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a Alexion Pharmaceuticals, Inc., Cheshire, Connecticut 06410
b Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211-5300
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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calcium, gamete biology, oocyte development, ovum, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ca2+ signaling seems to be crucial during oocyte maturation and fertilization as well. Mouse oocytes exhibit repetitive Ca2+ transients during meiotic maturation [3]. It has also been generally accepted that a global Ca2+ signal is necessary and sufficient to induce embryonic development [4].
In most instances, the elevated [Ca2+]i is required only for short periods of time; prolonged high intracellular Ca2+ levels lead to activation of proteases and DNA-fragmenting enzymes and, ultimately, to cell death [5]. The removal of Ca2+ from the cytosol is possible by means of two different mechanisms. The ATP-driven Ca2+ pumps transport Ca2+ against its chemical gradient into the SR/ER or extracellular space. The other ion transport protein, the Na+/Ca2+ exchanger, functions to translocate Ca2+ across the plasma membrane and couples the transport of Ca2+ to the "downhill" cotransport of Na+ [6]. In most cells, three Na+ are exchanged for one Ca2+.
The Na+/Ca2+ exchanger family belongs to the exchanger superfamily [7]. Three genes that code for the Na+/Ca2+ exchangers have been identified in mammals (NCX1, NCX2, and NCX3), and all share a striking degree of sequence identity. The NCX1 is the dominant gene in mammals; its product is widely distributed in cells, including cardiac, skeletal and smooth muscles, neurons, astrocytes, kidney, lung, and spleen. In contrast, the NCX2 and NCX3 gene products have been found only in brain and skeletal muscle. The open reading frame of NCX1 is alternatively spliced, giving rise to a number of isoforms [2]. The functional properties of the various Na+/Ca2+ exchangers are also similar: they have a relatively high affinity Ca2+ transport site at the intracellular membrane surface and a low affinity Ca2+ transport site at the extracellular surface [8]. Thus, under steady-state conditions, the primary function of the exchanger is to extrude Ca2+ out of the cell. However, under a variety of circumstances (e.g., high intracellular Na+ levels or decreased extracellular Na+), the exchanger can operate in a reverse mode, providing a potential source for Ca2+ influx [2].
The Na+/Ca2+ exchanger is expressed in the plasma membrane of many animal cells. Indications for its presence were found in hamster oocytes [9], and its presence was later reported in mouse oocytes, where it was demonstrated that the Na+/Ca2+ exchanger plays an active role in the regulation of [Ca2+]i [10, 11]. In the present study, we investigated whether a Na+/Ca2+ exchange mechanism exists in porcine oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocyte Maturation
Chemicals were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise indicated. Porcine oocytes were matured in vitro as described previously [12]. Oocyte-cumulus complexes were collected from porcine ovaries and rinsed three times in Hepes-buffered Tyrode medium containing 0.1% (w:v) polyvinyl alcohol (Hepes-TL-PVA). They were then matured for 42 to 44 h in a defined protein-free medium consisting of tissue culture medium 199 (TCM 199) supplemented with 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 10 ng/ml epidermal growth factor, 0.1% polyvinyl alcohol, 0.57 mM cysteine, 0.5 µg/ml LH, and 0.5 µg/ml FSH. After maturation, the oocytes were stripped of the attached cumulus cells by vigorous pipetting in the presence of 0.3 mg/ml hyaluronidase in 0.3 M mannitol solution.
Ca2+ Measurements
For fluorescence recordings of the [Ca2+]i, immature and mature oocytes were incubated in the presence of 2 µM fura-2 AM and 0.02% pluronic F-127 (both from Molecular Probes, Eugene, OR). After 40–50 min, the oocytes were rinsed and changes in the [Ca2+]i were measured using a Photoscan-2 photon counting fluorescent microscope system (Nikon, Tokyo, Japan). Fluorescence was recorded by calculating the ratio of fura-2 fluorescence at 510 nm excited by UV light alternatively at 340 and 380 nm. Intracellular free Ca2+ levels are presented as fluorescent ratio values with ratios of 1.2 and 6.5 representing 65 and 602 nM Ca2+, respectively [13].
