 min, 37°C) was used for polymerized actin detection. Specimens were analyzed and photographed with Zeiss IM-35 or Leitz Wetzlar “Orthoplan” fluorescence microscopes equipped with epifluorescence and phase contrast.

Evaluation of Meiotic Resumption Meiotic stages were evaluated on double-stained oocytes using various cytological parameters for nuclear and cytoplasmic maturation (see Table 1 legend for stage definition). The frequencies of GVBD, chromosome condensation, and spindle and aster formation were pooled from at least two replicate experiments. Proportions were compared with Fisher’s exact and x2 tests. To satisfy test conditions, one or more stages were pooled.

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C.E. PLANCHA AND D.F. ALBERTINI

TABLE 1. Time and Sequence of Early Nuclear and Cytoplasmic (Microtubule) Events Associated With In Vitro Resumption of Meiosis of Hamster Oocytes* Incubation time (hr) TO (0) T, (1-1.5) T, (2-2.5) T, (3-3.5) T4 (P4.5) T, (5-5.5)

No. oocytes 135 44 35 54 60 96

A 86.5 93

Nuclear events (%) GV' B C 12 7

~

-

-

-

-

-

~

~

17 ~

~

MT configurations (%) ~

GV(D) 1.5 -

83 100

100 100

SP -

SP +

E

F

G

H

I

J

85 7

15 91 37

2 63 76

-

-

-

-

2 84

~

~

-

-

-

-

~

5 5 1

17 95 15

-

*Data represent total percentages of oocytes taken from three separate experiments. Nuclear events as detected by DNA staining patterns with Hoechst 33258 have been classified as follows: A, GV containing one to four fully rimmed nucleoli;B, GV containing very small or no nucleolus (no chromosomes detected);C, GV containing condensed bivalents; D, no GV detected. Microtubule configurations as detected by immunofluorescence labelling have been classified as follows: E, MT network (interphase-like)present; F, no MT labelling detected or short cortical and/or perinuclear MT; G, only kinetochore MT detected; H, kinetochore MT and cytoplasmic asters detected; I, meiotic spindle and cytoplasmic asters detected;J, only meiotic spindle detected.

RESULTS Time Course of Hamster Oocyte Nuclear and Cytoplasmic Events During Resumption of Meiosis In Vitro To assess nuclear and cytoplasmic events simultaneously, the cells were DNA stained with Hoechst dye and labelled with antibodies against tubulin. The progression of nuclear and cytoplasmic events from 0 to 5-5.5 h r of in vitro-matured hamster oocytes is shown in Figures 1-6. At the time of isolation (To),most fully grown hamster primary oocytes present a prominent GV, usually containing one to four nucleoli (Fig. l a ) . In the cytoplasm, most GV-stage oocytes display a clear microtubule network (Fig. lb). At 1-1.5 h r of culture, all oocytes contain intact GV (Fig. 2a); however, most oocytes no longer exhibit cytoplasmic MT (Fig. 2b). At 2-2.5 hr, chromatin condensation, nucleolar dissolution, and phase contrast-assessed GVBD have occurred (Fig. 3a). Frequently, a t this time, a cage-like pattern of antitubulin labelling i s observed surrounding the condensing GV (Fig. 3b) or the chromosome set (not shown). At subsequent culture times (3-5.5 hr), GVBD and chromosome condensation occur in all oocytes (Figs. 4-6a). After a 3-3.5 h r culture period, kinetochore MT are seen associated with condensing chromosomes (Fig. 4b). At P 4 . 5 hr, culture antitubulin immunofluorescence reveals a prominent spindle and several cytoplasmic asters or MT organizing centers (MTOC) that appear to be cortical from through-focus analysis (Fig. 5b). Because fully condensed chromosomes appear to be congressing on formed spindles, this stage was classified as prometaphase I (PM,). Between 5 and 5.5 h r of culture, cytoplasmic MTOC are no longer evident in most cells, and the meiotic spindle is the only antitubulin-labelled structure (Fig. 6b). By this time chromosomes are continuing congression to the equatorial plates (Fig. 6a), and rhodamine-phalloidin staining reveals discrete cortical-positive regions overlying the

