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a Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003
b American Breeders Services, De Forest, Wisconsin 53532
c Department of Obstetrics/Gynecology and Anatomy/Cell Biology, Tufts University School of Medicine and New England Medical Center Hospital, Boston, Massachusetts 02111
d Biological Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan
e Laboratorium voor Fysiologie, Campus Gasthuisberg O/N, K.U. Leuven, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The signaling mechanism by which the sperm triggers and maintains this periodical Ca2+ release in oocytes is not fully elucidated, although inositol 1,4,5-trisphosphate (IP3), a widespread Ca2+-releasing molecule produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C, and its receptor (IP3R), are likely to be involved [5, 6]. IP3Rs are tetramers that act as ligand-gated channels facilitating Ca2+ release from internal stores/endoplasmic reticulum (ER) [7]. The involvement of this mechanism during fertilization is supported by previous findings: that in species in which measurements of intracellular levels of IP3 are possible in a few oocytes, i.e., Xenopus, the sperm triggers an immediate rise in IP3 mass [8, 9]; and in fertilized sea urchin eggs, increases in IP3 appear to coincide with important early events of fertilization [10]. In addition, in mammalian oocytes, in which measurements of IP3 mass are not yet possible due to size and lack of fertilization synchrony, maximal Ca2+ release in response to injection of agonists or fertilization, which is accomplished at the end of oocyte maturation at the MII stage [11, 12], coincides with a significant increase in the amount of IP3Rs [13–15]. Moreover, the abundant expression of the IP3R in oocytes [13, 16–18] and its spatial distribution, from cortical to perinuclear [18], suggest a significant role for the IP3R system in the generation of fertilization-associated [;t1Ca2+]i rises. Another Ca2+ channel that is expressed in mammalian oocytes is the ryanodine receptor (RyR) [19–21], although its contribution to Ca2+ release during fertilization remains to be demonstrated.
Several isoforms of the IP3R have been described in different cell types, and the pattern of expression is very different according to the cell type investigated [22–26]. The IP3R type 1 appears to be the most widely expressed (for review see [27]), while types 2 and 3, although less ubiquitous, may constitute the primary isoform in certain somatic cell types [23, 28, 29]. In oocytes, type-1 IP3R has been detected in several species [14, 15, 30–32], but the presence of the other isoforms has not been extensively studied and the extent of their contribution to fertilization-associated [;t1Ca2+]i rises is not known.
In addition to the expression of several isoforms, IP3R-mediated Ca2+ release can be regulated by several other mechanisms (for review see [27, 33]). One of them, IP3R down-regulation, has been observed in numerous cell lines exposed to continual cell surface receptor stimulation [25,34–36]; this down-regulation appears to be cell line, isoform, and agonist specific [25–37]. Down-regulation of the IP3Rs affects the sensitivity of IP3-induced Ca2+ release, and this in turn may limit Ca2+ responsiveness [25, 37]. The impact of this or other regulatory systems of IP3R-mediated Ca2+ release on fertilization-associated [;t1Ca2+]i oscillations in bovine oocytes is not known.
This study was conducted to further characterize the role of the IP3R system in fertilization-induced Ca2+ responses. We specifically investigated which isoforms of the IP3R are present in bovine oocytes/ovaries and whether fertilization- and/or agonist-triggered [;t1Ca2+]i oscillations induced IP3R down-regulation. Isoform-specific antisera were utilized to carry out these studies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocytes used in these studies were obtained by aspiration of 2- to 8-mm follicles collected at a slaughterhouse and transported at room temperature to the laboratory within 4–6 h of slaughter. Selection and maturation of oocytes were as previously described [38, 39]. In short, oocytes with an intact, compact cumulus mass were matured in TCM-199 medium (Gibco, Grand Island, NY), supplemented with 10% heat-treated calf serum (Gibco), 0.5 µg/ml FSH (ovine, oFSH-17; National Institute and Diabetes and Digestive and Kidney Diseases [NIDDK], Baltimore, MD), and 5 µg/ml LH (bLH-5 NIDDK). Cumulus-oocyte complexes were cultured for 22 h in groups of 10, in 50-µl drops under paraffin oil at 38.5°C in a humidified atmosphere containing 5.5% CO2. Oocytes were stripped of their granulosa cells by vortexing and were utilized for microinjection studies and for preparation of whole cell lysates for Western blotting procedures. Only oocytes that had extruded the first polar body and were assumed to have progressed to the MII stage were utilized for these studies. CR-1 salts [40], supplemented with BSA (6 mg/ml), Basal Medium Eagle's essential amino acids, 1 mM glutamine, and Minimal Essential Medium nonessential amino acids (all from Sigma Chemical Co., St. Louis, MO) were used as culture and fertilization medium. Mouse oocytes used to obtain data in Figure 5 were obtained as described previously [18]. Porcine oocytes were obtained directly from the oviduct after spontaneous ovulation.
