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Embryo |
a Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan
b Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka 814-0193, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Amino acids are taken up by the embryo via specific amino acid-transporting systems, including the Cl- channel [6], but little is known about the nature of these transporters. Glial [7] and endothelial cells [8] possess Cl- channels that are permeable for amino acids. Likewise, a swelling-activated Cl- channel has been shown to be present in early mouse embryos and to be permeable for amino acids under hypotonic conditions. Kolajova et al. [9, 10] demonstrated that these swelling-activated Cl- channels could be permeated by aspartate, taurine, and glycine; however, these channels are likely to be closed under iso-osmotic conditions. Relative permeability of amino acids versus Cl- was 0.2 in that study, which is in the same range as for most other swelling-activated Cl- channels (relative permeability, 0.1–0.3). Many types of Cl- channels have been identified in various cells, and all of these appear to have relatively low permeability (0.05–0.2) for organic anions, with the exception of canine vascular smooth muscle cell Cl- channels (relative permeability for aspartate-, 0.63) [11]. Thus, the question arises: Which type of Cl- channel is largely responsible for amino acid transport under physiological conditions?
In the present study, we characterized Cl- permeable channels present in early mouse embryos (1-cell stage) and found two different types: one that was highly permeable to amino acids and was osmotic pressure-sensitive, and one that was not.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Early stage embryos were collected from 6- to 10-wk-old B6C3F1 mice. Female mice were superovulated by i.p. injections with gonadotropin derived from pregnant mares (5 U; Serotropin; Teikoku Zoki, Tokyo, Japan) and hCG (4 U; Teikoku Zoki) at 48-h intervals. After the injection of hCG, females were mated with ICR males overnight and examined for the presence of vaginal plugs the next morning. Females were killed by cervical dislocation (
17 h after the hCG injection). Oviducts were excised, and embryos were collected and transferred into human tubal fluid (HTF) containing 101.6 mM NaCl, 4.69 mM KCl, 2.04 mM CaCl2, 0.2 mM MgSO4, 0.37 mM KH2PO4, 21.4 mM Na-lactate, 0.33 mM Na-pyruvate, 2.78 mM glucose, and 25 mM NaHCO3. Cumulus cells surrounding embryos were removed with HTF containing 0.3 mg/ml of hyaluronidase (Sigma, St. Louis, MO). The embryos, separated from cumulus cells, were cultured in HTF supplemented with 4 mg/ml of BSA (Sigma) at 37°C with 5% (v/v) CO2 in air. In some experiments, embryos were cultured in the presence or absence of chloride channel blockers in HTF from the zygote to the blastocyst stage.
Electrophysiological Recordings
Before all electrophysiological experiments, the zona pellucida was removed by incubation with either acidic Tyrode solution (Sigma) or 0.5% (w/v) protease (Sigma). The denuded embryos were then placed in a recording chamber (volume, 1 ml) mounted on an inverted microscope (TMD300; Nikon, Tokyo, Japan) and continuously superfused (1 ml/min) with control extracellular solution containing 134 mM NaCl, 6 mM KCl, 2.5 or 1.25 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 10 mM Hepes, adjusted to pH 7.3 with Tris-hydroxymethyl-aminomethane (Tris). In some experiments, extracellular concentration of Cl- ([Cl-]o) was altered by replacing NaCl with an equimolar concentration of Na-aspartate or Na-gluconate. The extracellular concentrations of Na+ ([Na+]o) and K+ ([K+]o) were adjusted by substituting NaCl with equimolar concentration of KCl. No detectable difference was observed on the outward rectifier current recorded in extracellular control solutions containing 1.25 versus 2.5 mM CaCl2.
For current recordings under different osmotic pressures, a hypotonic solution of 210 mOsm was superfused in the bath with the following ionic composition: 93.8 mM NaCl, 3 mM CsCl, 1.25 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 10 mM Hepes (pH 7.3 with Tris). Isotonic and hypertonic solutions (290 and 350 mOsm, respectively) were prepared by addition of appropriate amounts of mannitol to the hypotonic solution. All extracellular solutions prepared for electrophysiological experiments were adjusted to pH 7.3 throughout. Osmolality was monitored with a freezing-point depression osmometer (Osmometer Automatic; Knauer, Berlin, Germany). Osmolality of patch-pipette solutions used in the experiments was confirmed to be between 290 and 305 mOsm.