meiotic spindle (personal observations). This stage was classified a s late PM, or metaphase I (MI). Results of normal progression of untreated hamster oocytes are summarized in Table 1. Some degree of heterogeneity is detected at all time points. A small fraction (14%) of freshly isolated oocytes (To) lack nucleoli and/or cytoplasmic microtubules (Table 1, columns B and F). At T2, although most oocytes resemble T, oocytes regarding nuclear and MT configurations (Table 1, columns D and G), 35% possess a clear MT array around the chromosome set or surrounding the Hoechst-detectable GV (Fig. 3). At 4 4 . 5 hr, the spindle has not formed in a small percentage of oocytes, which, however, do exhibit cytoplasmic asters (Table 1). Two particular aspects of MT organization emerge from analysis of Table 1. First, MT disassembly occurs prior to GVBD. Second, multiple MTOC form late in PM, and seem to disappear in MI.

Effects of Protein Synthesis Inhibitors on Resumption of Meiosis To evaluate the protein synthesis requirements for resumption of meiosis, COC were cultured for 5 hr in the presence of either CX o r PUR. When inhibitors were added to the collection medium, identical results were obtained. Hoechst staining of treated oocytes reveals two cytologically distinct nuclear changes with respect to the extent of meiotic progression. The majority of oocytes undergo GVBD and exhibit full chromosome condensation (Fig. 7a; Table 2, GV- group). In -20-35% of the oocytes, depending on the treatment group, the GV is clearly visible (Fig. 8a), but different degrees of chromatin condensation are apparent. (Table 2, GV' group). Thus chromosome condensation and GVBD occur in the presence of protein synthesis inhibitors, although under these conditions a fraction (8-20%) of treated oocytes fail to undergo GVBD and chromosome condensation.

PROTEIN SYNTHESIS REQUIREMENTS IN MEIOSIS

Figs. 1-6. Sequence of early nuclear and microtubule configurations from time of collection (To) to 5 hr in vitro incubation (T5). Correlative Hoechst 33258 (a)and antitubulin (b) labelling patterns in whole-mount preparations from hamster oocytes. At the time of collection (To),oocytes present a prominent GV ( l a ) and a clear interphase-like MT network (lb).After 1hr of culture, a GV is still present (2a),but no tubulin is detected (2b). After 2 hr of culture, initial chromatin condensation is seen in some oocytes (3a),where a cortical

327

and a perperinuclear (arrowheads) array of MT can be seen (3b). Starting at T,, but mainly a t subsequent time points (T3-T5), GVBD has occurred (44a).At T,, the MT labelling is restricted to the chromosome region (4b), and 1 hr later a spindle is clearly identified, together with several MTOC (arrows in 5b).After 5 hr in culture (T5), the spindle is the only tubulin-positive structure identified in most oocytes (6b).Bar = 20 pm. x300.

328

C.E. PLANCHA AND D.F. ALBERTINI TABLE 2. In Vitro Effects of Protein Synthesis Inhibitors on Spontaneous Resumption of Meiosis of Hamster Oocytes MT configurations (%)

Nuclear events (%) Treatments (5-5.5 hr)