Immunocytochemistry
The Na+/Ca2+ exchanger protein in porcine oocytes was localized by immunocytochemistry. Mature oocytes were fixed in 3.7% paraformaldehyde for 30 min, washed in PBS with 3 mg/ml BSA, and permeabilized in 0.1% Triton X-100 for 5 min. Nonspecific binding sites were blocked in PBS containing 3 mg/ml BSA, 0.01% Triton X-100, and 5% goat serum for 1 h. The oocytes were then incubated in the presence of a monoclonal (mouse) anti-Na+/Ca2+ exchanger antibody (IgM; Affinity Bioreagents, Golden, CO) diluted 1:200 for 50 min. (The Na+/Ca2+ exchanger isoform that is recognized by the antibody was not specified by the producer.) After washing in PBS with 3 mg/ml BSA and 0.01% Triton X-100, they were incubated in fluorescein isothiocyanate (FITC)-labeled anti-mouse IgM (developed in goat) diluted 1:400 for 30 min. Following washing overnight in PBS with 3 mg/ml BSA and 0.01% Triton X-100, the oocytes were mounted in an antifade medium (Vectashield; Vector Laboratories, Burlingame, CA) under posted coverslips. The distribution of FITC-conjugated secondary antibody was visualized by laser-scanning confocal microscopy using a BioRad MRC-600 (BioRad, Richmond, CA) equipped with a krypton-argon ion laser and mounted on a Nikon Optiphoto II inverted microscope (Nikon).
Cortical granules were stained as described before [14]. After an induced [Ca2+]i increase, mature oocytes were cultured for 2 h in Hepes-TL-PVA. The zonae pellucidae were removed using a 0.1% pronase solution and, after three washes in PBS, the oocytes were fixed in 3.7% paraformaldehyde in PBS for 30 min. Then they were incubated in PBS containing 3 mg/ml BSA and 100 mM glycine for 15 min followed by a 5-min incubation in PBS with 0.1% Triton X-100. After washing the oocytes two more times in PBS, the cortical granules were stained with 100 µg/ml FITC-labeled peanut agglutinin for 30 min, and then the chromatin was labeled with 10 µg/ml propidium iodide. The stained oocytes were then washed in PBS containing 3 mg/ml BSA and 0.01% Triton X-100 and mounted on microscope slides. The presence of the cortical granules was examined by confocal microscopy. Nontreated oocytes and oocytes where cortical granule release was induced by a combined treatment with thimerosal/dithiothreitol [14] were used as controls. Confocal pictures of the oocytes were generated by selecting the most informative optical section separately for each filter; the images were then saved and superimposed. Thus, the red and green signals may come from different focal planes.
Chromatin Configuration
After a Ca2+ influx through the Na+/Ca2+ exchanger, the oocytes were cultured in Hepes-TL-PVA for 6 h, mounted under posted coverslips, and fixed in ethanol:glacial acetic acid (3:1) for >48 h. They were then stained with 1% (w:v) aceto-orcein, and the chromatin configuration was evaluated by using Hoffman Modulation Contrast Optics (Modulation Optics, Greenvale, NY) on an inverted Nikon Diaphot microscope at 400x magnification. Oocytes having one or more pronuclei were considered as activated.
Isolation of mRNA
Hybond-messenger affinity paper (Hybond-mAP; Amersham Pharmacia Biotech, Piscataway, NJ) was used to isolate poly(A) RNA from individual oocytes. Oocytes were incubated with a 3–4-mm2 piece of Hybond-mAP in guanidium isothiocyanate (GITC) lysis solution for 2 h. The lysis solution consisted of 4 M GITC; 0.1 M Tris-HCl, pH 7.4, and 1 M beta-mercaptoethanol, all in diethyl pyrocarbonate (DEPC)-treated water. The Hybond-mAP was then placed on Whatman filter paper (Fisher Scientific, St. Louis, MO) and the aqueous contents of the vials were carefully spotted onto the membrane. The Hybond-mAP was washed twice in 0.5 M NaCl + 0.1 M Tris-HCl, pH 7.4, in DEPC-treated water followed by two additional washes in 0.5 M NaCl in DEPC-treated water and two final rinses in 70% ethanol. The Hybond-mAP was then allowed to air dry for a few minutes and then immediately used for reverse transcription (RT).
