Molecular Human Reproduction, Vol. 8, No. 10, 912-918,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Inositol 1,4,5-trisphosphate receptor function in human oocytes: calcium responses and oocyte activation-related phenomena induced by photolytic release of InsP3 are blocked by a specific antibody to the type I receptor*
1 Infertility Centre, Department of Obstetrics and Gynaecology, 2 Department of Physiology and Pathophysiology, Faculty of Medicine, Ghent University, 3 Laboratory of Biochemistry and Molecular Cytology, Faculty of Agriculture, Ghent University, Ghent, Belgium, 4 Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Wako, Japan, 5 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology and 6 Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA
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Abstract |
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Type I inositol 1,4,5-trisphosphate-sensitive receptors (InsP3R) are expressed in human oocytes and may be involved in operating the Ca2+ release triggered by the fertilizing sperm. This study examines the contribution of type I InsP3R in operating Ca2+ release in human oocytes secondary to InsP3 itself, using a specific function-blocking antibody in conjunction with photolytic release of microinjected InsP3. Intracellular Ca2+ responses were assessed in oocytes microinjected with only caged InsP3 in experiment set A, while in experiment sets B and C, sibling oocytes were injected with caged InsP3 and the blocking antibody or a corresponding volume of medium, prior to flash photolysis. In experiment set C, certain fertilization-related phenomena (cortical granule exocytosis and chromatin configurations) were assessed using optical sections and three-dimensional image reconstructions obtained from a confocal laser scanning microscope. In experiment set A, photolytic release of InsP3 triggered a Ca2+ response (increase from
100 to 220 nmol/l followed by an exponential recovery, n = 8) and a wave in the oocytes that spread from the stimulation point to the opposite pole. In set B, photolytic InsP3 release generated Ca2+ responses in control oocytes (n = 9), but not in the antibody-injected oocytes (n = 7). In set C, cortical granule exocytosis and anaphase chromosome configurations were noted in the control oocytes after flash photolysis (n = 6). These changes were completely absent in antibody injected oocytes as their cortical granules were intact and the chromosomes were in metaphase. These oocytes had also lacked Ca2+ responses as in set B (n = 5). This study demonstrates the functional presence of type I InsP3R-operated Ca2+ channels in human oocytes and further suggests an active role of InsP3 in triggering the Ca2+ rise and secondary activation phenomena at fertilization. Ca2+ transients/caged IP3/fertilization/flash photolysis/inositol 1,4,5-trisphosphate receptors
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Introduction |
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A wavelike increase of intracellular Ca2+ followed by oscillations is the hallmark of mammalian fertilization (Miyazaki et al., 1993
). This event initiates the process of oocyte activation and related phenomena, which mark the onset of embryo development. Furthermore, the spatiotemporal characteristics of the Ca2+ release at fertilization are particularly vital for embryo development, possibly because they influence cellular processes and gene expression (Ozil, 1990; Bos-Mikich et al., 1997; Rout et al., 1997; Dolmetsch et al., 1998). Understanding the mechanisms determining the occurrence and spatiotemporal dynamics of Ca2+ response in the oocyte is therefore crucial.
Two types of receptor-operated channels are thought to mediate Ca2+ release in the oocytes, namely receptors that are sensitive to ryanodine (RyR) and those that are sensitive to the second messenger inositol 1,4,5-trisphosphate (InsP3R) (Berridge, 1993; Coronado et al., 1994). Of the two, the type I InsP3R are most prominently expressed in oocytes and hence, are likely to be the main Ca2+ channel-regulating receptors releasing Ca2+ from intracellular stores at fertilization (Miyazaki et al., 1992b; Fujiwara et al., 1993; Yue et al., 1995; Mehlmann et al., 1996; He et al., 1997). The functional role of type I InsP3R in operating Ca2+ release in oocytes at fertilization has been demonstrated in the golden hamster and mouse using a specific blocking antibody, and in frogs using a polyclonal antibody against a C-19 peptide of rat type I InsP3R (Miyazaki et al., 1992b; Xu et al., 1994; Runft et al., 1999). In human oocytes, the type I InsP3R are prominently expressed in oocytes, zygotes and embryos and are dynamically redistributed through maturation, fertilization and early embryogenesis (Goud et al., 1999). Nonetheless, there is no direct proof as to the functional role of the InsP3R in human oocytes and the information available in the literature is indirectly derived from studies on Ca2+ responses after exposure to various agonists/antagonists (Herbert et al., 1995, 1997; Sousa et al., 1996a,b).
