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a Departments of Obstetrics/Gynecology and Anatomy/Cell Biology, Tufts University School of Medicine and New England Medical Center Hospital, Boston, Massachusetts 02111
b Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003
c Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242
d Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115-5701
e Laboratorium voor Fysiologie, Campus Gasthuisberg O/N, K. U. Leuven, B-3000 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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In mature mammalian eggs at metaphase II (MII) of meiosis, 1,4,5-trisphosphate (IP3) is primarily responsible for the Ca2+ release and Ca2+-dependent events associated with fertilization, while the ryanodine receptor is believed to play a lesser role [5, 15–26]. In contrast to MII eggs, fully grown germinal vesicle (GV)-stage oocytes exhibit reduced calcium release when stimulated with IP3, sperm, or Ca2+ [27–31]. In GV-stage oocytes, these agonists also result in little or no cortical granule exocytosis [32–34], perhaps in part because of the reduction in Ca2+ released. One hypothesis to account for these deficiencies is that the calcium storage and release systems undergo important developmental changes during meiotic maturation, resulting in full competence to stimulate egg activation at MII [35–37].
Two maturation-associated changes that may account for the development of activation competence are an increase in the amount of the IP3R in mammalian oocytes [36, 38] and the reorganization of putative calcium stores. The fluorescent lipophilic dye DiI (which spreads into ER membranes) when injected into MII mouse oocytes becomes localized to cortical foci; however, labeled foci are reportedly absent in the deeper cytoplasm [35]. In contrast, GV-stage oocytes possess few cortical DiI-stained foci whereas larger dye accumulations are present deeper in the cytoplasm [35]. Immunolocalization [36] of the IP3R type 1 isoform was greater in the cortex of the mouse MII egg than in the GV oocyte, but little staining was observed in the interior cytoplasm at either stage. During hamster oocyte maturation, DiI and IP3R staining patterns also change, but in a manner differing from that observed in maturing mouse oocytes ([37]; see Discussion).
Other mechanisms may be responsible, at least in part, for changes in the sensitivity of the maturing egg to IP3 and the egg's ability to release Ca2+. In most somatic cells, more than one IP3R isoform is expressed and the relative ratios vary among cell types [39–42]. Also, the distribution of the various isoforms can be heterogeneous in some cells [43, 44]. Interestingly, the IP3 affinity of the three isoforms varies greatly, with the type 2 isoform (IP3R-2) having the highest [45, 46] and the IP3R-3 the lowest affinity [46]. Because the roles such isoforms may play in egg development and activation have not been explored, we have investigated the following questions: Are multiple IP3R isoforms present in oocytes and eggs? Is the maturation-associated acquisition of increased sensitivity to IP3 due to a change in the amount or distribution of the high-affinity type 2 isoform? Does the localization of a particular IP3R isoform provide a structural basis for the internal Ca2+ wave demonstrated by others? Do different isoforms have different spatial distributions?
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Swiss random-bred CF-1 mice (Charles River Breeding Labs., Wilmington, MA), 8–12 wk of age, were injected with 5 IU of eCG (Sigma Chemical Co., St. Louis, MO), and GV-stage oocytes were collected after 44–48 h by ovarian follicle puncture. Only oocytes ~80 µm in diameter and having surrounding normal condensed cumulus were used. After collection from oviducts, MII eggs with cumulus were prepared for microscopic studies, whereas cumulus-free eggs (0.1% hyaluronidase treated) were used for Western blot analysis. For each GV or MII collection, at least 4–8 females were employed.
Antibodies
The unique C-terminal region of the IP3R has been useful for generating specific antibodies in previous studies of eggs [13, 14, 36, 47, 48]. In this study, we used the isoform-specific rabbit polyclonal antibodies Rbt04 and Rbt02 raised against IP3R-1 and -2, respectively. They are directed against the C-terminal amino acids of the mouse IP3R-1 and IP3R-2 (type 1: LGHPPHMNVNPQQPA, type 2: SNTPHVNHHMPPH). The antibody against IP3R-3 is an IgG2a monoclonal antibody (Transduction Laboratories, Lexington, KY) directed against the N-terminal region of the human IP3R-3 (amino acids 22–230). These various isoform-specific antibodies were characterized previously and shown not to mutually cross-react [40, 46, 49].