As a positive control for RT-polymerase chain reaction (PCR), total RNA was isolated from porcine ovaries. Immediately after removal, the ovaries were flash frozen in liquid nitrogen and stored at -70°C until processed. For RNA isolation, they were placed into 20 ml of lysis buffer (STAT-60; Tel-Test, Friendswood, TX) and homogenized using a rotor-stator homogenizer. An additional 20 ml of lysis buffer was added to the homogenate, then a 1/10 volume of bromo-chloro-propane was pipetted into the solution. The mixture was shaken vigorously for 30 sec, left to sit for 2–3 min, and centrifuged at 10 000 x g for 15 min. The supernatant was then collected into a new tube and, by adding an equal volume of ice-cold isopropyl alcohol, the RNA was precipitated. The tube was shaken gently, stored at room temperature for 5 min, and centrifuged at 10 000 x g for 15 min. The isopropyl alcohol was then removed, the pellet was washed in ice-cold 80% ethanol, and the RNA was aliquoted in DEPC-treated water with 1 U/µl RNasin. Aliquots were stored at -70°C until use.
Reverse Transcription
In the RT reactions, Hybond-mAP with attached RNA was used. The reaction mixtures consisted of the following: 200 U M-MLV reverse transcriptase, M-MLV reverse transcriptase buffer, 2.5 µM random hexamers, 200 µM each dNTP, and 20 U RNasin (Promega, Madison, WI). Total RNA isolated from ovaries was reverse transcribed in a reaction mixture consisting of 200 U M-MLV reverse transcriptase, M-MLV reverse transcriptase buffer, 200 µM each dNTP, 2.5 µM reverse primer (see below), and 20 U RNasin. Milli-Q water (Millipore, Bedford, MA) was added to the reaction mixtures to make a final volume of 20 µl. The reactions were carried out under conditions of 42°C for 45 min followed by 95°C for 5 min using a PTC-100 Peltier effect thermocycler with a heated lid (MJ Research, Watertown, MA).
Polymerase Chain Reaction
The primers used to amplify a fragment of the Na+/Ca2+ exchanger from porcine oocytes were designed based on the nucleotide sequence of the porcine Na+/Ca2+ exchanger cDNA from the thick ascending limb cells of the porcine kidney [15]. The rationale behind this choice is that, to our knowledge, the above sequence is the only one that has been identified in the pig. The forward primer was 5'-TCCATTAGAATATTTGACCG-3' (bases 1812–1831) and the reverse primer was 5'-TTGGTGTGCTCTCCTAGGAT (bases 1998–2017). The primers were expected to amplify a 206-base pair (bp) DNA fragment. As an internal control, the following ß actin primers were used: forward primer 5'-GCTGTATTCCCCTCCATCGT-3' and reverse primer 5'-ACGGTTGGCCTTAGGGTTCA-3'. These primers were able to amplify a 220-bp fragment from porcine cDNA or a 350-bp fragment from genomic DNA. The cDNA from individual oocytes was amplified using the Expand High Fidelity PCR System (Roche Diagnostics, Indianapolis, IN). The 50-µl PCR reaction mixture contained 5 µl cDNA as a template, 200 µM each dNTP, 0.75 U enzyme, 1x reaction buffer with MgCl2, 4 nM of each primer, and Milli-Q water. When cDNA from ovaries was used for PCR, the reaction mixture was 25 µl, which consisted of 2 µl cDNA, 1 mM MgCl2, 2.5 U Taq polymerase, 1x reaction buffer, 1.8 nM forward primer, and the appropriate amount of Milli-Q water. The reactions started with 1 cycle of 95°C for 3 min, followed by 45 cycles each of 30 sec at 95°C to denature, 30 sec at 50°C for annealing, and 30 sec at 72°C for extension; the last cycle was followed by an 8-min extension.
