THE JOURNAL OF EXPERIMENTAL ZOOLOGY 260401-405 (1991)

An Increase of Intracellular Free Ca2+Is Essential for Spontaneous Meiotic Resumption by Mouse Oocytes MASSIMO DE FELICI, SUSANNA DOLCI, AND GREGORIO SIRACUSA Department of Public Health a n d Cell Biology, 2 n d University of Rome, 00173 Rome, Italy ABSTRACT The involvement of calcium ions in the mechanism of meiotic resumption has been studied in mouse oocytes made resistant to the lethal effects of calcium-free medium (CFM) by zona pellucida removal (De Felici et al., '89). We show here that such oocytes undergo meiotic resumption in CFM (as evaluated by germinal vesicle breakdown, GVBD) a t a rate comparable to that shown by oocytes cultured in medium containing 1.7 m M Ca2+.The addition to CFM of 50 u M Quin2/AM (a membrane permeable, high affinity Ca2+chelator) totally prevents GVBD, while purported antagonists of Ca2' release from intracellular stores, such a s 150 u M 8-(N,Ndiethy1amino)octyl-3-4-5 trimethoxybenzoate (TMB-8) or 300 u M chlortetracycline, only cause a slight meiotic delay. On the other hand, if the oocytes are pre-incubated for 30 min in CFM supplemented with 100 u M TBM-8 plus 0.2 m M dibutyryl-cyclic AMP (dbcAMP, a reversible inhibitor of GVBD), and then cultured in the same medium, without dbcAMP, a sustained inhibition of meiotic maturation is obtained. Our observations suggest that a n increase in intracellular free Ca2+ is essential for meiotic resumption by mouse oocytes; in the experimental absence of external Ca2+,release of the cation from internal stores is sufficient to allow meiotic resumption.

Considerable evidence exists to show that calcium plays a central role in the resumption of meiosis in invertebrate (Rosenberg and Lee, '81; Meijer and Guerrier, '81) and amphibian oocytes (Kostellow et al., '80; Moreau et al., '80). Although a calmodulin-dependent step appears t o be involved in the preovulatory resumption of meiosis in mammalian oocytes (bovine: Jagiello et al., '82; Maruska et al., '84; mouse: Jagiello et al., '82; Bornslaeger et al., '84j, the role played by calcium ions in such phenomenon remains unclear. For instance, conflicting results have been reported on external Ca2+ requirement for in vitro meiotic maturation (rat: Tsafriri and BarAmi, '78; Batta and Knudsen, '80; hamster: Racowsky, '86; swine; Bae and Channing, '85; bovine: Leibfried and First, '79; Jagiello et al., '82; Maruska et al., '841, nor is it clear if meiotic resumption requires an increase in cytoplasmic free Ca2+ (mouse: Jagiello et al., '82; Powers and Paleos, '82; hamster: Racowsky, '86; rat: Tsafriri and Bar-Ami, '78). In the mouse the study of Ca2+ involvement in the resumption of meiosis is complicated by the fact that fully grown oocytes degenerate within 1.5 hr when cultured in calciumfree medium (De Felici and Siracusa, '82). In the present paper the role of Ca2+ in the resumption of meiosis by mouse oocytes has been Q 1991 WILEY-LISS, INC.

studied by using isolated oocytes that had been made resistant t o the lethal effects of CFM by removing their zona pellucida (De Felici et al., '89). The purpose of the present work was t o determine: 1)if external calcium is required for spontaneous meiotic resumption by mouse oocytes in culture, and 2) if an elevation of intracellular free calcium is required for such phenomenon.

MATERIALS AND METHODS Collection of oocytes and culture conditions Fully grown mouse oocytes, with an intact germinal vesicle (GVj and freed of cumulus cells, were obtained from 8-12 week old CD-1 mice (Charles River, Italy) by puncturing the ovaries with fine needles in a Hepes-buffered medium containing 1.7 mM Ca2+(MH [De Felici and Siracusa, '821); when Ca2+was omitted (calcium-free medium, CFM), 5 mM EGTA was added to the medium. Following isolation, the oocytes were freed of their zona pellucida by a brief treatment with acidified Tyrode's solution (pH 2.5); zona removal makes the oocytes resistant to the lethal effects of CFM (De Felici et al., '89).

