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a Loeb Research Institute and Departments of Obstetrics and Gynecology (Division of Reproductive Medicine), and
b Cellular and Molecular Medicine, University of Ottawa, and
c Human IVF Program, Ottawa Hospital, Ottawa, Ontario, Canada K1Y 4E9
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In many cells, the anion channel that is activated during RVD is permeable not only to inorganic anions, principally Cl-, but also to organic osmolytes [4–6]. This property may be especially important to zygotes. First, embryo development can be rescued from the deleterious effects of higher osmolarities [7, 8] by any of a number of organic osmolytes [9–11]. These are imported into the cell to provide osmotic support; mouse zygotes accumulate several-fold more of the model osmolyte, glycine, when external osmolarity is increased [12]. Presumably, zygotes regulate the intracellular levels of such osmolytes not only by importing them but also by using some mechanism for releasing them when the concentration is too high. A swelling-activated channel that is permeable to organic osmolytes would serve this function. Second, Lane and Gardner [13] have shown that a lack of nonessential amino acids, a group that includes several organic osmolytes, is detrimental to mouse zygote development, and that even a 5-min exposure to medium devoid of amino acids has a marked negative effect on development. This could be due to a loss of accumulated amino acids via the swelling-activated channel. Indeed, glycine and taurine accumulated by early embryos are both rapidly lost upon cell swelling [12, 14], and a large swelling-induced increase in permeability to several amino acids has been demonstrated in mouse zygotes or early embryos ([1,12] and unpublished results). Therefore, the activity of a swelling-activated anion channel in zygotes could have a large impact on embryo development, not only by controlling cell volume but also by affecting the intracellular stores of amino acids and other organic compounds.
The electrophysiological characteristics of swelling-activated osmolyte/anion channels in other cells have been extensively investigated [4, 5]. These currents are outwardly rectifying, and they are more permeable to I- than to Cl-. Significant currents can be carried by negatively charged organic osmolytes such as aspartate and taurine [4–6]. Current is blocked by pharmacological Cl- channel blockers and also by external ATP in millimolar amounts [15]. Since mouse zygotes may have such channels, based on the demonstrated increase in their permeability to organic compounds upon swelling and the apparent dependence of RVD upon Cl- channel activity, we have used electrophysiological methods to investigate the properties of any swelling-activated currents in zygotes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice were maintained on a 12L:12D cycle. Zygotes and oocytes were obtained from female CF1 strain mice (Charles River Canada, St. Hyacinth, PQ, Canada), which were superovulated by i.p. injection of 5 IU eCG 4 h before the start of the dark period, followed 47.5 h later by 5 IU hCG (both hormones from Sigma Chemical Co., St. Louis, MO). For zygotes, the mice were caged with BDF strain males (Charles River Canada) immediately following hCG, and left overnight. Zygotes were removed from oviducts into Hepes-KSOM medium (see below) at 19–23 h post-hCG, and fertilization was confirmed by the presence of two pronuclei; oocytes were similarly obtained at approximately 16 h post-hCG. The cumulus was removed, when necessary, using hyaluronidase (0.3 mg/ml for 5–10 min). Zygotes and oocytes were used immediately or kept for up to several hours in KSOM embryo culture medium under oil at 37°C in 5% CO2/air. Zonae were removed immediately before electrophysiological measurements by a 15-min exposure to 1.0 mg/ml pronase, after which they were washed in Hepes-KSOM and transferred to the experimental chamber (below).
