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Biology of Reproduction 66, 667-674 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Capacitative Calcium Entry Mechanism in Porcine Oocytes1

Zoltán Macháty2,a, Jagdeece J. Ramsoondara, Aaron J. Bonkb, Kenneth R. Bondiolia, and Randall S. Pratherb

a Alexion Pharmaceuticals, Inc., Cheshire, Connecticut 06410 b Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of the capacitative Ca2+ entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca2+-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca2+ entry. A similar divalent cation influx could also be detected with the Mn2+-quench technique after inositol 1,4,5-triphosphate-induced Ca2+ release. In both cases, lanthanum, the Ca2+ permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca2+ influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca2+ entry mechanism might help in refilling the intracellular stores after the release of Ca2+ from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca2+ entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca2+ entry mechanism and thus contributes to Ca2+ influx.

calcium, embryo, fertilization, ovum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Signal transduction at fertilization of mammalian oocytes includes a series of Ca2+ transients that are responsible for stimulating meiotic resumption in the oocyte and activating its developmental program [1, 2]. The activating signal is believed to be the Ca2+ oscillation itself, whose frequency, amplitude, and duration are thought to encode important information that influences subsequent development [3, 4]. The oscillation is generated by the cyclic release of Ca2+ from the internal store through specialized Ca2+ release channel receptors. The released Ca2+ is then resequestered into the stores by sarcoplasmic endoplasmic reticulum Ca2+ ATP-ase (SERCA) pumps followed by additional release/replenishment cycles. In addition to the release from internal stores, it has been suggested that a continuous influx of Ca2+ through the plasma membrane is necessary to maintain the oscillation [5]. The contribution of Ca2+ influx accounts for the sensitivity of the oscillation frequency to variations in the level of external Ca2+. Ca2+ oscillation was inhibited in mouse oocytes by a decrease in the extracellular Ca2+ concentration and was totally blocked in the absence of extracellular Ca2+ or in the presence of Ca2+ influx channel antagonists [6].

Ca2+ signaling in many cell types involves Ca2+ oscillations. In excitable cells, oscillations arise primarily from the fluctuation in the entry of external Ca2+ via voltage-activated calcium channels [7]. However, agonist stimulation of many nonexcitable cells triggers Ca2+ release from intracellular stores followed by a Ca2+ influx across the plasma membrane [8]. In the latter case, the extracellular Ca2+ is probably required to refill the Ca2+ pools, because the majority of Ca2+ released from the store is extruded from the cell across the plasma membrane [9]. The Ca2+ influx pathway seems to be activated by depletion of the intracellular Ca2+ stores and is termed capacitative Ca2+ entry [10]. Capacitative Ca2+ entry is thought to play a role in sustaining Ca2+ oscillation that accompanies fertilization in mammalian oocytes [7, 11], and the presence of such a Ca2+ entry was observed in mouse oocytes during the Ca2+ spikes induced by fertilization or various artificial stimuli [12].

The capacitative Ca2+ entry pathway has not yet been identified. There are a number of channels that can bring Ca2+ into cells as a result of store depletion; these channels are generally called store-operated channels [7,13]. Previously, the transient receptor potential (trp) gene product in Drosophila photoreceptors has been suggested as a promising candidate [14]. The Drosophila trp locus encodes a protein consisting of 1275 amino acids with 6 putative transmembrane segments; it displays significant similarity to voltage-gated Ca2+ channels but lacks the charged amino acids that make up their voltage sensor. Trp appears to be a key element in the inositol 1,4,5-triphosphate (InsP3)-dependent phototransduction process in invertebrates by serving as a Ca2+ entry channel [15]. Homologues of trp have been described in several species (reviewed in [16]); however, they have never been identified in mammalian oocytes. We searched for the presence of the capacitative Ca2+ entry pathway in porcine oocytes and the presence of a trp homologue that could serve as a Ca2+ influx channel after store depletion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte Maturation

Experiments were conducted according to institutional Animal Care and Use Committee guidelines. All chemicals were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise indicated. Oocyte-cumulus complexes were collected from porcine ovaries and rinsed 3 times in Hepes-buffered Tyrode medium containing 0.1% (w/v) polyvinyl alcohol (Hepes-TL-PVA). The complexes were matured in groups of 50 in 500 µl NCSU-23 medium [17] supplemented with 10% porcine follicular fluid, 0.1 mg/ml cysteine, 10 ng/ml epidermal growth factor, 10 IU/ml eCG, and 10 IU/ml hCG. After 22 h, the complexes were transferred into a culture dish containing the same medium without hormones and were cultured for an additional 22 h. The cumulus cells were then removed by vigorous pipetting in Hepes-TL-PVA in the presence of 0.3 mg/ml hyaluronidase.

