MOLECULAR REPRODUCTION AND DEVELOPMENT 29:163-171(1991)

Cytochalasin D Treatment Induces Meiotic Resumption in Follicular Sheep Oocytes V. DE SMEDT AND D. SZOLLOSI I.N.R.A., Unit6 de Biologie de la Fkcondation, Station de Physiologie animale, Jouy-en-Josas, France

ABSTRACT Ovine cumulus-enclosed oocytes collected from antral follicles (3-5 mm in diameter) were cultured in vitro with 2 x lo6 granulosa cells/ml in the presence or absence of gonadotropins or in the presence of cytochalasin D (CD). The maturation rate was assessed after 24 h of culture. In the control group, in the presence of gonadotropins (follicle-stimulatinghormone-luteinizing hormone (FSH-LH; 10 pg/ml) 100% of the oocytes reached metaphase II. Whereas intercellular junctions were no longer present after 6-7 h of culture, germinal vesicle breakdown (GVBD) occurred by the same time. In contrast, in the absence of gonadotropin, the majority of the oocytes (59%)remained blocked in GV stage. The inhibition exerted by the granulosa cells on meiotic resumption was overcome when the cumulus-oocytecomplexes (COCs)were incubated in CD (5 pg/ml)for 6 h at the beginning of the culture. Under these conditions, 85%of the oocytes matured with extrusion of the first polar body. Cytological analysis by cytofluorescence (NBD phallacidin) and electron microscopy showed that, after 6 h of treatment, CD provoked a redistribution of the microfilaments, mainly in the cumulus cells and to a lesser extent in the oocyte cortex. Intercellular junctions disappeared concomitantly with a significant decrease of the intercellular transport of tritiated uridine. The initiation of GVBD occurred at the same time. These results indicate that the resumption of meiosis was correlated with a loss of both junctional complexes (intermediate and gap junctions) between the cumulus cells and the oocyte. Key Words: In vitro maturation, Ovine, Oocyte, Germinal vesicle breakdown

Larsen et al., 1987). The stability of this junctional system is reenforced by large adhesive junctions (macula adherens). During the resumption of meiosis in vitro and in vivo, important nuclear and cytoplasmic changes occur in the cumulus oocyte complexes (COCs) (Szollosi et al., 1978). In previous experiments of in vitro maturation (IVM) of sheep oocytes, we have shown that, after 6-7 h of culture in the presence of gonadotropins, dispersion of the cumulus and a progressive retraction of FP occurred. The dissociation of the intercellular junctions began concomitantly with germinal vesicle breakdown (GVBD) (Szollosi et al., 1988). Similar observations have been reported by Hyttel et al. (1986). The loss of gap junctions consequent to the FP retraction suggested an important remodelling of cytoskeletal elements, such as the microfilaments (MF),present in the cumulus cells as well as in the oocyte cortex of sheep (Le Guen and Crozet, 19891, mouse (Maro et al., 19841,and rat (Battaglia and Gaddum-Rosse, 1986). Therefore, we investigated the effect of cytochalasin D (CD), known to affect specifically the MF system, during IVM of sheep oocytes. Following a 6 h of treatment with the drug, we observed that all the FP were withdrawn and that the intercellular junctions were no longer existing. Interestingly, resumption of meiosis occurred in most of the treated oocytes simultaneously, whereas in the controls GV remained intact and heterologous junction complexes were conserved. Our observations suggested that the two events were temporally closely associated.

MATERIALS AND METHODS Animals Cyclic and noncyclic ewes from different breeds were injected three times with 4 mg (Armour) of folliclestimulating hormone (pFSH) 48, 40, and 24 h before slaughter. The cyclic ewes treated were slaughtered on days 10 and 12 of the estrous cycle.