Solutions and Chemicals
All solution components were obtained from Sigma. Osmolarity was measured using a vapor pressure osmometer (model 5520; Wescor, Logan, UT). External solutions were based on KSOM embryo culture medium [16]. KSOM, which is bicarbonate/CO2-buffered, was used for short-term storage of zygotes as described above. All experiments were done in variants of Hepes-KSOM, in which 21 mM of the 25 mM NaHCO3 was replaced by Hepes and the pH adjusted to 7.3–7.4 at room temperature using NaOH. The hypotonic medium used for cell swelling was 180 mOsM Hepes-KSOM, whose composition is shown in Table 1 (designated 70 Cl-); 250 and 330 mOsM Hepes-KSOM was usually produced by the addition of 60 mM or 150 mM mannitol to the external solutions shown in Table 1, although in one set of experiments the osmolarity was increased by adding 30 mM or 80 mM NaCl. In several experiments, some or all of the NaCl was omitted from the external solution and replaced with NaI, sodium aspartate, or taurine. The compositions of these external solutions are shown in Table 1 (designated by their aspartate, I-, and Cl- concentrations). These external solutions were used with KCl/potassium gluconate-based internal (pipette) solutions containing either 17 mM or 65 mM Cl- (internal solutions designated 17 Cl- and 65 Cl- in Table 1).
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In one set of experiments, most of the usual components of Hepes-KSOM were omitted to produce a simplified solution without significant potential current carriers except the anion of interest (Cl-, aspartate, or taurine) and Na+ as the major cation. These were designated 80 Cl-, 80 asp, and 155 taurine (Table 1). They were used with the CsCl- and cesium methanesulfonate-based internal solutions designated 80 Cl- and 0 Cl-, respectively (Table 1).
Drugs were added to media from stock solutions immediately before an experiment. The Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) were prepared as 0.1 M stock solutions in dimethyl sulfoxide (DMSO). DIDS stocks were kept for up to 1 mo at -20°C, while NPPB stocks were prepared daily. The final amount of DMSO in media was 
0.1%. ATP was prepared as a 125 mM stock in deionized H2O and adjusted to pH 6–7 with NaOH.
Chamber and Solution Changes
All electrophysiological measurements were done in an open chamber with a coverslip bottom (Biophysica, Sparks, MD) mounted on a Zeiss Axiovert (Carl Zeiss, Thornwood, NY) inverted microscope and maintained at room temperature. Zygotes and eggs adhered to the clean glass bottom strongly enough to allow solution changes. Solutions were superfused at a rate of 2 ml/min through an 18-gauge stainless steel tube connected to a gravity-driven perfusion system in which the choice of superfusion solution was controlled by solenoid valves (BPS-4 Bath Perfusion System; Adams and List Assoc., Westbury, NY). Solution was withdrawn using suction provided by a Barnant (Barrington, IL) model 470–5942 pressure/vacuum station to maintain a constant fluid level.
Whole-Cell Current Measurements
The whole-cell voltage-clamp technique was used to record membrane currents from mouse zygotes or eggs. Currents and imposed voltages were monitored continuously with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). A Digidata 1200B digitizer with p-CLAMP (v. 6) software (Axon Instruments) was used online to sample current signals.
Patch microelectrodes were pulled from glass tubes (1.65-mm outer diameter; Garner Glass Company, Claremont, CA) on a two-stage vertical puller (model L/M-3P-A; List-Medical, Darmstadt, Germany) and fire polished before use. After the pipettes had been filled with the appropriate internal solution (see below and Table 1), electrodes had resistances of 2–5 M
. Recordings were made only if the total series resistance (electrode and access resistances) was less than 8 M. Typically, 70–80% of the series resistance was compensated using the amplifier. The reference electrode was either a 3 M KCl agar bridge or AgCl-coated silver wire.
At the start of each experiment, the zygote or oocyte was placed into modified Hepes-KSOM at 250 mOsM, with CaCl2 added to bring the total Ca2+ concentration to 10 mM. This higher extracellular Ca2+ greatly facilitated gigaseal formation [17]. Recordings were made from cells only where the seal resistance exceeded 5 G. At the end of each experiment, the patch was deliberately excised to obtain an outside-out patch. Whenever the excised patch seal resistance was < 1 G, the integrity of the seal was assumed to have been compromised during the experiment, and the data from that zygote were not used. After achievement of the whole-cell mode, superfusion was begun with solution containing the normal (1.7 mM) Ca2+ concentration. Subsequent external solution changes were made by superfusing different solutions, as described above.
