Molecular Human Reproduction, Vol. 5, No. 5, 441-451,
May 1999
© 1999 European Society of Human Reproduction and Embryology
Presence and dynamic redistribution of type I inositol 1,4,5-trisphosphate receptors in human oocytes and embryos during in-vitro maturation, fertilization and early cleavage divisions
1 Infertility Centre, Department of Obstetrics and Gynaecology, University Hospital, De Pintelaan 185, Ghent 9000, and 2 Laboratory of Biochemistry and Molecular Cytology, Faculty of Agriculture, University of Ghent, Ghent, Belgium
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We studied the presence and distribution of the intracellular calcium channel regulating type I inositol 1,4,5-trisphosphate receptors (IP3R) in human immature and mature oocytes, pronuclear zygotes and cleaved embryos using a specific antibody. Two approaches were used: (i) fluorescence immunocytochemistry using a confocal laser scanning microscope (CLSM) and (ii) Western blotting. With confocal microscopy, the receptors were found in the oocytes, fertilized zygotes as well as cleaved embryos at all stages studied. The pattern and distribution of the receptor staining in the oocytes changed gradually from a diffuse granular patchy one at the germinal vesicle (GV) stage to a reticular and predominantly peripheral one through the metaphase I and metaphase II (MII) stages. After fertilization, the distribution changed gradually to both, peripheral and central in the zygotes and early 2–4-cell embryos and predominantly perinuclear in the 6–8-cell embryos. Furthermore, an overall increase in the staining intensity was observed from GV to MII stage oocytes and from zygotes to 6–8-cell embryos. We also studied the spatial distribution of the receptor in detail by constructing three-dimensional images from the serial optical sections obtained on the CLSM. Peculiar peripheral aggregates of receptor clusters were noted in the MII stage oocytes, zygotes and some blastomeres from early cleaved embryos. Finally, Western blots performed on the extracts of 72 in-vitro matured oocytes and 50 spare cleavage stage embryos showed positive bands at ~260 kDa. These findings coincide with and thus possibly represent the dynamic changes occurring in the cellular Ca2+ release systems through oocyte maturation, fertilization and early embryogenesis. Thus, type I IP3R are likely to play a role during these stages of early development in the human.
calcium channels/embryogenesis/immunohistochemistry/inositol trisphosphate/oocytes
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Introduction |
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Repetitive oscillations in the intracellular calcium concentration, [Ca2+]i, are known to control a number of different cellular activities including metabolism, cell cycle progression and differentiation (Whitaker and Patel, 1990
; Berridge, 1993). In mammalian oocytes, spontaneously occurring Ca2+ oscillations mark the acquisition of meiotic competence and possibly also play a role in driving the final stages of oocyte maturation (Carroll and Swann, 1992; Homa et al., 1993; Carroll et al., 1994). The mechanisms responsible for the Ca2+ release evolve further during oocyte maturation so that a mature oocyte is capable of mounting an oscillatory Ca2+ response typically seen at fertilization (Fujiwara et al., 1993; Miyazaki et al., 1993; Mehlmann and Kline, 1994; Herbert et al., 1997). The Ca2+ oscillations continue to occur also during embryogenesis (Sousa et al., 1996a; Stacheki and Armant, 1996a) and the spatiotemporal nature of these oscillations may be critical in regulating embryo development (Ozil, 1990; Stachecki and Armant, 1996a,b; Bos-Mikich et al., 1997). Due to this vital role played by [Ca2+]i oscillations during oocyte maturation, fertilization and embryogenesis, their molecular mechanisms assume a great importance.
Two types of channels are known to mediate the intracellular Ca2+ release, namely the inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) operated channels (Berridge, 1993; Coronado et al., 1994). The IP3R are known to play a central role in triggering the Ca2+ release at fertilization in a number of different non-human species (Miyazaki et al., 1992; Fissore et al., 1995). The RyR have been demonstrated in oocytes from some species but their level of expression is comparatively much lower than that of the IP3R and the contribution of RyR to Ca2+ release during oocyte maturation and fertilization is as yet speculative (Ayabe et al., 1995; He et al., 1997; Machaty et al., 1997; Yue et al., 1998).