Fluorescent Recordings

The oocytes were loaded with the Ca2+ indicator dye fura-2 by incubation in the presence of 2 µM acetoxymethyl ester form of the dye and 0.02% pluronic F-127 (both from Molecular Probes, Eugene, OR) for 40–50 min. After incubation, the oocytes were rinsed and exposed to various treatments, and the changes in the intracellular free Ca2+ concentration ([Ca2+]i) were followed using a Photoscan-2 photon-counting fluorescent microscope system (Nikon Corp., Tokyo, Japan) as described previously [18]. Fluorescence was recorded by calculating the ratio of fura-2 fluorescence at 510 nm excited by ultraviolet light at 340 and 380 nm. The [Ca2+]i levels are presented as fluorescent ratio values with ratios of 1.2 and 6.5 representing 65 and 602 nM Ca2+, respectively.

Microinjection

To induce the release of Ca2+ from the intracellular stores, the second messenger InsP3 was injected into the oocyte cytoplasm using a microinjector (Narishige Co. Ltd., Tokyo, Japan). InsP3 was dissolved in carrier medium consisting of 10 mM Hepes and 100 µM EGTA buffered at pH 7.0. The amount injected was about 40 pl, which is 4% of the total cytoplasmic volume of ~1000 pl. Microinjection was performed in Hepes-TL-PVA on a heated stage of a Diaphot inverted microscope (Nikon).

In Vitro Transcription

The plasmid vector pBluescript KS containing the Drosophila trp cDNA ctrp-9 downstream of the T7 promoter (a generous gift from C. Montell) was transfected into Escherichia coli DH5{alpha} cells. Plasmid DNA was isolated and linearized with the restriction endonuclease KpnI (Promega Corp., Madison, WI), and mRNA was transcribed from the cDNA with T7 polymerase using the RiboMAX Large Scale RNA Production System (Promega), following the manufacturer's recommendations. To produce capped RNA transcripts, the reaction was performed in the presence of 3 mM m7G(5')ppp(5')G (Boehringer-Mannheim Corp., Indianapolis, IN). Purified RNA was precipitated with 0.3 M sodium acetate and ethanol. The pellet was resuspended in diethylpyrocarbonate (DEPC)-treated water containing RNasin (1 IU/µl; Promega) to a final concentration of approximately 800 ng/µl, and the samples were stored in 3-µl aliquots at -70°C.

Western Blot

Oocytes injected with ctrp-9 mRNA and control oocytes (injected with DEPC-treated water) were lysed in groups of 20 in 5 µl in denaturing Laemmli sample buffer and boiled for 1 min. The proteins in the lysate were separated with SDS-PAGE (10% w/v polyacrilamide), and separated proteins were electrophoretically transferred for 2 h onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA) for subsequent probing. Immunodetection was achieved by incubating the blots with {alpha}zctrp antiserum (an antibody raised in rabbit against the trp protein; a gift from C. Montell) diluted 1:2000 in PBS with 0.01% Tween-20 and 5% nonfat dry milk. To detect the primary antibody, blots were incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG antibody diluted 1:5000 in PBS, 0.01% Tween 20, 5% nonfat dry milk, washed thoroughly in PBS with 0.01% Tween 20, and exposed to enhanced chemiluminescence reagents for 1 min. Subsequently, the blots were exposed to X-OMAT AR film (Eastman Kodak Co., Rochester, NY).

Messenger RNA Isolation

Poly(A) RNA was extracted from individual oocytes using Hybond-messenger affinity paper (Hybond-mAP; Amersham Pharmacia Biotech, Piscataway, NJ). Oocytes were incubated with a 3- to 4-mm2 piece of Hybond-mAP for 2 h in guanidium isothiocyanate (GITC) lysis solution (4 M GITC, 0.1 M Tris-HCl, pH 7.4, 1 M beta-mercaptoethanol; all in DEPC-treated water). After incubation, the Hybond-mAP was placed on Whatman filter paper (Fischer Scientific, St. Louis, MO), and the aqueous contents of the vials was carefully spotted onto the membrane. The Hybond-mAP was then washed twice in 0.5 M NaCl plus 0.1 M Tris-HCl, pH 7.4, in DEPC-treated water, twice in 0.5 M NaCl in DEPC-treated water, and twice in 70% ethanol. The Hybond-mAP was then allowed to air dry for a few minutes and immediately used for reverse transcription (RT).