During each experiment, zygote or oocyte diameter was measured periodically using the eyepiece reticle in the microscope with a x20 objective to obtain a record of changes in cell diameter. The eyepiece reticle scale was calibrated using a stage micrometer, and each division was found to correspond to 4.84 µm. Diameters were recorded to the nearest half-division (approximately 2.4 µm).
In one set of experiments, cell-attached perforated-patch recordings were made with an electrode containing the ionophore nystatin [18]. The tips of these micropipettes were first partly filled by dipping for about 30 sec in nystatin-free internal pipette solution and then back-filled with the same solution containing nystatin (225 µg/ml). The stock solution of nystatin (30–60 mg/ml) was made in DMSO prior to the experiment. The final concentration of DMSO never exceeded 0.1%, and DMSO itself was found to have no effect.
The same command voltage protocols were followed for every experiment. During each recording from a zygote or oocyte in whole-cell mode, the holding voltage was maintained at -20 mV (uncorrected for any liquid junction potential) and the current was sampled at a rate of 20 Hz (i.e., every 50 msec). Every 50 sec, the voltage was ramped from -60 to +80 mV (imposed voltage uncorrected for liquid junction potential) at a rate of 0.093 mV/msec (1.5-sec total duration) to generate current spikes that allowed the whole-cell current at -60 and +80 mV to be visualized.
At points during the recording where current-voltage plots were to be generated for analysis (usually after whole-cell currents had stabilized after an experimental manipulation), the current was sampled at an increased rate of 100 Hz (i.e., every 10 msec), and the voltage was ramped at a slower rate of 0.05 mV/msec (2.8-sec total duration), to yield current measurements at each 0.5-mV increment of voltage for the duration of the voltage ramp. This allowed more precise determinations of reversal potentials and conductances. All current-voltage plots shown below were obtained using this protocol.
Liquid junction potentials for each combination of internal pipette solution and external solution were determined as described by Neher [19]. Voltages given throughout have been corrected for liquid junction potential. Measured liquid junction potentials were not affected by changes in external mannitol concentration. Measured liquid junction potentials were +7 to +12 mV when the internal pipette solution contained 17 mM Cl-, +5 mV when the internal Cl- was 65 mM, and -2.5 to +3.5 mV when 80 mM CsCl or 120 mM cesium methanesulfonate was used as internal solution.
Data Analysis
Off-line analyses were performed using Clampfit version 6 (Axon Instruments) to generate current-voltage plots. Currents were obtained directly from current-voltage data. Conductances were obtained as the slope of the current-voltage relation at the stated potential, calculated by linear regression. Data in the text are expressed as mean ± SEM. Data were compared using ANOVAs followed by the Tukey-Kramer multiple comparison test, t-tests, or paired t-tests, where appropriate, as specified in the text (Instat; GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The resting membrane potential (Vm) was measured immediately upon breakthrough into the whole-cell mode (in 10 mM Ca2+ medium) for each of a total of 58 mouse zygotes. The mean Vm was -32.1 ± 0.4 mV (± SEM) after correction for the liquid junction potential. This is comparable to the value previously reported for resting potential in mouse zygotes (-31.8 ± 1.0; [20]). The mean whole-cell current, measured at +60 mV after the external medium had been replaced with normal, 1.7 mM Ca2+ medium, was 2.4 ± 0.1 nA, and the mean conductance at +60 mV was 53 ± 4 nA (n = 58). The mean diameter of these zygotes in 250 mOsM medium was 71.6 ± 0.5 µm.