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Fertilization procedures were as previously described [39, 41]. In brief, frozen semen, donated by American Breeders Association (De Forest, WI), was processed by the Percoll method, and the separated motile sperm were added at a final concentration of 500 000 sperm/ml. Heparin (10 µg/ml; Sigma) was added to the fertilization medium to induce sperm capacitation [42]. Cumulus-intact oocytes were used for this procedure and were removed from the fertilization drop 8 h after insemination. The oocytes were then cultured for an additional 8 h. Immediately afterward, cumulus cells were removed by vortexing and oocytes washed several times to release the few remaining granulosa and/or sperm cells. These oocytes were then used for crude lysates for Western blotting. Control fertilized oocytes handled in a similar manner were fixed and stained with Hoechst 33258 [39] to evaluate fertilization rates. Upon evaluation, 85% (212 of 250) of the inseminated oocytes exhibited at least two pronuclei 16 h after inseminations and were assumed to be fertilized. These results were obtained from four different replicates.
Microinjection Techniques and [;t1Ca2+]i Monitoring
Microinjection of MII bovine oocytes was carried out as previously reported [41]. In brief, oocytes were microinjected in 50-µl drops of Tyrode's lactate-Hepes supplemented with 2.5% sucrose (w:v) using Narishige (Tokyo, Japan) manipulators mounted on a Nikon Diaphot microscope (Nikon, Garden City, NY). Glass micropipettes were loaded by suction with one of the following: 0.5 mM fura-2 dextran (fura-2D, dextran 10 kDa; Molecular Probes, Eugene, OR) or 10 µM adenophostin A, a generous gift of Dr. K. Tanzawa (Sankyo Co., Ltd., Tokyo, Japan). Both reagents were prepared in a buffer that consisted of 75 mM KCl and 20 mM Hepes (pH 7.0) and were expelled into the oocyte's cytoplasm by pneumatic pressure (PLI-100 picoinjector; Harvard Apparatus, Cambridge, MA). Each oocyte received approximately 7–15 pl of each reagent (calculated according to Lee [43]), and this amount represented approximately 1–2% of the total oocyte volume.
Monitoring of fura-2D fluorescence was done as previously described [41]. Briefly, a 75-watt xenon arc lamp provided illumination, and the excitation wavelengths were at 340 and 380 nm. The intensity of UV light was attenuated by the presence of neutral density filters, and the emitted light was quantified by a photomultiplier tube after passing through a 500-nm barrier filter. The fluorescent signal was averaged for the whole oocyte. A modified Phoscan 3.0 software program (Nikon) on a 486 IBM-compatible computer controlled the rotation of a filter wheel and a shutter apparatus to alternate wavelengths. [;t1Ca2+]i was determined from the 340/380 ratio of fluorescence according to Grynkiewicz et al. [44]. Rmax, Rmin, and ß were calculated using 10 µM fura-2D in Ca2+-free Dulbecco's PBS supplemented with 2 mM CaCl2 (Rmax) or 2 mM EDTA (Rmin) and 60% sucrose (w:v) to correct for intracellular viscosity [45]. The same solution was also used alone for background subtraction. Ratios of 0.15 and 1.5 were obtained for Rmin and Rmax, respectively.
[;t1Ca2+]i monitoring started approximately 30 min after injection of fura-2D, and oocytes were measured individually in 40 µl of medium placed on a glass coverslip on the bottom of a culture dish covered with paraffin oil and maintained at room temperature. Readings were taken for 1 sec at each wavelength, and ratios were obtained every 4 sec. Recordings of baseline [;t1Ca2+]i values were carried out for 2–5 min and were briefly stopped to microinject adenophostin A. Fluorescence ratios were then monitored for 30 min.