Because mammalian trp is expressed at high levels in ovarian tissues [19, 20], total RNA was isolated from porcine ovaries to be used as a positive control for RT polymerase chain reaction (PCR). Ovaries were flash frozen in liquid nitrogen immediately after removal and stored at -70°C until processed. For RNA isolation, ovaries were removed from the liquid nitrogen, placed into 20 ml lysis buffer (STAT-60; Tel-Test, Inc., Friendswood, TX), and homogenized using a rotor-stator homogenizer. An additional 20 ml of lysis buffer was added to the homogenate followed by a 1/10 volume of bromo-chloro-propane. The mixture was then shaken vigorously for 30 sec and allowed to sit for 2–3 min. Following centrifugation at 10 000 x g for 15 min, the supernatant was collected into a new tube and the RNA was precipitated by adding an equal volume of ice-cold isopropyl alcohol. 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 poured off, the pellet was washed in ice-cold 80% ethanol, and the RNA was aliquoted in DEPC-treated water with 5 µl/ml RNasin. Aliquots were stored at -70°C until use.

Reverse Transcription

Hybond-mAP with attached RNA was used in the RT reactions, which 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, Inc., Watertown, MA). The reaction mixtures consisted of the following: 200 IU M-MLV reverse transcriptase, M-MLV reverse transcriptase buffer, 2.5 µM random hexamers, 200 µM each dNTP, and 20 IU RNasin (Promega). Milli-Q water (Millipore) was added to the reaction mixtures to make a final volume of 20 µl.

Total RNA isolated from ovaries was reverse transcribed in a reaction mixture consisting of 200 IU M-MLV reverse transcriptase, M-MLV reverse transcriptase buffer, 200 µM each dNTP, 2.5 µM reverse primer, and 20 IU RNasin. The final volume of 20 µl was achieved by adding Milli-Q water. The RT reaction was carried out by incubating the reaction mixture at 42°C for 45 min followed by incubation at 95°C for 5 min.

Polymerase Chain Reaction

The primers used to amplify a trp homologue from porcine oocytes were designed based on conserved regions of the murine (Mtrp3) and human (Htrp3) trp homologues [21]. The forward primer was 5'-AAGGACATATTCAAGTTCAT-3' (bases 2147–2166 of the Htrp3 sequence), and the reverse primer was 5'-CCATTCTACATCACTGTCAT-3' (bases 2460–2479 of the Htrp3 sequence). The primers were expected to amplify a 333-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. When cDNA from individual oocytes was amplified, the 50-µl PCR mixture contained 5 µl cDNA as a template, 2 mM MgCl2, 200 µM each dNTP, 2.5 IU Taq polymerase, 1x reaction buffer, 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 IU 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 of denaturation for 30 sec at 95°C, annealing for 30 sec at 56°C, and extension for 1 min at 72°C, with an 8-min extension following the last cycle.

Experiment 1

First we investigated whether a Ca2+ influx can be generated in porcine oocytes by the depletion of the intracellular Ca2+ stores. Thapsigargin, a tumor-promoting plant sesquiterpene lactone, inhibits the endoplasmic reticulum Ca-ATPases (Ca2+ pumps) with little effect on the plasma membrane Ca-ATPase. It is routinely used to drain the intracellular stores of their Ca2+ content. Fura-2-loaded oocytes were incubated in Ca2+-free Hepes-TL-PVA medium in the presence of 10–50 µM thapsigargin for 3 h to deplete intracellular Ca2+ stores [22, 23]. After washing in Ca2+-free medium (to remove thapsigargin and ensure that the intracellular stores remain empty), normal Ca2+-containing medium was added to the oocytes, and the changes in the [Ca2+]i were measured. Oocytes incubated in Ca2+-free Hepes-TL-PVA for 3 h without thapsigargin were used to show the Ca2+ entry under normal conditions, when the intracellular Ca2+ stores were full.