INTRODUCTION From the beginning of follicle formation, the oocyte is intimately associated with follicle cells by gap and intermediate junctions. After the differentiation of the granulosa and of the cumulus cells (and its innermost cellular layer, the corona radiata), they remain associOocyte Treatments ated with the oocyte by means of foot processes (FP) of The ovaries were dissected and the largest nonatretic the cumulus corona cells that cross the zona pellucida. follicles, between 3 and 5 mm in diameter, were seThe bulbous termini of these FP indent deeply the oocyte plasmalemma in sheep (Szollosi et al., 1988) and in cow oocytes (Hyttel et al., 1986). In the contact area, Received October 9, 1990; accepted January 28, 1991. small gap junctions provide electrotonic and ionic com- Address reprint requests to Dr. D. Szollosi, I.N.R.A., Unite de Biologie munications between the two cell types (Anderson and de la Fecondation, Station de Physiologie animale, 78352 JouyAlbertini, 1976; Gilula, 1977; Szollosi et al., 1978; en-Josas Cedex, France.

0 1991 WILEY-LISS, INC.

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lected under dissecting microscope in Dulbecco’s-phosphate-buffered saline (PBS) at room temperature. The COCs and granulosa cells (GC) were released from the follicles and washed in TC 199 (Ref. 10-201-20 Flow Laboratories, France) containing 4 mM NaHC03 and buffered at pH 7.35 with 20 mM Hepes (Sigma). The same medium, enriched with 10% heat-inactivated fetal calf serum (FCS) and 1 pgiml estradiol (EPI7),was used for the culture. In every experiment the COCs were cocultured with 2 x lo6 GC/ml (Staigmiller and Moor, 1984) at 39°C with gentle agitation under three different conditions: 1)the basic medium (control); 2) in the basic medium, in the presence of 10 pgiml pFSH-LH (Combarnous, France); 3) in the basic medium, with the addition of 5 bgiml CD (Sigma). After incubation for 6 h with CD, some COCs were rinsed and the coculture was continued in basic medium for 18 h.

Cytological Procedures Electron microscopy (EM). COCs were fixed and prepared following the procedure employed and reported previously (Szollosi et al., 1988). Fluorescence microscopy (FM). COCs were fixed for 50 min in 3.7% formaldehyde in phosphate buffer (PBS). After washing in PBS, the COCs were quickly passed through gelatin (10% in PBS buffer) and then embedded in 15% gelatin (in PBS). The samples were cooled in isopentane (-130°C) (Rime et al., 1987). Cryostat sections (10 pm) were hydrated and permeabilized for 30 min in PBS-0.03% saponin and then incubated for 1 h with NBD phallacidin (Molecular Probes Inc.; 5 IUiml). After washing in PBS, the preparations were mounted in PBS-glycerol (City University, London, United Kingdom). Observations were made via epifluorescence microscopy. After 24 h of culture. COCs were mechanically denuded and were fixed in acetic acid-ethanol(1:3VIV) for 24 h. They were then stained with lacmoid to determine the nuclear stages reached by the oocytes. Incorporation of Tritiated Uridine Measurements of the transfer of tritiated uridine, a specific marker of intercellular coupling (Moor et al., 1980), were compared with COCs treated or not with CD in five experiments (7-14 COCs per group). COCs were incubated for 6 h with or without CD. Ten microcuries of [5-6 3H]uridine (Amersham) was added for the duration of the last hour of the incubation. Some oocytes were mechanically denuded at the beginning of the culture under exactly the same conditions as control oocytes. The oocytes were quickly mechanically denuded in the first rinse. The last rinse (2 ml of PBS buffer) was split into two equal parts. One served as a blank; the second, containing the oocytes, represented the experimental group. Both groups were then transferred to Packard flasks and treated in 100 p1 of soluene (Packard) to dissolve the oocytes overnight at room temperature. Scintillation liquid was added, and the samples were counted with a scintillation counter.

For each group, the mean of the incorporated radioactivity per oocyte was calculated (see Table 1).