Membrane Preparations and Western Blots
Microsomes from bovine ovaries were prepared as previously described [15]. In brief, ovaries were homogenized with a glass-silicone-coated homogenizer in 0.3 M sucrose, 1 mM EDTA, 1 mM 2-mercaptoethanol, 200 µM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 50 mM Tris-HCl (pH 8.0; all chemicals from Sigma) and centrifuged at 2000 x g for 10 min to remove the heavy particulate. The supernatant was recentrifuged at 105 000 x g for 45 min, and the microsomal precipitates were resuspended in 0.3 M sucrose, 1 mM EDTA, 1 mM 2-mercaptoethanol, 50 mM Tris-HCl (pH 8.0). Crude lysates from oocytes were prepared from 10 or 400 denuded bovine oocytes collected in Tyrode's lactate-Hepes supplemented with 3 mg/ml polyvinylpyrrolidone (Mr 40 000; Sigma).
Microsomal preparations or crude oocyte lysates were combined with double-strength electrophoresis sample buffer [46]. Samples were boiled for 5 min and loaded into 4% SDS-polyacrylamide gels. The separated proteins were then transferred onto nitrocellulose membranes (Micron Separation, Westboro, MA) using a Mini Trans Blot Cell (Bio-Rad, Hercules, CA). The membranes were first washed in PBS-0.005% Tween 20 (PBS-T) and then blocked in PBS-T supplemented with 6% nonfat dry milk. After additional washes in PBS-T, the membranes were incubated overnight at 4°C with the assigned primary antibody, washed again in PBS-T, and incubated for 1 h with a secondary horseradish peroxidase-coupled antibody. After three additional washes in PBS-T, membranes were developed using the enhanced chemiluminescence (ECL) detection system according to the manufacturer's instructions (Amersham, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ). Quantification of immunoreactive IP3R-1 was carried out using Adobe Photoshop (Mountain View, CA). The intensity of the band in MII oocytes was used as the baseline value and arbitrarily assigned the value of 1. The intensity of the IP3R-1 band for untreated control oocytes matured for 40 h, or fertilized, or injected with adenophostin A and collected 40 h after initiation of maturation was calculated relative to 1, and statistical comparison was performed. Prestained SDS-PAGE molecular weight markers, broad range (Bio-Rad), were run in parallel to estimate the molecular weight of the immunoreactive bands. Western blot analysis for identification of isoforms was repeated at least two times, and those Western procedures in which quantification of the type-1 isoform was carried out were repeated at least three different times.
Antibodies
Polyclonal IP3R isoform-specific antisera have been generated by utilizing the unique C-terminal region of the IP3R [47]; these antibodies also recognize the oocyte/egg's IP3Rs [14, 15, 18, 30–32]. In this study, isoform-specific rabbit polyclonal antibodies raised against the IP3R-1 (Rbt04) and IP3R-2 (Rbt02) were used. They were raised against the following amino acid sequence in the C-terminal end of IP3R molecule: (type 1) LGHPPHMNVNPQQPA, (type 2) SNTPHVNHHMPPH. The antibody used to recognize the type-3 IP3R isoform was obtained from Transduction Laboratories (Lexington, KY) and is an IgG2a monoclonal antibody raised against the N-terminal region (amino acids 22–230) of the human IP3R-3. These isoform-specific antibodies were previously shown not to mutually cross-react [23, 47, 48]. In this study, the anti-IP3R-1, -2, and -3 antibodies were used at 1:4000, 1:200, and 1:500, respectively. Negative controls were obtained by using preimmune serum (type 1) and by omission of the primary antibody (type 3). For IP3R-2, the Rbt02 antiserum was preincubated for 1 h with 5 mg/ml of the C-terminal peptide used to raise the antibody (a generous gift of Dr. Frank Longo, University of Iowa, Iowa City, IA, and prepared by Genemed Biotechnologies Inc., South San Francisco, CA) before dilution to 1:200 for probing the membrane.