Received July 17, 1990: revision accepted November 28, 1990.

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Cultures were carried out in air at 37°C in 50 or 20 ul drops of medium under paraffin oil, in Petri dishes or in Terasaki wells (Falcon 30341, respectively. Germinal vesicle breakdown (GVBD), as detected under a stereomicroscope, was chosen as criterion of meiotic resumption. GVBD was monitored every 30-60 min during the first 3 hr of culture. Degenerated cells were not included in the calculations. Plan of experiments Three series of experiments were carried out as follows:

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Series I Fig. 1. Time course of meiotic resumption (GVBD) by To determine if external Ca2+ is required for zona-free mouse oocytes cultured in Ca2'-containing medium the resumption of meiosis, for each mouse the oo- (MH) ( 0 )or in calcium-free medium plus 5 mM EGTA (CFM) (0). Each point represents the average of at least three differcytes from one ovary were collected in MH (con- ent experiments (vertical bars, S.E.). Total numbers of ootrol group), those from the other ovary in CFM. cytes examined: MH: 88; CFM: 102. Following zona pellucida removal the oocytes were cultured for 3 hr in the two media. Series I1 In this series of experiments the oocytes from one ovary were collected in CFM (control oocytes) and those from the other ovary in CFM containing various concentrations of acetoxymethyl Quin2 (QuinB/AM), 8-(N,N-diethylamino)octyl3-4-5-trimethoxybenzoate(TMB-8),or chlortetracycline (CTC). After zona pellucida removal, the oocytes were cultured for 3 hr in the same media to determine the GVBD time course. Stock solutions (10 mM) of Quin2/AM (Calbiochem) were made up in DMSO and stored at - 20°C until final dilution with CFM. Concentrated fresh solutions of TMB-8 and CTC (Sigma) were made up in water and diluted with culture medium at the appropriate concentrations immediately before use.

RESULTS Series I The majority of zone pellucida-free oocytes (about 70%, results not shown) remained viable after 3 hr of culture in CFM, thus confirming our previous findings (De Felici et al., '89). In Figure 1 it is shown that such oocytes resume meiosis in CFM at a rate similar t o oocytes cultured in Ca2+ -containing medium.

Series I1 The results obtained in Series I experiments did not rule out the possibility that a meiosistriggering increase of intracellular free calcium, released from internal stores, was still occurring in our experimental conditions. If such possibility is prevented by chelating intracellular free calcium (by culture in CFM additioned with Quin2/ Series I11 AM, a high-affinity Ca" indicator and chelator For each mouse, the oocytes from one ovary that readily permeates cell membranes [Tsien et (control group) were collected in CFM additioned al., '821), or by preventing its release from interwith 0.2 mM dibutyryl-cyclic AMP (dbcAMP) nal membranes (by culturing the oocytes in CFM (Boehringer), and those from the other ovary in additioned with TMB-8, a calcium antagonist CFM containing dbcAMP plus various concentra- that purportedly blocks the release of Ca2+from tions of TMB-8 or CTC. After zona pellucida re- isolated sarcoplasmic reticulum vesicles [Chiou moval the oocytes were cultured for 30 min at and Malagodi, '751, or with CTC, an antibiotic 37°C in CFM containing the compounds indicated that chelates Ca2+ and Mg2+ ions while inabove, and finally washed and cultured for 3 hr tercaled into the lipid bilayer of cell membranes in the same media, without dbcAMP. In some ex- [Caswell, '72; Chandler and Williams, '78]), then periments CFM was replaced by MH during col- a Ca2+-dependent meiotic resumption should be inhibited or delayed. lection and initial (30 min) incubation.