Statistical Analysis The results were tested by x2 for the status of nucleus and two-way analysis of variance for the measurement of transfer of tritiated uridine. RESULTS In Vitro Oocyte Maturation In the control group, all oocytes reached metaphase I1 after 24 h of culture in the presence of gonadotropins. In contrast, in the absence of the gonadotropin, only 40% of the COCs resumed meiosis and only 2% of them reached Metaphase 11, whereas 60% remained blocked in GV stage. Under the same conditions in the presence of CD during the first 6 h, 85% of the oocytes completed meiotic maturation (Table 2). The matured oocytes obtained following this treatment were comparable to the controls cultured with gonadotropin. The extrusion of the first polar body proceeded normally. This was not the case when the COCs were exposed to the drug continuously over 24 h (data not shown). Measurement of Tritiated Uridine Incorporation The ratio of treatedhntreated was 0.2. Thus the amount of tritiated uridine was reduced by 80% with CD treatment compared with the controls. The same rate was obtained in control experiment when the zona pellucida was removed before counting (data not shown). Ultrastructural Studies These studies were carried out on COCs cultured for 0, 1, 2, 6, and 12 h under different conditions. Culture in gonadotropin-free basic medium. The GV remains intact (Fig. la) in the oocytes cultured with gonadotropic hormones. The perivitellin space is narrow and the bulbous termini of corona cell FP indent deeply the oocyte plasma membrane. Numerous intercellular junctions are present along the apposing membranes. The most obviousjunctions are characterized as adherent (intermediate) junctions (Stevenson and Paul, 1989). Small gap junctions are intercalated among the adherent junctions (Fig. 2a). Thin filaments about 6-8 nm in diameter occupy the entire volume of the FP. In the corona cell body (not shown), narrow bundles are close to the plasma membrane. No difference was observed after 24 h of the culture in a hormone-free medium (Fig. 3). A thin layer of filaments of the same diameter is located in the oocyte cortex (Fig. a), which is usually accompanied by a small group of microtubules (Fig. 2a). Most cellular details shown are similar to controls at the onset of the culture. Culture in a gonadotropin-containingmedium. The first signs of meiotic resumption (maturation) are visible after 6 h of culture. The GV had either a very irregular shape, as the inner leaflet of the nuclear envelope (NE) adhered to itself and to the condensing chromosome bivalents to form the circular bivalent stage (Fig. lb), or the NE was broken in many places

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TABLE 1. Influence of CD on Coupling Between Cumulus Cells and Oocytes No. of CD (min-max)a Control CD/control experiments cpm/min/oocyte (min-max)b (min-max) 5

17b (4-39)

82 (25-188)

0.21 (0.09-0.37)

aMinimum and maximum observed values. bAddition of CD reduced significantly the uptake of uridine (P < 0.01). TABLE 2. Meiotic Stages of Sheep Oocytes After 24 h of Culture* FSH LHb (%) CDc (%) Control" (%)

+

No. of oocytes No. of exp. Metaphase I1 Telophase-Anaphase I Metaphase I Prometaphase I GV

56 5 1 (2)

-

16 (28.5) 6 (10.5) 33 (59)

37 4 37 (100) -

-

40 3 34 (85) 2 (5) 4 (10)

-

aControl groups of COCs cocultured for 24 h in gonadotropin (FSH-LH)-freemedium. bThe COCs were cocultured for 24 h in the presence of FSH LH (10 pg/ml). CTheCOCs were exposed to CD for 6 h, followed by an incubation for 18 h in medium free FSH and LH. * P < 0.01.

