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Transmembrane Regulation of Intracellular Calcium by a Plasma Membrane Sodium/Calcium Exchanger in Mouse Ova¹

^a Department of Obstetrics and Gynecology, Women and Infants Hospital, Brown University, Providence, Rhode Island 02905 ^b Marine Biological Laboratory, Woods Hole, Massachusetts 02543

	ABSTRACT

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Regulation of cytoplasmic free calcium concentration ([Ca²⁺)]_i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca²⁺]_i is known to undergo oscillations, but it is unknown how basal [Ca²⁺]_i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca²⁺]_i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 ± 5.2 fmol/cm² per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 µM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca²⁺]_i. The presence of a Na⁺/Ca²⁺ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na⁺/Ca²⁺ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca²⁺]_i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na⁺/Ca²⁺ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca²⁺]_i.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Intracellular calcium is a ubiquitous element that is implicated in many cell processes, including secretion, mitosis, and meiosis [1–3]. Furthermore, sustained elevated intracellular calcium concentrations induce cell death [4, 5]. Hence, all mammalian cells precisely control intracellular levels of calcium during normal cell function. In resting cells, the cytoplasmic free calcium concentration ([Ca²⁺]_i) is typically 50–200 nM, which represents an approximate 10 000-fold gradient compared to that of the extracellular milieu that is in the 2 mM Ca²⁺ range. Under certain circumstances, such as hormonal stimulation of endocrine cells [6] and fertilization of ova [7, 8], [Ca²⁺]_i undergoes transient elevations that can reach 1–2 µM. A number of mechanisms return such elevations to the resting levels and maintain low [Ca²⁺]_i levels against the extracellular concentration gradient. These mechanisms include sequestration by intracellular organelles, principally the endoplasmic reticulum (ER) and mitochondria, as well as extrusion across the plasma membrane. Normally, the plasma membrane has at least two systems that maintain low [Ca²⁺]_i: a Na⁺/Ca²⁺ exchanger powered by the Na⁺-K⁺-ATPase, and a Ca²⁺-ATPase [9, 10]. In most cells, a large proportion of total cellular ATP is used to power these transmembrane ion gradients. Disruption of Na⁺/Ca²⁺ antiport or Ca²⁺-ATPase causes cell dysfunction and death in many cells, such as neurons and muscle, and some diseases of these tissues involve perturbed regulation of [Ca²⁺]_i [9]. Furthermore, pathological processes, such as free radical attack and hypoxia, inactivate the Na⁺-K⁺-ATPase and Na⁺/Ca²⁺ antiporter and may even reverse the latter, thus increasing cytoplasmic free calcium levels [11, 12]. Excessively high free cytosolic calcium levels disrupt cell function by activating intracellular calcium-sensitive enzymes and proteases and increasing mitochondrial calcium levels [9, 13, 14].

During fertilization of the ovum, intracellular free calcium follows a pattern of transient elevations that likely lead to reduced maturation-promoting factor activity, thus permitting progression of the meiotic cell cycle and fertilization [15]. The mechanism of sperm-induced calcium oscillations is unknown, although a soluble sperm-derived substance is able to initiate calcium oscillations within the oocyte [16, 17]. The identity of this substance is controversial, since a protein termed oscillin [18], which was believed to be the soluble factor, is now characterized as glucosamine-6-phosphate deaminase [19], and it is unable to mimic the calcium mobilization effects of oscillin [20]. However, calcium waves are essential for normal development of the fertilized zygote. Correspondingly, the transient nature of the [Ca²⁺]_i elevations implies that active calcium extrusion and/or sequestering mechanisms also must be essential for fertilization. It is known that Na⁺/K⁺-ATPase is important for normal development of mouse preimplantation embryos [21, 22]; however, the mechanisms by which the mouse oocyte regulates [Ca²⁺]_i are not fully understood. In the present study, we have begun to investigate the contribution of Na⁺/Ca²⁺ exchange across the plasma membrane as a regulator of [Ca²⁺]_i in the mouse oocyte.