Swelling-Activated Current in Zygotes
A swelling-induced increase in whole-cell current was observed in every zygote assessed (n = 58). Hypotonic swelling was induced by replacing the initial 250 mOsM Hepes-KSOM with 180 mOsM Hepes-KSOM. Since the internal solutions were 230 mOsM, reducing the external osmolarity to 180 resulted in osmotic swelling, evidenced by an increase in diameter (e.g., Fig. 1, top). In some experiments the 180 mOsM medium was replaced with 330 mOsM medium after maximal swelling had been achieved, to rapidly decrease volume. Figure 1 (bottom) shows a representative current trace from one such experiment using a zygote in whole-cell patch-clamp mode, where the external osmolarity was first decreased from 250 mOsM to 180 mOsM and then increased to 330 mOsM (osmolarity controlled with mannitol). The external solution used was that designated 70 Cl-, and the internal solution used was that designated 17 Cl- (Table 1). Each spike in the trace is generated by a 1.5-sec-long voltage ramp that cannot be resolved as a ramp with the time scale used in the figure. The extent of the spikes is a measure of the whole-cell current. When the external osmolarity was reduced from 250 mOsM to 180, this zygote swelled to approximately 1.15 times its initial diameter (Fig. 1, top), and the total current increased about 3-fold. The increase in current was reversed when the cell volume was again decreased; here, decreasing the diameter to a value below the initial diameter (to about 0.93 times the initial diameter) by introducing 330 mOsM medium caused the current to decrease below its initial level as well. Returning the zygotes to 250 mOsM rather than 330 mOsM medium after swelling in 180 mOsM medium caused a similar reduction in current to the pre-swelling value (not shown).
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A representative current-voltage plot for a single zygote (Fig. 2A) shows current as a function of applied voltage in isotonic (250 mOsM), hypotonic (180 mOsM), and then hypertonic (330 mOsM) media (same solutions and protocol as in Fig. 1). The current-voltage plots shown were recorded after each cell had reached a constant diameter in each solution. The current is outwardly rectifying under these conditions; and current greatly increased upon swelling, and then decreased upon shrinkage, over the entire range of test voltages.
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Whole-cell current at about +60 mV was determined as a function of zygote diameter in 250, 180, and 330 mOsM Hepes-KSOM, with osmolarity varied using mannitol, for a number of zygotes. The current increased by an average of almost 3-fold when the average diameter increased by an average of 1.13 times upon exchange of 250 mOsM medium for 180 mOsM (Fig. 2B). The conductance, measured at approximately +60 mV (as the local slope of the current-voltage relation), increased similarly (Fig. 2B). After swelling, introducing 330 mOsM medium caused a decrease in both current and conductance (Fig. 2B). Similar data were obtained in experiments in which NaCl rather than mannitol was used to control osmolarity (not shown), indicating that osmolarity, rather than changes in mannitol concentration, was the relevant parameter. Thus, there is a substantial swelling-activated current in mouse zygotes.
The reversal potential was not greatly affected when the current was activated by swelling: at 250, 180, and 330 mOsM (altered by changing mannitol concentrations), the mean (± SEM) reversal potentials were -29.8 ± 0.6 (n = 58), -28.5 ± 0.6 (n = 58), and -33.1 ± 3.1 (n = 7) mV, respectively. This difference just reached significance (p = 0.04).
In the whole-cell configuration, the interior of the cell and pipette are continuous, and therefore the normal diffusible constituents of the cytoplasm are dialyzed away and largely replaced by the internal pipette solution. While this allows the desired control over intracellular ionic contents, it might result in the loss of important metabolites or components of signaling cascades. Therefore we performed several experiments using the nystatin-permeabilized patch technique rather than the whole-cell patch configuration, with the same internal and external solutions as those specified above. With this method, only small ions permeate the patch, and the intracellular contents of other constituents are preserved. The results were nearly identical to those obtained in whole-cell mode: both zygote diameters (70.2 ± 1.5 µm in 250 mOsM, 80.0 ± 1.5 µm in 180 mOsM, and 63.9 ± 1.9 µm in 330 mOsM, n = 3) and whole-cell currents (2.7 ± 1.2 nA at 250 mOsM, 6.2 ± 1.7 nA at 180 mOsM, and 1.0 ± 0.1 nA at 330 mOsM, measured at +60 mV as before) were essentially indistinguishable from those obtained in whole-cell mode (Fig. 2B). Therefore, all subsequent experiments were carried out in the whole-cell configuration.