Experiment 2

The onset of a divalent cation influx after a Ca2+ transient was investigated by using the manganese (Mn2+)-quench technique [24]. Release of Ca2+ from the intracellular stores was stimulated by intracellular injection of approximately 40 pl of 2.5 µM InsP3, the InsP3 receptor agonist. As a Ca2+ surrogate, Mn2+ was added to the external medium. Mn2+ is thought to be able to translocate across the plasma membrane, bind fura-2, and quench its fluorescence [24]. This technique enables measurement of divalent cation influx even when Ca2+ release from the internal stores is coincident. The entry of Mn2+ into the cell was monitored by imaging the resulting quench in fura-2 fluorescence at 510 nm excited at 340 and 360 nm. Although the signal resulting from the 340 nm excitation is [Ca2+]i sensitive, at 360 nm fura-2 fluorescence is independent of [Ca2+]i and any decrease in fluorescence is due only to Mn2+ entry.

Experiment 3

The Drosophila trp protein was expressed in porcine oocytes by injecting approximately 32 pg mRNA made by in vitro transcription of the cDNA and allowing 15 h for translation, which was enough time in our previous experiments [25]. Control oocytes were injected with the carrier medium (DEPC-treated water). The injected oocytes were stained with the Ca2+ indicator dye fura-2 AM and incubated in Ca2+-free Hepes-TL-PVA with 50 µM thapsigargin for 2 h. Because in experiment 1 a 3-h thapsigargin incubation stimulated very distinct capacitative Ca2+ entry, probably because of complete store depletion, the incubation time in this experiment was reduced to 2 h so that any difference between injected and noninjected oocytes would be more apparent. The baseline fluorescence of the oocytes was then recorded in Ca2+-free Hepes-TL-PVA, and changes in [Ca2+]i were measured for 20–30 min after the addition of Ca2+-containing medium.

Experiment 4

In this experiment, we determined whether RNAs homologous with trp were present in the porcine oocyte. Poly(A) RNA was isolated from the oocytes, and cDNA was prepared by RT-PCR. The primers used for the PCR were designed as described above. The PCR products were electrophoresed on a 1.8% 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 contained a mammalian homologue of trp, and the sequences were used to determine the homology between human (and mouse) trp and the trp found in porcine oocytes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Depletion of Ca2+ Stores Generates a Ca2+ Influx

When applied in Ca2+-free medium, thapsigargin (10–50 µM) caused the depletion of Ca2+ stores and induced an increase in [Ca2+]i in pig oocytes. Figure 1 shows the response of an oocyte treated with 50 µM thapsigargin; the increase consisted of a slowly rising and falling peak. Concentrations of 10 and 20 µM thapsigargin caused slightly smaller increases in [Ca2+]i. In 15 of 18 oocytes, emptying the intracellular Ca2+ stores promoted Ca2+ entry after the readdition of Ca2+, which was detected as a rise in the [Ca2+]i (Fig. 2A). The increase in [Ca2+]i started 0–300 sec after adding the Hepes-TL-PVA medium and went on until the end of the measurements, because the blocked pumps could not reaccumulate Ca2+ and the empty stores kept sending the activating message to the Ca2+ entry pathways infinitely. However, the intracellular Ca2+ levels of the control oocytes that were not treated with thapsigargin were not affected by the presence of extracellular Ca2+; in these oocytes (12/12), no observable increase in the [Ca2+]i was detected (data not shown).



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  		FIG. 1. Ca2+ release in a porcine oocyte induced by 50 µM thapsigargin. The oocyte was held in Ca2+-free medium, and then thapsigargin (arrow) was added



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  		FIG. 2. Capacitative Ca2+ entry in porcine oocytes. The intracellular stores were depleted by incubation of the oocytes in Ca2+-free medium for 3 h in the presence of 50 µM thapsigargin. After baseline measurement in Ca2+-free medium, Ca2+ was added (arrow) to the oocytes (A). The Ca2+ entry evoked by store depletion was totally inhibited by 1 mM La3+ (B). Each figure represents 1 oocyte

In Xenopus oocytes, the capacitative Ca2+ entry pathway could be blocked reversibly by the application of 1 mM Zn2+ [26], whereas in other cells lanthanum (La3+) and nickel (Ni2+) were reported to block the capacitative Ca2+ influx [27–29]. In accordance with these reports, the thapsigargin-evoked Ca2+ entry in 11 of 11 porcine oocytes was completely blocked by 1 mM La3+ (Fig. 2B). Thus, store depletion triggers Ca2+ entry in porcine oocytes, indicating the presence of a capacitative Ca2+ entry pathway.