	MATERIALS AND METHODS

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Animals

Female mice (B6C3F1) purchased from Charles River Breeding Labs (Wilmington, MA) were injected i.p. with 10 IU eCG, followed 48 h later by 5 IU hCG. The following morning at 13–15 h post-hCG, animals were killed, and oocyte-cumulus cell complexes (OCC) were harvested from the ampullae. The OCC were denuded of cumulus cells by a 2-min incubation with hyaluronidase (250 U/ml) in modified Human Tubal Fluid (Hepes buffered; Irvine Scientific, Santa Ana, CA) containing BSA (0.4%) and antibiotics; this was followed by washing and trituration in modified Human Tubal Fluid to remove residual cumulus cells. Denuded oocytes at the metaphase II stage of development were placed in drops (50 µl) of Human Tubal Fluid containing BSA (0.4%) and antibiotics that had been previously equilibrated under light paraffin oil (Sigma Chemical Co., St. Louis, MO) in a humidified incubator at 37°C and 5% CO₂.

The use of animals in these studies was in strict accordance with Institutional Animal Care and Use Committee Guidelines that adhere to the principles set out in the NIH guide, "Guide for the Care and Use of laboratory Animals," DHEW Publication No. (NIH) 80-23, Revised 1985.

Extracellular Calcium Flux Determination

Calcium flux was measured at the zona pellucida of mouse ova by means of a self-referencing, calcium-selective, microelectrode that was based on previous publications [23, 24]. A calcium-selective electrode was prepared by extruding a thin-walled, single-barrel, borosilicate glass capillary tube (World Precision Instruments, Sarasota, FL) using a Flaming-Brown P97 micropipette puller (Sutter Instrument Co., Novato, CA) to produce a tip diameter of approximately 2–4 µm. The glass capillary was dried and sialinized at 270°C with N,N-dimethyltrimethylsilyamine (Fluka, Ronkonkoma, NY). After further drying, the glass capillary was back-filled with 100 mM CaCl₂ and front-filled with calcium ionophore I cocktail A (Fluka). The electrode was placed in a holder with silver pellet and Ag/AgCl wire (World Precision Instruments), and a potassium chloride-agar salt bridge (3 M KCl, 3% agar) was used to complete the electronic circuit. Amplified calcium-dependent voltage signals were fed to a computer via an A/D board (CIO-DAS1600; Computer Boards Inc., Mansfield, MA) that allowed storage and off-line analysis of the data. Microelectrodes were calibrated in a series of CaCl₂-containing solutions, and only electrodes that produced the expected Nernstian slope of 28 ± 2 mV/decade change in concentration were used.