The relative permeability of anions (X-) against Cl- (PX/PCl) was calculated by an equation derived from the Goldmann-Hodgkin-Katz equation:
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Membrane currents and potentials were measured using the whole-cell configuration of the patch-clamp technique [12] with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 1 kHz and digitized at a sampling frequency of 2–5 kHz. Membrane currents and potentials were monitored with a chart recorder and stored on computer disk or on videotape through a pulse-code modulator. Data acquisition and analysis were performed with pCLAMP software (version 8.0; Axon Instruments).
Patch pipettes were prepared using a pipette puller (PP83; Narishige, Tokyo, Japan), and the tips were heat-polished using a microforge (MF83; Narishige). The standard K+-rich pipette solution contained 95 mM K-aspartate, 47.5 mM KCl, 3 mM MgCl2, 2 mM ATP (disodium salt), 0.3 mM EGTA, and 10 mM Hepes adjusted to pH 7.28 with Tris. The Cs+-rich patch-pipette solution contained 95 mM Cs-aspartate, 47.5 mM CsCl, 3 mM MgCl2, 2 mM ATP (disodium salt), 0.3 mM EGTA, and 10 mM HEPES, adjusted to pH 7.28 with Tris. Patch-pipette resistance was 2–5 M
. Series resistance compensation (70–90%) was used to reduce the error in the clamped potential. A 1 M KCl/3% (w/v) agar bridge was used as an indifferent electrode. Liquid junction potentials caused by replacement of Cl- with gluconate and aspartate were 2 mV, and the membrane potentials were corrected by subtraction of this value. The liquid junction potentials produced by Cl- replacement with I- and Br- were negligible. The zero-current potential before formation of the gigaseal was regarded as 0 mV. In the present experiments, the mean membrane capacitance of embryos was 257 ± 31 pF (n = 95), and current density was calculated by dividing membrane currents by membrane capacitance.
Membrane potentials of embryos were also recorded with conventional microelectrode technique. Glass capillary microelectrodes were filled with 3 M KCl and had a resistance of 30–40 M. Membrane potentials were measured with a microelectrode amplifier (MEZ-8201; Nihon Kohden, Tokyo, Japan) and recorded on a chart recorder.
All electrophysiological experiments were performed at room temperature (24–26°C).
Chemicals
4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; Sigma), 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB; Calbiochem, La Jolla, Calif., USA), and 2-[3-(trifluoromethyl)-anilino] nicotinic acid (niflumic acid; Sigma) were used to block Cl- channels. Except for DIDS, these blockers were dissolved in dimethyl sulfoxide (DMSO; Sigma) and then diluted in the extracellular solution. The final DMSO concentration in the extracellular solution was less than 0.1% (v/v), even when more than two drugs were simultaneously applied. We confirmed that 0.1% DMSO in the extracellular solution had no effect on the membrane currents and potentials of mouse embryo. The DIDS was directly dissolved into the extracellular solution. Unless specified, all other chemicals used were obtained from Sigma.
Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was extracted from early mouse embryos using S.N.A.P. total isolation kit (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized in a reverse transcription (RT) reaction mixture containing 500 ng of total RNA with Super Script II reverse transcriptase (Gibco BRL, Rockville, MD). The reaction proceeded at 25°C for 10 min and at 42°C for 50 min and was terminated at 70°C for 15 min. The DNA was amplified in a polymerase chain reaction (PCR) mixture containing cDNA, PCR buffer, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.2 mM each forward and reverse primer, and 0.025 U/ml of recombinant Taq DNA polymerase (Takara Shuzo, Tokyo, Japan). The PCR was incubated at 95°C for 1 min, at 53°C for 1 min, and at 72°C for 30 sec in 40 cycles. The PCR products were separated on 2% (w/v) agarose gel and stained with SYBR Gold (Molecular Probes, Eugene, OR). The fluorescence of the PCR products was detected with an image analyzer (fluoroimage analyzer FLA-2000F; Fuji Film, Tokyo, Japan) at 473-nm excitation using 520-nm emission.