Therefore, as an initial step towards exploring the role of the InsP3R in human oocytes, we investigated the Ca2+ responses to InsP3 in oocytes injected with or without a specific function-blocking antibody to the type I InsP3R (18A10 mAb) (Miyazaki et al., 1992a,b). A novel method of photolytic release of injected InsP3 was employed and the responses were studied by assessing the calcium release as well as early phenomena related to oocyte activation such as cortical granule release and exit from the meiotic metaphase. Photolytic release of the injected caged InsP3 resulted in Ca2+ release, which spread globally in the oocyte in a wavelike manner. This phenomenon was followed by cortical granule release as well as metaphase–anaphase transition. On the other hand, blockade of the type I receptor in the antibody-injected oocytes showed both complete absence of the calcium responses as well as maintenance of the chromosomes in the meiotic metaphase without any cortical granule release.
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Materials and methods |
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Study design
In experiment set A, oocytes were loaded with a Ca2+-sensitive dye and injected with caged InsP3 at the desired concentration and Ca2+ responses were recorded during and after flash photolysis. In experiment set B, Ca2+ measurements were performed in a similar way in sibling oocytes that were either sham-injected (controls) or injected with a blocking antibody (18A10mAb), then injected with caged InsP3 and subjected to flash photolysis. In experiment set C, sibling oocytes were treated as in set B and were subsequently subjected to fluorescent staining for cortical granules and chromatin after flash photolysis of injected caged InsP3. Assessment for cortical granules and chromatin was performed under a confocal laser scanning microscope (CLSM).
Source of oocytes
The project was approved by the Institutional Ethical Review Board of Ghent University. Accordingly, donated spare germinal vesicle (GV) and metaphase I (MI) stage oocytes from patients undergoing ICSI were subjected to in-vitro maturation (Goud et al., 1998). The oocytes that matured to the metaphase (MII) stage at the end of 30–32 h of culture without gross size, shape and cytoplasmic abnormalities were selected for the study. As the experiment sets B and C involved a comparison of two groups, care was taken to assign sibling oocytes with similar features into groups that would be compared subsequently.
Fluo-3 loading and microinjection of caged InsP3 and antibody
The mean diameter of each oocyte was measured using an ocular grid in three perpendicular axes at x400 magnification, and the approximate volume of each oocyte was deduced from this, assuming a spherical shape. This helped to estimate the amount of caged InsP3 and/or antibody to be injected into each individual oocyte in order to attain a desired concentration. The oocytes in either group were then incubated for 1 h in human tubal fluid medium (HTF; Irvine Scientific, Irvine, CA, USA) containing 10 µmol/l of the Ca2+-sensitive cell permeable dye fluo-3-AM (Molecular Probes, Eugene, OR, USA). The dye loading was continued after further steps involving the microinjection of caged InsP3 and/or 18A10 mAb.