The PCR products were electrophoresed on a 1.5% agarose gel, isolated, and cloned into the plasmid vector pCR2.1 (Invitrogen, Carlsbad, CA). Plasmids containing inserts of the correct size were sequenced by MWG Biotech (High Point, NC). Sequencing of the PCR product was expected to show whether porcine oocytes contain the porcine homologue of the Na+/Ca2+ exchanger.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the experiments, mature porcine oocytes were used unless otherwise indicated. First, the oocytes were placed in 10 µl Hepes-TL-PVA, and 1 ml Na+-free medium was added that resulted in an approximately 100x dilution of the original Na+ concentration (or alternatively, they were placed directly into Na+-free medium) and the changes in the [Ca2+]i were recorded. The Na+-free medium consisted of choline chloride 114 mM, KCl 3.2 mM, KH2PO4 0.4 mM, MgCl2 x 6H2O 0.5 mM, Hepes 9.2 mM, CaCl2 x 2H2O 2 mM, PVA 0.1%, pH 7.0. The decrease in the extracellular Na+ concentration triggered a reverse Na+/Ca2+ exchange in 17 out of 20 oocytes, which was detected as an increase in [Ca2+]i. The increase started 117.0 ± 11.8 sec after the addition of the Na+-free medium, and the duration of the Ca2+ transient was 158.1 ± 6.17 sec. In three oocytes, the elevation occurred as repetitive Ca2+ transients (Fig. 1, A and B).
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We also tested whether the Na+/Ca2+ exchange mechanism could be demonstrated in immature oocytes. Porcine oocytes at the germinal vesicle stage were held in 10 µl Hepes-TL-PVA medium to determine the steady-state [Ca2+]i levels, and then Na+-free medium was added to the oocytes. A large increase in the [Ca2+]i could be measured in these oocytes, indicating that the Na+/Ca2+ exchanger operates at the germinal vesicle stage (n = 6; Fig. 1C).
Inhibition of the Na+/K+-ATPase with ouabain is known to increase intracellular Na+ concentration. Porcine oocytes were preincubated in the presence of 0.4 mM ouabain for 2 h, and the oocytes with high intracellular Na+ content were placed into Hepes-TL-PVA. The Na+ overload induced by ouabain triggered a Na+-dependent Ca2+ influx in five out of eight oocytes, of which four showed repetitive Ca2+ transients (Fig. 1D).
The [Ca2+]i elevation was totally abolished in the absence of extracellular Ca2+ (n = 9). In Ca2+-free medium, the decreased extracellular Na+ concentration or the ouabain pretreatment failed to induce a Ca2+ influx (Fig. 2A). Amiloride (1 mM), which is able to block the Na+/Ca2+ exchanger, inhibited the Ca2+ influx caused by the absence of extracellular Na+ (n = 7; Fig. 2B). Another inhibitor of the Na+/Ca2+ exchange, bepridil, also blocked the Ca2+ influx induced by the Na+-free medium. If the changes in the [Ca2+]i were monitored in the presence of 50 µM bepridil, the Na+-free medium did not induce changes in the intracellular Ca2+ levels (n = 11; data not shown).
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Finally, we investigated the possibility of whether the Ca2+ entry can be mediated by other Ca2+ influx channels that might be present in the oocyte's plasma membrane. We found that both verapamil (50 µM) and pimozide (50 µM), inhibitors of L- and T-type voltage-gated Ca2+ channels, respectively, were without any effect (n = 18; Fig. 2, C and D). Large Ca2+ influxes triggered by Na+-free media were detected even in the presence of these Ca2+ channel blockers.
Effects of the Ca2+ Influx
Because we found that exposure of the oocytes to Na+-free medium induced a large increase in the [Ca2+]i concentration, we investigated the effect of the Ca2+ transients on the oocyte. We measured the changes in the [Ca2+]i after the addition of 1 ml Na+-free medium to 10 µl Hepes-TL-PVA, and when a Ca2+ influx was detected, the measurements were stopped and the oocytes were transferred into Hepes-TL-PVA for culture. In this way, we were trying to verify that only oocytes that showed a Ca2+ influx were used for further testing. After a 2-h incubation, the oocytes were fixed and the cortical granules were labeled. Confocal microscopy revealed that the Ca2+ influx via the Na+/Ca2+ exchanger did not induce cortical granule exocytosis, the granules were present under the plasma membrane in 19 out of 20 oocytes examined. In control oocytes, where a Ca2+ transient was induced by a combined treatment with 200 µM thimerosal/8 mM dithiothreitol, the granules were not visible (n = 20; Fig. 3).