The head-stage with microelectrode was attached to a three-dimensional stepping motor apparatus (Applicable Electronics, Forrestdale, MA), controlled by ASET software (ScienceWares, East Falmouth, MA) that provides resolution of 0.05 µm/step. Amplifiers and motion controllers were originally developed at the Biocurrents Research Center, Marine Biological Laboratory, Woods Hole (see www.mbl.edu/BioCurrents). The apparatus was mounted on a platform attached to an inverted microscope (Axiophot; Nikon Corp., Melville, NY) that was equipped with a CCD camera (Cohu, San Diego, CA) and micromanipulator (Narishige USA Inc., East Meadow, NY), and the microscope was placed on a vibration isolation table within a closed Faraday cage (Technical Manufacturing Corp., Peabody, MA). Individual ova were transferred to a glass-bottomed Petri dish and bathed in a Modified M2 medium (NaCl, 94.66 mM; KCl, 4.78 mM; KH₂PO₄, 1.19 mM; MgSO₄·7H₂O, 1.19 mM; NaHCO₃, 4.15 mM; CaCl₂·2H₂O, 0.05 mM; Hepes, 20.85 mM; sodium lactate, 23.38 mM; sodium pyruvate, 0.33 mM; glucose, 5.56 mM). To further examine the influence of sodium on oocyte calcium regulation, we studied the effect of rapid sodium replenishment following a period of depletion on trans-zona calcium flux. Ova were placed in sodium-free Modified M2 (N-methyl-D-glucamine, 140 mM; KH₂PO₄, 1.19 mM; MgSO₄·7H₂O, 1.19 mM; MgCl₂·6H₂O, 1.77 mM; NaHCO₃, 4.15 mM; CaCl₂·2H₂O, 0.05 mM; choline bicarbonate, 3.8 mM; K-Hepes, 20 mM, pH 7.3; lactic acid, 2.6 mM; sodium pyruvate, 0.33 mM; glucose, 5.56 mM) for 10 min prior to the addition of a 10-fold volume of sodium-replete Modified M2, and calcium-dependent potential was recorded at the zona. The Petri dish was placed on the microscope stage, and the CCD camera imaged the oocyte that was positioned by means of a micromanipulator and holding pipette, or in some cases by means of a poly-lysine-coated glass-bottomed microwell (high molecular weight fraction; MatTek Corp., Ashland, MA). Computer-controlled positioning of the Ca²⁺-selective electrode placed it tangentially within 1 µm of the zona pellucida, and a free Ca²⁺-dependent potential was recorded. The software then controlled the movement of the electrode to a point 30 µm perpendicularly to the original tangential position, where a second Ca²⁺-dependent potential measurement was made. Previous studies had determined the optimum conditions for electrode oscillation frequency and move parameters [24]; hence oscillations were at a frequency of 0.3 Hz, and software recorded the digital output of potential after a wait period following travel of the microelectrode. The difference in Ca²⁺-dependent potential at the two points was used to assess Ca²⁺ flux at the zona pellucida. By determining the calcium concentration at two points 30 µm apart, the Fick equation can be applied to compute net calcium flux (see [24]): J = D(dc/dx), where J is an ion flux in the x direction, dc/dx is its concentration gradient, and D is the diffusion constant. Since the diffusion constant of calcium is known, measurement of calcium concentration gradient near the oocyte's surface reflects its net flux across the oocyte's surface. Thus, the difference in potential recorded between the two points is proportional to ionic flux. This technique, including theory and application, is described in detail by Kuhtreiber and Jaffe [23] and by Smith et al. [24] (see also www.mbl.edu/BioCurrents).

Intracellular Calcium Determination

To measure [Ca²⁺]_i, ova were incubated at 37°C for 30 min with the [Ca²⁺]_i indicator fura-2 acetoxymethyl ester (5 µM) in modified Human Tubal Fluid supplemented with BSA (0.4%). The ova were washed (three times with 1 ml each time) with Dulbecco's PBS containing BSA (0.1%) and transferred onto poly-lysine (high molecular weight fraction; Calbiochem, San Diego, CA)-coated coverslips that replace the plastic base of a culture dish. The culture dish containing the oocyte and 2 ml Modified M2 was placed onto the stage of an inverted microscope equipped for epifluorescence microscopy (IMT-2; Olympus, Tokyo, Japan). The microscope was coupled to a dual-excitation spectrofluorometer (Deltascan; Photon Technology International, Monmouth Junction, NJ), and the fluorescence of the cells was monitored at 510 nm (long-pass filter) during dual excitation at 340 nm and 380 nm (5-nm band pass). The [Ca²⁺]_i was calculated from the ratio of fluorescence at 340/380 nm as previously described [6, 25]. The ratio of fluorescence of fura-2 maximally bound to calcium was estimated experimentally by measuring fluorescence of ova after exposure to ionomycin (10 µM), while minimal calcium-dependent fluorescence was obtained by addition of EGTA (10 mM), as based on the previously described methodology [26].

Chemicals

Culture media, Human Tubal Fluid, and modified Human Tubal Fluid were obtained from Irvine Scientific (Santa Ana, CA). Bepridil was supplied by Research Biochemical International (Natick, MA); fura-2 acetoxymethyl was purchased from Molecular Probes (Eugene, OR); thapsigargin and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Drugs were added to ova in a volume equal to that present in the bathing media to ensure rapid mixing.

Statistics

Data are expressed as mean ± SEM. Comparison between two means was made through use of the Student's t-test, while three or more groups were compared with ANOVA and Tukey's all pair-wise comparison. Calculated differences of p < 0.05 were considered to be statistically significant.