+

and long segments of the NE are in the proximity of condensed chromatin. Concomittant with GVBD, the relationship of the FP and their intercellular contacts with the oocyte are modified. The FPs are exteriorized during the contraction and dispersion of the cumulus (Fig. lb,), which either brought the corona cell termini to the level of the oocyte plasma membrane (Fig. 3c) or the oocyte cortex is deformed and at each point where the FP is still attached a small cytoplasmic protuberance formed and projected into the enlarging perivitellin space (Fig. 3d) (Thibault et al., 1987). The adherens-type intercellular junctions remain intact between some of the corona cell processes and the plasmalemma of the oocyte. Very few gap junctions remained between cellular FP and oocyte. Culture in basic medium containing CD. In most of the treated samples analyzed, meiotic maturation and GVBD were initiated or had taken place. The remaining GV was morphologically very distorted (Fig. lc) and identical to the GV after 6 h of culture with gonadotropins. Most of the nucleoplasm was displaced and adjacent segments of the inner leaflet of the NE stuck together. The orthogonal corona cells were very tightly associated with each other and with the zona pellucida even though FP were rare and were generally withdrawn. Some corona cell fragments remained in the perivitellin space, but corona cell-oocytejunctional complexes were rare (Fig. 4).In corona radiata cells, very large, irregular MF bundles were present in the cytoplasm, close to the nucleus (Fig. 4).The MF layer in the oocyte cortex either is not demonstrable or in certain limited areas it appears to be unchanged (Fig. 4a).

Immunofluorescence Studies The EM studies clearly show certain details of the distribution of cellular organelles with the COCs but are not well suited for appreciating the overall remodelling of the MF system. The organization of the actin filaments was therefore assessed by specific cytofluorescent staining using NBD phallacidin (Barak et al., 1980). Coculture in gonadotropin-free medium. The MF are localized in the periphery of the cumulus (corona cells) and are obvious in the entire FP crossing the ZP (Fig. 5). In the oocyte, thin F actin layer underlines the plasma membrane uniformally. F actin in both FP termini and oocyte cortex formed a regular fluorescent layer against the ZP. Coculture in gonadotropin medium. In the presence of gonadotropins, the intercellular contact area label by NBD phallacidin became more diffuse and irregular. In the cumulus cells, some MF were visible. They were dispersed throughout the entire cytoplasm (but a clearly distinct layer was seen close to the cell periphery). The number of the fluorescing FP crossing the ZP declined (Fig. 5c,d). Coculture in basic medium containing CD. Drastic changes occur in the MF system in the presence of CD. FP are no longer seen across the ZP. Large aggregates of MF are formed in the cumulus cell cytoplasm (Fig. 5e,fJ. Fluorescent patches are present under the ZP, originating probably from FP fragments of irregular form and size in the perivitellin space (Fig. 50. Most of the MF material is within the cell bodies of the corona cells surrounding the oocyte. In the cortex of