As internal control for RNA quantity, the same cDNA was amplified using primers specific for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The following specific PCR primers for mRNA of Cl- channels (CLCs) and GAPDH mRNA were used: for CLCA1 (accession no. AF047838), forward primer was 1936–1955 (5'-GACATCAAGTCACCTTGGAG-3') and reverse primer was 2457–2476 (5'-GGTATCAGACTGGAAGCATT-3'); for CLC2 (accession no. AF097415), forward primer was 1171–1189 (5'-GGAAGATTGTCCAGGTGAT-3') and reverse primer was 1464–1483 (5'-GCAGACATCCAGAACTTCAT-3'); for CLC3 (accession no. AF029347), forward primer was 1170–1789 (5'-TGTGTCTCTGGTGGTTATTG-3') and reverse primer was 2206–2225 (5'-GGAAGAGATGGAGTATGCTG-3'); for CLC4 (accession no. Z49916), forward primer was 258–276 (5'-GCTGAAGAGAGGAGGATGA-3') and reverse primer was 588–607 (5'-AGATCGATGACTCCAGCTAA-3'); for CLC5 (accession no. AF134117), forward primer was 326–345 (5'-GCACCGAGAGATTACCAATA-3') and reverse primer was 689–708 (5'-CCTTGACAAGAGATACAGCA-3'); for CLC6 (accession no. NM011929), forward primer was 1244–1263 (5'-TCCAGGTCACATCAGAAGAT-3') and reverse primer was 1624–1643 (5'-GACTCGATCAGGATGACTGT-3'); for CLC7 (accession no. NM-011930), forward primer was 1418–1436 (5'-CTTCTGTGCAGATGGTGAA-3') and reverse primer was 1864–1883 (5'-CTGGATGTGCATGTCATAGA-3'); and for GAPDH (accession no. NM-008084), forward primer was 792–811 (5'-AACCTGCCAAGTATGATGAC-3') and reverse primer was 962–981 (5'-TACCAGGAAATGAGCTTGAC-3').
Statistical Analysis
Data are expressed as mean ± SD and with the number of cells (n). Statistical significance was determined by Student t-test, and P values less than 0.05 were considered to be significant. In some experiments, chi-square test was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mouse embryos cultured in HTF medium developed to the blastocyst stage in more than 70% of embryos collected. To determine any effect of Cl- conductance on the cleavage of developing embryos, various Cl- channel blockers were applied, and the cleavage ratio under each condition for 2-cell, 4-cell, morula, and blastocyst stages was measured 0.5, 1.5, 2.5, and 3.5 days after the beginning of culture, respectively (Fig. 1). Addition of NPPB (500 µM) reduced the cleavage ratio, and none of these embryos survived to the blastocyst stage. Addition of DIDS (100 µM) or niflumic acid (200 µM) also reduced the cleavage ratio to the same extent as NPPB (500 µM; data not shown). A higher concentration of DIDS (1 mM) or niflumic acid (300 µM) further suppressed cleavage, and no embryo survived to the 4-cell stage (Fig. 1).
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Contribution of K+ and Na+ Currents in Whole-Cell Currents and Resting Membrane Potential
Under whole-cell voltage-clamp configuration, membrane currents were recorded at a holding potential of -25 mV. A time-independent current was elicited in response to either depolarizing or hyperpolarizing pulses (duration, 300 msec) when the patch pipette contained K+-rich solution. This current showed prominent outward rectification (Fig. 2A). Application of tetraethylammonium (TEA) or 4-aminopyridine (4AP) slightly reduced this outward current, and simultaneous application of TEA (10 mM) and 4AP (1 mM) inhibited the outward current by 20.2 ± 12.7% (n = 10) of control at +70 mV (Fig. 2B). The reversal potential of TEA- and 4AP-sensitive current was -60 mV. Using a Cs+-rich solution in the pipette, the inhibitory effect of TEA and 4AP was eliminated, indicating a minor contribution of the TEA- and 4AP-sensitive K+ current to the outward rectifier current. Replacement of extracellular Na+ with N-methyl-D-glucamine resulted in a current decrease of 6.3 ± 1.0% of control at +70 mV (n = 4; data not shown).
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To confirm the importance of Cl- conductances on the membrane potential, measurements using conventional microelectrode technique under various ionic conditions were performed. Under normal ionic condition (control extracellular solution), the mean resting membrane potential in mouse embryos was -22.3 ± 9.4 mV (n = 8). This value was the same as that measured by whole-cell current-clamp technique with standard K+-rich pipette solution (-22.9 ± 6.7 mV, n = 95). These values of resting membrane potential of early mouse embryos were very close to the calculated Cl- equilibrium potential. Decreasing [Cl-]o (replaced with aspartate) depolarized the membrane (Fig. 2C), and the straight line obtained from a least-squares fit of membrane potential versus [Cl-]o had a slope of 43 mV per 10-fold change in [Cl-]o (Fig. 2D). Either increase in [K+]o or decrease in [Na+]o also depolarized the membrane; however, a 10-fold change in either [K+]o or [Na+]o only shifted the membrane potential by 7 and -13 mV, respectively (Fig. 2D). These results indicate that Cl- current is the major determinant of resting membrane potential in early mouse embryos.