	RESULTS

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Determination of Intracellular-Free Calcium in Mouse Ova

We used fura-2 and microspectrofluorimetry to measure [Ca²⁺]_i in ova in order to establish the effects of known regulators of intracellular free calcium in our experimental model. Under basal conditions, [Ca²⁺]_i was determined to be 137 ± 20 nM (n = 6). Although the mechanisms of calcium mobilization induced by sperm are not the same as seen during parthenogenetic activation, we have used ethanol to increase intracellular free calcium, presumably arising by temporary opening of membrane calcium channels and also release of calcium from intracellular pools [27, 28]. To examine the effect of ethanol on intracellular free calcium, we exposed mouse ova to 7% ethanol, a concentration previously used to activate ova [28] and which, in our hands, stimulates cell division (data not shown). We found that ethanol stimulated a transient increase to 1152 ± 156 nM (n = 3) within 60 sec, which fell to 467 ± 142 nM after 2 min, followed by a second transient increase to 720 ± 200 nM at 5 min and thereafter an elevated plateau phase of 434 ± 84 nM. The data of Figure 1 show an intracellular free calcium trace from a representative experiment.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Elevation of [Ca2+]i by ethanol. Representative trace showing [Ca2+]i in a fura-2-loaded oocyte in response to a parthenogenetic stimulation with ethanol (7%).

Since ethanol can affect plasma membrane calcium channels, we wished to establish a model that increased intracellular free calcium independently of plasma membrane effects. Thapsigargin (Tg), an inhibitor of the ER Ca²⁺-ATPase, causes increased intracellular calcium in most cell types and has been previously shown to increase [Ca²⁺]_i in mouse oocytes [29]. Figure 2 shows the intracellular free calcium dose response of mouse ova to Tg, and Table 1 summarizes the data of basal [Ca²⁺]_i before Tg addition, as well as resulting peak and plateau [Ca²⁺]_i (mean ± SEM of n replications). In addition, the response time to the peak and plateau phase of [Ca²⁺]_i are summarized. There was a lag-time and amplitude effect with different concentrations of Tg, such that a low dose (25 nM) produced a small, transient increase in [Ca²⁺]_i that peaked at 226 ± 42 nM (n = 6) after approximately 15 min. In the case of 250 nM Tg, the lag period was reduced and [Ca²⁺]_i peaked after approximately 10 min at 438 ± 67 nM (n = 5). When Tg was increased to 2.5 µM, there was no lag phase, and the transient increase in [Ca²⁺]_i reached a maximum amplitude of 575 ± 34 nM (n = 12) after approximately 5 min, which was a significantly shorter period to the peak than that induced by 25 nM Tg (p < 0.05).

View larger version (28K): [in this window] [in a new window]   FIG. 2. Release of intracellularly stored Ca2+ by Tg. Mouse ova were loaded with fura-2, and the dose-response effects of Tg on [Ca2+]i were determined. Tg was added to achieve a) 25 nM, b) 250 nM, and c) 2.5 µM final concentration. Representative traces are shown.

Determination of Extracellular Calcium Flux in Mouse Ova

Before investigating the effects of ethanol and Tg on calcium flux at the zona pellucida of individual mouse ova, we determined the presence of flux under basal conditions. In each experiment, calcium-dependent potential was continuously recorded and averaged at 1-min intervals for the duration of the experiment. The data obtained from 69 ova showed a calcium flux of 20.6 ± 5.2 fmol/cm² per sec, which was significantly different (p < 0.002) from the background value of -3.3 ± 3.4 fmol/cm² per sec obtained at a minimum distance of 100 µm from the zona pellucida. These observations are consistent with net calcium efflux from the oocyte. Figure 3 summarizes data to show the time course of basal calcium efflux over a 10-min period.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Net calcium flux was measured at the zona pellucida of mouse ova over a 10-min period. The signal from each oocyte was averaged at 1-min intervals and pooled with similar data from other ova. The graph represents data accumulated from 69 ova, and each data point is the mean ± SEM.