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The entry of Ca2+ did not induce the resumption of meiosis either. Six hours after the Ca2+ influx mediated by the Na+/Ca2+ exchanger, the oocytes were fixed in 25% (v:v) acetic ethyl alcohol and stained for chromatin configuration. The rate of oocytes that formed pronuclei was 2.1% (3/140) in the group that was exposed to Na+-free medium, 2.4% (4/165) in the low Na+-treated group, and 1.9% (3/155) in the ouabain-pretreated group. The rest of the oocytes remained in metaphase II.
Localization of the Na+/Ca2+ Exchanger
Exposure of porcine oocytes to the monoclonal antibody followed by the FITC-conjugated secondary antibody resulted in a distinct staining pattern (Fig. 4). In 45 out of 45 oocytes, the fluorescent labeling had a patchy distribution in the plasma membrane, with areas of intense fluorescence on some parts of the membrane and little or no fluorescence in other areas. No labeling appeared to take place in the cytoplasm, indicating that the Na+/Ca2+ exchangers are located predominantly in the oocyte's plasma membrane. This corresponds with the role of the exchanger, i.e., to remove Ca2+ from the cell. Control oocytes (n = 35) did not show this staining pattern.
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Molecular Cloning of the Na+/Ca2+ Exchanger
PCR amplification revealed the expected 206-bp band from both oocyte and ovary cDNA (Fig. 5). Sequence analysis of the PCR product indicated that the band amplified from the porcine oocyte and ovary cDNA corresponded with the porcine Na+/Ca2+ exchanger reported earlier [15]. The percent of homology within the consensus region (bases 446–653) was 98.1% (Fig. 6). This indicates that pig oocytes express a porcine homologue of the Na+/Ca2+ exchanger.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fertilization is also accompanied by an elevation in the oocyte's cytosolic free Ca2+ levels that, in mammals, take place as repetitive Ca2+ transients. In the pig, as in most species, a transient is generated by the release of Ca2+ from the internal stores, and as a result, the steady-state [Ca2+]i of 60–100 nM rises to approximately 600 nM [13, 19]. The elevated [Ca2+]i then declines to the basal level fairly rapidly; some of the Ca2+ is used to replenish the stores, while in mouse oocytes, it was also shown to be pumped out of the cell [10].
The importance of Na+/Ca2+ exchange in maintaining low [Ca2+]i in oocytes is not clear. In mouse oocytes, a homeostatic coupling between the Na+/Ca2+ exchanger and plasmalemmal Ca2+-ATPase was suggested [10]. However, others found that the net direction in which the exchanger moves Ca2+ depends on the membrane potential as well (see [7]). Because the reversal potential of the exchanger is similar to the resting membrane potential, it indicates that the driving force for Ca2+ extrusion by the exchanger is very low. Moreover, mouse oocytes could recover from Ca2+ increases in the presence of reverse-mode Na+/Ca2+ exchange, arguing against a major role of the exchanger in maintaining Ca2+ homeostasis in the mouse oocyte [11]. These data suggest that, although Na+/Ca2+ exchange may contribute to the Ca2+ buffering power of the oocyte, most of the Ca2+ is probably removed by the plasma membrane Ca2+-ATPase.
Depending on the prevailing electrochemical driving forces (i.e., the Na+ and Ca2+ concentrations and the membrane potential), the exchanger can function in either the Ca2+-efflux or Ca2+-influx mode. As in several cell types, a decrease in the extracellular Na+ concentration triggered reverse Na+/Ca2+ exchange in porcine oocytes; in some cases, the increase in the [Ca2+]i occurred as repetitive Ca2+ transients. Although the role of the reverse mode, i.e., Ca2+-influx, remains controversial [20], it can be detected in many cells and provides convenient means to reveal Na+/Ca2+ exchange. Increasing the driving force for reverse-mode exchange by elevating intracellular Na+ levels with ouabain also induced Na+-dependent Ca2+ influx. The source of Ca2+ for the [Ca2+]i elevation was clearly extracellular since it was totally abolished in the absence of extracellular Ca2+. Although no pharmacologic agent is available that is specific for the Na+/Ca2+ exchanger [21], amiloride and bepridil are widely used to block the Na+/Ca2+ exchanger [22, 23]. The fact that both amiloride and bepridil inhibited the Na+-induced Ca2+ influx is consistent with the idea that the exchanger is present in porcine oocytes. Moreover, verapamil and pimozide, inhibitors of L- and T-type voltage-gated Ca2+ channels, respectively [24], could not block the Ca2+ entry induced by the absence of extracellular Na+. The presence of L- and T-type voltage-gated Ca2+ channels has been confirmed in a variety of oocytes [25–27]; our results, however, rule out the possibility that Ca2+ influx took place through such channels.