In human oocytes, [Ca2+]i oscillations are found to occur during both fertilization and embryogenesis (Taylor et al., 1993; Tesarik et al., 1994; Sousa et al., 1996a). Also, the Ca2+ signalling mechanisms of the human oocyte are known to evolve during the final stages of maturation (Herbert et al., 1997). However, the molecular mechanisms responsible for this phenomenon remain elusive. One possible mechanism may be related to the development of IP3R-mediated Ca2+ release systems as seen in the mature oocytes of golden hamsters and mice (Fujiwara et al., 1993; Shiraishi et al., 1995; Mehlman et al., 1996). However, direct proof about the presence or distribution of the IP3R in the human oocytes is lacking and the current information is based mainly on the results of experiments evaluating the [Ca2+]i response of human oocytes and embryos exposed to various agonists/antagonists (Herbert et al., 1995, 1997; Sousa et al., 1996a,b; 1997). In fact, most of the information on the presence, distribution and role of the InsP3R in oocytes is based on studies in experimental and farm animals (Miyazaki et al., 1992; Kume et al., 1993 Kume et al., 1997; Mehlman et al., 1996; He et al, 1997; Yue et al., 1998). Similarly, information about the presence and role of IP3R in mammalian embryos is sparse. Therefore, our study was aimed at obtaining a direct information about the presence and distribution of the IP3R in the human oocytes and embryos through meiotic maturation, fertilization and early cleavage divisions. We used two approaches, namely immunocytochemical localization with a confocal laser microscope, aided with three-dimensional image reconstruction software and Western blotting. A polyclonal antibody specific for the type I IP3R was employed for both approaches (Mehlmann et al., 1996). A dynamic redistribution of the IP3R was observed through the stages of oocyte maturation, fertilization and early embryonic cleavage divisions.
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Materials and methods |
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Source of oocytes and embryos
The project involving the in-vitro maturation and in-vitro fertilization (IVF) of spare germinal vesicle (GV) stage oocytes from patients was sanctioned by the institutional ethical review committee. Oocytes obtained from patients undergoing intracytoplasmic sperm injection (ICSI) cycles were examined for their maturation status. Those oocytes with a GV that were unused clinically and were donated for research by consenting patients were used for the study. The metaphase I (MI) and metaphase II (MII) oocytes were obtained after culturing these GV oocytes in a maturation medium as described below. Also, zygotes bearing more than two pronuclei (>2 PN) after both clinical IVF and ICSI and cleaved embryos unused for transfer or cryopreservation were donated for this study by consenting patients. Some 2 PN zygotes and cleaved embryos were obtained after ICSI on the in-vitro matured oocytes (Goud et al., 1998a
). The number of oocytes and embryos used and their sources are displayed in Table I. For Western blotting, pooled lysates from 50 embryos and 72 in-vitro matured MII oocytes were used.
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In-vitro maturation
The spare GV stage oocytes were either fixed immediately or transferred to a maturation medium supplemented with gonadotrophins, oestradiol and epidermal growth factor (Goud et al., 1998a
) for 12–14 h or 30–36 h to obtain MI or MII stage oocytes respectively. The oocytes were closely examined under an inverted microscope equipped with Hoffman contrast to confirm the maturation status prior to any further processing. Oocytes with gross abnormalities in the cytoplasm were eliminated from the study.
Intracytoplasmic sperm injection
ICSI was carried out on in-vitro matured oocytes with donor spermatozoa (Goud et al., 1998a). The resultant pronuclear zygotes were fixed at 18–20 h after ICSI using a procedure described below. Some of the 2PN zygotes were allowed to cleave by culturing for another 24–36 h to obtain cleaved embryos.
Positive and negative controls
Oviductal MII stage oocytes were obtained from 6–8-week old female Syrian golden hamsters (IFFA Credo, Brussels, Belgium) in which ovulation had been stimulated using a previously described method (Goud et al., 1998b). A total of 17 MII stage hamster oocytes having normal morphology were used as positive (n = 7) and negative (n = 10) controls. Crude lysates were prepared from rat cerebella of adult male rats using a previously published method (Mehlmann et al., 1996). Five in-vitro matured MII stage oocytes, four pronuclear zygotes (two with 2PN and two with 3PN) and three cleavage stage embryos were used as negative controls (Table I
).