To examine the effects of raising [Ca²⁺]_i by means of ethanol and Tg on calcium flux at the zona pellucida, experiments were conducted after a 10-min equilibration period. Neither ethanol nor Tg had any effect on the calcium microelectrode characteristics. In Figure 4, data are summarized from experiments with 13 ova to show the effect of ethanol (7%) on calcium flux. The addition of ethanol stimulated an immediate increase in calcium efflux that reached 129.0 ± 35 fmol/cm² per sec within the first minute and slowly fell back toward zero but that was maintained at a positive efflux compared to the flux prior to the addition of ethanol.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Effect of parthenogenetic activation on net calcium flux. Mouse ova were equilibrated for 10 min prior to the addition of ethanol (7%) to parthenogenetically activate them. The data in the figure were obtained from 14 ova, and calcium-dependent potential was continuously recorded and averaged at 1-min intervals. Each point is the mean ± SEM.

We confirmed that Tg elevated [Ca²⁺]_i, and we wished to determine whether Tg also affected extracellular calcium flux. The data in Figure 5 illustrate that all concentrations of Tg were found to increase calcium-dependent potential when compared to buffer control, although different patterns were seen dependent upon concentration. At the highest concentration used (2.5 µM), net calcium efflux was immediately stimulated and was significantly different from the control value by the second minute of exposure (p < 0.05). Net calcium efflux reached a maximum of 87.8 ± 19.4 fmol/cm² per sec (n = 11) by 3 min of exposure before slowly returning toward 40 fmol/cm² per sec over the next 10 min. In contrast, after the addition of 25 and 250 nM Tg, calcium efflux showed a slow increase that reached a significantly elevated value compared to the control values after 9–10 min of exposure and that was maintained at approximately this level.

View larger version (34K): [in this window] [in a new window]   FIG. 5. Intracellular calcium release and its effect on calcium efflux at the zona pellucida. After an equilibration period of 10 min, ova were treated at time zero with Tg or buffer, and calcium-dependent potential was recorded. Potential was averaged at 1-min intervals, and each data point is the mean ± SEM of experiments with n ova. (2.5 µM Tg, n = 13; 250 nM Tg, n = 14; 25 nM Tg, n = 18; buffer, n = 10). Data points labeled with a, b, or c are significantly different (p < 0.05) from control values at the same time point (a = 25 nM vs. Control; b = 250 nM vs. Control; c = 2.5 µM vs. Control).

Since Ca²⁺ efflux could be regulated by a Na⁺/Ca²⁺ exchange protein, we investigated whether Na⁺/Ca²⁺ exchange occurs in ova. Bepridil is an inhibitor of Na⁺/Ca²⁺ exchange and has been shown to have an ID₅₀ of 30 µM in cardiac sarcolemmal membranes [30]. In the present studies we investigated the dose response to bepridil of [Ca²⁺]_i in mouse ova, and the data are illustrated in Figure 6. Bepridil did not affect the calcium microelectrode characteristics. Table 2 summarizes basal [Ca²⁺]_i, bepridil-induced peak and plateau phase [Ca²⁺]_i, and the response time to the peak and plateau phase [Ca²⁺]_i (mean ± SEM of n replications). When ova were treated with 12.5 µM bepridil, [Ca²⁺]_i increased from 122 ± 6 nM to 232 ± 51 nM, which was not a statistically significant increase. In the case of 25 µM bepridil, [Ca²⁺]_i increased to a peak value of 469 ± 102 nM (n = 5) after approximately 10 min before falling to a plateau level of 241 ± 31 nM. When the concentration was increased to 50 µM, bepridil stimulated an immediate transient increase to 746 ± 166 nM (n = 7) that reached a peak after approximately 10 min before falling to an elevated plateau level of 300 ± 86 nM. These doses of bepridil were used to examine calcium flux at the zona pellucida, and Figure 7 shows that all concentrations of bepridil (12.5, 25, and 50 µM) induced an increase in net calcium influx, which was followed within 4–6 min by a recovery toward a calcium efflux.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Representative traces depicting the effects of bepridil on [Ca2+]i in mouse ova. Fura-2-loaded ova were treated with 12.5 µM (a), 25 µM (b), or 50 µM bepridil at the arrow. (Discontinuous traces due to closure of shutter during addition of bepridil.)