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Fig. 2. The effect of various concentrations of Quin2iAM on the time course of meiotic resumption by zona-free mouse oocytes cultured in CFM: (0)CFM, (D) 10 u M Quin2/AM, ( 0 )30 u M Quin2/AM, (A) 50 uM QuinZiAM. Each point represents the average of at least three different experiments (vertical bars, S.E.). Total numbers of oocytes examined CFM: 150; Quin2iAM 10 uM: 58; 30 uM: 68; 50 uM: 90.

Fig. 3. The effect of 300 uM chlortetracycline (CTC) ( 0 )or 150 uM 8-(N,N-diethylamino)octyl-3-4-5-trimethoxybe~oate (TMB-8) (A) on the time course of meiotic resumption by zona-free mouse oocytes cultured in CFM (0). Each point represents the average of a t least three different experiments (vertical bars, S.E.). Total numbers of oocytes examined: CFM: 120; CTC: 85; TMB-8: 102.

The results showed that GVBD was completely inhibited in oocytes cultured in CFM i n the presence of 50 u M Quin2/AM, but only delayed if the chelator was used at lower concentrations (Fig. 2). Most of the oocytes (72190) transferred to complete medium after 3 h r of incubation in CFM plus 50 u M Quin2/AM appeared viable 5 hr later (as evaluated by microscopic observation). Moreover, about 60% of these (42/72) had undergone GVBD. TMB-8 or CTC, added to CFM at concentrations that did not significantly affect oocyte viability (up to 150 u M for TMB-8 and up to 300 u M for CTC), were only able to delay by approximately 1 hour the resumption of meiosis (Fig. 3).

without dbcAMP, resulted in more than 90% GVBD inhibition (Fig. 4). The same effect (8590% inhibition of GVBD) was equally seen when, during oocyte collection and preincubation, CFM was replaced by MH (Ca2'-containing medium) (two experiments). The effect of TMB-8 was reversible: 50/55 of the oocytes were viable 5 hr after transfer to drugfree MH, and about 80% of them (39/50) had undergone GVBD. Preincubation of oocytes with dbcAMP and 300 u M CTC did not cause any additional inhibitory effect on meiotic resumption over that seen (Fig. 3) in the absence of preincubation.

Series I11 This series of experiments was carried out to determine if the failure of TMB-8 and CTC to inhibit spontaneous meiotic resumption (as seen in Series I1 experiments) was due to the fact that the depletion of intracellular free Ca2+ was not sufficiently rapid (e.g., because too slow was the effect of the drugs on internal Ca2+ release and/ or the efflux of Ca2+from the oocyte) to prevent a calcium-dependent meiosis-triggering event. The results showed that a 30 min preincubation of oocytes in CFM supplemented with 0.2 m M dbcAMP (a reversible inhibitor of the resumption of meiosis in vitro [Cho et al., '741) and 0.1 m M TMB-8, followed by culture in the same media

DISCUSSION The results of the experiments in Series I demonstrate that mouse oocytes are able to resume meiosis in the absence of a n influx of extracellular calcium: 90% of the oocytes made resistant to the lethal effects of Ca-free medium by zona pellucida removal are able to spontaneously resume meiosis within 3 hr of culture in such medium (Fig. 1). The data appear in contradiction with previous reports showing a calcium dependency for meiotic resumption in mammalian oocytes cultured in Ca-free, EGTA containing media (cumulusenclosed porcine and mouse oocytes: Bae and Channing, '85, and Jagiello et al., '82, respectively; cumulus enclosed and denuded hamster oocytes: Racowsky, '86). Species differences in the

M. DE FELICI ET AL.

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Fig. 4. The effect of various concentrations of TMB-8 on the time course of meiotic resumption by zona-free mouse oocytes cultured in CFM: (0) CFM, (A)25 uM TMB-8, ( 0 ) 50 uM TMB-8, (m) 100 u M TMB-8. In these experiments (Series 111; see text for experimental details), the oocytes were pre-incubated for 30 min in CFM containing the indicated concentrations of TMB-8 and 0.2 mM dbcAMP before transfer to CFM containing TMB-8 alone. Each point represents the average of at least three different experiments (vertical bars, S.E.).Total numbers of oocytes examined: CFM: 115; TMB-8 25 uM: 87; 50 uM: 48; 100 u M 55.