The caged InsP3 (Calbiochem, San Diego, CA, USA) was dissolved at 5 mmol/l in an intracellular buffer containing 134 mmol/l KCl, 7.8 mmol/l NaCl, 7.8 mmol/l Na2HPO4, and 1.4 mmol/l KH2PO4, pH 7.2. About 10–15 pl (11.8 ± 0.1 pl) of caged InsP3 was microinjected into each oocyte to be studied, using a glass ICSI micropipette with a shaft of 10 µm internal diameter that was constant for 
500 µm length. The injection technique was similar to ICSI (Goud et al., 1997) and a predetermined amount of caged InsP3 was injected by calculating the length of the column and consequently the volume within the micropipette segment. Leakage of caged InsP3 from the micropipette during the procedure was avoided by protecting the column with 10 µm columns (0.5–1.0 pl) of mineral oil on either side transiently before microinjection. The first mineral oil column was expelled just prior to the penetration of the oolemma, and microinjection was stopped and the micropipette withdrawn as the second column reached the tip of the injection pipette. This allowed a fair amount of accuracy in microinjecting the desired quantity of caged InsP3 in the oocytes. In both the experimental sets, the approximate intracellular concentration of caged InsP3 was 50 µmol/l (Table I).
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In experiment sets B and C, oocytes were pre-injected with the 18A10 antibody (estimated final concentration
70 µg/ml, Table I
) using the same technique described above. The control oocytes in both these experiment sets received injection of the corresponding volume of the vehicle (phosphate-buffered saline, 0.4% bovine serum albumin). The oocytes were allowed to recover over the next 30 min before injection of the caged InsP3. Fluo-3 loading then continued after caged InsP3 microinjection until the time when the oocytes were subjected to flash photolysis.
Flash photolysis
The oocytes were transferred to the warm stage (37°C) of a fluorescence microscope equipped to perform Ca2+ imaging and flash photolysis. The flash photolysis light was generated with a band-pass filtered mercury-arc bulb focused to a small spot. The UV light exposure was controlled with a diaphragm and an exposure time of 250–500 ms was used. The Ca2+ responses were recorded on a PC or VCR and were subsequently analysed.
Staining for cortical granules and chromatin
In experiment set C, oocytes and their sibling controls were subjected to flash photolysis of caged InsP3 using the same protocol used for experiment set B, but were allowed to recover for 30–45 min in HTF at 37°C. They were then subjected to zona removal with acid Tyrode’s solution (Sigma-Aldrich NV/SA, Bornem, Belgium), attached to poly-L-lysine-coated coverslips, fixed for 1h in freshly prepared 4% paraformaldehyde, blocked overnight in a 3% blocking solution (Sigma), stained with rhodamine-conjugated lens culinaris agglutinin (LCA; Vector Laboratories, Burlingame, CA, USA) (Ghetler et al., 1998) and mounted in Vectashield containing 4,6-diamidino-2-phenylindole (DAPI; Vector). The oocytes along with the suitable negative and positive controls were subjected to confocal microscopy (Bio-Rad 1024 UV). Image processing and three-dimensional (3-D) image reconstruction were done with Imaris and Huygens system-2 (SVI, Hilversum, The Netherlands).
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Oocyte Ca2+ responses following flash photolysis of caged InsP3 (experiment set A)
In experiment set A, eight MII stage oocytes were obtained from three patients and were successfully injected with caged InsP3. All oocytes survived the injection procedure. The estimated volume of caged InsP3 within the oocytes was 11.8 ± 0.1 pl (mean ± SE). Estimates of oocyte volume, concentrations of caged InsP3 and 18A10 mAb, as well as the fluorescence changes (
F/F%) are presented and illustrated in Table I and Figure 1 respectively. Overall, flash photolysis of caged InsP3 (250 ms duration) triggered a Ca2+ response in all the oocytes, and the relative fluorescence change corresponded roughly to a Ca2+ rise from 100 to 220 nmol/l. The Ca2+ response was followed by an exponential recovery characterized by a time constant of 34 ± 9 s (Figure 1A,B). In seven oocytes, application of the flash at the oocyte periphery demonstrated a Ca2+ wave spreading from the stimulation point to the opposite side of the cell at a speed of 19.3 ± 1.5 µm/s (Figure 1B–D). Oocyte Ca2+ changes did not propagate to the polar bodies.