View larger version (29K): [in this window] [in a new window]   FIG. 7. Effects of bepridil on net calcium influx. Ova were treated with bepridil (12.5 µM, n = 12; 25 µM, n = 6; 50 µM, n = 6), and calcium-dependent potential was recorded at the zona pellucida, averaged over 1 min. Each point is the mean ± SEM of n replications.

The results of the study on the effect of rapid sodium replenishment following a period of depletion are shown in Figure 8, which demonstrates that net calcium efflux at the zona pellucida was greatly stimulated by the replenishment of external sodium and was sustained for up to 15 min. However, at a background distance (BG) of at least 500 µm from the ovum, no significant flux was detected.

View larger version (22K): [in this window] [in a new window]   FIG. 8. Net calcium efflux stimulated by replenishment of external sodium. Mouse ova were placed in sodium-free Modified M2 for 10 min before addition of a 10-fold volume of sodium-replete Modified M2. Calcium-dependent potential was recorded at the zona pellucida and averaged at 1-min intervals, and a background recording (BG) was made at a minimum distance of 500 µm from the ova. Data are the mean ± SEM of 8 replications.

	DISCUSSION

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In the present studies we found that metaphase II-arrested ova exhibit an apparent net calcium efflux, a finding consistent with data for other cell types using similar technology [31, 32]. The temporal resolution of the microelectrode is such that a rapid, transient, influx of calcium associated with the opening of plasma membrane calcium channels is unlikely to influence the recording, whereas relatively steady state transmembrane effluxes are measured. When calcium channels open, inward flux tends to be rapid and transient because Ca²⁺ flows into the cell down a 10 000-fold concentration gradient. On the other hand, Ca²⁺ must be pumped out of the cell against this large gradient. In the red blood cell, transmembrane transporters and exchangers, which drive a calcium efflux, will pump calcium out for about 5–1/2 h for each second that a single calcium channel remains open [33]. Thus, healthy cells would be expected to constantly pump calcium out across their plasma membranes. Indeed, based on our previous observations with two-cell mouse embryos, calcium efflux may be a marker for cell viability, since we found a positive correlation between the presence of calcium efflux and in vitro developmental potential of embryos. No efflux was detected in early embryos that failed to develop [34]. A physiological test of oocyte and early embryo viability determined noninvasively, as in the present methodology, could have significant implications for in vitro fertilization programs that currently depend on nonfunctional, morphological appearance of ova and embryos for use in their selection procedures.

During fertilization, a calcium wave is propagated as the sperm fuses with the oocyte, and this wave is followed by oscillatory transient increases in [Ca²⁺]_i that vary in frequency among species, and somewhat among ova [16, 35]. The mechanisms of this calcium response are not fully understood, although release of calcium from intracellular stores that are sensitive to inositol trisphosphate is likely [16]. Little is known, however, about the mechanisms of calcium regulation in fertilized ova. In the present studies we used ethanol to elevate [Ca²⁺]_i, since this is known to be a parthenogenetic stimulus in mouse ova. Indeed, we found that ethanol stimulated a rapid, transient increase in [Ca²⁺]_i that returned to an elevated plateau phase and persisted for at least 10 min. Correspondingly, when ova were treated with ethanol, the calcium ion-selective microelectrode detected an immediate increase in net calcium efflux at the zona pellucida that paralleled the [Ca²⁺]_i response. A similar parallel increase in [Ca²⁺]_i and calcium efflux occurs in the marine animal Phallusia mammilata during fertilization [23]. These observations indicate that the opening of calcium channels in the plasmalemma results in an influx of calcium that is countered by calcium-removal mechanisms across the cell membrane. However, the elevated plateau phase of [Ca²⁺]_i following ethanol treatment also suggests that a new balance is reached between influx, efflux, and sequestration.