Statistical Analysis
Statistical comparisons of the intensity of the IP3R-1 bands after use of different experimental conditions were performed using one-way ANOVA; if differences were observed, multiple comparisons between means were carried out by applying the Tukey-Kramer method using the JMP IN software (SAS Institute, Cary, NC). In all cases, significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The exit of mammalian oocytes from MII arrest following fertilization is made possible by the generation of [;t1Ca2+]i oscillations. Although IP3Rs are likely to be involved in this Ca2+ release, it is not known which of the IP3R isoforms is present and may contribute to Ca2+ release in bovine oocytes. Thus, using isoform-specific antibodies, we determined which isoforms are expressed in bovine oocytes and ovaries. As shown in Figure 1, the IP3R-1, detected as a band of approximately 260 kDa, was abundant in as few as 10 oocytes and also richly present in microsomal preparations from ovaries (Fig. 1), as observed previously [15].
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Expression of the IP3R-2 was also detected in bovine oocytes and ovarian samples, although in much reduced amounts. Approximately 400 oocytes and 10 µg of microsomal ovarian preparations were required to detect its presence (Fig. 2). This represented 40 times the number of oocytes and three times the amount of ovarian tissue required to detect the presence of the type-1 isoform. IP3R type 2 exhibited a slightly smaller molecular mass than IP3R type 1, approximately 250 kDa, which is similar to the mass reported in rat and mouse cell lines [47]. In oocytes, the type 2-specific polyclonal antiserum Rbt02 had to be utilized at low dilutions (1:200), and this resulted in recognition of several proteins (Fig. 2). To confirm whether the band suspected to be the type-2 isoform was indeed the IP3R-2, the primary antibody was incubated, prior to probing the membrane, with the C-terminal peptide utilized to raise the antibody. As can be observed in Figure 2, the ovarian lane probed in this manner lacked the IP3R-2 band (Fig. 2), suggesting that the lower molecular weight proteins are recognized in a nonspecific way by the antiserum.
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The IP3R-3 was also detected in bovine oocytes and ovaries (Fig. 3). As for the type-2 isoform, larger amounts of material, 400 oocytes and 10 µg of ovarian microsomal proteins, were needed for its detection. Taken together, the data indicate that the three IP3R isoforms are present in bovine oocytes and ovaries. However, in oocytes there appears to be a clear predominance of the type-1 isoform.
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IP3R-1 Was Down-Regulated During Fertilization
Fertilization-associated [;t1Ca2+]i oscillations last for several hours in oocytes of all mammalian species studied to date [5, 49]. Invariably, as pronuclear formation approaches, these Ca2+ responses cease or decrease in amplitude [3,50–52]. It is possible that this diminishing Ca2+ release may correlate with a similar decrease in the number of IP3Rs. Thus, the relative amounts of the IP3R-1 in control-aged and fertilized bovine oocytes were evaluated. We chose to monitor the IP3R-1 because, from the aforementioned experiments, it appeared to be present in oocytes in overwhelmingly larger amounts than the other isoforms. As shown in Figure 4, IP3R-1 amounts decreased significantly (~ 40%) 16 h after insemination (40 h postinitiation of maturation; P < 0.05). In contrast, control oocytes aged for 40 h under the same conditions exhibited a nonsignificant decline of IP3R-1 levels. These results showed that IP3R-1 was down-regulated during fertilization.
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Adenophostin A, a High-Affinity Analogue of IP3, Induced Down-Regulation of IP3R-1 Similar to That During Fertilization
Adenophostin A, a new powerful IP3R agonist from Penicillium brevicompactum [53], has been shown to trigger long-lasting [;t1Ca2+]i oscillations in mouse oocytes [54]. Furthermore, coinjection of adenophostin A with round spermatids resulted, after transfer into recipients, in the delivery of live pups [54]. Thus, the ability of adenophostin A to trigger [;t1Ca2+]i oscillations in other mammalian oocytes was tested by injecting different concentrations of the agonist into bovine and porcine oocytes. Mouse oocytes were also injected and used as controls. As shown in Figure 5, injection of adenophostin A induced high-frequency [;t1Ca2+]i oscillations in all mammalian oocytes tested. In mouse oocytes (Fig. 5A; 19 of 19 oocytes), the [;t1Ca2+]i oscillations exhibited higher frequency than those observed in bovine oocytes (7 of 8; Fig. 5B) and in porcine oocytes (5 of 5; Fig. 5C). In bovine oocytes the frequency of oscillations was dose dependent (compare Fig. 6A, 10 µM, and Fig. 5B, 20 µM). Moreover, to consistently trigger oscillations in porcine oocytes, doses in excess of 100 µM adenophostin A were required, and in spite of this, the oscillations were short-lived (Fig. 5C).