Fluorescence immunocytochemistry
All the reagents were obtained from Sigma, (St Louis, MO, USA) unless specified otherwise. The oocytes and embryos were treated with acid Tyrode's solution to remove the zona, rinsed in phosphate-buffered saline (PBS) and attached to a poly-L-lysine-coated coverslip. Fixation was performed in a freshly prepared 4% paraformaldehyde solution in PBS containing 0.1% (w/v) polyvinyl alcohol (PVA) for 45 min followed by permeabilization in PBS containing 1% bovine serum albumin (BSA) and 0.05% Triton X-100. The cells were treated overnight at 4°C in a 3% blocking solution and incubated for 1 h with a primary antibody [affinity purified, polyclonal antibody (C-19) generated in rabbit against a synthetic peptide comprising the terminal 19 amino acids of the rat type I InsP3R, 1:500 (kind courtesy of Dr. Barbara Ehrlich)] (Mehlmann et al., 1996). After three 5 min washes in the permeabilizing solution, the samples were incubated with the secondary antibody [fluorescent isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin (Ig), 1:500, 1 h]. The samples were washed again three times in the permeabilizing solution and 20 µg/ml propidium iodide (PI) was included in the last wash before mounting the samples in Vectashield (Vector Laboratories, NTL, Brussels, Belgium). The positive controls were treated in the same way as the test samples and negative controls were treated only with the secondary antibody. Edges of the coverslips were sealed with nail polish and the specimens were examined under a Bio-Rad 1024 UV confocal laser microscope and the images were processed using Lasersharp software version 3.0 (Bio-Rad, Eeke, Belgium).
Western blotting
For Western blotting, zona-free oocytes and embryos were lysed in groups of 2–10 in 2–5 µl of lysis buffer, the constituents of which were as described before (Mehlmann et al., 1996) and frozen at –70°C. The lysates from 72 oocytes and 50 embryos were thawed, pooled and reduced to a volume of 5 µl. The crude extract of rat cerebellum was used as a positive control. The samples were run separately on a one-dimensional 5% sodium dodecyl sulphate (SDS) polyacrylamide gel for electrophoretic separation of proteins (Laemmli, 1970). The protein concentrations were predetermined by Lowry's method using a kit (kit for microdetermination of total protein, Sigma Diagnostics). The total protein concentrations used to load in each of the lanes were as follows: Lane 1A (rat cerebellar crude lysate corresponding to 6 µg), lane 2A (lysate from 50 embryos corresponding to 17 µg), lane 1B (rat cerebellar lysate corresponding to 2 µg) and lane 2B (lysates from 72 oocytes corresponding to 21.6 µg). After the run, the proteins from the gel were transferred onto a nitrocellulose membrane (Hybond, Amersham, Ghent, Belgium), subjected to 5% blocking solution overnight at 4°C and incubated with the primary (C-19, 1:7500, 1 h) followed by the secondary (horseradish peroxidase conjugated donkey anti rabbit Ig, Amersham, 1:1000, 1 h) antibody. Finally, the membranes were treated with a reagent from the enhanced chemiluminiscence kit (ECL, Amersham) prior to a 15 sec-10 min exposure to an X-ray film.
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Germinal vesicle stage oocytes
The GV was centrally located in five oocytes, while in the other seven, the location of the GV was more or less peripheral. All the 12 GV stage oocytes showed the presence of the green FITC signal within the oocyte. The spatial resolution of the receptor was aided due to the possibility of examining optical sections passing through several cortical, subcortical and equatorial planes of the oocyte. The pattern of the staining was patchy and the distribution was diffuse. The large areas or patches of the receptor were found mainly in the central ooplasm and the periphery of the oocytes was relatively less abundant in the receptor concentration. The perinuclear zone showed the presence of the receptor but it was not particularly more prominent than that in the rest of the oocyte (Figure 1A–E
). With slight differences in intensity, all the oocytes showed similar findings. The GV was well demarcated as a region free of the FITC signal. The chromatin within the GV was visualized as a red fluorescent signal of PI (Figure 1A, F).
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Metaphase I stage oocytes
All six MI oocytes were positive for the FITC fluorescence. The pattern of receptor distribution changed from the diffuse, patchy type into a more reticular type (Figure 2A–F
). However, in the centre of the MI oocytes, there were certain zones with patches of diffuse staining. Furthermore, the receptor was now also noted to be concentrated in the cortex forming a peripheral rim (Figure 2A, B and F). Three-dimensional reconstructed images did not reveal any new findings other than those seen in the optical sections. The chromosome metaphase plate of the first meiosis was well visualized in all the oocytes due to the red fluorescent signal of PI.