Tg is known to inhibit the ER Ca²⁺-ATPase, which results in release of Ca²⁺ from the ER but has no effect on the plasmalemmal Ca²⁺-ATPase [36]. Treatment of mouse ova with Tg induced a dose-dependent increase in [Ca²⁺]_i that was time and amplitude sensitive. The maximal concentration of Tg (2.5 µM) used in the present studies induced a rapid spike in [Ca²⁺]_i, similar to that observed with ethanol, while the lowest concentration (25 nM) also transiently increased [Ca²⁺]_i, but to a lesser degree and only after a lag of approximately 5 min. The intermediate concentration of Tg (250 nM) produced an intermediate amplitude and lag to increased [Ca²⁺]_i. Since Tg inhibits Ca²⁺ sequestration by the ER, the return toward basal values of [Ca²⁺]_i is likely due to extrusion across the plasma membrane and to sequestration by other stores, such as those in mitochondria. Indeed, the importance of Ca²⁺ extrusion as a mechanism to maintain low [Ca²⁺]_i in mouse ova was indicated by the calcium ion-selective microelectrode that recorded stimulation of Ca²⁺ efflux, which paralleled the amplitude and/or time effects of Tg on [Ca²⁺]_i.

Sodium/calcium exchange has been shown to take place in retinal rod outer segments [37], brain synaptosomes [38], and cardiac myocytes [39], and is an important mechanism that transports substantial quantities of calcium in these cells. It is not clear to what extent the calcium efflux observed in the present studies is driven by a plasma membrane ATPase versus a Na⁺/Ca²⁺ exchanger. In the absence of a specific inhibitor of the plasma membrane Ca²⁺-ATPase, this is a difficult issue to resolve. On the other hand, although bepridil is a calcium channel blocker in addition to being an inhibitor of Na⁺/Ca²⁺ exchange, the present data are consistent with the latter mechanism's being an important regulator of [Ca²⁺]_i in the oocyte. When ova were treated with bepridil (25, 50 µM), [Ca²⁺]_i underwent a transient elevation, consistent with inhibition of calcium extrusion, and net increase in influx of calcium down its concentration gradient. It is not clear why no increase in [Ca²⁺]_i was detected in ova treated with 12.5 µM bepridil although increased calcium influx was detected. It is possible that the influx of calcium induced by 12.5 µM bepridil was compartmentalized or sequestered to a site other than free cytosolic Ca²⁺ recorded by fura-2. In the present studies, we found that elevations in [Ca²⁺]_i induced by the higher concentrations of bepridil were transient. It is likely that the reduction in the elevated [Ca²⁺]_i was brought about through the actions of the plasma membrane Ca²⁺-ATPase stimulating extrusion, in addition to sequestration of Ca²⁺ by intracellular organelles, including mitochondria. Studies in other cell types have also indicated the profound homeostatic coupling between Na⁺/Ca²⁺ exchange and plasmalemmal Ca²⁺-ATPase for maintenance of low [Ca²⁺]_i when one of these extrusion mechanisms is compromised [40]. Further evidence for a functional sodium/calcium exchanger in mouse ova comes from our studies in which replenishment of extracellular sodium, after depletion, induced a large calcium efflux. These findings suggest that removal of extracellular sodium reduces extrusion of Ca²⁺, leading to intracellular accumulation of calcium. When extracellular sodium was made rapidly available to the oocyte, the Na⁺/Ca²⁺ exchanger was activated, stimulating calcium efflux.

We have shown that elevation of [Ca²⁺]_i stimulates calcium efflux across the zona pellucida. This flux is likely driven, at least in part, by a Na⁺/Ca²⁺ exchange mechanism, since flux across the zona was affected by the sodium content of the media and bepridil, and also bepridil-regulated [Ca²⁺]_i. The Na⁺/Ca²⁺ exchanger may be an important contributor to homeostatic mechanisms that maintain [Ca²⁺]_i at low levels during the fertilization process.

	ACKNOWLEDGMENTS

 
We appreciate Dr. H.R. Behrman for the use of the microspectrofluorimeter (Deltascan) at the Reproductive Laboratory Section, Yale University, New Haven, CT.

	FOOTNOTES

 
¹ This work was supported in part by NIH KO8 01099-01 to D.L.K.

² Correspondence: J.R. Pepperell, Department of Obstetrics and Gynecology, Women and Infants Hospital, Brown University, 101 Dudley St., Providence, RI 02905. FAX: 401 453 7599; jpeppere{at}wihri.org

Accepted: December 11, 1998.

Received: September 9, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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