Pharmacology of Swelling-Activated Current
We tested whether the Cl- channel blockers DIDS and NPPB would affect swelling-activated current in zygotes. The same external and internal solutions were used as specified in the previous section. Figure 3 shows representative current-voltage plots from individual zygotes. In all cases, current had greatly increased from that in the initial 250 mOsM medium (not shown) upon swelling in 180 mOsM medium, as described above. At concentrations of 100 µM, either DIDS (Fig. 3A) or NPPB (Fig. 3B) greatly inhibited this swelling-activated current. DIDS was more effective at blocking outward current than inward current, so that the current-voltage relation showed a characteristic sag at positive test potentials, while NPPB appeared equally effective at all test potentials.
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Inhibition was quantitated by comparing the current and conductance at +60 mV before and after introduction of the inhibitor for each zygote tested. The mean (± SEM) ratio of the whole-cell current in 100 µM DIDS to that before the introduction of DIDS was 0.35 ± 0.04 (n = 4), and the mean ratio of the conductance was 0.39 ± 0.05. These decreases were highly significant (p = 0.0007 and 0.0014, respectively, by paired t-test). The data for inhibition by 100 µM NPPB were similar, with current and conductance decreasing to 0.35 ± 0.05 and 0.34 ± 0.08 (n = 4), respectively, relative to the values before introduction of NPPB. These decreases were also highly significant (p = 0.001 and 0.004, respectively). The diameters of the hypotonically swelled cells were unaffected by application of the inhibitors: 81.8 ± 1.5 µm (mean ± SEM, n = 4) both before and during DIDS exposure, and 80.0 ± 1.5 µm before, and 81.3 ± 1.0 µm during, 100 µM NPPB exposure (n = 4). In contrast, current and conductance were essentially unchanged upon the introduction of 10 µM NPPB (relative mean current and conductance of 1.07 ± 0.05 and 0.95 ± 0.08, respectively, which are not significantly changed from before the addition of NPPB: p = 0.26 and 0.60, respectively, by paired t-test, n = 3). This indicates a Ki for NPPB inhibition approximately between 10 and 100 µM.
External ATP also inhibited the swelling-activated Cl- current in mouse zygotes, as reported for other cell types [15]. External ATP at 5 mM greatly attenuated the outward current without affecting the inward current significantly (Fig. 3C). These experiments, however, were technically difficult, since introducing ATP at 5 mM invariably disrupted the seal between the zygote membrane and the patch electrode. Therefore, we gradually increased the ATP in a step-wise fashion from 0.1 mM until 5 mM was reached, preserving the seal (steps were 0.1, 0.5, 1.0, 3.0, and 5.0 mM). Because of the significant time that elapsed while this was done, we compared the current in 5 mM ATP to that immediately after ATP was washed out, rather than to the current before any ATP had been introduced. Data obtained from two zygotes showed ratios of current in ATP relative to those after washout of 0.33 and 0.38. ATP at 5 mM was then reintroduced to confirm that it would again inhibit the current to the same extent (not shown). During the step-wise increase in ATP concentration, no decrease in the current was observed in either zygote until 3.0 mM ATP was reached (not shown), indicating that the Ki for ATP inhibition was between approximately 1.0 and 3.0 mM.
Effect of Changes in Anion Composition onSwelling-Activated Currents
A series of experiments were performed in which Cl- concentrations were altered and in which other anions were substituted for Cl-. Where external Cl- concentration was changed from its usual value of 70 mM, whole-cell current measured at approximately +60 mV was compared to current measured in the same cells before external Cl- had been altered (Fig. 4A). In the experiment in which the Cl- concentration in the pipette was increased to 65 mM, the current was compared to that measured in other zygotes with the usual value of 17 mM Cl- in the pipette. In addition, the reversal potential for each condition is shown, after correction for liquid junction potential (Fig. 4B).
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Increasing the internal Cl- concentration from 17 to 65 mM with 70 mM external Cl- caused a decrease in current and shifted the reversal potential toward more positive potentials (Fig. 4). Substituting external Cl- with aspartate decreased current and shifted the reversal potential toward more positive potentials; a greater effect on each parameter was seen when all 70 mM external Cl- was replaced with aspartate than when only 50 mM Cl- was replaced with aspartate and 20 mM Cl- remained (Fig. 4). In contrast, similar replacement of external Cl- with I- (Fig. 4) resulted in an increase in current, and the reversal potential shifted toward a more negative potential when all 70 mM external Cl- was replaced with I-.