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Metaphase II stage oocytes
All 10 MII stage oocytes exhibited the reticular pattern of the FITC signal. However, the distribution of the receptor was different from that in the GV and the MI stage oocytes. Also, the overall the intensity of fluorescence was higher in all the MII oocytes in comparison with the GV and MI oocytes. From the optical sections going through various planes of the oocyte, a conspicuously higher concentration of the receptor was observed in the cortex than in any other region in the oocyte (Figure 3D–F
). However, a small region around the metaphase chromosome plate was free of the receptor (Figure 3A–C). A hallmark of the MII oocytes was the formation of prominent clusters of the receptor in the cortex. These clusters measured ~2–5 µm in size, as opposed to the much less conspicuous and relatively smaller clusters seen in the GV and the MI stage oocytes (Figure 3D–F, Figure 4A). These receptor clusters also showed a tendency to form localized aggregates as seen from the 3-D reconstructed images (Figure 3G–I). In two oocytes, polar bodies were retained through the process of fixation, which on examination were found to be rich in the receptor.
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Pronuclear and metaphase zygotes
Both the two pronuclear as well as tripronuclear zygotes showed the presence of the receptor. In comparison with the MII stage oocytes, the receptor distribution was somewhat diffuse but the receptor presence was still slightly higher in the periphery than at the centre. However, the prominent receptor clusters noted in the MII stage oocytes seemed to be replaced by smaller, not so prominent clusters in the periphery of the zygotes. The pronuclei were clearly visible even with single channel recordings as they were free of receptor staining (Figure 4C
). The receptor presence was also noted around the pronuclei, but the fluorescence intensity was not higher than that in the cortex. There was no difference in the distribution and intensity of the receptor signal among the 2PN and 3PN zygotes. In one zygote, the fixation was delayed up to 24 h post-ICSI. This zygote had already entered the mitotic metaphase as the pronuclei had broken down and the chromosomes were arranged on a metaphase plate in the centre of the zygote. In this case, the receptor presence, distribution, as well as intensity were similar to that seen in the pronuclear zygotes and the region around the mitotic chromosomes was free of the receptor signal (Figure 4D).
Cleavage stage embryos
Of the 12 embryos subjected to immunocytochemistry, four were at the 2–3-cell stage, five were at the 4–6 cell stage and three were at the 7–8 cell stage. All these embryos were virtually free of fragmentation and blastomere size asynchrony was obsereved only in two 3-cell embryos. Six out of the 12 embryos had at least one multinucleated blastomere. However, the pattern and distribution of the receptor signal did not differ between blastomeres with a single or multiple nuclei. Blastomeres from all the embryos stained positive for the green FITC fluorescence indicating the presence of the receptor. The regions of nuclei were free of the receptor signal. Two types of distribution patterns were seen in the blastomeres. In the first type, seen in all the blastomeres from the 2–4-cell embryos, the receptor was present in the periphery as well as the centre and also in the rest of the inner cytoplasm in a diffuse pattern, as also confirmed on the reconstructed 3-D images (Figure 5A–C and G–I). There were particular sites in the embryos with a higher receptor density. These sites consisted of the blastomere cleavage furrows and the regions of contact between the blastomeres. A higher receptor density also appeared at specific sites in the blastomere periphery (Figure 5H). These receptor rich sites were located more or less symmetrically on the opposite poles in the blastomeres so that an imaginary plane passing through these sites could divide the blastomere into approximately two halves. Therefore, these sites may probably be serving as the sites of origin of the future cleavage divisions. The receptor presence was also evident in the polar bodies. In the second type, as seen from the blastomeres of the 4–6 and the 6–8-cell embryos, the receptor presence was prominently noted around the nuclei as well as in the inner cytoplasm (Figure 5D–F). The pattern was diffuse granular although some receptor clusters could be noted around the blastomere nuclei (Figure 5D–F). The receptor concentration was clearly more in the perinuclear zone than the periphery. Also, the overall intensity of staining was clearly more in the embryos at these developmental stages than the zygotes and the 2–3-cell embryos. Thus, the IP3R appeared to become more prominent around the blastomere nuclei following the third and the following embryonic cleavage divisions. Similar findings were also noted in the cleaved embryos derived from 3PN zygotes after IVF as well as ICSI.