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Flash photolysis of caged InsP3 in antibody-injected and control oocytes (experiment sets B and C)
In experiment set B, 22 sibling oocytes from nine patients were assigned for injection with 18A10 mAb (test subgroup, n = 11) or sham injection (control subgroup, n = 11) in addition to caged InsP3 injection. In the control group, nine of the 11 oocytes survived microinjection of caged InsP3 and medium. The estimated volume of caged InsP3 in control oocytes was 12.9 ± 0.2 pl, similar to that for the test subgroup (12.9 ± 0.3 pl). Also, the estimated oocyte volumes and concentrations of InsP3 were similar in the test and control groups (Table I
). The volumes of the injected 18A10 antibody and medium (sham injection) were also similar (data not shown). Flash photolysis of caged InsP3 (500 ms duration) resulted in relative fluorescence increase in all nine control oocytes indicating a Ca2+ response (Table I, Figure 1A–D), corresponding to a Ca2+ increase from a baseline value of 100 to 300 nmol/l. In the test subgroup, seven of 11 oocytes survived microinjection of caged InsP3 and 18A10 antibody. Flash photolysis of the caged InsP3 resulted in no fluorescence increase in any of the oocytes injected with the 18A10 antibody, indicating a complete lack of a Ca2+ response in all of the seven surviving oocytes.
Spatiotemporal characteristics of the Ca2+ response
In experiment set B, the control and antibody-injected oocytes were monitored for fluorescence increase for 10 min subsequent to photolytic release of caged InsP3. In the control group, a single Ca2+ rise was noted in six out of nine oocytes and no subsequent Ca2+ rises were noted. However, in the other three control oocytes, we observed at least three spontaneous Ca2+ rises during the 10 min after flash photolysis of caged InsP3 (Figure 1E). Such secondary spontaneous Ca2+ rises were never observed in the oocytes injected with the 18A10 antibody.
Cortical granule and chromatin status in oocytes after flash photolysis (experiment set C)
In experiment set C, test and control oocytes were injected with 18A10 antibody (n = 6) or were sham-injected with medium (n = 6), before injection of caged InsP3 as in experiment set B. They were subjected to flash photolysis and subsequently processed for cortical granule and chromatin staining. In the control group, all six oocytes survived the injection procedure while in the test group five out of six oocytes survived the microinjection procedure. Flash photolysis resulted in a Ca2+ response in the control group, similar to that in experiment set B (Table I). Also, the antibody-injected oocytes exhibited a complete lack of Ca2+ response as in experiment set B.
Analysis of the individual 2–5 µm optical sections of the oocytes as well as the 3-D reconstructions revealed cortical granule exocytosis. This was evident from the extrusion of cortical granule contents in the perivitelline space (Figure 2A,B). In some control oocytes, the cortical granule contents formed aggregates just outside the oolemma and these were clearly noted in 3-D reconstructed images (Figure 2D). On the other hand, the cortical granules in the 18A10 mAb-injected oocytes were found to be intact (Figure 2C,E). The chromosomes in all the control oocytes were found to be in anaphase (n = 6, Figure 2A,B inset). However, the chromosomes in the antibody-injected oocytes were found to be in metaphase configuration (Figure 2C). Thus, the control oocytes showed both cortical granule exocytosis as well as metaphase–anaphase transition, whereas the antibody-injected oocytes lacked both of these phenomena.
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Discussion |
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A rise of intracellular calcium in the oocytes is known to play a key role during fertilization and possibly also during oocyte maturation (Miyazaki et al., 1993
; Homa, 1995; Mattioli et al., 1998; Pesty et al., 1998). The Ca2+ release during fertilization in mammals is peculiar as it takes the form of repetitive oscillations. This is in contrast to the single rise in Ca2+ that occurs in other species such as the amphibians (Swann and Parrington, 1999). Furthermore, spatiotemporal characteristics of the Ca2+ oscillations are thought to directly influence embryonic development (Ozil, 1990; Bos-Mikich et al., 1997). Therefore, elucidation of the intracellular Ca2+ release mechanisms is vital in mammalian oocytes.