Membrane Preparations and Western Blots
Microsomes from mouse ovaries were prepared as described previously [13, 38]. In brief, tissue samples were homogenized with a glass-polytetrafluoroethylene homogenizer in 0.3 M sucrose, 1 mM EDTA, 0.1 mM PMSF, 2 µg/ml pepstatin, 2 µg/ml leupeptin, 1 mM 2-mercaptoethanol, 50 mM Tris-HCl (pH 8.0) and centrifuged at 2000 x g for 10 min. 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 GV oocytes or MII eggs were made after the zona pellucida was removed by acid Tyrode treatment [36] for < 1 min at 20°C. Immediately upon zona dissolution in a concave well, the well was flooded with an excess of Earle's balanced salt solution (25 mM Hepes buffer, pH 7.3, 0.1% polyvinyl pyrrolidone), and the eggs were removed promptly and transferred into sample buffer [50].
Microsomal preparations or crude mouse oocyte lysates in sample buffer were boiled for 4 min and loaded onto 4% SDS-polyacrylamide gels. The separated proteins were then transferred onto nitrocellulose membranes (Micron Separation Inc., Westboro, MA) using a Mini Trans Blot Cell (Bio-Rad, Hercules, CA). The membranes were first washed in PBS-0.05% Tween 20 (PBS-T) and then blocked in the same solution supplemented with 6% nonfat dry milk (blocking solution). 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 washes in PBS-T, membranes were developed using the ECL (enhanced chemiluminescence) detection system according to manufacturer instructions (Amersham, Arlington Heights, IL). Anti-IP3R-1, -2, and -3 antibodies were used at 1:4000, 1:100–1:1000, and 1:500, respectively. Negative controls were obtained by using preimmune serum, omission of the primary antibody, or preincubation of the Rbt02 antiserum for 1 h with 5 mg/ml of the C-terminal peptide (Genemed Biotechnologies Inc., South San Francisco, CA) before dilution to 1:100–1:1,000 for probing the membrane. Positive controls were obtained by using cell lines with a known pattern of IP3R-isoform expression (data not shown). IP3R-1 and -3 are expressed in A7r5 rat smooth muscle cells, C3H10T1/2 mouse fibroblasts, and CHO-K1 chinese hamster ovary cells. In addition, the latter two cell lines express significant amounts of IP3R-2. Total microsomal fractions from these cell lines were therefore prepared as previously described for immunoblot positive controls [46]. The quantification of immunoreactive IP3R was carried out using Adobe Photoshop (Mountain View, CA). The intensity of the IP3R band in GV oocytes or from nonstimulated ovaries was used as the baseline value and arbitrarily assigned the value of 1. The intensity of the IP3R bands of MII oocytes or from eCG-stimulated ovaries was calculated relative to 1 and statistically compared, and the molecular masses were estimated by using prestained, broad range, SDS-PAGE molecular weight markers (Bio-Rad).
Immunofluorescence
Ovulated mouse cumulus masses were rapidly washed in two changes of PBS and pipetted into a droplet of OCT compound (Miles Inc., Kankakee, IL). The droplet was snap-frozen by rapid immersion in isopentane cooled in liquid nitrogen and stored at -80°C. Ovaries from mature mice, trimmed of adnexa, were also snap-frozen as described above. Frozen 5-µm sections from eggs and ovaries were mounted on glass slides and fixed for 15 min in one of the following solutions: a) acetone (-20°C), b) methanol (-20°C), or c) 1% paraformaldehyde (4°C) in PBS (as indicated in Results). After fixation in acetone or methanol, the specimens were allowed to dry; paraformaldehyde-fixed specimens were washed in two changes of PBS (5 min each). Specimens were blocked in 3% BSA in PBS.