Swelling-Activated Current Carried byAspartate and Taurine
Because substitution of external Cl- with aspartate did not completely abolish outward currents (Fig. 4 and above), we performed further experiments to determine whether aspartate or taurine would conduct swelling-activated currents in mouse zygotes. To minimize the possibility of confounding outward K+ currents, we used internal pipette solutions that contained Cs+ as the only monovalent cation (Table 1). In the first set of experiments, internal CsCl was used with a total internal Cl- concentration of 80 mM. We first confirmed that swelling-activated currents were present with CsCl internal solutions. With 80 mM Cl- external solution used to produce symmetric Cl-, an outwardly rectified, swelling-activated current was evident, and this swelling-activated current was inhibited by DIDS (Fig. 5A); outward current measured at +60 mV increased upon swelling from a mean of 2.0 ± 0.5 nA (in 250 mOsM medium) to 5.7 ± 1.3 nA (180 mOsM), and then decreased to 1.9 ± 0.6 nA after the introduction of DIDS (n = 5). Current at 180 mOsM was significantly greater than at 250 mOsM or in the presence of DIDS (p < 0.01 by repeated measures ANOVA followed by Tukey-Kramer test). Thus, swelling-activated currents similar to those observed with pipette solutions based on KCl or potassium aspartate were still seen with CsCl pipette solutions.
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We next tested the effect of substituting external Cl- with aspartate. Using the same CsCl internal solutions with 80 mM Cl-, current was measured after zygotes were swelled in 180 mOsM external solution containing 80 mM Cl-, and then subsequently when external Cl- was replaced with 80 mM aspartate (Table 1). The current at +60 mV was 1.6 ± 0.3 nA in 250 mOsM medium before swelling, increasing to 6.5 ± 0.5 nA after swelling (n = 4; p < 0.001). When all external Cl- was then replaced with aspartate, outward current decreased, and reversal potential shifted in the positive direction (Fig. 5B). For the four zygotes tested, mean current decreased from 6.5 ± 0.5 nA to 1.0 ± 0.1 nA (p < 0.001), and reversal potential shifted from -7.0 ± 0.7 mV to +41 ± 2 mV (p = 0.0002 by paired t-test). However, despite the decrease, significant outward current still remained even after all external Cl- was substituted with aspartate.
In a separate set of experiments, we used an internal pipette solution based on cesium methanesulfonate (Table 1). In this set of experiments, zygotes were swelled in 80 mM external aspartate solution at 180 mOsM, and their volume was then rapidly decreased by increasing the external osmolarity to 320 mOsM with mannitol while maintaining the aspartate concentration at 80 mM (Fig. 5C). In swelled zygotes in 180 mOsM medium, the mean current at +60 mV was 1.6 ± 0.4 nA. Current decreased significantly to 0.6 ± 0.1 nA at 320 mOsM (n = 5; p = 0.03 by paired t-test). Reversal potential shifted from +5 ± 5 to -9 ± 2 mV (p = 0.04). In other experiments, zygotes were swelled in 180 mOsM medium with 80 mM aspartate, and then 100 µM DIDS was introduced with aspartate and osmolarity unchanged (Fig. 5D). DIDS inhibited the outward current, which decreased from a mean of 1.5 ± 0.3 nA to 0.9 ± 0.2 nA with DIDS (n = 3; p = 0.04). There was no significant change in reversal potential. Thus, even in the absence of internal and external Cl-, a swelling-activated current that was inhibited by DIDS was evident in external aspartate.