300 uM, respectively) (Fig. 2 and 3). Failure of TMB-8 and CTC t o prevent GVBD might perhaps be attributed t o their effect in antagonizing an intracellular Ca2+ rise being too slow. A complete block of GVBD was indeed obtained with 100 u M TMB-8 (but not with CTC), provided spontaneous meiotic resumption was artificially delayed by preincubating the oocytes for 30 min in a medium containing, in addition to TMB-8, also the GVBD-preventing compound dbcAMP (Fig. 4). A plausible alternative explanation of these results is that the preincubation period is required to allow efflux of the existing intracellular free calcium from the oocyte, rather than deployment of the inhibitory action of TMB-8 on internal Ca2+ stores. The finding that the inhibitory effect of TMB-8 was equally seen if Ca2+was present during the preincubation period rules out the former hypothesis. It should be pointed out that a 3 hr incubation in CFM additioned with Quin2/AM or TMB-8 is apparently not deleterious for zona pellucida-free oocytes; in both cases most of them (about 80%) were still viable 5 hr after transfer to drug-free MH, and about 60% and 80% of them, respectively, were able to resume meiosis. TMB-8 has been shown t o inhibit a number of cell functions thought t o be controlled by Ca2+,such as mitochondrial movements during the ascidian sperm reaction (Lambert and Lambert, '83), bud formation in Funaria (Saunders and Helper, '83), and cortical granule exocytosis in sea urchin eggs (Stapleton et al., '85), possibly by inhibiting the release of Ca2+from sarcoplasmic reticulum-like organelles (see Rubin, '81,for review), although other action mechanisms are also possible (Simpson et al., '84). In conclusion, the results reported here indicate that an increase in intracellular free Ca2+ is essential for meiotic resumption by mouse oocytes; in the experimental absence of external Ca2+,release of the cation from internal stores is sufficient t o allow meiotic resumption.

ability t o accumulate and retain Ca2+ ions, and the use of the complex McCoy's 5A medium in the study on mouse oocytes (Jagiello et al., '82) might, at least in part, explain the discrepancy. On the other hand, meiosis resumption has been reported to occur in the absence of external Ca2+ in cumulus-enclosed bovine and rat oocytes (Leibfried and First, '79, and Tsafriri and Bar-Ami, '78, respectively). Moreover, the results reported by Paleos and Powers ('81) showing that drugs that block calcium uptake (verapamil or tetracaine) do not prevent meiotic resumption by mouse oocytes cultured in a simple Ca2+-containing medium, are consistent with our observations. We then studied if an increase of intracellular free Ca2+ is involved in this phenomenon. When intracellular Ca2+is chelated by culturing the ooACKNOWLEDGMENTS cytes in CFM supplemented with 50 u M Quin2/ AM, a high-affinity calcium chelator which inhibThis work was supported by grants from the its various cell functions probably controlled by a National Research Council (FATMA and BBS) Ca2+rise (Affolter et al., '84; Lew et al., '84; Rao and the M.U.R.S.T. (grants 60% and 40%). et al., '86; Pershandsingh et al., '871, a complete inhibition of GVBD is obtained (Fig. 2). The reLITERATURE CITED sumption of meiosis was only delayed by about 1 Affolter, H., P. Erne, E. Burgisser, and A. Pletscher (1984) hr in oocytes cultured in CFM supplemented with Ca2+as messenger of 5HT,-receptor stimulation in human lower Quin2/AM concentrations or with nontoxic blood platelets. Naunyn-Schmiedebergs Arch. Pharmacol., 325~337-341. concentrations of TMB-8 and CTC (150 u M and

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