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Embryo |
Human Reproduction Unit, Royal North Shore Hospital,3
Developmental Physiology Laboratory,4 Department of Physiology, University of Sydney, Sydney, New South Wales, 2065 Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-agatoxin; -conotoxin MVIIC), N-type (-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the 
1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (±) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.
calcium, developmental biology, early development, embryo, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ontologically, the first of the defined autocrine loops to be activated in the embryo is mediated by PAF. PAF is an ether phospholipid (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) that is released by the zygote of all mammalian species studied to date (e.g., mouse [1], human [6], rabbit [7], and sheep [8]). PAF is produced de novo from simple substrates by the zygote [9, 10], and its release requires extracellular albumin [11, 12]. On release, it acts back on the preimplantation embryo in an apparently receptor-dependent manner to enhance embryo metabolism and the rate of cell-cycle progression [13–15], and it results in enhanced embryo survival and development [2, 5, 16–18]. Embryo-derived PAF must act by the embryonic 2-cell stage for normal embryo survival to ensue [19].
The preimplantation mouse embryo expresses mRNA for a G-protein linked receptor with specificity for PAF [20, 21]. This receptor is known to activate a number of G-proteins, and, in many cell types, a common result is the induction of transient increases in intracellular calcium concentration ([Ca2+]i) [22]. Both embryo-derived [23] and exogenous PAF [20, 23] could induce [Ca2+]i in the early mouse embryo. Transients were single global events that resulted in desensitization of [Ca2+]i to a subsequent challenge with PAF [23]. This desensitization persisted for 30–60 min, by which time the embryo regained responsiveness. The [Ca2+]i response of the mouse embryo to embryo-derived PAF required the presence of exogenous albumin [23], and albumin was also required for the release of PAF from the embryo [11, 12].
[Ca2+]i signaling induced by embryo-derived PAF was developmentally regulated [23]. Responsiveness to PAF was first observed at 10 h after fertilization (zygote stage) and persisted until 41–43 h postfertilization (late 2-cell stage) [23]. The onset of responsiveness to PAF by the mouse zygote required new transcription from the zygotic genome. Embryos were most consistently responsive, and showed responses of greatest amplitude, to PAF in the mid-2-cell stage (
31–33 h postfertilization). The attenuation of PAF-induced [Ca2+]i responses in the late 2-cell stage was due to a loss in the capability of the embryo to respond to the available PAF [23].
The [Ca2+]i transients induced by embryo-derived PAF were blocked by exposure of mouse embryos to exogenous PAF acetylhydrolase [23]. This inhibition was reversible since subsequent treatment with exogenous PAF induced [Ca2+]i transients in a concentration-dependent manner. The response was blocked by PAF-receptor antagonists and was not elicited in response to biologically inactive PAF analogues. Although the PAF-induced [Ca2+]i transients were sensitive to inhibition of the phospholipase C/inositol trisphosphate (PLC/IP3) pathway, they were also entirely dependent on the presence of exogenous calcium [23], which may infer the requirement for calcium influx. The [Ca2+]i transients seem important for normal embryo development since buffering of intracellular calcium (with BAPTA-AM), such that baseline levels were unaffected but transients were suppressed, caused inhibition of embryo development, and this could be ameliorated by excess PAF [23].
There is currently little knowledge of the nature of signal transduction in the early mammalian embryo. Responses to this early autocrine trophic/survival factor may provide an important exemplar and therefore warrants detailed investigation. Signal transduction by calcium influx is common in excitable cells; however, cells of the preimplantation embryo are not currently recognized as electrically excitable. The aim of the current study was to investigate the role of extracellular calcium in the production of a PAF-induced calcium transient in the 2-cell mouse embryo. The results indicate a requirement for calcium influx primarily via a channel with the characteristics of an L-type channel.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Swiss albino mice (Laboratory Animal Services, University of Sydney, NSW, Australia), 6–9 wk old, were superovulated by intraperitoneal injection of 10 units of equine chorionic gonadotrophin (Folligon, Intervet International, Boxmeer, The Netherlands) followed 48 h later by 10 units of human chorionic gonadotrophin (Chorulon, Intervet). Females were then paired with males of proven fertility. Day 1 of pregnancy was confirmed by the presence of a copulation plug. The use of animals was in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and was approved by the Institutional Animal Care and Ethics Committee.
Collection Medium and Embryo Collection
All components of media were tissue culture grade from Sigma (St Louis, MO). Unless otherwise stated, all media were supplemented with 3 mg bovine serum albumin (BSA)/ml (Fraction V, CSL Ltd, Melbourne, VIC, Australia).