Ca2+ Transient Induces a Divalent Cation Influx

In 16 of 16 oocytes, InsP3 induced a transient elevation in fluorescence with excitation at 340 nm, indicating an increase in the [Ca2+]i. After the Ca2+ transient, the signal returned to the resting value. Simultaneous measurement at 360 nm revealed only a slight instability in fluorescence (Fig. 3A); at this wavelength fura-2 fluorescence is insensitive to changes in [Ca2+]i. When the oocytes were microinjected with InsP3 in the presence of 3 mM Mn2+ in the external medium (or alternatively Mn2+ was added subsequent to microinjection), there was a rapid decline in fluorescence well below the basal value (14/14 oocytes). This decrease in the fluorescence intensity was due to extracellular Mn2+ that entered the oocyte after the InsP3-induced Ca2+ transient and quenched the fluorescence of the intracellular dye (Fig. 3B). The basal rate of fluorescence quenching due to Mn2+ translocation across the plasma membrane in the control noninjected oocytes was considerably less.



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  		FIG. 3. Divalent cation influx triggered by an InsP3-induced Ca2+ release in porcine oocytes. The injection of 2.5 µM InsP3 (arrow) triggered an elevation in fluorescence with excitation at 340 nm (lower trace), indicating an increase in [Ca2+]i. Simultaneous measurement at 360 nm (upper trace) revealed only a slight instability in fluorescence; at this wavelength fura-2 fluorescence is insensitive to changes in [Ca2+]i (A). In the presence of 3 mM Mn2+ in the external medium, InsP3 caused a rapid decline in fluorescence (B). This decrease in the fluorescence intensity was due to extracellular Mn2+ that entered the oocyte after the InsP3-induced Ca2+ transient and quenched the fluorescence of the intracellular dye at both wavelengths. The arrow marks the addition of Mn2+; the y-axis shows fluorescence in arbitrary units

La3+, the inhibitor of Ca2+ entry channels, totally blocked the cation influx and hence the decline in fluorescence at both wavelengths. When InsP3 was microinjected in the presence of 1 mM La3+, the fluorescence intensities stayed near the resting values, even after the addition of Mn2+ in all oocytes (7/7; data not shown). These results support the idea that a capacitative Ca2+ entry mechanism exists in porcine oocytes, i.e., the discharge of Ca2+ from intracellular stores stimulates an inward Ca2+ current that might play a role in refilling the stores.

Heterologous Expression of trp Channels Increased Ca2+ Influx

The Drosophila trp protein was expressed in porcine oocytes when approximately 32 pg mRNA encoding the trp channel was injected. The existence of an approximately 150-kDa protein was demonstrated in the mRNA-injected oocytes by Western blot analysis using {alpha}zctrp, an antiserum raised against the Drosophila ctrp-9 protein. In the control oocytes, this protein was not present (Fig. 4). Application of external Ca2+ after thapsigargin treatment to carrier medium-injected oocytes induced a Ca2+ influx, indicating the presence of the endogenous capacitative Ca2+ entry mechanism. However, the increase in the [Ca2+]i caused by Ca2+ entry occurred more quickly in oocytes expressing Drosophila trp. The time required for the baseline Ca2+ to reach its maximum value and begin to oscillate was significantly shorter in the mRNA-injected oocytes than in the carrier medium-injected oocytes (8.0 ± 2.3 sec vs. 27.0 ± 2.8 sec, P < 0.001; Fig. 5, A and B). The Ca2+ entry-evoked [Ca2+]i increase was completely blocked by 1 mM La3+ (data not shown). These findings suggest that trp homologues expressed in porcine oocytes may function as Ca2+ entry channels.



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  		FIG. 4. Western blot analysis of porcine oocytes injected with mRNA encoding for the Drosophila ctrp-9 protein. An approximately 150-kDa protein was present in the mRNA-injected oocytes but was absent in the oocytes injected with the carrier medium



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  		FIG. 5. The effect of trp expression on capacitative Ca2+ entry. Oocytes were incubated with 50 µM thapsigargin in Ca2+-free medium for 2 h. Following baseline measurement in Ca2+-free medium, Ca2+ was added (arrow) to the oocytes. The Ca2+ entry in the mRNA-injected oocytes (A) was faster than that in the control oocytes (B) because of the higher number of Ca2+ entry pathways in the plasma membrane

Porcine Oocytes Contain trp mRNA

PCR amplification revealed the expected 333-bp band from both oocyte and ovary cDNA (Fig. 6). Sequencing of the PCR product showed that the band amplified from porcine oocyte cDNA corresponded with the murine (Mtrp3) and human (Htrp3) trp sequences; the product had 92.0% identity with Mtrp3 and 96.2% identity with Htrp3 (Fig. 7; GenBank accession no. AF420483). These results indicate that porcine oocytes express a trp homologue.