Primary antibodies against IP3R-1 and IP3R-2 were used at a 1:100 dilution in 1% BSA in PBS; monoclonal antibody to IP3R-3 was employed at a dilution of 1:10–1:1000. After incubation for 1 h, the specimens were washed three times in PBS (5 min each) and incubated for 1 h in secondary antibody conjugated to fluorescein isothiocyanate (goat anti-rabbit, 1:16 dilution for IP3R-1 and -2; goat anti-mouse, 1:20–1:100 for IP3R-3). Specimens were washed two times in PBS (15 min each) and stained in 4,6-diamidino-2-phenyl indole (DAPI; Sigma) for 5 min. After washing two times in PBS (5 min each), specimens were mounted in Vectashield (Vector Labs., Burlingame, CA), covered with a glass coverslip, and viewed with epi-illumination. Specimens were photographed with Kodak (Eastman Kodak, Rochester, NY) T-MAX film with an ASA of 800. Negative control experiments were performed with specimens incubated with preimmune serum to either isoform, or in the absence of primary antibodies, or with a mixture of IP3R-2 antibody and C-terminal peptide prepared similarly as described above for Western blots, diluted 1:100. In each case, labeled secondary antibody was subsequently added. Observations of specimens subjected to IP3R-1, -2, and -3 were made from over 100 sectioned ovulated oocytes and approximately 30 sectioned ovaries.
Electron Microscopy (EM)
Cumulus-egg masses and ovaries were fixed in 2.5% glutaraldehyde, 0.5% paraformaldehyde in 0.05 M phosphate buffer. They were then osmicated (0.5%) for 60 min at 4°C, washed in buffer and water, en bloc stained in 0.5% uranyl acetate for 15 min at 4°C, dehydrated, and embedded in Epon-Araldite (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were stained briefly with uranyl acetate followed by lead [51]. In an effort to characterize cytoplasmic membranes, 10 MII and 10 GV-stage oocytes were thin-sectioned, resulting in about 100 electron micrographs per stage (5 x 7 inches in size; x17 000). These were analyzed for membrane systems in the vicinity of the cell cortex [51].
Statistical Analysis
Statistical comparisons of the intensity of IP3R bands were performed using one-way ANOVA followed by Student's t-test if significance was found (p < 0.05) in the ANOVA analysis. The Student's t-test was used to compare mean distances between IP3R-2-stained foci.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During meiotic maturation, mammalian oocytes develop an increased sensitivity to IP3. To determine whether this is associated with a change in the amount or ratio of IP3R isoforms, lysates from equal numbers (n = 50) of mouse GV oocytes or MII eggs were evaluated by immunoblotting with isoform-specific antibodies. Indicative of the IP3R in mammalian oocytes [19, 36, 38], a band of approximately 260 kDa was found in both the GV and MII stages with type 1 antibodies (Fig. 1). In zona-free specimens, the density of the IP3R-1 increased about 1.9-fold during meiotic maturation (Fig. 1A). The IP3R-1 was detected in as few as 20 MII eggs. As a negative control, the primary antiserum to the IP3R-1 was replaced by preimmune serum and the corresponding IP3R-1 band was not observed (data not shown).
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In order to determine whether there is an increase in the IP3R-1 in ovarian tissue during follicular growth in response to gonadotropin, mice were treated with 5 IU of eCG, and ovaries were harvested 44–48 h later. Equine CG alone was used in order to maximize the number of follicle cells. When equal amounts of total microsomal protein were compared from samples from unstimulated (control) and eCG-treated mice, it was found that gonadotropin stimulation did not result in a significant increase in the amount of the IP3R-1 (Fig. 1B).
IP3R-2 appeared to be less abundant than IP3R-1, because it was not detected in samples of ~1000 pooled MII mouse eggs. In contrast, the IP3R-2 was easily detectable in ovarian microsomal samples (Fig. 2A), although it was less abundant and had a slightly smaller molecular mass (250 kDa) than the IP3R-1 (not shown), as previously reported for other mouse cell types [46]. In addition, the amount of IP3R-2 increased ~50% after gonadotropin stimulation (Fig. 2, B and C; p < 0.05). The gonadotropin sensitivity of follicle cells, combined with their strong immunofluorescence staining in granulosa cells (see below), suggests that the IP3R-2 is present primarily in follicle cells. In a negative control experiment, the IP3R-2 band was not detected when the primary antibody was preincubated with the C-terminal peptide. In additional control studies (data not shown), the presence or absence of IP3R-2 was confirmed in cell lines known to be positive or negative for IP3R-2 (see Materials and Methods and [46]).