Mice were killed by cervical dislocation. Embryos were flushed from the reproductive tract using Hepes-buffered modified human tubal fluid medium (Hepes-modHTF- 102 mM NaCl, 4.6 mM KCl, 0.20 mM MgSO4, 0.4 mM KH2PO4, 21.4 mM sodium lactate, 1 mM glutamine, 0.33 mM sodium pyruvate, 2.78 mM glucose, 2.0 mM CaCl2, 4 mM NaHCO3, 21 mM Hepes buffer, pH 7.35; 285 mOsm/L).
Calcium Imaging
Embryos were washed three times in Hepes-modHTF and incubated with Fura-2 AM (1 µM; Molecular Probes, Eugene, OR) in BSA-free perfusion medium for 30 min and then washed three times in BSA-free perfusion medium. Perfusion medium was the same composition as Hepes-modHTF with 3 mg BSA/ml. In some experiments, embryos were incubated in recombinant plasma-type PAF acetylhydrolase (rPAF acetylhydrolase, 175 µg/ml; ICOS Inc., Bothell, WA) in Hepes-mod HTF for 15 min and washed in Hepes-modHTF medium before calcium imaging.
Embryos in BSA-free perfusion medium were placed onto a Cell-Tak (Collaborative Biomedical Products, Bedford, MA) treated glass coverslip that was attached to the perfusion chamber. During the study it was found that embryos adhered more effectively to the Cell-Tak if the zona pellucida was removed. After we established that the response was the same in the presence or absence of the zona pellucida (not shown), all subsequent imaging experiments were performed on zona-free embryos. Experiments in Figure 1 were on zona-intact embryos. The zona pellucida was removed by brief treatment with 0.5% pronase, followed by extensive washing in Hepes-modHTF. After establishing baseline readings and subtracting background, perfusion was initiated. Relative changes in [Ca2+]i were measured using fluorescence ratiometric imaging of Fura-2 at excitation wavelengths of 340 and 380 nm. One frame (0.04 sec) was captured at each wavelength every 5 sec. The [Ca2+]i was averaged over an entire 2-cell embryo and thus does not reflect the peak calcium concentrations achieved within regions of a cell. Results were recorded with a Panasonic video camera (model WV-BP 310/A) linked to a Macintosh computer via a Pixelpipeline frame grabber. Images were captured and analyzed with Ionvision software (Improvision, Coventry, UK). All imaging was performed on a Nikon Diaphot microscope using 100 W Xenon illumination and a 20x Olympus DPlan Apo UV lens. Up to 10 embryos were within the field of view of this objective and were imaged simultaneously.
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Calcium imaging was performed as previously described [23] with modification. Briefly, the chamber contained 0.5 ml of medium and was perfused with medium at 37°C at a rate of 1 ml/min. Treatment drugs were added to the perfusion medium unless otherwise stated.
Calibration Procedure for Measuring Intracellular Calcium Concentration
Intracellular calcium concentrations were calculated using the equations of Grynkiewicz et al. [24]. Rmax (340/380 nm ratio value at high calcium concentrations) was determined in the presence of 2 mM calcium and ionomycin (1 µM, Calbiochem, Alexandria, NSW, Australia). Rmin (340/380 nm ratio value in calcium-free conditions) was determined by perfusing with Ca2+-free perfusion medium until a stable baseline was achieved. Calcium-free media had CaCl2 replaced by NaCl and contained 50 µM 1,2-bis(2-aminophenoxyl)-ethane-N,N,N',N'-tetra-acetic acid tetrakis (BAPTA). BAPTA was used rather then EGTA since EGTA caused [Ca2+]i transients in its own right in 2-cell embryos [23].
Manganese Quench Analysis
Analysis of calcium influx by quenching of Fura-2 fluorescence at 360 nm was performed as previously described [25], with modification. Replacement of calcium in extracellular medium with Mn2+ provides a means of analyzing the activation of cation influx channels in response to PAF. Preparation of embryos and imaging of Mn2+ quenching was performed in a similar manner to calcium imaging, except that the perfusion medium had CaCl2 replaced with 0.1 mM Mn2+ and osmotically adjusted with NaCl. Imaging was performed at excitation wavelengths of 340 and 360 nm, and the epifluorescence at each wavelength was recorded separately as arbitrary units of fluorescence. The imaging chamber containing embryos was initially perfused with calcium and protein-free perfusion media for 60 sec. The medium was then changed to the Mn2+-containing medium with BSA, and perfusion was performed for 50 sec prior to the application of PAF (200 ng/ml) in the same medium. As a control, parallel experiments were performed in which perfusion medium contained Ca2+ instead of Mn2+.
Patch-Clamp Methods
Standard whole-cell patch-clamp techniques were used to study Ca2+ currents in 2-cell embryos collected fresh from the reproductive tract. All patch-clamp studies were performed on zona-free embryos. Three different groups of embryos were used in these experiments: 1) embryos that were untreated before patch clamping, 2) embryos that were treated with rPAF acetylhydrolase (175 µg/ml) at 37°C for 15min immediately before patch clamping, and 3) embryos that were treated with PAF (200 ng/ml) at 37°C for 10min immediately before patch clamping.