Oscillatory release of Ca2+ has also been noted during human fertilization (Taylor et al., 1993; Tesarik et al., 1994; Tesarik and Sousa, 1995). However, the mechanisms involved in this process are as yet unclear. A sperm-specific factor has been suggested to initiate Ca2+ oscillations in oocytes, but the exact nature of this factor in not known (Parrington et al., 1996; Sette et al., 1997; Dale et al., 1999). According to an alternate theory proposed to explain the Ca2+ release at fertilization, an interaction between a sperm surface ligand and putative oocyte surface receptors activates Gq-protein mediated mechanisms. This generates the secondary messenger, InsP3, via the activation of phospholipase C (Schultz and Kopf, 1995). However, even this theory is not entirely accepted as a Gq protein antibody fails to block Ca2+ release at fertilization (Williams et al., 1998).
Recently, a combination of the above-mentioned two theories was proposed (Swann and Parrington, 1999). Accordingly, phospholipase C activity in the sperm factor is thought to be involved in generation of InsP3 in the oocytes (Jones et al., 1998). Therefore, involvement of InsP3 and its receptors may still be important in the generation of Ca2+ transients in oocytes.
InsP3R are the major Ca2+ channel operating receptors in human as in other mammalian oocytes (Shiraishi et al., 1995; Mehlmann et al., 1996; He et al., 1997; Macháty et al., 1997; Goud et al., 1999). Moreover, between the ryanodine-sensitive receptors (RyR) and the InsP3R, the latter seem to be the main contributors of Ca2+ release at fertilization. This is supported by the prominent expression of InsP3R compared to the very low or absent expression of the RyR seen in various species (Ayabe et al., 1995; Yue et al., 1995; He et al., 1997). Furthermore, InsP3 itself, and its agonists can induce an intracellular Ca2+ rise in human and other mammalian species (Fujiwara et al., 1993; Fissore et al., 1995).
Among the various subtypes of the InsP3R identified so far, the type I receptor is most prominently expressed in oocytes (Parrington et al., 1998; Fissore et al., 1999). In the human oocytes, as in other species, the type I InsP3R undergoes an increase and a dynamic redistribution through maturation (Shiraishi et al., 1995; Mehlmann et al., 1996; He et al., 1997; Goud et al., 1999). Finally, in the MII stage oocyte, the type I InsP3R is expressed in close proximity with the oolemma, which encounters the sperm after zona penetration and also with the cortical granules, which are released at fertilization (Kline, 2000).
We have previously shown that human oocytes express the type I InsP3R as seen from Western blots and immunocytochemistry (Goud et al., 1999). In continuation of this study, we aimed to investigate the functional presence of the type I InsP3R in human oocytes. We preferred the use of InsP3 itself and its specific antibody rather than the various agonists used by others (Sun et al., 1994; Fissore et al., 1995; Sousa et al., 1996a,b). However, InsP3 in its native form is likely to be rapidly metabolized, thus requiring immediate recording of cellular Ca2+ changes. We avoided this problem by using InsP3 in a caged, inactive form, which was injected into oocytes, whereas the active InsP3 was released from the inactive caged compound after exposure to UV light (Callamaras and Parker, 1998; Leybaert and Sanderson, 2001). This method allowed us to monitor the effects of InsP3 immediately after its release into the oocytes. Furthermore, we used the specific function-blocking antibody 18A10, which was previously shown to block InsP3 thimerosal as well as sperm induced Ca2+ oscillations in hamster oocytes (Miyazaki et al., 1992a,b).