We also tested whether outward current persisted when all external Cl- was replaced with taurine. These experiments were carried out at pH 8.2, where the concentration of taurine in the anionic rather than zwitterionic form is about 50 mM vs. only about 8 mM at pH 7.4 (of 155 mM total; Table 1). CsCl internal pipette solutions with 80 mM Cl- were used, in a protocol identical to that for the first set of aspartate experiments described above. At external pH 8.2, a swelling-activated current was again evident in symmetric 80 mM Cl-, with mean current increasing from 1.9 ± 0.6 nA at 250 mOsM to 5.3 ± 0.8 nA at 180 mOsM (n = 3; p < 0.01). The magnitude of the swelling-activated current that was elicited at pH 8.2 was similar to that at pH 7.4 (6.0 ± 0.7, n = 9). Replacing all external NaCl with taurine reduced, but did not completely eliminate, outward current (Fig. 5E); mean current decreased from 5.3 ± 0.8 to 0.27 ± 0.1 nA (n = 3; p < 0.01). The reversal potential shifted from -7 ± 1 mV to +51 ± 2 mV when external Cl- was replaced with taurine (p = 0.0002).
Swelling-Activated Current in Ovulated Oocytes
Whole-cell current in ovulated oocytes was measured in 250 mOsM medium, then in 180 mOsM medium to induce hypotonic swelling, and finally in 180 mOsM medium with 100 µM DIDS. These experiments were identical to those carried out with zygotes and described above, with 17 mM internal Cl- and 70 mM external Cl-. At breakthrough, the measured resting membrane potential (Vm) of ovulated oocytes was -32.3 ± 1.7 mV (n = 6). This is essentially identical to the -32.1 mV measured for zygotes (above), but different from values of -44.1 ± 1.2 mV [20] or -17.7 ± 0.8 mV [17] reported previously in patch-clamp studies. The mean whole-cell current in oocytes at +60 mV was 0.9 ± 0.2 nA (n = 6) in 250 mOsM medium, which then increased significantly to 1.9 ± 0.2 nA after hypotonic swelling in 180 mOsM medium (p < 0.05 by Tukey-Kramer test). After the subsequent addition of DIDS, the mean current decreased slightly to 1.7 ± 0.3 nA, but this decrease was not significant (p > 0.05). Mean oocyte diameter increased from 76.0 ± 1.0 µm in 250 mOsM medium to 83.2 ± 1.5 µm in 180 mOsM medium and remained unchanged in 180 mOsM medium with DIDS. The increase in diameter is highly significant (p < 0.001 by Tukey-Kramer test, with no difference between 180 mOsM with and without DIDS). Compared to the currents measured in zygotes under identical conditions (means of 2.4 nA in 250 mOsM and 6.6 nA in 180 mOsM medium; see above and Fig. 2B), the currents measured in ovulated oocytes (0.9 and 1.9 nA) were significantly smaller (p = 0.0005 and < 10-5, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The characteristics of the swelling-activated current in mouse zygotes resemble those of swelling-activated currents that have been described in other types of cells and that are mediated by channels permeable to Cl- and organic osmolytes. These channels have been termed volume-sensitive organic osmolyte/anion channels (VSOAC [4]) or volume-expansion sensing, outwardly rectifying channels (VSOR [5]). The molecular identity of the channel protein is not yet certain, although it may be the ClC-3 isoform of the ClC family of Cl- channels, which has very similar properties [21]. VSOAC/VSOR currents are outwardly rectifying, which is also a property of the swelling-activated current in the mouse zygote. This contrasts with the presence of inward rectification or lack of rectification characterizing some other putative swelling-activated Cl- currents [4]. VSOAC/VSOR currents are inhibited by DIDS, which is reported to be effective at a concentration of 100 µM [5, 21–23], with greater inhibition of outward than of inward current [5, 21–23]. This is similar to what was seen here in mouse zygotes (Figs. 3A and 5A). NPPB is reported to inhibit VSOAC/VSOR currents in a more voltage-independent fashion than DIDS [23, 24], a result that was also seen in mouse zygotes (Fig. 3B). The effective concentration of NPPB in mouse zygotes (10 µM was ineffective while 100 µM substantially inhibited) is consistent with the Ki values reported in other cell types [5, 25]. Millimolar levels of external ATP are reported to block outward, but not inward, current passing through VSOAC/VSOR channels [4, 5, 15, 21]; this was also observed for the swelling-activated current in mouse zygotes (Fig. 3C). We found inhibition of currents in zygotes by ATP at concentrations 
3 mM, consistent with the levels reported to inhibit VSOAC/VSOR currents in other cell types [4, 5, 15, 23]. The VSOAC/VSOR-mediated current is also characterized by a greater permeability to I- than to Cl- [4, 5, 21], as was the swelling-activated current in mouse zygotes (Fig. 4). Finally, VSOAC/VSOR channels are permeable to organic osmolytes such as aspartate and taurine [4–6]. The significant outward current that remained even after all external Cl- had been replaced with aspartate or taurine (Fig. 5), in the absence of other ions that could carry significant outward current, indicated that the swelling-activated channels in zygotes are similarly permeable to aspartate and taurine. Furthermore, the aspartate current was volume-sensitive and DIDS-inhibitable, as expected if it is carried by such anion channels. Therefore, we believe that the swelling-activated current in mouse zygotes is mediated by anion channels that are similar to the VSOAC/VSOR channels described in other cells [4, 5].