Patch-clamp pipettes were manufactured from borosilicate microhematocrit tubes (Modulohm, Herlev, Denmark). A List EPC-7 patch-clamp amplifier (List, Darmstadt, Germany) was used to measure whole-cell currents. We have previously demonstrated [26] the presence of a low-voltage-activated T-current in the 2-cell embryo. This current was not apparently involved in PAF-induced transients [23]. In order to favor the recording of high-voltage-activated currents and to minimize recording low-voltage-activated T-type currents, the membrane potential was held at -60 mV, and depolarizing voltage pulses of 1 sec duration were then applied to voltages between -20 and +80 mV at intervals of 5 sec. Currents were low-pass filtered, sampled, and digitized at 0.2 kHz with a MacLab-4 data acquisition interface (AD Instruments, Sydney, Australia) attached to a Macintosh-IIvx computer. Ba2+ was used as the charge carrier. The currents at each voltage step were recorded before and after treatment of embryos with different kinds of L-type Ca2+ channel blockers, namely, diltiazem (75 µM), nifedipine (80 µM), and verapamil (80 µM). Inward currents were measured as the difference between the whole-cell currents before and after the addition of a drug or control solution to the bath solution (NaCl 55 mM, KCl 4.69 mM, MgCl2 0.2 mM, Na2EDTA 0.11 mM, glucose 5 mM, CaCl2 2.04 mM [1.94 mM free Ca2+]), Hepes 20.4 mM, BaCl2 50 mM (49.99 mM free Ba2+), adjusted to pH 7.4, 300 mOsM/kg—a physiological concentration of extracellular Ca2+ (2.04 mM)—was included in the bathing solution to prevent the permeation of calcium channels by Na+. The patch pipette had a resistance of 4–10 M
when filled with 115 mM pipette solution (NaCl 25 mM, N-methyl-D-glucamine [NMDG] 115 mM, MgCl2 1 mM, Hepes 10 mM, glucose 10 mM, EGTA 0.5 mM, EDTA 0.01 mM, adjusted to pH 7.2 with glutamic acid). EDTA was included in pipette and bath solutions to remove any contaminating heavy metals and minimize oxidative damage to the embryos (the bath solutions is modeled on normal embryo culture media, which includes EDTA [5]). EGTA was included in the pipette solution to buffer intracellular Ca2+. All patch-clamp experiments were performed at 36°–37°C by constant perfusion of the bath solution. The rate of perfusion was 1 ml/min.
Treatments
PAF (Sigma, equal mixture of 1-o-octadecyl/hexadecyl-2-acetyl-2-sn-glyceryl-3-phosphocholine) was prepared as a 1-mg/ml stock solution in chloroform. Aliquots were removed to a siliconized glass test tube, reduced to dryness under a stream of N2, and dissolved in perfusion medium to the desired concentration.
Some inhibitors and antagonists were also used. They were initially prepared as 2000-fold concentrated stocks in either perfusion medium or dimethylsulfoxide (Me2SO) and then diluted to working concentrations in perfusion medium. In all experiments in which Me2SO was used as a solvent, control medium contained the same concentration of Me2SO.
To investigate the dependence of transients on extracellular calcium, responses to exogenous PAF were examined in perfusion medium free of calcium (with osmolality adjusted with NaCl). The role of calcium channels was assessed by the actions of the following channel modulators: diltiazem; -agatoxin IVA; -conotoxin GVIA; -conotoxin MVIIC; nimodipine, nifedipine (all dissolved in perfusion medium and from Calbiochem); SKF 96365 (medium); (±) BAY K 8644 (medium); verapamil; pimozide; 2-aminoethyl diphenyl borate (2-APB); BAPTA-acetoxymethyl ester (AM); econazole (all dissolved in Me2SO) (all from Sigma). The following modulators of signal transduction and calcium homeostasis were also used: thapsigargin (Me2SO), LaCl3 (medium), and cadmium chloride (medium).
Detection of mRNA for L-Type Channel
Evidence for the expression of mRNA for some -subunits of the voltage-gated channels in the early embryos was sought using reverse transcriptase polymerase chain reaction (RT-PCR). Primers were designed and purchased from Fisher Biotech (Perth, WA, Australia). The primers were 1-C subunit (GenBank accession no. M57682: 5'-TGT CTA TTC CAT CCC CTT C-3' and 5'-CCT CAC CAA AAA AAT CTC C-3'), 1-D subunit (GenBank accession no. M57682: 5'-GTC AGA TTC TTA ACA CCT CC-3' and 5'-TTC TTC CTC TCC TTT TCC-3'), and ß-actin [27] (5'-CGT GGG CCG CCC TAG GCA CCA-3' and 5'-GGG GGA CTT GGG ATT CCG GTT-3'). Control tissue (brain) was examined in parallel experiments.