Effects of DIDS and [Cl-]o on the Cl Current in Mouse Embryos
Figure 3, A and B, shows effects of DIDS on the current-voltage relationship and amplitude of outward rectifier current. Addition of DIDS (500 µM) inhibited the outward rectifier current in a voltage-dependent manner (Fig. 3, A and B). The reversal potential of DIDS-sensitive current was -30 mV. Addition of DIDS inhibited the outward rectifier current in a concentration-dependent manner, and maximum inhibition of the current was observed with addition of 100 µM DIDS (n = 11) (Fig. 3B). Other Cl- channel blockers, NPPB and niflumic acid, also inhibited the outward current in a concentration-dependent, but not in a voltage-dependent, manner (NPPB, n = 10; niflumic acid, n = 7) (Fig. 3, C and D).
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To investigate the [Cl-]o dependence of the outward rectifier currents, extracellular solutions containing various [Cl-]o were superfused in the bath. When [Cl-]o was reduced from 143.5 to 9.5 mM (replaced with gluconate), the outward current was suppressed slightly (Fig. 4A). However, when cells were pretreated with DIDS (300 µM), the reduction of [Cl-]o markedly inhibited the outward current (Fig. 4B). At +75 mV, suppression of outward current by [Cl-]o reduction to 9.5 mM in the presence of DIDS (n = 5) was significantly larger than that in the absence of DIDS (n = 10, P < 0.05).
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Effects of Osmotic Pressure on Outward Currents of the Mouse Embryos
Osmolality of extracellular solution was varied to determine whether the DIDS-sensitive outward current in mouse embryos is swelling-activated. Increasing the osmolality of external solution to 354 mOsm reduced outward current, and addition of DIDS (300 µM) did not affect the remaining outward current. On the other hand, niflumic acid (300 µM) further suppressed the outward current recorded under hypertonic condition (Fig. 5A). Reduction of osmolality to 210 mOsm increased the outward current, and addition of DIDS (300 µM) inhibited the current to a larger extent than was seen under isotonic condition without DIDS (Fig. 5B).
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Anion Permeability of the Cl- Channel
As shown in Figure 4, replacement of Cl- with gluconate suppressed the outward current in the presence or absence of DIDS; however, suppression of this current was much larger in the presence of DIDS than in its absence. This difference may indicate that relatively large molecules can permeate DIDS-sensitive Cl- channels. Thus, we investigated the permeability for amino acids using Cs+-rich pipette solution (146 mM Cl-). Replacement of [Cl-]o with gluconate- shifted the reversal potential to a more positive direction, and replacement with either [I-]o or [Br-]o shifted the reversal potential to a more negative direction. The relative permeabilities of anions versus Cl- were calculated with the Goldmann-Hodgkin-Katz equation, and values for I-, Br-, and gluconate- were 1.4, 1.2, and 0.46, respectively (Fig. 6). When the same experiments were performed in the presence of DIDS, relative permeabilities obtained were 1.4 for I-, 1.2 for Br-, and 0.13 for gluconate-.
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Detection of mRNAs of Cl- Channels by RT-PCR
The RT-PCR was performed to establish what types of Cl- channel mRNA are expressed in early mouse embryos. As shown in Figure 7, all CLCs examined were expressed in mouse embryos, except for the Ca2+-sensitive Cl- channel-1 (CLCA1). No products were obtained in the absence of reverse transcriptase, indicating that our RT-PCR yielded specific products derived from the target mRNA. These results show that many CLC Cl- channels are expressed in early mouse embryos.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We obtained a very close agreement between measured membrane potential and calculated equilibrium potential of Cl- in our experiments, demonstrating the importance of these channels for setting resting membrane potential. Changes in [Cl-]o dominantly shifted the membrane potential. Our measured values did not fit exactly those predicted by the Nernst equation, probably because of a contribution of highly amino acid-permeable Cl- channels as well as some contribution of K+ and Na+ permeabilities under our experimental conditions.