We also examined unfertilized, ovulated mouse oocytes for swelling-activated currents. However, only very small swelling-activated currents could be elicited in oocytes. These currents were also not significantly inhibitable by DIDS, in contrast to those in zygotes. Therefore, while there is some swelling-activated current in oocytes, this current is much smaller and may have different pharmacological properties than that in zygotes. Further work will clearly be needed to determine exactly when the large, DIDS-inhibitable, swelling-activated current evident in zygotes develops after fertilization.
The relative permeabilities of the swelling-activated current in mouse zygotes to aspartate, taurine, and Cl- can be estimated using the Goldman-Hodgkins-Katz (GHK) equation with the measured values of the reversal potential, if it is assumed that there is no significant contribution to the whole-cell current from conductances other than the swelling-activated current and no contribution from ions other than these three. This condition was approached in the experiments in which CsCl was used in the internal pipette solution (Fig. 5), although some of the current may have been due to other conductances. In this case, and when the external Cl- and internal amino acid concentrations are both zero, the GHK equation gives the reversal potential as:
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We have previously shown that mouse zygotes can recover from hypotonic swelling and are therefore capable of RVD [2]. RVD was blocked by DIDS or NPPB, as well as by millimolar external ATP. Thus, the pharmacological properties of RVD in mouse zygotes are similar to those of the swelling-activated current we have described here (although NPPB inhibited RVD at a somewhat lower concentration than current [2]). Therefore, it appears likely that volume regulation in the mouse zygote depends upon swelling-activated anion channels that are permeable to Cl- and also to organic compounds such as aspartate, taurine, and glycine, and that efflux of one or more such osmolytes from the cytoplasm underlies the zygote's ability to recover from an increase in volume.
A swelling-induced increase in permeability to a group of organic compounds may also underlie the observed detrimental effect of even very short exposures of zygotes to amino acid-free media [13]. Upon flushing from the oviduct into Hepes-KSOM, we have observed that zygotes immediately swell by a small, but significant, amount (unpublished results). Thus, when zygotes are removed from the oviduct into similar media, permeability to a number of anionic and neutral amino acids will be increased, and accumulated amino acids could then be rapidly lost from the zygote. Since mouse zygotes accumulate large amounts of some amino acids in vivo [27], a loss of these compounds as the embryo is placed into in vitro culture could be deleterious.
Further investigations are needed to determine which amino acids and other organic compounds, in addition to aspartate, taurine, and probably glycine, can permeate through the swelling-activated pathway in zygotes. Also, the presence of this mechanism in later-stage embryos still remains to be demonstrated, as does its presence in the zygotes and embryos of other species. Finally, the mechanism of its regulation, and its relationship to other osmolyte transporters in zygotes, remain to be investigated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jay M. Baltz, Loeb Research Institute, 725 Parkdale Ave., Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5327; jbaltz{at}lri.ca
Accepted: November 24, 1998.
Received: August 11, 1998.
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