For all RT-PCR assays, the following controls were always undertaken: 1) Mouse ß-actin was used as a positive control for the effectiveness of RNA extraction and RT-PCR reactions (the ß-actin primer pair was designed to span the first intron (87 bp in length) of the rodent ß-actin gene; thus, contaminating genomic DNA could be detected using these primers); 2) to control for false positive PCR amplification of contaminating genomic DNA, some samples did not include reverse transcriptase; 3) water was added instead of sample to test for contamination with extraneous DNA; and 4) some samples were randomly treated with RNase I (Promega Corp., Madison, WI) prior to RT, confirming the RNA origin of positive RT-PCR reactions (controls 3 and 4 not shown in results).
RNA was extracted with TRIzol Reagent (Life Technologies Inc., Gaithersburg, MD) containing 50 µg of carrier RNA (yeast transfer RNA, Sigma) as previously described [27]. Isolated RNA was treated with DNase to eliminate possible contamination with genomic DNA by resuspending the RNA pellet in 20 µl of resuspension solution (Tris-HCl 40 mM, NaCl 10 mM, and MgCl2 6 mM, pH 7.9) containing 2 units of RQ1 DNase (Promega) and incubated at 37°C for 30 min. Following the addition of a second equal volume of resuspension solution, RNA was phenol-chloroform reextracted. The RNA pellet was dissolved in double-autoclaved Milli-Q water in the presence of RNase Inhibitor (Promega) (final concentration 1 unit/ml).
RNA was reverse transcribed at 42°C for 30 min with 1.5 units of murine leukemia virus reverse transcriptase primed with 0.25 µM oligo (dT) in 20 µl of reaction mix containing 3 mM MgCl2, 60 mM KCl, 50 mM Tris-HCl, pH 8.3, 1 mM each dNTP and 1 unit of RNase Inhibitor (all reagents supplied by Perkin-Elmer Life Sciences, Foster City, CA). The RT reaction was then terminated by heating at 99°C for 5 min and cooling to 5°C.
Twenty microliters of RT reaction volume were used for test sample in a final PCR reaction volume of 50 µl containing 2 mM MgCl2, 10 mM KCl, 50 mM Tris-HCl, pH 8.3, 0.2 mmol each dNTP; 2.5 units of AmpliTaq DNA polymerase and 0.4 µM each of a specific primer pair were subjected to 35 rounds of amplification in a Corbett Thermal Reactor. PCR reaction products were analyzed by electrophoresis on 2% agarose gel stained with ethidium bromide to visualize PCR product on a UV transilluminator. Fragments were verified by size, the product was extracted, and the sequence analyzed to confirm they were from the target gene (ABI PRISM Dye terminator Cycle Sequencing Ready Reaction Kit from Perkin-Elmer, performed by SUPAMAC, Redfern, NSW, Australia).
Immunofluorescence
Embryos were recovered fresh from the reproductive tract and washed extensively in protein-free Hepes-mod HTF and then fixed in 2% formaldehyde in phosphate-buffered saline at 25°C for 30 min. Goat heat-inactivated serum in phosphate-buffered saline (20% v/v) with 2% BSA at 25°C for 1 h was used to block nonspecific binding. Embryos were incubated in primary antibody (1:100; rabbit anti-calcium channel 1-C; Sigma) or nonimmune rabbit serum at 4°C for 15 h, followed by goat anti-rabbit IgG (FITC-labeled; Sigma) at 25°C for 1 h. Embryos were viewed with a BioRad Radiance confocal microscope using a Nikon Plan Apo 60x/1.40 oil immersion objective. Images were captured with LaserSharp 2000 Version 4.0 (build 365) software (BioRad) using identical laser and microscope settings for all images shown. All experiments incorporated several negative control treatments: incubation of embryos in nonimmune immunoglobulin, no primary antibody, no secondary antibody, and nonfluorescent secondary antibody.
Statistical Analysis
Statistical analyses were performed on SPSS statistical package (version 9, SPSS Inc., Chicago, IL). Responses over time were analyzed by repeated measures analysis of variance. Comparisons of peak amplitude of [Ca2+]i responses were by t- test and the proportion of embryos expressing an L-type current by chi-square test. In all cases experiments were repeated a minimum of three times, and the results are the total number of embryos observed (numbers shown in figures) or representative traces of an embryo from each treatment. Mn2+ influx was analyzed by linear regression analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study confirmed that exogenous PAF induced a transient increase in [Ca2+]i in the 2-cell embryo and that extracellular calcium (2 mM) was required for expression of this transient (Fig. 1A). The amplitude of the transient was not different over the range of 0.25, 0.5, 1, and 2 mM external calcium Ca2+ (not shown). PAF-induced transients did not occur in calcium-free medium (Fig. 1A), either with and without the addition of the calcium chelator BAPTA (1 µM) to the media (not shown). The requirement for external calcium for PAF-induced [Ca2+]i transients implicates a plasma membrane calcium-influx channel(s). The addition of La3+ (200 µM) or Cd2+ (10 µM) (Fig. 1B) both caused partial inhibition of the transients, which suggests an influx of calcium was through channel(s) with at least partial ion selectivity.