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To determine whether [;t1Ca2+]i oscillations that are exclusively initiated by IP3R activation can induce down-regulation of IP3R-1 [55], 10 µM adenophostin A was injected into bovine oocytes, and the amounts of IP3R-1 in these and uninjected control oocytes were tested by Western blotting 16 h postinjection. As shown in Figure 6 (B and C), IP3R-1 was significantly down-regulated by the Ca2+ responses induced by adenophostin A, and the magnitude of the degradation was similar to that observed during fertilization. Together, these results strengthen the possibility that IP3R-1 participates in the generation of fertilization-associated Ca2+ responses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The three IP3R isoforms that have been described show a high degree of structural homology but exhibit different affinities for IP3 (IP3R-2 > IP3R-1 > IP3R-3) [24, 47, 56] and may be regulated in different ways [25, 29, 57]. Thus, the relative expression of these isoforms may influence the pattern and profile of Ca2+ release. In Xenopus oocytes, a subtype similar to the type-1 isoform is exclusively expressed [30]. The expression of the IP3R isoforms in mouse oocytes was recently investigated in two separate studies. A first report indicated that in mouse oocytes the mRNA for all three IP3R could be detected, although Western blotting gave evidence only for the presence of IP3R-1 [17]. In a second study, the presence of all three IP3R isoforms was demonstrated by Western blotting and/or immunofluorescence analysis, although here also it was clear that the IP3R-1 was the most prevalent isoform [18]. In the current study on bovine oocytes, all three IP3R isoforms were present at the protein level with type 1 the most abundant isoform detected, suggesting that type 1 is responsible for the majority of the Ca2+ release during bovine fertilization. The other two isoforms may also contribute to Ca2+ release during fertilization. A role for type 2 in oocytes deserves further investigation because of its higher affinity for IP3 [24,47], its presence in the oocyte cortex [18], and its role in the initiation [57] or amplification [58] of intracellular Ca2+ signals. In addition, IP3R-2 and -3 have the potential to form heterotetramers with type 1 [59, 60] and in this manner further contribute to Ca2+ release during fertilization.
The apparent predominance of the type-1 isoform by Western analysis in bovine oocytes is consistent with its important functional role as deduced from other studies using different methods. First, a well-known, function-blocking antibody (18A10), specific for the type-1 isoform, inhibits the fertilization-associated Ca2+ release in mouse and hamster oocytes [14, 61]. Second, the "global" cytoplasmic immunostaining of type 1, in contrast to the sparse punctuate staining of type 2, makes type 1 the most likely candidate responsible for intracellular Ca2+ release from the cortex to the perinuclear region [18]. Thus, the predominant amount and distribution of type 1 could provide the structural basis for the Ca2+ wave that traverses the cytoplasm at fertilization [2, 5, 6, 62] and initiates mammalian development.
Although injection of the 18A10 antibody failed to inhibit fertilization-associated [;t1Ca2+]i oscillations in bovine oocytes [52], injection of heparin, an IP3R competitive inhibitor [63], completely inhibited fertilization-induced [;t1Ca2+]i oscillations [41]. It remains to be explained whether the absence of antibody inhibition was due to technical difficulties (insufficient 18A10 concentration or time of injection before insemination) and/or contributions to Ca2+ release by other isoforms/Ca2+ channels. In this regard, the RyR has also been identified in mammalian oocytes in studies using RyR agonists [64–66], Western blotting [19], and immunofluorescence [19, 21]. Although RyR may mediate a [;t1Ca2+]i rise in mouse, bovine, and porcine oocytes [64–66], the current evidence indicates that the temporal and spatial pattern of fertilization-associated [;t1Ca2+]i oscillations cannot be accounted for on the basis of this receptor alone. For example, ryanodine does not elicit [;t1Ca2+]i oscillations [64–66]; relatively low amounts of the RyR are detected (requiring > 1000 mouse or 400 bovine oocytes) [19, 20]; and the spatial distribution of the RyR appears to be exclusively cortical [19, 21].