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  		FIG. 6. RT-PCR products for detecting the presence of a trp homologue in porcine oocytes. RNA was extracted from cells, and first strand cDNA was reverse transcribed. PCR was performed for 45 cycles. Lane 1: molecular size marker; lane 2: no-template control with trp primers; lane 3: ovarian cDNA with trp primers; lanes 4–7: trp cDNA fragment from oocytes; lane 8: no-template control with ß actin primers; lane 9: ovarian cDNA with ß actin primers; lane 10: oocyte cDNA with beta; actin primers



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  		FIG. 7. Nucleotide sequence of the PCR product from porcine oocytes together with known human and mouse sequences. The 333-bp fragment amplified from porcine oocytes showed 96.2% identity with the human sequence (Htrp3) and 92.0% identity with the mouse sequence (Mtrp3).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of these experiments clearly indicate the presence of a capacitative Ca2+ entry mechanism in porcine oocytes. This mechanism was demonstrated with the use of thapsigargin, the plant sesquiterpene lactone. Thapsigargin induces a passive depletion of intracellular Ca2+ stores by inhibiting the SERCA pumps. In porcine oocytes, it also induced a small Ca2+ transient in the absence of extracellular Ca2+, indicating depletion of the Ca2+ pools. Consistent with the capacitative entry model, thapsigargin activated a substantial Ca2+ influx after the readdition of Ca2+. The thapsigargin concentration used in these experiments is higher than that normally used in somatic cells; it is comparable to the concentrations reported in a study of mouse oocytes [22]. Because thapsigargin acts directly on the SERCA pumps without generating any Ca2+-releasing second messengers, such a result indicates that depletion of Ca2+ stores provides sufficient signal for the activation of Ca2+ entry. This hypothesis has been supported by findings in a large number of cells where the Ca2+ influx pathways also remained activated as long as the intracellular pools were not permitted to refill [16]. Originally it was postulated that Ca2+ influx pathways would take Ca2+ directly into the Ca2+ stores without elevating free Ca2+ levels in the cytosol [30]. However, later experiments indicated that repletion Ca2+ first enters the cytoplasm (thus the entry is associated with an increase in [Ca2+]i), and SERCA pumps then transport it into the endoplasmic reticulum [31]. Our results are also in accordance with findings in mouse oocytes, where the addition of Ca2+ to the oocytes after the thapsigargin-induced Ca2+ transient was able to induce a Ca2+ influx [12, 22].

The presence of capacitative Ca2+ entry was also demonstrated after intracellular injection of the Ca2+ signaling molecule InsP3. Normally, InsP3 is generated by the hydrolysis of membrane phospholipids; it then binds to its receptor located in the endoplasmic reticulum, which results in a rapid release of Ca2+ to the cytoplasm. The Ca2+ release induced by InsP3 stimulated an immediate divalent cation entry, as revealed by the Mn2+ quench technique. Because InsP3 was implicated in intracellular Ca2+ release during fertilization [4], the Mn2+ influx activated by the InsP3-induced Ca2+ release indicates that capacitative Ca2+ entry can be stimulated with physiological second messengers in porcine oocytes. This stimulation was clearly demonstrated in mouse oocytes, where the stimulation of cation influx was associated with the fertilization Ca2+ spikes [12].

The identity of the capacitative Ca2+ entry channels is not known. There are various pathways by which extracellular Ca2+ can enter the cell, including channels operated by voltage, receptors, or second messengers [4]. To distinguish it from other Ca2+ entry channels, the term Ca2+ release-activated Ca2+ current (ICRAC) was used to refer to the current flowing through the capacitative Ca2+ entry channels [32]. ICRAC is probably the most meticulously characterized Ca2+ influx current, but the entry channel has not yet been classified at the molecular level. A very promising candidate for a CRAC-like protein has been the mammalian homologue of the Drosophila protein trp. During visual signal transduction in invertebrates, light induces the release of Ca2+ from intracellular stores [33], followed by photoreceptor depolarization and the development of the so-called receptor potential. It is also followed by the activation of 2 membrane channels, trp and trp-like (trpl), which in turn admit Ca2+ and other cations into the cell and depolarize it. In wild-type flies, if light persists, the receptor potential is sustained by this Ca2+ influx [34]. In trp-deficient flies, photostimulation causes only a transient receptor potential (trp) because the photoreceptors are unable to sustain an influx of Ca2+ through the membrane channels.