Characterization of the Calcium Influx
Three different classes of L-type calcium channel blockers significantly inhibited PAF-induced signal transduction: benzothiazepine (diltiazem), phenylalkylamine (verapamil), and dihydropyridine (nimodipine) (Fig. 2). Diltiazem caused a marked dose-dependent inhibition of the amplitude of the PAF-induced transient (Fig. 2A). Verapamil also caused a significant reduction in the calcium transients (Fig. 2B), but its inhibition was not complete. The use of diltiazem and verapamil had an additive effect (Fig. 2C), being consistent with their different sites of action on the L-channel. When treated with diltiazem (75 µM), 54% of embryos showed no detectable [Ca2+]i transient in response to PAF, compared with only 3% in vehicle-treated controls. For combined diltiazem/verapamil treatment (60/80 µM), the proportion of embryos not reacting to PAF increased to 83% (P < 0.01). Nimodipine (a dihydropyridine) also caused significant inhibition of PAF-induced [Ca2+]i transients (Fig. 2D). Inhibitors of the following membrane voltage-activated calcium channels had no effect on PAF-induced transients: T-type (pimozide, 10 µM), P/Q-type (-agatoxin IVA, 70 nM, -conotoxin MVIIC, 200 nM), and N-type (-conotoxin GVIA, 200 nM) (results not shown).
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To determine whether an L-type channel is present in the 2-cell embryo, RT-PCR was performed for the 1-C and 1-D subunits of L-type channels. RNA for the 1-C but not 1-D subunits were found in 2-cell-stage embryos, while mRNA for both subunits were routinely detected in brain extracts (Fig. 3A). Embryos treated with nonimmune serum (Fig. 3B) failed to show any specific immunostaining. By contrast, confocal imaging of indirect immunofluorescence labeling with an antibody directed to the 1-C subunit protein (recognizes an intracellular loop of the channel) showed a high level of staining in the 2-cell embryo (Fig. 3, C–F). The most intense staining was localized to the region of the apical membranes of each blastomere. The staining was not uniform over the membrane surface with some small areas having relatively little expression. There was also essentially no staining of the apposing lateral membranes between blastomeres. Some channel protein expression was observed within the cytoplasm, and this was most intense within the cortical regions of the cytoplasm. In most cases there was no staining within the nucleus (Fig. 3, C and D), although in a small number of cases some blastomeres showed nuclear staining (Fig. 3, E and F). Presumptive nucleoli were never observed to be stained. The second polar body was commonly stained by the antibody, but membrane antigen localization was not observed in this meiotic remnant. The perivitelline space was commonly seen to stain slightly for channel protein; this may have been sourced from the degenerating polar bodies. It was confirmed [23] that the L-type channel agonist (±) BAY K 8644, a dihydropyridine agonist for L-type channels, induced a [Ca2+]i transient in the 2-cell embryo (Fig. 4). It was shown for the first time that this response was dependent on the presence of extracellular calcium (Fig. 4A), inferring that the resulting transient was indeed a consequence of calcium influx through the BAY K 8644-activated channel. The response of the 2-cell mouse embryo to 100 µM BAY K 8644 was desensitized following repeated BAY K 8644 challenges (not shown). The desensitization of the influx response to BAY K 8644 also occurred if the prior challenge was with PAF (Fig. 4B); conversely, an initial challenge with BAY K 8644 caused heterologous desensitization of the [Ca2+]i response to PAF (Fig. 4C). This heterologous desensitization reaction, and the demonstration that the [Ca2+]i response by the embryo to both agents required external calcium, argues that both BAY K 8644 and PAF activate the same calcium channel.