Cycloheximide Puromycin

No. oocytes

A

GV+ B

C

GV-

D

E

F

G

H

168 38

20 8

6 3

8 10

66

2 3

15 10

-

82 82

79

SP+

SP -

5

I

J

1

-

-

-

With respect to microbubule alterations, spindle for- increased with longer recovery times (Table 3, columns mation is inhibited completely in treated oocytes (Table I and J; Fig. 9b). 2, sp+ group). Typically, oocytes that fail to undergo DISCUSSION GVBD exhibit a perinuclear MT network (Fig. Bb), These results demonstrate that spontaneous in vitro whereas, in oocytes that undergo GVBD, MT arrays surround the condensed chromosome set (Fig. 7b). In resumption of meiosis in the hamster oocyte involves a both groups, most oocytes exhibit cortical MTOC topo- series of nuclear and cytoplasmic rearrangements that graphically distinct from chromosomes (Figs. 7,8c; Ta- specifically include changes in MT organization. Conble 2, column H). Thus meiotic spindle assembly is com- trary to the case in the mouse, a n interphase-like MT pletely blocked in the presence of protein synthesis network is observed in GV-intact, fully grown hamster inhibitors, although microtubule changes associated oocytes. Early cytoplasmic modifications defined in with meiotic resumption occur, as indicated by the dis- this study include loss of interphase MT, formation of appearance of interphase MT, formation of perinuclear perinuclear MT arrays, kinetochore MT assembly, and MT arrays, and emergence of numerous cortical MTOC. emergence of cortical MTOC prior to the formation of Analysis of Table 2 points to the occurrence of a sim- the meiotic spindle. Furthermore, we demonstrate that ilar response observed with a 5 h r incubation in 100 protein synthesis is not required for the initial cytoplaspg/ml CX or PUR regarding both nuclear (P = 0.23) mic and nuclear events associated with meiotic reand MT configurations (P = 0.75). Approximately 6 6 sumption under these conditions but is required for 79% of treated oocytes undergo GVBD (GV- group; col- assembly of the first meiotic spindle. umn D), while 21-34% retain a GV (GV' group, Table Nuclear and Cytoplasmic Dynamics During 2, columns A-C). Spindle formation is inhibited in all Resumption of Meiosis of Fully Grown drug-treated oocytes (Table 2, columns I and J), and Hamster Oocytes even lower doses of PUR were effective a t inhibiting The protocol used in this study for obtaining fully spindle assembly (83%, 10 pgiml, 4.5 hr, personal obgrown hamster oocytes, i.e., collection 68-78 h r followservations). ing PMSG administration, is a valid approach since it Treatment Reversibility Assessment produces a morphologically and functionally homogeTo evaluate the recovery response of treated oocytes neous population. This study shows that spontaneous with respect to nuclear and microtubule changes, COC meiotic resumption occurs almost synchronously in this were washed free of inhibitors and evaluated by double- system and that oocytes progress to metaphase of meiolabelling fluorescence microscopy. Following a 5 h r sis I during a n initial culture period of 5-5.5 hr. The treatment for each inhibitor, oocytes were washed time course of this progression is comparable to that three times in excess medium without inhibitors, incu- described by Racowsky and Satterlie (1985). Although i t is known that nuclear events occur conbated for either 3.5 or 5 h r in inhibitor-free medium, and fixed. The results are shown in Figure 9 and in comitant with cytoplasmic changes during oocyte maturation (Szolosi, 1972; Van Blerkom and Runner, 1984; Table 3. Regarding nuclear progression, recovery is efficient, Albertini, 1987; Van Blerkom, 1991), usually only nusince already by 3.5 h r all oocytes have undergone clear status is assessed to evaluate resumption of meioGVBD and complete chromatin condensation sis and maturation (Wassarman et al., 1976; Racowsky, (P < 0.005; Table 3, column D; see also Fig. 9a). The 1984,1986;Racowsky and Satterlie, 1985; Motlik et al., extent of MT recovery, however, is related to the inhib- 1990; Motlik and Rimkevicova, 1990; Downs, 1990; itor used and to the time of recovery analyzed (3.5and 5 Mattioli et al., 1991). To evaluate oocyte maturation hr). Specifically, PUR-washed oocytes exhibit a faster fully, both cytoplasmic and nuclear events should be recovery than CX-washed oocytes, since no significant monitored simultaneously by methods that afford a differences could be found between 3.5 and 5 h r after clearer image of these compartments (Mattson and AlPUR removal (P = 0.31; Table 3, columns E-J). In con- bertini, 1990; Wickramasingue et al., 1991; Gavin et trast, MT recovery of CX-washed oocytes increased sig- al., 1991; Messinger and Albertini, 1991). The concurnificantly from 3.5 to 5 h r of incubation (P = 0.00003; rent use of tubulin and DNA-specific fluorescent probes Table 3, columns E-J). Moreover, the percentage of has allowed us to detect a previously unrecognized seinhibitor-washed oocytes exhibiting spindle formation ries of events.

PROTEIN SYNTHESIS REQUIREMENTS IN MEIOSIS

329

Fig. 7-8. Correlative Hoechst 33258 (a)and antitubulin (b,c) labelling patterns in protein synthesis inhibitor-treated oocytes for 5 hr. Tubulin patterns are shown for two distinct optical planes: equatorial (a,b) and tangential (c). Most commonly as depicted in 7, GVBD has occurred (7a), and although no spindle is present, strong MT labelling is seen around the chromosome set (7b). Figure 8 depicts an oocyte where chromosome condensation has occurred (Sa), and the GV is

surrounded by a n array of MT (arrowheads in 8b).Cortical MTOC are apparent in both cases (arrows in 7c and 8c). Bar = 20 pm. X330. Fig. 9. Correlative Hoechst 33258 (a)and antitubulin (b)labelling pattern in protein synthesis inhibitor-treated oocytes, following a 5 hr incubation in inhibitor-free medium. Condensed chromosomes are apparent (a) on the meiotic spindle (b); note absence of cytoplasmic MTOC. Bar = 20 pm. ~ 5 8 5 .