In experiment set A, we investigated the Ca2+ responses in human oocytes that were matured in vitro from the GV stage. We found an instantaneous response to photolytic release of InsP3 in the form of a fluorescence change indicating Ca2+ release. This Ca2+ release signal propagated from the stimulation point to the rest of the oocyte in a wavelike manner, as demonstrated by the fluorescence recordings performed at four different points within the oocyte. The peak Ca2+ change associated with this wave was nearly the same throughout the oocyte, indicating that Ca2+ by itself is not the diffusing ion. It is known that the diffusion of Ca2+ ions in the cytoplasm proceeds at a slower rate as compared to InsP3, because Ca2+ is bound to less mobile cytoplasmic Ca2+ binding molecules (Allbritton et al., 1992). The wavelike propagation of Ca2+ changes is therefore more likely the result of the diffusion of InsP3 through the oocyte cytoplasm. Compatible with this conclusion is the fact that the overall Ca2+ wave propagation velocity of 19.3 µm/s is in the order of the root-mean-square velocity which, assuming diffusion in one direction, was calculated to be in the order of 24 µm/s (square root from 2·D/t, with D[InsP3] = 283 µm2/s). The waveform Ca2+ release was subsequently followed by an exponential recovery. Overall, the characteristics of the Ca2+ wave generated in response to photolytically released InsP3 were similar to the initial Ca2+ response occurring at fertilization in human and other mammalian species (Miyazaki et al., 1993; Taylor et al., 1993). However, in experiment set A, no subsequent spontaneous transients occurred in any of the oocytes, which is contrary to the oscillatory Ca2+ release characteristically seen during mammalian fertilization. Oscillatory Ca2+ release in oocytes has also been noted after InsP3 injection. Thus the subsequent Ca2+ responses in oocytes in experiment set A were different compared to earlier reports (Galione et al., 1994; Jones et al., 1998). These differences may be related to the quantity of InsP3 released after the flash, as we used 250 ms exposure time, which may cause a release of InsP3 that is inadequate for mounting secondary responses as the primary response may not reach the threshold (Miyazaki et al., 1993). Hence we increased the UV flash exposure time to 500 ms in our subsequent experiment sets. We found that the initial peak Ca2+ response was higher in experiment sets B and C compared to that in experiment set A (300 versus 220 nmol/l respectively). As increased exposure time increases the relative concentration of photolytically released InsP3, this could lead to a proportionately higher Ca2+ response. Furthermore, increased exposure time also leads to the occurrence of spontaneous Ca2+ transients in some oocytes at a frequency of 1–2/min.
Another possible contributor to differences in the peak primary and the subsequent spontaneous Ca2+ responses could be related to the oocyte’s InsP3 sensitivity and ability to generate spontaneous Ca2+ transients. This may be compromised in oocytes matured in vitro from the GV stage as opposed to being retrieved at the MII or MI stage. In experiment sets B and C, we included oocytes matured from MI stage in addition to those matured from the GV stage. Interestingly, all the oocytes showing secondary spontaneous Ca2+ transients had been retrieved at the MI stage and were subsequently matured in vitro, as compared with the other oocytes which were matured from the GV stage in vitro. These differences in spontaneous Ca2+ transients indicate that these two types of oocytes may differ in terms of their Ca2+ release mechanisms.
In experiment sets B and C, our main objective was to investigate the contribution of type I InsP3R in controlling intracellular Ca2+ release and oocyte activation in oocytes respectively. In both experiment sets B and C, the specific function-blocking antibody to type I InsP3R completely blocked the InsP3-induced Ca2+ release (IICR) in oocytes that were injected with the antibody prior to flash photolysis.
The 18A10 mAb recognizes an epitope close to the proposed Ca2+ channel region in the COOH-terminus of the receptor protein and inhibits IICR in mouse cerebellar microsomes (Furuichi et al., 1989; Nakade et al., 1991). Furthermore, it also blocks the Ca2+ release induced by InsP3 and spermatozoa in hamster and mouse oocytes (Miyazaki et al., 1992b; Xu et al., 1994).