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Mn2+ Quench Studies
At a wavelength of 360 nm, Mn2+ has the effect of quenching (reducing) fluorescence by Fura-2. Thus, the substitution of Mn2+ for Ca2+ in the bathing media allows Mn2+ influx to be directly visualized and thus indirectly measures influx channel activity. Mn2+ had no effect on baseline Fura-2 fluorescence prior to PAF challenge. On addition of PAF, a rapid quenching at 360 nm occurred (Fig. 5A). This was a consistent response by embryos and was inhibited by diltiazem (not shown). There was no corresponding change in Fura-2 fluorescence at 340 nm for the first 40 sec after PAF challenge, indicating that over this time the influx of Mn2+ did not correspond with an increase in [Ca2+]i. Given the lag between the addition of PAF to the perfusion media and its equilibration through the perfusion chamber, the lag of 15–20 sec between PAF addition and Mn2+ influx probably reflects an almost immediate Mn2+ influx response by embryos to PAF. By contrast it was approximately 60 s after addition of PAF to media before a detectable rise in [Ca2+]i occurred (measured at 340 nM). The rise in [Ca2+]i corresponded with an increase in the slope of the quench curve at 360 nm. It was noted that in calcium-containing media (in the absence of Mn2+) (Fig. 5B), the first phase of Fura-2 quenching observed in Mn2+ media did not occur. This supports the conclusion that this early response was due to the activation of an inward cation channel. However, the quench that occurred coincidentally with the rise in [Ca2+]i was still observed in the absence of Mn2+. Since Ca2+ can also cause some quenching at 360 nm [28], it is likely that this secondary quench curve was a consequence of the release of internal Ca2+ stores. There was no difference (P > 0.05) in the slope of this second phase curve in media with and without Mn2+, indicating that its activity may be explained entirely by the release of internal calcium stores. The data from these experiments show unequivocally that PAF causes the rapid opening of a cation influx channel that was inhibited by diltiazem.
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Patch-Clamp Analysis
Stepwise membrane depolarization of the 2-cell embryo by increasing the media extracellular concentration of KCl to 75 mM caused a [Ca2+]i transient (detected by Fura-2 analysis) (Fig. 6). Smaller depolarization caused by 25 and 50 mM KCl did not induce a [Ca2+]i (not shown). We previously showed [29] that 30 mM KCl caused a 35-mV depolarization of the 2-cell embryo, and it is assumed in this study that 75 mM induced complete depolarization. The transient induced by 75 mM KCl was significantly reduced (P < 0.01) by treatment with diltiazem and verapamil but not by pimozide (Fig. 6) a putative inhibitor of a T-type Ca2+ current found in the 2-cell embryo [26]. Given this indication of an electrically active current in the 2-cell embryo, patch-clamp analysis was performed to further characterize the Ca2+ channel responsible for the depolarization-induced Ca2+ influx.
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Whole-cell patch-clamp experiments detected a current that was activated by membrane depolarization. In these experiments Ba2+, and not Ca2+, was used as the charge carrier since we were unable to record any voltage-dependent inward current in the presence of 50 mM extracellular Ca2+. This result is not unprecedented and probably stems from the rapid Ca2+-dependent inhibition of the 1-c subunit but not other subunits [30]. Figure 7A shows representative traces of changes in whole-cell current during depolarizations. The average response of a number of embryos is shown in Figure 7B, as is its detection by the inhibition with the calcium channel blocker diltiazem. The current was maximal at a voltage of 36.94 ± 2.59 mV (mean ± SEM) with a current of 0.23 ± 0.03 nA. Two other L-type channel blockers (nifedipine and verapamil) were equally effective in identifying the channel within the 2-cell embryo (Fig. 7B). Of 31 embryos measured with diltiazem, a detectable current was observed in 12 (39%). It was previously shown that endogenous PAF desensitized, for a time, subsequent responses to PAF by the embryo [23]. Thus, to assess whether the small proportion of embryos in which the L-type current was observed may reflect desensitization as a consequence of prior action of embryo-derived PAF, embryos were initially treated with rPAF acetylhydrolase (that degrades embryo-derived PAF stores [23]). rPAF acetylhydrolase pretreatment resulted in detection of current activity in a significantly (P < 0.05) greater proportion of embryos, and the peak current in these embryos was also significantly increased (P < 0.05; Fig. 7C). In contrast, prior treatment of embryos with exogenous PAF resulted in a significant (P < 0.05) reduction in the proportion of embryos expressing the current compared with those pretreated with rPAF acetylhydrolase. These results show that 2-cell embryos possess a depolarization-activated membrane channel, with the properties of an L-type calcium channel. The activity of the channel was desensitized by prior action of either embryo-derived or exogenous PAF, providing support for a role for this channel in the signal transduction induced by PAF.
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Investigation of Store-Operated Channels
Notwithstanding this demonstration of activation of an L-type channel, it was shown [23] that the generation of a PAF-induced [Ca2+]i transients were dependent on the release of IP3-sensitive stores as assessed by inhibition by U73122 and Xestospongin C. The role of these stores was further investigated by use of a cell-permeable inhibitor of IP3 receptors (IP3R), 2-APB. This inhibitor blocked PAF-induced transients (Fig. 8A), supporting a role for the PLC/IP3 pathway in this pattern of PAF-induced signal transduction. To assess whether extracellular calcium was required for the release of IP3-sensitive stores, 2-cell embryos were treated with thimerosal, a sulfhydryl agent that causes the release of IP3-sensitive internal stores [31]. A similar marked [Ca2+]i transient in response to thimerosal occurred in both calcium-free and calcium-containing medium (Fig. 8B), suggesting that the failure of embryos to respond to PAF in calcium-free medium was not secondary to the loss or inactivation of these internal stores. In the presence of extracellular calcium, a secondary peak was observed in response to thimerosal that was not observed in calcium-free medium. This peak, which was modest in amplitude, may be caused by an influx of calcium in response to store depletion and therefore reflect the actions of a store-operated channel (SOC) in the 2-cell embryo.