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The IP3R-3 was easily detected in mouse ovarian microsomes and as a faint band in a sample of 1100 mouse MII eggs (Fig. 3). It appeared as a band at approximately its predicted size of 248 kDa. The lower molecular mass band (about 177 kDa) present in ovaries was also previously detected in other tissues and believed to represent a proteolytic fragment [40]. GV-stage oocytes were not examined for IP3R-2 and -3 because of the difficulty in obtaining such a large number of oocytes completely free from granulosa cell contamination, coupled with the difficulty in detecting types 2 and 3 bands in MII eggs.
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Immunofluorescence of IP3R Isoforms
The spatial localization of calcium release channels has profound implications for the initiation and spread of the fertilization-associated Ca2+ wave. Because the IP3R-1 has not been reported in the mouse egg interior and because the distribution of other IP3R isoforms is not known in these cells, an immunofluorescence localization study was undertaken with isoform-specific antibodies. The IP3R-1 antibodies had a reticular staining pattern throughout the egg (Fig. 4, a–c). This staining pattern was similar in acetone- and methanol-fixed preparations. Paraformaldehyde-fixed specimens were inconsistent in their reactivity to the antibodies employed here. The descriptions that follow are from methanol-fixed specimens. Cumulus cells also had a bright cytoplasmic fluorescence with antibodies to the IP3R-1 (Fig. 4, a and c).
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With antibodies to IP3R-1, follicular GV-stage oocytes demonstrated fluorescence throughout the cytoplasm (Fig. 4d). Differences in the intensity and pattern of staining were not apparent in non-antral and early to late antral follicles. Follicle cells had diffuse and punctate staining, identified by staining outside of the nucleus (compare Fig. 4, d and d').
In contrast to the staining pattern seen with IP3R-1 antibodies, bright punctate deposits of cytoplasmic fluorescence were observed in ovulated MII eggs incubated with IP3R-2 antibodies (Fig. 5, a and a''; Fig. 5a', chromatin staining). The larger stained deposits varied in size with a range of 1–3 µm in diameter. In the region 0–5 µm from the cell surface, these deposits had an average distance apart of about 3 µm in both GV and MII stages (Table 1). The deposits were not strictly localized to any specific region within the egg; both cortical and subcortical regions contained the stained bodies. Cumulus cells were observed to have stained with the IP3R-2 antibody in a pattern much like that described for the IP3R-1 antibody; i.e., staining was prevalent throughout the cytoplasm (Fig. 5, a and a''). Starting with secondary follicles and up to and including large antral follicles, the periphery of the GV itself reacted with the IP3R-2 antibody (Fig. 5, b and c). In antral follicles, oocyte staining was present in discreet areas scattered throughout the cytoplasm and was reminiscent of that in ovulated eggs (Fig. 5c; Fig. 5c', chromatin staining).
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Negative control preimmune sera for both IP3R-1 and-2 antibodies, or specimens treated with only secondary antibody, provided identical results; i.e., virtually no fluorescence was detectable in ovulated eggs and ovarian oocytes (see Materials and Methods) (Figs. 4e, 5d; Figs. 4e', 5d', chromatin staining). Preparations immunodepleted with the IP3R-2 C-terminal-specific polypeptide sequence did not demonstrate reactivity to sectioned eggs and ovaries. With a spectrum of antibody dilutions (1:10–1:1000), it was not possible to conclusively demonstrate specific localization of IP3R-3 relative to control antibodies in oocytes and cumulus cells.
EM
In order to identify structures that would be isomorphic with those staining with IP3R antibodies (above) and to determine the distribution of the ER, several electron microscopic approaches were undertaken. Conventional EM demonstrated discrete small (0.1- to 0.3-µm diameter) membrane-bound vesicles that formed a larger aggregate in both GV-stage oocytes and MII eggs (large arrows, Fig. 6). These vesicles appeared more spherical than tubular in shape, and some were interconnected. An aggregate of vesicles was round to oval in shape and usually 1–2 µm in diameter. Aggregates were not observed in all sections (50–100 nm in thickness), indicating that they are discrete foci. In the cortex of GV oocytes and MII eggs, aggregates were spaced a mean distance of 21 µm and 3–4 µm apart, respectively (Table 1).