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Controls
All the seven hamster oocytes processed in a similar manner (positive controls) exhibited the fluorescent receptor signal in a pattern that was similar to the MII stage human oocytes. The pattern was reticular, and the distribution was predominantly cortical, with the formation of clusters. The chromosome metaphase plates of the positive controls were well visualized and the region around the metaphase chromosomes was free of fluorescence in all the positive control oocytes. All 10 hamster oocytes, five human MII stage oocytes (Figure 4B
), three cleavage stage embryos (Figure 4E) and two 3PN and the two 2PN zygotes that were treated only with the secondary and not the primary antibody, were negative for fluorescence.
Western blotting
A positive band corresponding to ~ 260 KDa appeared on the blots in both the lanes corresponding to human oocytes as well as embryos. A similar band was also noted in the lane for positive controls (Figure 6).
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Changes in the Ca2+ release systems during oocyte maturation
Oocyte maturation involves a co-ordinated process of nuclear and cytosolic events, regarded as nuclear and cytoplasmic maturation. The nuclear events include chromatin condensation and spindle formation whereas cytoplasmic maturation includes organelle redistribution, activation of cell cycle factors and maturation of the Ca2+ release systems. The latter is particularly important as it confers the oocyte with the capability to undergo a repetitive oscillatory Ca2+ release at fertilization which is critical for the completion of meiosis and activation of embryo development (Miyazaki et al., 1986
; Whitaker and Swann, 1993; Taylor et al., 1993; Tesarik et al., 1994). The components of the Ca2+ release machinery of the oocyte are the Ca2+ stores and the channels which regulate the release of Ca2+ through these stores. Smooth endoplasmic reticuli (SER) are the major stores of Ca2+ in the oocytes (Eisen and Reynolds, 1984; Clapham, 1995) and type I IP3R are the most abundant Ca2+ channel regulators in Xenopus as well as mammalian oocytes (Parys et al., 1992; Kume et al., 1993; 1997a; Mehlman et al., 1996; He et al., 1997). Both the SER as well as IP3Rs are known to undergo changes that supposedly bestow the activation and fertilization capability on the oocytes of various non-human species (Mehlman et al., 1995Mehlman et al., 1996; Shiraishi et al., 1995; He et al., 1997; Kume et al., 1997). Therefore, changes in the IP3Rs during oocyte maturation could possibly reflect the changes occurring in the oocyte Ca2+ release system even in the humans. Hence, we studied the pattern and distribution of the IP3R through oocyte maturation.
Inositol 1,4,5-trisphosphate receptors in GV stage oocytes
In the GV stage oocytes, the findings of diffuse patchy distribution throughout the oocyte are similar to those in the Xenopus, mouse and hamster oocytes (Kume et al., 1993Kume et al., 1997; Shiraishi et al., 1995; Mehlmann et al., 1996). The receptor in these studies was found to be predominantly in patches in the inner cytoplasm and also around the nucleus. In the GV stage oocytes of mice, the IP3R are possibly involved in operating the spontaneous Ca2+ oscillations occurring during maturation (Carroll and Swann, 1992; Carroll et al., 1994). Although similar Ca2+ oscillations have not been demonstrated in higher mammals, heparin, which is an antagonist of the IP3R, and also chelators of Ca2+ can inhibit oocyte maturation (Homa, 1991; Kaufman and Homa, 1993). This inhibition is most probably related to the inhibition of the activation of cell cycle factors that should occur during maturation. In fact, a direct relation between IP3R activation and increased activity of H-1 kinase and mitogen-activated protein (MAP) kinase was recently found in bovine oocytes during in-vitro maturation (He et al., 1997). Hence, the IP3R detected in the human GV stage oocytes may also be involved in operating the Ca2+ release, thereby contributing to the process of maturation by the activation of H-1 kinase and MAP kinases.
We also found that the receptor was diffusely distributed around but not in the interior of the GV, as also found in immature hamster oocytes. The IP3R around the GV may be actively involved in operating the Ca2+ release within the nucleoplasm during oocyte maturation (Shiraishi et al. 1995; Pesty et al., 1998). Thus, the type I IP3R are present in human GV oocytes and their distribution is similar to that described for the other non-human species. Possibly, the IP3R may also play an important role during oocyte maturation in humans just as in the other non-human species studied earlier.