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As well as blocking IP3R receptors, 2-APB and Xestospongin can block calcium influx via SOCs [32]. SOCs can also be blocked by SKF 96365 [33] and econazole [34]. However, PAF-induced [Ca2+]i transients in the 2-cell embryo were not inhibited by 25 µM SKF 96365 or 40 µM econazole (Fig. 8C). Emptying of internal stores with thapsigargin caused a calcium transient in the absence of external calcium (Fig. 8D). On addition of calcium to media in the presence of thapsigargin, there was a small increase in [Ca2+]i compared with similar embryos in the absence of extracellular medium, indicative of SOC activity in response to store depletion. This activity confirms the presence of SOC in the 2-cell embryo, and their action is consistent with the modest putative SOC activity observed in response to thimerosal treatment of 2-cell embryos (Fig. 8A). The failure of SKF 96365 and econazole to block PAF-induced transients and the relatively small SOC activity measured in embryos suggest that 2-APB and xestospongin were unlikely to have been exerting their effects on PAF-induced transients via their actions on SOCs.
Given that PAF-induced calcium influx was entirely blocked by selective L-type channel blocker and the absence of evidence for a role of SOC in the formation of the transient, it is concluded that calcium influx is primarily via L-type channels. The inhibition of PAF-induced transients by inhibitors of mobilization of Ca2+ from IP3-sensitive stores suggests an interdependence between calcium influx and release of internal calcium stores for the formation of the PAF-induced transient.
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DISCUSSION |
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The observation that inhibition of the PLC/IP3 pathway (by U 73122 and xestospongin) blocked PAF-induced transients was extended in the current study by the observation that another putative membrane-permeable IP3R antagonist (2-APB) also inhibited PAF-induced [Ca2+]i transients. The Mn2+ quench experiments showed that influx via the channel was a rapid early response to PAF, occurring approximately 40 sec prior to a detectable increase in [Ca2+]i. A well-known feature of the release of calcium from IP3-sensitive stores is that the IP3R is sensitive to both Ca2+ and IP3 but can be activated only by IP3. Thus, the receptors' response to IP3 is sensitized by calcium. Indeed it is suggested that the IP3R acts as a computation switch capable of integration of inputs from several sources that lead to summative conformational changes that modulate its response to IP3 [16, 17]. Thus, highly localized increases in [Ca2+]i due to calcium influx in response to PAF may allow store release to occur in response to relatively low concentrations of IP3 generated by PAF action. The resulting release of internal IP3-sensitive stores of Ca2+ might then sensitize neighboring IP3 receptors, leading to a rapid release of Ca2+ stores and the generation of a substantive [Ca2+]i transient throughout the embryo. Such coupling between calcium influx and calcium release from internal stores is well recognized to occur in excitable cells [37].
The voltage-gated Ca2+ channels consist of 1-subunits that form the ion-pore forming unit of the channel and regulatory 2
- and ß-subunits (in some channels there is an additional 
-subunit) [38, 39]. The different functional subclasses of the channels are defined by the type of 1-subunit present. At least 10 1-subunits have been described. Each -subunit is a single protein (190–250 kDa) consisting of four homologous domains (I–IV), each containing six putative transmembrane segments (S1-S6). The major forms of L-type channels each possess either an 1-C (cardiac, smooth muscle, neurons), 1-D (endocrine cells and neurons) or an 1-S-subunit (skeletal muscle).
The concentration of the range of highly selective inhibitors of L-type channels (diltiazem, verapamil, and nimodipine) required to inhibit [Ca2+]i transients in the 2-cell embryo was higher than is commonly required in somatic cells. However, it should be noted that the actions of these inhibitors are strongly use and state dependent [40, 41]. For example, dihydropyridine has a high affinity for inactivated channels that are favored in depolarized cells but have a much lower affinity for resting channels, which occur at resting membrane potentials. Thus, at depolarizing potentials, dihydropyridines have affinities in the low nanomolar range, but at hyperpolarization potentials, the affinity may be in the range of 10 µM. The affinity and actions of benzethiazapines and phenylalkylamines are also highly state dependent [42]. These large state-dependent differences in affinity make conventional dose-response analyses difficult to interpret in a complex setting such as whole-embryo superfusion, as performed in this study. A detailed understanding of the action of these inhibitors in the model must await a more detailed analysis of the electrical properties of the influx channel in the embryo.