Fully grown hamster GV-stage oocytes obtained from large antral follicles (2700 km) present a microtubule network typical of interphase cells, clearly contrasting to the case in the mouse, where oocytes display a n M phase-like MT network (Mattson and Albertini, 1990). In the mouse, previous work has shown that, a t the time of meiotic competence acquisition, distinct M

phase alterations occur in cytoplasmic MT involving loss of long interphase MT and the emergence of cytoplasmic MTOC (Wickramasinghe et al., 1991). In contrast, meiotically competent GV-stage hamster oocytes (Plancha and Albertini, in preparation) retain a n interphase MT network (Fig. l a ) , and dynamic alterations in MT organization in this species of rodent do not ensue

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C.E. PLANCHA AND D.F. ALBERTINI

TABLE 3. Reversibility of Protein Synthesis Inhibitor Treatments on Spontaneous Resumption of Meiosis of

Hamster Oocytes

MT configurations (%)

Nuclear events (%) Incubation time (postinhibitors) Post-5 hr cycloheximide incubation 3.5 hr 5 hr Post-5 hr puromycin

No.

GV ' B

oocytes

A

59 58

-

-

-

-

32 34

-

-

-

-

C

GV(D)

E

F

G

H

I

J

2

100 98

-

56 26

29 16

2 17

10 19

22

-

100 100

-

3 12

31 15

19 12

28 38

19 23

~

SP

SP+

~

4

incubation 3.5 hr 5 hr

until meiotic resumption. Recently, Messinger and Albertini (1991) reported that dynamic changes in the distribution, number, and MT nucleation capacity of oocyte centrosomes occur in the course of meiotic maturation in the mouse. Specifically, two populations of centrosomal MTOC were observed, one that contributed to spindle pole assembly and another that exhibited stage-specific interactions with the oocyte cortex (Messinger and Albertini, 1991). In the present study, we show that MT disassembly and the emergence of antitubulin-detectable MTOC occur in a stage-specific manner in maturing hamster oocytes. However, the loss of interphase MT and MTOC detection follows resumption of meiosis in the hamster, in contrast to the mouse, where these events precede meiotic resumption. These results imply species-specific differences in MT dynamics, since identical fixation and processing conditions were employed, as described in earlier studies on the mouse (Mattson and Albertini, 1990; Wickramasinghe et al., 1991; Messinger and Albertini, 1991). Since the MT alterations noted above precede spindle assembly and are influenced by the activation of MPF a t M phase entry (Verde, 19901, it is likely that different mechanisms exist in these different species for the activation of MPF. Differences in the state of MT organization in GV-stage oocytes from these two species may be due to variations in the time course of MPF activation relative to resumption of meiosis. As discussed previously, GV-stage mouse oocytes exhibit M phase-like alterations prior to GVBD, whereas the present study illustrates that these alterations occur somewhat coincident with GVBD in the hamster. Collectively, these observations suggest that meiotic arrest may be imposed at different points in the first meiotic prophase for different species. The extent to which protein synthesis is required for meiotic resumption and critical MT events has not been fully evaluated and prompted further analysis of this problem.

Protein Synthesis Requirements for Nuclear and Microtubule Changes Associated With Resumption of Meiosis in the Hamster Sensitivity of mammalian oocytes to protein synthesis inhibition varies from species to species (for review, see Motlik and Kubelka, 1990). In mouse and rat