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Within aggregates, individual small vesicles (~0.1 µm diameter) had a somewhat electron-dense interior and were significantly smaller and less dense than cortical granules (Fig. 6). The vesicle aggregates were absent from the cortex underlying the meiotic spindle (data not shown), which is known to be an actin-rich region with a reduced number of organelles [52, 53]. In contrast, vesicles surrounding the aggregates were larger, were irregularly shaped, and had a more lucent interior with membrane-associated internal projections of greater density (Fig. 6). As previously observed, some of these large vesicles were within a tenth of a micron of the egg plasma membrane [51]. Specific labeling with anti-IP3R primary vs. preimmune antibodies at the ultrastructural level was unsuccessful, likely due to problems associated with paraformaldehyde fixation as also noted in fluorescence studies.
Quantification of Vesicle Aggregates
In order to determine whether there were maturation-associated changes in cortical aggregate number and similarities with the stained IP3R-2 foci, aggregates were quantified. More vesicle aggregates were observed at the ultrastructural level in MII eggs than in GV-stage oocytes (Tables 2 and 3). Also, a larger number of aggregates were observed in the cortex than in the subcortex (2–5 µm from the cell surface) for both stages (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This is the first report documenting the expression and localization of multiple IP3R isoforms in mammalian oocytes at the protein level and leads us to propose plausible functional roles for these isoforms. One or more cortical isoforms may play a role in initiating Ca2+ release, perhaps in conjunction with IP3 and a factor from sperm [54–57]. Because the IP3R-1 and ER are present throughout the cytoplasm, they are likely to provide a continuous network for the spread of the Ca2+ wave in MII-stage mouse eggs [58] (e.g., IP3-induced Ca2+ release sensitizes adjacent IP3-sensitive stores that in turn perpetuate the Ca2+ wave [5]). Due to the nearly complete inhibition of Ca2+ release by the IP3R-1-specific antibody 18A10 (see below), the other IP3R isoforms may not release significant amounts of Ca2+ relative to the total released during egg activation, but they may play an indirect role. For example, localizations of the IP3R-2 may represent spatially disconnected relay stations with a higher affinity for IP3 than the IP3R-1 [45, 46]. These foci may facilitate the propagation of the Ca2+ wave through Ca2+-mediated local sensitization of the IP3R-1 [47, 59–65]. It is also possible that, in the immediate microdomain of sperm-egg interaction, the IP3R-2 may play a role in initiating Ca2+ release that may not be detected if the global release via the IP3R-1 is inhibited by 18A10. An alternate function for the IP3R-2 and -3, which is also compatible with the 18A10 inhibition results, would be as a component of a heterotetrameric receptor [66–68].
The Western, fluorescence, and 18A10 results suggest that the IP3R-1 is the most prevalent and functionally important isoform in vertebrate eggs. On Western blots, IP3R-1 was easily detected in 20 or 50 eggs, whereas 1100 eggs were required for a relatively faint band for IP3R-3. In addition, in Xenopus oocytes, the available evidence points to an exclusive or nearly exclusive presence of IP3R-1 (reviewed in [59]). Consistent with these findings are previous studies with the monoclonal function-blocking antibody 18A10 to the C-terminal region of the IP3R-1 [69]. Injection of 18A10 into mouse eggs blocks IP3-induced Ca2+ release to near basal levels at 88 µg/ml [36] and inhibits Ca2+-dependent egg activation events [20] occurring after fertilization or IP3 injection [70]. In hamster eggs, 18A10 (~300 µg/ml) blocks all detectable Ca2+ elevations activated by sperm or by amounts of injected IP3 sufficient to elicit a fertilization-like Ca2+ rise [5, 19].
Although evidence was presented at the mRNA level for the expression of the three IP3R isoforms [71], and both Western and immunofluorescence studies were undertaken with well-characterized antibodies to all three isoforms, the IP3R-2 Western and the IP3R-3 immunofluorescence was not identified by Western and immunofluorescence analysis, respectively. First, this is very likely due to the findings strongly suggesting that the vast majority of the receptor is IP3R-1 (i.e., only 20 eggs were needed for a strong IP3R-1 Western signal vs. a weak signal from 1100 eggs for IP3R-3; and complete cytoplasmic fluorescence was found for IP3R-1 vs. only small punctate regions for IP3R-2). Second, it is well known that some antibodies, especially at the limit of detection, work well for either Western blot or fluorescence, but not necessarily both methods.