IP3R distribution in MI and MII oocytes
We found that IP3R underwent a remarkable change in their distribution through the final stages of meiotic maturation. At the MII stage, the oocytes showed a peripheral dominance of receptor distribution. This change occurred gradually through the MI stage and formation of prominent receptor clusters in the cortex was typically seen in the MII oocytes. The dimensions and the peculiar appearance of the receptor clusters in the cortex, and the reticular morphology of the receptor in the inner cytoplasm are similar to those of SER in the MII stage human oocytes (our unpublished data). SER is known to undergo a redistribution and change in structure during maturation of starfish, sea urchin, mouse, and hamster oocytes (Terasaki and Jaffe, 1991; Jaffe and Terasaki, 1993; Mehlmann et al., 1995; Shiraishi et al., 1995). Therefore, the changes of peripheral migration and cortical clustering of IP3R in our study most likely reflect the changes in the distribution and ultrastructure of the SER. Finally, in a mature unfertilized oocyte both the IP3R and the SER clusters are preferentially located in the cortex, possibly close to the site of sperm–oocyte membrane contact (Shiraishi et al., 1995; Mehlmann et al., 1996). These changes could contribute to the increase in the oocyte sensitivity to oscillogens that occurs during oocyte maturation (Fujiwara et al., 1993; Herbert et al., 1997).
We also found a phenomenon of formation of localized aggregates of the receptor in the oocyte cortex. This phenomenon was noticed in all the MII stage oocytes although the extent of the aggregation varied to some extent. Visualization of this phenomenon was aided by examining the 3-D images reconstructed from serial optical sections on the CLSM to create images representing the whole oocyte. The localized aggregation of the cortical IP3R may possibly be related to polarization of the IP3R in mature oocytes. Although the issue of polarity in human oocytes and embryos is yet unresolved (Edwards and Beard, 1997), certain proteins are now known to be expressed in human oocytes and embryos in a polarized manner (Antczak and Van Blerkom, 1997). However, this phenomenon needs a further close evaluation, possibly by studying the expression of certain other proteins. The approach of 3-D image reconstruction with a CLSM may be helpful in this regard.
One other difference between the GV and MII stage oocytes was a remarkable increase in the intensity of fluorescence observed in the latter. This phenomenon occurred despite the use of the same concentrations of the primary and secondary antibodies and the same settings of the microscope during excitation of the cells with the laser. However, this is an overall impression and confirmation of this finding on quantitative protein studies was beyond our scope. One of the reasons for this was the unavailability of a much larger number of oocytes that are needed for such studies. But quantitative studies on the IP3R have been performed on the oocytes from other species and a relative increase in the receptor number was found in the mouse oocytes during maturation (Mehlmann et al., 1996). This increase in number and peripheral migration of the IP3R through maturation may explain the increase in the oocyte sensitivity to oscillogens. Thus, the same explanation may also be applicable to the human oocytes.
Type I IP3R in human zygotes and cleavage stage embryos
The InsP3R were found also in the pronuclear zygotes and the zygote metaphase. The pattern seen in zygotes was different from that noted in the MII oocytes. These findings are in agreement with the redistribution of the SER from periphery to centre reported in human zygotes (Sousa et al., 1997). Recently, sinusoidal Ca2+ rises were observed during the first embryonic cell cycle in human zygotes (Sousa et al., 1996a). Similar Ca2+ rises have also been reported during the pronuclear breakdown in the zygotes of rabbits and mice (Tombes et al., 1992; Fissore and Robl, 1993; Kono et al., 1996). Based on the theory that Ca2+ released from the ryanodine sensitive stores exerts an inhibitory influence on the IP3 sensitive stores, Sousa et al. (1996a) proposed that Ca2+ oscillations occurring in the human zygote are mediated via IP3-sensitive stores. In this context, our finding of the presence of type I IP3R in human zygotes is more direct and supports the possibility that these receptors are involved in mediating the Ca2+ release in the human zygotes.