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DISCUSSION The present study demonstrates that the resumption of meiosis does not take place spontaneously or is delayed considerably, when COCs are cocultured with 2 x lo6 GC/ml in the absence of gonadotropins. The results are in good agreement with those obtained by Tsafriri and Channing (1975) in the domestic pig. Furthermore, we have shown that, parallel with the GV stage block, the junctional complexes and the intercellular communications between the cumulus and the oocyte are unchanged. These observations suggest a synergistic effect (Downs and Eppig, 1984; Thibault et al., 1987) of the inhibitory factors to maintain in the metabolic coupling intact permitting the passage of small molecules responsible for the oocyte meiotic arrest (Channing et al., 1982). The preovulatory surge in vivo or the addition of gonadotropins in vitro overcomes the follicular inhibition, inducing resumption of meiosis. The in vitro maturation system we employed gave a high rate of maturation and normal development after in vitro fertilization (IVF) (Crozet et al., 1987). Furthermore, we have shown a progressive loss of gap functions regulating the intercellular communications and the transfer of inhibitory substances, which is interrupted from the follicle cells to the oocyte, thus reinitiating the meiotic maturation (Dekel et al., 1988). The present studies show that CD triggers the resumption of meiosis. It disturbs the actin filament network in COCs by affecting the attachment of actin filaments to the plasma membranes probably by way of other minor cytoskeletal proteins within the plasma membrane, Probable candidates are talin and/or vinculin and (Y actinin or the more recently characterized calpactins, whose role may be precisely to bind actin to the plasma membrane. Other adherens substances between the FP and oocyte must also be affected at the same time. The action of CD thus would be indirect, affecting in the first instance the distribution and attachment of actin in the proximity of the junctions of the FP and secondarily the stability of the entire junctional complex. The preovulatory gonadotropin discharge would not have any direct action on the oocyte but rather on other metabolic cascades in the COCs, since mammalian oocytes lack receptors for gonadotropins. Similar results were recently reported for the rat by Dekel et al. (1988) using CB and colchicine. This Fig. 1. a: Semithin section of COC cultured for 6 h in the absence interpretation is further supported by recent results of of gonadotropin showing the germinal vesicle (GV). b: Semithin section of COC cultured for 6 h in the presence of gonadotropin Dekel et al., who showed that heptanol, an agent showing the initiation of germinal vesicle breakdown (GVBD). c: interfering with gap junctions integrity, induces oocyte Semithin section of COC cultured for 6 h in the presence of CD maturation in isolated rat COCs. Our EM and IF showing the initiation of GVBD. X 1,700. studies provide additional detail on the effects of CD on cumulus cells, such as relocalization and aggregation (or clumping) of F actin in the periphery of the cumulus the oocyte, no continuous MF layer remains. Most of cells, leading to the formation of irregular filamentous the FP are withdrawn, and no or few cellular commu- structures. The same type of F actin aggregates has nications remain between the corona cells and the been described in 3T3 cells after CB treatment (Weber et al., 1976).The interruption of intercellular commuoocyte.

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Fig. 2. Oocyte before culture. a: Electron micrograph showing a cumulus cell foot process (FP) indenting deeply in the oocyte plasmalemma. Note the presence of gap junctions (arrows). x75,OOO. b: Electron micrograph showing a microfilament (MF)layer underlying the oocyte plasma membrane. x 45,000.

nications occurs progressively starting after 3 h of CD lesser degree than the MF in corona cells. The regiontreatment and was reaching its maximum effectiveness ally remaining MF observed with EM did not represent at 6 h (data not shown). In contrast, cortical MF sufficient material to be demonstrated with the fluoorganization of the oocyte seems to be affected to a rescent phallacidin. Nevertheless, when CD treatment

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Fig. 3. Electron micrographs showing interactions between cumulus cell foot process (FP) and oocyte cultured 6 h. a , b In the absence of gonadotropin. Note the presence of gap junctions (GJ) and adherent junctions (AJ) between the two compartments. a: The abundant microfilament (MF) network in the bulbous termini of the

FP. x75,OOO. b: Bulbous termini of F P indenting deeply in the oocyte (arrowhead). x 15,000. c , d In the presence of gonadotropin. AJ still remained between F P and oocyte. Note the deformations of the ooplasmic membrane a t each contact point (arrowheads). c, ~75,000; d, ~10,000.

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Fig. 4. a: Electron micrograph showing the perivitellin space ofan oocyte treated for 6 h with CD. Note the absence of interaction between corona radiata foot processes and the oocyte plasmalemma. x 18,500. b,c: Electron micrographs showing large bundle of MF (arrowhead) in the nucleus periphery of a cumulus cell. b, ~ 1 3 , 5 0 0c,, ~ 5 0 , 0 0 0 .

is prolonged for 24 h, polar body (PB) extrusion is inhibited. The difference of the filament sensitivity to the drug in the two cellular compartments could be due to the difference in the status of dynamic equilibrium of MF (Maro et al., 1984; Battaglia and Gaddum-Rosse, 1986) or to posttranscriptional modifications of the oocyte actin filaments. Also, during maturation with gonadotropins, the cumulus cells change their shape, visible at first at the moment of retraction of the FP, followed by expansion of the cumulus. These signs imply an active remodelling of the filamentous component of the cytoskeleton. In view of these results, the most important effect of the CD is to isolate the oocyte from the cells surrounding it. The isolation results in a