After the cloning of the Drosophila trp gene [15], the presence of trp homologues was identified in several species [21, 26, 35, 36]. Its presence was also shown in porcine aortic endothelial cells [37]. Trp was first suggested to be a capacitative Ca2+ entry channel by Hardie and Minke [14]. Expression of trp in insect Sf9 cells resulted in a depletion-activated inward current [38]. When expressed in Xenopus oocytes, trp enhanced Ca2+ influx after thapsigargin treatment [26]. Moreover, the rat trp homologue, when expressed in Xenopus oocytes, also stimulated increased Ca2+ conductance [39], and the human trp homologue expressed in a mammalian cell line enhanced store-operated Ca2+ entry [21]. Our findings are consistent with these results. In trp-expressing porcine oocytes, the increase of the Ca2+ concentration due to Ca2+ influx reached maximum levels significantly faster than in control oocytes. This higher rate of increase is probably due to the increased number of Ca2+ entry channels in the plasma membrane.

To date, 7 mammalian trp homologues have been identified [40]. This present study is the first to confirm the existence of a trp homologue in a mammalian oocyte. The cDNA fragment from porcine oocytes showed 92.0% identity with mouse and 96.2% identity with the human trp sequences. Electrophysiological studies on single channel activity are needed to verify whether this trp channel can serve as a capacitative Ca2+ entry pathway after depletion of intracellular stores or whether the Ca2+ influx through these channels simply represents additional Ca2+ entry. Although several researchers have shown that trp1, trp4, and trp5 may function as store-operated channels [41–43], others demonstrated that mammalian trp channels are not activated by store depletion, at least when heterologously expressed [20, 44]. Moreover, data suggest that trp3 functions as a Ca2+-activated nonselective cation channel and that the thapsigargin-induced Ca2+ entry in trp3-expressing cells is due to activation of this channel by Ca2+ entering through the endogenous capacitative entry pathway [45, 46]. Similarly, trp6 transfected COS.M6 cells showed augmented Ca2+ entry only after surface receptor activation and not after store depletion by thapsigargin [47]. As revealed by cell-attached patch recordings used to monitor trpl single-channel activity, thapsigargin induced an increase in trpl activity in the presence of extracellular Ca2+ when expressed in Sf9 cells [48]. However, the increase in trpl activity was blocked by low-micromolar concentrations of La3+ that previously completely inhibited endogenous capacitative Ca2+ entry but had no effect on cation flux via trpl, suggesting that trpl channel activity requires Ca2+ entry via the endogenous capacitative Ca2+ entry pathway. Heterologous expression of trpl also gave rise to cation currents that are not activated by the depletion of internal stores but are stimulated following activation of membrane receptors linked to phosphoinositide turnover [40, 49, 50]. The trp channel and CRAC, the typical capacitative Ca2+ entry channel, also have different permeability properties: trp has a higher conductance and is much less specific than the CRAC channel [11]. Thus, the role of trp proteins as capacitative Ca2+ entry channel molecules is still not proven.

In summary, porcine oocytes have a capacitative Ca2+ entry mechanism that is activated after depletion of intracellular stores by SERCA pump inhibition or following a Ca2+ transient induced by the second messenger InsP3. Heterologous expression of the Drosophila trp protein in these oocytes increases Ca2+ influx following store depletion. Porcine oocytes also contain mRNA homologous with mouse and human trp molecules, indicating that the oocytes express a trp homologue. Functional characterization is needed to determine whether the trp channel serves as the capacitative entry pathway.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Robert L. Matteri for his valuable assistance in designing primers for PCR.


   FOOTNOTES
 
First decision: 29 August 2001.

1 This report is based on work supported by the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture, under agreement 99-35203-7675. Back

2 Correspondence and current address: Zoltán Macháty, Columbus Farming Corporation, P.O. Box 1160, Sherburne, NY 13460. FAX: 607 674 6309; machatyz{at}columbusfarming.com Back

Accepted: October 12, 2001.

Received: June 26, 2001.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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