oocytes, GVBD and chromatin condensation occur (Wassarman e t al., 1976; Schultz and Wassarman, 1977; Ekholm and Magnusson, 1979), but meiotic spindle assembly is impaired (Hashimoto and Kishimoto, 1988). In contrast, pig and cattle oocytes are unable to undergo GVBD under similar conditions but do exhibit partial chromatin condensation (Motlik et al., 1990; Motlik and Kubelka, 1990; Mattioli et al., 1991).To our knowledge, there is no information available with respect to the effect of protein synthesis inhibition on MT events prior to formation of the meiotic spindle. Chromatin analysis of hamster oocytes treated with protein synthesis inhibitors indicates that the majority of hamster oocytes undergo GVBD and chromatin condensation in the presence of inhibitors (Table 21, resembling observations on oocytes from other rodent species (Wassarman et al., 1976; Schultz and Wassarman, 1977; Ekholm and Magnusson, 1979). However, 2035% of oocytes were unable to resume meiosis based on GVBD (Table 2; columns A-C), although reversibility experiments show that all GV+-inhibited oocytes undergo GVBD upon drug removal (Table 3). Thus, regarding nuclear rearrangements, this is an intermediate situation, with the mouse and rat a t one end, representing protein synthesis independence, and the pig and cow at the other end, representing protein synthesis dependence for GVBD (for review, see Motlik and Kubelka, 1991). Since a similar response was observed in both inhibitor-treated groups, data were pooled, revealing a proportion of 68% (n = 206) that undergo GVBD (Table 2, column D). The 99% confidence interval for that proportion is 5 5 4 1 % ; therefore, hamster oocytes exhibit a sensitivity to protein synthesis inhibitors that is intermediate between the two behaviors described so far (for review, see Motlik and Kubelka, 1990). Species differences in the initial response to inhibitors could be due to differences in the turnover rate of proteins required for meiotic resumption. The extent to which these effects are related to the activation of MPF is deserving of further study. This study has also shown that a t the level of the cytoplasm a n ordered series of changes in MT organization occur prior to formation of the meiotic spindle. Interestingly, whether GVBD had occurred or not, loss of interphase MT, perinuclear MT array formation, and

PROTEIN SYNTHESIS REQUIREMENTS IN MEIOSIS cortical MTOC appearance occurred in most drugtreated oocytes. Since these events are normally associated with meiotic resumption, this indicates that some degree of cytoplasmic maturation can occur independent of nuclear maturation and active protein synthesis. Spindle formation, however, was shown to be totally dependent on protein synthesis, in a reversible fashion. For CX, the MT recovery rate increased with longer recovery times. The MT recovery rate was also shown to be faster and more complete with PUR, a less effective inhibitor (Table 3; see also Downs, 1990; Motlik and Rimkevicova, 1990). While few data are available with respect to the role of MPF in directing these early events of meiotic resumption in mammals, recent studies in Xenopus oocytes shed some light on this problem (Rime et al., 1990; Minshull et al., 1991). Complete ablation of cyclin mRNAs following antisense oligonucleotide injection did not imapir the ability of frog oocytes to mature in response to progesterone stimulation (Minshull et al., 1991). This indicates that, in the frog, maternal stores of the cyclin protein component of MPF are sufficient to drive meiotic progression. More interesting perhaps is the finding that okadaic acid, a protein phosphatase inhibitor, induces MPF activation in Xenopus oocytes in the presence of protein synthesis inhibitors (Rime et al., 1990). This treatment was found to support nuclear envelope breakdown, lamin depolymerization, and chromatin condensation, but typical meiotic spindles failed to form (Rime et al., 1990).Comparable observations were recently made using the mouse oocyte (Gavin et al., 1991). In conjunction with the present findings, these observations raise the possibility that the early events of meiotic resumption are posttranslationally regulated through the actions of protein phosphatases. Later events required for meiotic spindle assembly may involve a more complex type of regulation, such as translational control mechanisms, for meiotic progression to proceed. Further studies will be needed to clarify the regulation of MTOC function at this protein synthesis-dependent stage of meiotic progression.

ACKNOWLEDGMENTS We are grateful to Prof. J.F. David-Ferreira for constant support; to Dr. Antonio Gouveia for statistical advice; and to Drs. J a n Motlik, Sergio Gulbenkian, Dineli Wickramasinghe, Susan Messinger, and Solveig Thorsteinsdottir for enthusiasm and helpful discussions. This study was partially supported by NIH grant HD 20068, by the Instituto Nacional de Investigaqao Cientifica (contract No. 89/SAD/12), and by the Fundaqao Luso-Americana para o Desenvolvimento. REFERENCES Albertini DF (1987): Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes. Dev Biol 120:121-131. Albertini DF, Wickramasinghe D, Messinger S, Mattson BA, Plancha CE (1992):Nuclear and cytoplasmic changes during oocyte matura-

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