In the cleavage stage embryos, we found a further gradual change in the receptor pattern, distribution as well as intensity of the staining. In the 2–3 cell embryos, the receptor distribution was diffuse and there were particular receptor rich peripheral sites in the blastomeres. These sites were mainly within the cleavage furrows, at the regions of blastomere to blastomere contact and also at peripheral sites where future cleavage could possibly occur. Calcium waves that can be inhibited by heparin have been reported to occur along the cleavage furrows in Xenopus embryos (Muto et al., 1996). Therefore the concentration of the IP3R within the cleavage furrows and at sites of possible future cleavages in the human embryos may also serve as sites of beginning of similar Ca2+ waves.
IP3-sensitive receptor presence at the sites of blastomere contact is of particular interest. Blastomeres from human embryos are known to be connected to one another by gap junctional complexes (Hardy et al., 1996). Although the relation between the gap junctions and Ca2+ oscillations or IP3R in the blastomeres is as yet unknown, propagating increases in intracellular free Ca2+ in neighbouring cells are generally believed to be a form of a mechanism of cell–cell communication (Sanderson, 1996).
In most blastomeres from the 4-cell stage embryos, the receptors were mainly located around the nuclei and the pattern of the staining was more diffuse granular than reticular. Also, the intensity of the IP3R staining was much higher in the blastomeres from these embryos in comparison to the pronuclear stage zygotes and the 2–3-cell embryos. The finding of the perinuclear IP3R in the blastomeres is similar to the finding in the early cleavage stage embryos of Xenopus (Kume et al., 1997b) and suggests a close correlation between the IP3/Ca2+ signalling system and progression of cell cycle in these embryos (Kubota et al., 1993; Ciapa et al., 1994). Similarly, it might even explain the Ca2+ transients occurring in the cleavage stage mammalian embryos and their consequent acceleration of the preimplantation embryo development (Stachecki and Armant, 1996a,b; Bos-Mikich et al., 1997; Rout et al., 1997).
One possible explanation of the prominent presence of the IP3R around the nuclei of the blastomeres is that it may have been newly synthesized by the embryo. Therefore, the expression of the newly synthesized IP3R may coincide with embryonic genome activation. This theory explains the differences in the distribution as well as characteristics of the receptor in the oocytes and embryos. An increase in the expression of the IP3R has been reported to occur during development in Xenopus embryos (Kume et al., 1997b). A similar increase of the expression may also occur during the early cleavage divisions of the human embryo. Thus, prominently expressed IP3Rs in the cleavage stage human embryos may be involved in operating Ca2+ release in the embryos.
Western blots on oocytes and embryos
Finally, we identified the IP3R in the lysates from both human oocytes and embryos as a protein of ~260 kDa. This finding is similar to that reported in the mouse and bovine oocytes (Mehlmann et al., 1996; He et al., 1997). However, the number of cells required to obtain a blot was different in mouse (500) and bovine (20) oocytes. Also, there were minor differences in terms of the size of the protein, which may possibly be attributed to post-translational modifications or interspecies differences. The size of the receptor protein in the embryos was similar to that of the oocytes. To the best of our knowledge, this is the first report on the immunocytochemical and Western blot detection of the IP3R in human oocytes and embryos.
Our findings indicate that in the human, the IP3R are dynamically redistributed throughout the final stages of oocyte maturation, fertilization and early embryonic cleavage divisions. The findings in the maturing oocytes are concordant with the findings reported in the other species. But the information obtained in the embryos is new even for mammalian embryos. The receptor has been extensively studied in the Xenopus, where it plays a role in cellular differentiation during advanced stages of embryo development (Kume et al., 1997b,c). Confirmation of a similar role in the human embryo is still a distant task, complicated by the unavailability and ethical restrictions. Our findings do indicate that the type I IP3R in human embryos undergo a change in distribution during the early cleavage divisions. But the functional significance of these changes can be proved only by further evaluation. Nevertheless, demonstration of the presence and distribution of the type I IP3R in human oocytes, zygotes and cleaved embryos is pivotal and encourages us to probe further into this area.
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Acknowledgments |
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The authors thank Dr Barbara Ehrlich, University of Connecticut Health Center, Farmington, CT, USA, for providing the primary antibody used for this study. The study was supported by a grant awarded by the University of Ghent, Belgium.
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Notes |
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3 To whom correspondence should be addressed
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References |
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Submitted on October 29, 1998; accepted on February 15, 1999.
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