The expression of 1-c subunit mRNA and protein in the 2-cell embryo, the induction of a [Ca2+]i transient following membrane depolarization that was inhibited by L-type channel inhibitors, and the electrophysiological measurements of a high-voltage-gated channel that was inhibited by the three classes of L-type channel blockers taken together provide strong evidence for the presence of an electrically excitable L-type calcium channel in the 2-cell embryo. We have also previously demonstrated that the 2-cell embryo possesses a low-voltage-activated T-type calcium channel [26]. Such results raise the possibility that the early embryo has some of the properties of an electrically excitable cell. It has been previously shown [43] that treatment of the early embryo with the L-type channel blocker diltiazem caused marked inhibition of embryo development, implicating a requirement for channel function in embryo development.
The fact that [Ca2+]i transients did not occur in calcium-free media but did when Mn2+ was present in media argues that Mn2+ acted as both a suitable cation substitute for influx and also for activation of codependent release of internal calcium stores. The use of the manganese quench technique provides direct confirmation that PAF causes activation of a cation influx channel, implying that this is the requirement for external calcium. A number of lines of evidence indicate that the influx is via an L-type channel: 1) PAF-induced Mn2+ quenching was blocked by diltiazem; 2) PAF-induced [Ca2+]i transients were blocked by diltiazem, verapamil, nimodipine, and nifedipine [23]; 3) PAF and BAY K 8644 caused mutual heterologous desensitization of each other's capacity to generate [Ca2+]i transients; 4) exogenous and embryo-derived PAF caused desensitization of voltage-activated L-type channel function; and 5) pharmacological inhibition of other major classes of membrane calcium channels did not inhibit PAF-induced calcium influx. While this study implicates the L-type channel as a major calcium influx channel activated by PAF, the nature of the study cannot conclusively exclude the action of other channels. Indeed the possibility that the channel is electrically activate suggests that its actions are secondary to changes in membrane potential, which would require the action of other channels, such as K+ channels.
Given the evidence for the action of the L-type calcium channel, it may seem curious that 1-C subunit gene null embryos survive the preimplantation with apparent defect; they die on Day 14.5 postcoitum [44]. It is noteworthy, however, that expression of gene products in the 2-cell embryo is almost exclusively from maternal gametic (oocyte) stores of mRNA and protein [45]. Given that the production of null embryos was by mating of heterozygous parents, even null embryos will most likely have wild-type normal allelic products present in the 2-cell embryo from the oocyte. Thus, in the absence of information on 1-C mRNA inheritance and RNA stability in the knockout strain, analysis of the early embryo phenotype of null embryos from heterozygous crosses is equivocal.
The current study does not define the mechanism of activation of the L-type channel by PAF. PAF is known to act via a G-protein linked receptor in many cell types [22], and mRNA for this receptor is present in the 2-cell embryo [21]. The inhibition of PAF's actions by a PAF-receptor competitive antagonists (WEB 2086) and the pattern of agonist-induced desensitization of the response indicates that PAF's actions are receptor mediated [23]. The activation of the L-type channel may be via membrane depolarization. A significant depolarization of the mouse embryo occurs during the first cell cycles, and this may contribute to the cell-cycle dependence of PAF's actions [29, 46]. The classical intracellular secondary messenger pathways may also have a role in L-type channel activation. Phosphorylation of the channel by protein kinase C stimulates Ca2+ influx [47], and this apparently occurs as a result of a shift in the channel's activating voltage. The 1-C-subunit also contains a proline rich domain that acts as a SH3-binding domain [48]. The SH3 domain from several tyrosine kinases (Src, Lyn, Hck) binds to this proline-rich domain [48]. This binding of SH3 domains seems unique to the 1-C subunit. Ca2+/calmodulin binding to an IQ motif of the 1-C-subunit is also reported to act as a point of regulation, acting to both facilitate and inactivate channel activity [49, 50].
The results of the study indicate that further detailed investigation of the nature and regulation of ligand-induced calcium channel activation and release of calcium stores promises new insights into the nature of early embryo development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 61 2 9926 6343; chriso{at}med.usyd.edu.au
Received: 18 December 2002.
First decision: 22 January 2003.
Accepted: 11 February 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and absence (
) of diltiazem (75 µM). The bars show a similar net change in current when nifedipine (80 µM) or verapamil (80 µM) were used as the channel blocker. C) The proportion of embryos displaying Ba2+ influx and the mean peak amplitude of the influx is shown for embryos collected fresh from the reproductive tract and patched immediately (n = 31); those that were subjected to treatment with rPAF acetylhydrolase (PAF-AH), to degrade embryo-derived PAF, prior to patching (n = 28); and embryos exposed to PAF immediately prior to analysis (n = 21)