The ultrastructural results extend previous fluorescence studies by directly visualizing cortical membrane structures fitting reported characteristics of the ER, the proposed site of Ca2+ stores involved in the Ca2+ wave at fertilization. The size, spacing, and morphology of aggregates of smaller vesicles by EM are similar to the focal cortical localizations of the ER stained by DiI [35]. In addition, in both cases, these structures are not observed in the cortex over the meiotic spindle. The small vesicles within an aggregate appear to be interconnected. There may be indirect continuity with other aggregates or subcortical ER by way of vesicle trafficking, as evidenced by the various stages of apparent vesicle fusion and vesicular structures between aggregates. It is also possible that there is direct continuity not visualized by EM, as previously indicated by 1–2-µm foci that appeared to be interconnected by fine irregular bridges in the DiI study [35]. It is of note that no other cortical membranes were observed in our study that could account for the specific characteristics of the DiI-stained large foci in MII eggs. In hamster oocytes, cortical collections of tubular profiles of ER are observed, although these appear to undergo a change to a less focal arrangement during maturation to the MII stage [37].
In MII eggs, the shape, diameter (1–2 µm), and spacing (3–4 µm) of the vesicle aggregates by EM are remarkably similar to the shape, diameter (1–3 µm), and spacing (3.3 µm) of the foci stained by the IP3R-2 antibody. This congruence strongly suggests but does not prove that the vesicle aggregates are a site of IP3R-2 localization. In addition, the high electron density and granularity of the contents of small vesicles within aggregates, by analogy with the density of the mitochondrial matrix (a Ca2+ sequestration site with electron-dense granules), may reflect a high concentration of protein and Ca2+ (Fig. 6).
This study supports previous investigations showing that the maturation-associated increase in sensitivity to IP3 is correlated with an increase in the mass of the receptor and an increase in cortical membrane foci in the cytoplasm [35, 36]. Similar to our previous finding of a 3- to 4-fold increase in individual vesicle profiles in the cortex during maturation [51], a 5-fold increase was observed in vesicle aggregates. However, an increase in the number of IP3R-2 antibody-staining foci was not observed in terms of the distance between foci. It is possible that not all vesicle aggregates identified by EM are stained by IP3R-2 antibodies and that some aggregates contain other receptor isoforms. For example, because the maturation-associated increase in vesicle aggregates is accompanied by a 90% increase in IP3R-1, the additional vesicle aggregates may represent new localizations of IP3R-1.
This study also demonstrated that the egg's companion granulosa cells possess IP3R-1 and -2, and possibly type 3 as well. These cells employ IP3 as a signaling molecule in response to protein hormones [72]. LH stimulates not only cAMP production but also an increase in phosphatidylinositol [73] and IP3 [74] via an LH receptor-mediated activation of phospholipase C [75]. Consistent with the ability of FSH to increase the number of granulosa cells and LH receptors [72], our findings are that gonadotropin priming via eCG significantly increases the amount of IP3R-2 but not IP3R-1 in ovarian microsomal fractions. This result suggests that the type 2 receptor may play an important role in responding to LH after FSH priming. The IP3R in the granulosa cells immediately surrounding the oocyte allows us to speculate that IP3 changes, mediated by gonadotropin receptor signaling, may regulate cytosolic Ca2+-mediated events that in turn influence the maturing oocyte through follicle cell-oocyte gap junctions [76].
In conclusion, this work provides evidence that heterogeneity at the level of IP3R-isoform expression exists in mouse oocytes and in granulosa cells. It is suggested that this heterogeneity may have a functional role in the initiation and propagation of intercellular Ca2+ signals that regulate oocyte-follicle cell interactions and intracellular signaling that initiates mammalian development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Tom Ducibella, Dept. Ob/Gyn, Tufts University, School of Medicine, 136 Harrison Ave., Boston, MA 02111. FAX: 617 636 2917; tducibel{at}opal.tufts.edu
Accepted: August 18, 1998.
Received: May 1, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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