Our findings in human oocytes are similar to those in hamster oocytes with regard to IICR, although the peak Ca2+ levels and the 18A10 mAb concentrations required to block Ca2+ release were relatively lower (300 versus 500–550 nmol/l and 70 versus 165 µg/ml respectively) (Miyazaki et al., 1992b). Nevertheless, these differences can be explained by the difference in the InsP3 injection amount and technique and possibly by an interspecies difference. Miyazaki et al. (1992b) have used an iontophoretic technique, in which the relative amount of InsP3 administered is represented by the magnitude of the square of the current pulse applied. Increasing the magnitude of the pulse led to an increase in the peak Ca2+ rise and also proportionately increased the amount of 18A10 mAb required to block the Ca2+ release completely. Therefore our micromolar concentrations of InsP3 released after photolytic release cannot be directly compared with those of Miyazaki et al. (1992b). Nevertheless, the Ca2+ responses were completely blocked by the 18A10 mAb concentrations of 70 µg/ml in human oocytes in our study. Thus, even presuming that there are no species differences in terms of InsP3 sensitivity between human and hamster oocytes, it is possible that the amount of InsP3 released photolytically in our experiments may be lower than those reported by Miyazaki et al. (1992b).
One small finding in our study was that the oocytes injected with the 18A10 mAb had a slightly higher rate of damage than the sham-injected control oocytes. Although the oocyte numbers were too small to draw conclusive evidence, considering the role of intracellular Ca2+ in repairing the membrane disruption (McNeil and Terasaki, 2001), InsP3 and its receptor may play a role in membrane wound repair.
In experiment set C, in addition to monitoring the Ca2+ responses, we also studied two fertilization-related phenomena, namely cortical granule exocytosis and meiotic progression. Close examination of the optical sections obtained on the CLSM allowed us to evaluate the occurrence of cortical granule (CG) exocytosis as well as the chromatin configurations. All the control oocytes showed signs of CG exocytosis, although there were minor differences in the degree of its occurrence. The CG exocytosis in the control oocytes was so distinct that quantitative studies were deemed unnecessary. On the other hand, the 18A10 mAb-injected oocytes distinctly revealed intact cortical granules. Furthermore, study of the chromatin configurations revealed that in the control oocytes, the chromosome groups separated, indicating a metaphase–anaphase transition. The 18A10 mAb-injected oocytes, however, showed that their chromosomes were distinctly in meiotic metaphase. Thus, these oocyte activation-related phenomena were related to the Ca2+ release that was induced by InsP3 and mediated via the type I InsP3R. These findings are in agreement with the occurrence of CG exocytosis and cell cycle progression examined by others (Whitaker and Patel, 1990; Xu et al., 1994; Macháty et al., 1997).
Thus, photolytic release of InsP3 in MII stage human oocytes resulted in a Ca2+ response, which was blocked by the specific 18A10 mAb. The occurrence and spatiotemporal characteristics of Ca2+ release were dependent on the amount of InsP3 released and also on the source of the oocyte. Blockade of the Ca2+ release in 18A10 mAb-injected oocytes also resulted in the blockade of subsequent fertilization-related phenomena of CG exocytosis and meiotic exit. Collectively, these phenomena indicate an active role played by type I InsP3R and InsP3 in controlling the Ca2+ release in human oocytes and raise the likelihood of InsP3 and type I InsP3R playing an active role during human fertilization.
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Acknowledgements |
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Financial support was provided by the Ghent University, Ghent, Belgium.
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Notes |
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* Presented at the 56th Annual Meeting of the American Society for Reproductive Medicine, San Diego, CA, USA in October 2000.
7 To whom correspondence should be addressed: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 4707 St Antoine Boulevard, Detroit, MI 48201, USA. E-mail: pgoud{at}med.wayne.edu
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Submitted on March 11, 2002; resubmitted on May 23, 2002; accepted on July 31, 2002.
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