Since degradation of the IP3Rs is associated with a concurrent decrease in sensitivity of IP3-mediated Ca2+ release in other cells [36, 37], this mechanism may also be responsible, in part, for the cessation of or decrease in amplitude in Ca2+ responses that is observed with time after fertilization [3, 50–52]. In fact, fertilized mouse oocytes at the pronuclear stage exhibited a marked reduction in both the amounts of IP3R and IP3-inducible Ca2+ release [17]. Consistent with these findings, our results demonstrate both a fertilization- and adenophostin A-induced, time-dependent decrease in the IP3R-1 in bovine oocytes. Thus, [;t1Ca2+]i oscillations induced by IP3R activation may cause IP3R down-regulation in fertilized oocytes. However, it is important to note that at the pronuclear stage, the stage at which all these studies were carried out, IP3R desensitization may also occur from concomitant decreases in the activities of mitogen-activated protein and histone H1 kinases [67] that may affect the phosphorylation status and function of the IP3R channel.
IP3R down-regulation is thought to be caused primarily by proteasome-mediated proteolysis and possibly by a reduction in IP3R promoter activity [34–36]. In fertilized oocytes, proteolysis may be the most important of the two mechanisms, given the low transcriptional activity observed in early cleavage stages in mammals [68]. It was speculated that Ca2+ release would activate proteases in the vicinity of the IP3R and trigger receptor degradation [36]. However, it has been recently demonstrated that binding of IP3 to its receptor may be required for receptor down-regulation. Binding of IP3 may induce conformational changes making the receptor proteins susceptible to protease degradation and/or to ubiquitin conjugation [55]. The precise mechanism of IP3R down-regulation during fertilization is not known, and our present results do not discriminate between these two possible pathways. However, the finding that injection of adenophostin A, an IP3R agonist, induced IP3R-1 down-regulation similar to that after fertilization strengthens the possibility that the sperm stimulates IP3 production during mammalian fertilization.
Interestingly, although the IP3R-1 appears to be the predominant isoform in bovine microsomes from whole ovaries, the other isoforms are well represented and may actively participate in the regulation of Ca2+ release in ovarian cells. Granulosa cells represent a major ovarian cell type that utilizes Ca2+ release to stimulate steroidogenesis [69] as well as undergoing LH-mediated production of phosphatidylinositol and IP3 [70, 71]. Cumulus cells have IP3Rs [18] and are coupled to the oocyte by gap junctions [72]. Because IP3 can propagate Ca2+ signals between coupled cells [73], it is tempting to speculate that IP3 from cumulus cells may modulate Ca2+ levels in the oocyte. In support of this, the addition of LH to cumulus-oocyte complexes triggered Ca2+ release in cumulus cells, followed by a [;t1Ca2+]i rise in the oocyte [74].
In conclusion, our results demonstrate that bovine oocytes and ovary express all three IP3R isoforms. In ovaries, expression of these receptors suggests a role in granulosa cell signaling that may regulate follicular development as well as oocyte maturation. In oocytes, the predominance of IP3R-1 in the unfertilized mature oocyte, and its regulated demise following sperm penetration, may represent an important developmental mechanism for changing the response to IP3 during very early mammalian development. Finally, despite the predominance of IP3R-1, additional studies will be required to determine whether the various other IP3R isoforms and Ca2+ channels in the oocyte subserve different functions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: R.A. Fissore, Department of Veterinary and Animal Sciences University of Massachusetts, Amherst, MA 01003. FAX: 413 545 6326; rfissore{at}vasci.umass.edu
Accepted: May 17, 1999.
Received: March 18, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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22–24 h postinitiation of maturation (MII), or 16 h later (40 h), or at 16 h postinsemination (40 h Fert). Molecular weight standards (x 10-3 of actual) appear at left. B) The immunoreactive IP3R-1 band was quantified by densitometry and statistical comparison was performed. Quantification of the type-1 isoform was carried out in at least three different blots. The results show a significant reduction of type-1 IP3R as detected in fertilized oocytes in comparison to MII oocytes (P < 0.05). There was also less IP3R-1 in fertilized oocytes than in control-aged oocytes, but the differences were not significant (P > 0.05). Bars with common superscripts are not significantly different