In embryos of the B6C3F1 strain, two types Cl- channels are active under normal osmotic conditions. One is activated by low osmolality and is suppressed in high osmotic conditions. This osmotic pressure-sensitive Cl- current was inhibited by DIDS, because superfusion of a hypertonic solution (354 mOsm) abolished the DIDS-sensitive component of the current. The other Cl- current is not sensitive to osmotic pressure or DIDS but is blocked by niflumic acid. The presence of swelling-activated Cl- current in mouse embryos had been reported previously in CF1 strain mice [9, 10]. Although there appears to be little difference in the pharmacological and physiological properties of the swelling-activated Cl- currents in the CF1 and B6C3F1 mice embryos, the swelling-activated Cl- channels observed in our study are active under isotonic conditions, suggesting a physiological role.
Amino acid permeability has been reported for swelling-activated Cl- channels in many cell types [7–9]. The relative permeability of amino acids versus Cl- ranges between 0.1 and 0.3, and a ratio of 0.2 was reported for the swelling-activated Cl- current in CF1 mouse embryos [9]. One exception is vascular smooth muscle cells from the rat with an amino acid-permeability ratio of 0.63 [11]. Compared to the CF1 strain, the B6C3F1 mice embryos used in the present study displayed a significantly larger amino acid permeability. Although significant variance was found in the proportion of DIDS-sensitive (swelling-activated) and DIDS-insensitive Cl- current among individual embryos, the mean value of relative permeability of amino acids versus Cl- in whole-cell current was significantly larger than that calculated for the DIDS-insensitive Cl- current alone. If we assume 50% of the whole-cell current to be DIDS-sensitive (Fig. 4B), then the amino acid permeability of DIDS-sensitive Cl- current should be around 0.79.
Because no ion substitute for Cl- is completely impermeable for Cl- channels, it is uncertain whether the residual current observed in Cl--deficient solutions in the presence of DIDS is passing though Cl- channels. If a Cl- impermeable channel is involved in this residual current, then the above value for amino acid permeability of the DIDS-sensitive Cl- current would be an underestimate.
The reason for the observed difference of amino acid permeability in CF1 and B6C3F1 embryos is unclear. In CF1 mice embryos, Kalajova and Baltz [9] speculated that either ClC3 or pICln proteins may be candidates for the swelling-activated Cl- channels, because mRNAs for both ClC3 and pICln were expressed in these embryos. Indeed, electrophysiological characteristics of the currents through the CLC3 are reported to be outwardly rectifying, blocked by DIDS, and more permeable to I- than to Cl- [16]. The RT-PCR experiments using B6C3F1 embryos also indicated that various ClC messengers were expressed in our embryos (Fig. 7). In rat renal and pulmonary arterial smooth muscle cells, Yamazaki et al. [11] reported a swelling-activated Cl- channel that is highly permeable to aspartate (relative permeability ratio, 0.63). However, to our knowledge, no evidence exists for the presence of ClC3 in the membrane of embryonic cells. Thus, molecular biological identification of swelling-activated Cl- channels expressed in different mice strains will be essential.
Several physiological roles for Cl- channels have been suggested in various cell types [16–19]: 1) pathway for amino acid transport, 2) regulation of cell volume, 3) contribution to membrane potential, and 4) role for cell proliferation. Dawson et al. [20] suggested that swelling-activated Cl- channels may constitute a release pathway for amino acids to regulate the cell volume in early mouse embryos, because glycine accumulation was observed in high-osmolality extracellular solution compared to control. Because glycine transport into cells occurred via a volume-dependent pathway, the swelling-activated Cl- channels appeared to be important for regulation of Cl- concentration in the cell [7]. It is uncertain regarding the low (<0.3) permeability ratio whether swelling-activated Cl- channels found in many cell types play a significant role for water and amino acid transport. On the other hand, the swelling-activated Cl- channels found in B6C3F1 cells in the present study have a very high permeability to amino acids (0.79) and are active under isotonic conditions, suggesting that these channels may play a significant role in amino acid transport.
In summary, we report two types of Cl- channels in early mouse embryos: DIDS-sensitive channels and DIDS-insensitive channels. The DIDS-sensitive Cl- channels possess a very high permeability to amino acids, are osmotic-sensitive, and are active under isotonic conditions. The DIDS-insensitive channels have low amino acid permeability and are not osmotic-sensitive.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Momoyo Sonoda, Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku Fukuoka 814-0180, Japan. FAX: 81 92 865 4114; e-mail: momo-s{at}cis.fukuoka-u.ac.jp
Received: 19 June 2002.
First decision: 12 August 2002.
Accepted: 1 October 2002.
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