high rate of maturation, nearly as high as with gonadotropins. The role of the gonadotropins, therefore, appears as an indirect one, affecting some metabolic events involving the maintenance of the actin filament function (attachment to the plasma membrane), which secondarily terminates the junctional complexes. Thus maturation of fully grown sheep oocytes from antral follicles is probably the result of its isolation from the cytoplasmic inhibitory effects of the somatic cells (cumulus and mural granulosa cells) surrounding it and transported by way of gap junctions to the oocyte. This may suggest that somatic cells surrounding the oocyte could maintain the pre-MPF in the oocyte cytoplasm and that the rupture of cell contacts is necessary to

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Fig. 5. Distribution of F actin (arrowheads) shown by NBD phallacidin staining shown in COCs cultured during 6 h. a,b: In the absence of gonadotropin. Cumulus cells (CC) are apposed against the zona pellucida (ZP). Numerous foot processes (FP) cross the ZP. a, ~ 1 , 2 0 0b, , X3,600. c,d: In the presence of FSH-LH (10 pgiml): CC

begin to react. F P still cross the ZP. c, ~ 1 , 6 0 0d, , x4,SOO. e,E In the presence of CD ( 5 Fgiml). Patches of F actin in the cytoplasm of the CC and in the contact area between cumulus and oocyte. Note the absence of labeling in the ZP. e, x 1,200, f, X3,600.

activate the cell cycle. Meiotic maturation thus may depend on a negative function, rather than on the existence of a positive signal inducing maturation. In our experiments with CD, tritiated uridine transfer was reduced by 80% of the original rate at the time

when we found the dismantling of junctional complexes between the FP and the oocyte. Thus the sum of our results can be interpreted in the sense of cause and effect concerning the loss of gap junctions and GVBD. In contrast, Moor et al. (1980)reported for sheep that in

CYTOCHALASIN D AND MEIOTIC RESUMPTION vivo or in culture of entire follicles in vitro the intercellular coupling decreased several hours after GVBD. Moreover, it has been demonstrated that metabolic coupling declines more slowly in vivo that in vitro in mouse (Salustri and Siracusa, 1983) and in pig (Motlik et al., 1986). Also, it is possible that after GVBD another kind of transport system is activated, which could involve the coated vesicle system. Coated vesicles are present in large numbers during the entire time of meiotic maturation and if activated could account for the reported residual uridine transport.

ACKNOWLEDGMENTS We thank Prof. C. Thibault for critical reading of the manuscript, Mrs. D. Huneau for skillful technical assistance in preparing ultrathin sections, Mrs. A. Solari for statistical analysis, and Mr. C. Slagmulder, and Mr. R. Scandolo for the photographs. REFERENCES Anderson A, Albertini DF (1976): Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J Cell Biol 71:680-686. Barak LS, Yocum RR, Nothnagel EA, Webb WW (1980):Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz2-0x9-1-3 diazole phallacidin. Proc Natl Acad Sci USA 77:980-984. Battaglia DE, Gaddum-Rosse P (1986): The distribution of polymerised actin in rat eggs and its sensibility to cytochalasin B during fertilization. J Exp Zoo1 237:97-105. Channing CP, Anderson LD, Hoover DJ, Kolena J , Osteen KG, Pomerantz SH, Tanase K (1982): The role of the non steroidal regulators in control of oocyte and follicular maturation. Rec Progr Hormone Res 38:331-408. Crozet N, Huneau D, De Smedt V, Theron MC, Szollosi D, Torres S, Sevellec C (1987):In vitro fertilization with normal development in the sheep. Gamete Res 16:159-170. Dekel N, Aberdam E, Sherizly I(1988):The functional significance of the cytoskeleton in regulation of oocyte maturation. In H Parvinen, I Huhtaniemi, U Pellimiemi (eds); Development and function of reproduction, Vol. 11.Serono symposium review No. 14. pp. 305-316.

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