Generally, it is believed that, during fertilization, the Ca2+ released from the internal stores is responsible for stimulating cortical granule release [28]. Microinjection of Ca2+ buffers into sea urchin eggs [29] or exposure of isolated fragments of egg cortex to Ca2+ [30] have been demonstrated to induce exocytosis. Pig oocytes also released their cortical granules after microinjection of Ca2+ [31]. Interestingly, the Ca2+ influx induced by low extracellular Na+ concentration did not induce cortical granule exocytosis. The reason for this is not clear. There is a possibility that Na+-free medium or ouabain pretreatment could stimulate the release of cortical granules, although it is not very likely because we demonstrated previously that similar, or even less dramatic, changes in cytoplasmic Ca2+ levels could trigger the release of cortical granules in porcine oocytes after treatment with thimerosal [14] or Ca2+ ionophore [32]. It is conceivable that the kinetics of cortical granule exocytosis in these oocytes was slower and did not take place by the time fixation started. Moreover, the Ca2+ entering the oocytes through the exchanger did not induce meiotic resumption either. From this point of view, it did not matter whether medium with low Na+ concentration, ouabain pretreatment (data not shown), or Na+-free medium was used to induce the influx. One possible explanation for the lack of effect is that the action of Ca2+ was localized to certain areas under the plasma membrane. The results may also indicate that not all Ca2+ transients have equal effects; the Ca2+ passing through the exchanger was probably not able to induce the changes that normally occur after intracellular Ca2+ release. This, however, needs further verification because it was also reported that prolonged exposure of mouse oocytes to Ca2+-free medium prior to their return to complete medium induced activation [33], probably through a Ca2+ influx via the Na+/Ca2+ exchanger.
As a membrane transporter, the Na+/Ca2+ exchanger is anchored in the plasma membrane with nine transmembrane segments [34]. Immunocytochemical studies have revealed that, in a number of cells, including smooth muscle, astroglial cells, and neurons, the exchanger was confined to regions of the plasma membrane that are closely apposed to underlying SR or ER [2]. In the neuron cell body, it was distributed over the surface in a reticular pattern that resembled the pattern of underlying reticulum [35]. In pig oocytes, the label was also restricted to the cell surface; this distribution is consistent with the fact that the exchanger serves as a Ca2+ transporter across the plasma membrane.
The nine transmembrane segments of the exchanger are arranged in two sets that are separated by a large intracellular loop containing a high-affinity Ca2+ binding site and a specific region where the extensive alternative splicing occurs [7]. The latter region of the intracellular loop is encoded by six small exons, and the different combinations of these exons give rise to numerous mRNAs encoding different isoforms. The primers used in the PCR reactions were designed from the regions flanking the alternative splicing site. Using RT-PCR, we amplified a gene transcript that encodes a portion of the intracellular loop of the exchanger that showed high homology with the NACA3 isoform of the Na+/Ca2+ exchanger isolated from porcine kidney [15].
These results confirm our earlier findings that porcine oocytes possess a functional Na+/Ca2+ exchanger. Its presence was demonstrated via Ca2+ influx induced by altering the Na+ concentration gradient across the plasma membrane, identifying gene transcripts by RT-PCR and detecting the expressed transporter protein by immunocytochemistry. The Ca2+ influx generated via the exchanger during reverse-mode operation was not effective in triggering cortical granule exocytosis nor meiotic resumption. The function of the exchanger in the mechanism of Ca2+ homeostasis needs further investigation.
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FOOTNOTES |
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2 Correspondence: Zoltán Macháty, Columbus Farming Corporation, P.O. Box 1160, Sherburne, NY 13460. FAX: 607 674 6309; machatyz{at}columbusfarming.com
Received: 7 February 2002.
First decision: 28 February 2002.
Accepted: 2 May 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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