1 CReATe (Canadian Reproductive Assisted Technology) Program Inc., Toronto, Ontario, Canada, 2 Department of Anatomy and Cell Biology, University of Toronto, 3 Department of Obstetrics and Gynecology, Sunnybrook and Womens College Health Sciences Center, University of Toronto and 4 Canadian Institutes of Health Research Group in Molecular Biology of Membrane Proteins and the Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada
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Abstract |
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Key words: calcium release receptors/calnexin/calreticulin/calsequestrin/human embryos
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Introduction |
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Regarding intracellular Ca2+ release channels in oocytes, it has been firmly established that in sea urchin oocytes both InsP3R and RyR are involved in Ca2+ changes associated with fertilization (McPherson et al., 1992). In contrast, no RyR have been detected in Xenopus and hamster oocytes, implying that in these species Ca2+ release may be entirely dependent on the function of a single InsP3-sensitive store (Miyazaki et al., 1992
; Parys et al., 1992
; Kume et al., 1993
). In addition, current evidence suggests that both receptor families are present in murine (Ayabe et al., 1995
; Mehlmann et al., 1996
; Pesty et al., 1998
; Fissore et al., 1999
), bovine (Yue et al., 1995
, 1998
), porcine (Machaty et al., 1997
) and human oocytes as well as in human embryos (Tesarik et al., 1995
; Sousa et al., 1996a
,b
; Goud et al., 1999
). Despite the recognition of a role for Ca2+ in oocyte maturation, fertilization and early embryo development (Jones, 1998
; Kline, 2000
; Carroll, 2001
), little is known about essential ER/SR proteins that are involved in the Ca2+ signalling system of human oocytes and embryos.
In addition to controlling Ca2+ homeostasis, the ER/SR membrane plays a critical role during protein synthesis, and folding, post-translational protein modification and degradation (Ellgaard et al., 1999). Many ER lumenal proteins, including calreticulin, function as Ca2+-binding/storage molecules and as molecular chaperones. Calnexin, a protein similar to calreticulin, is an integral ER membrane Ca2+-binding chaperone, which shares many substrates with calreticulin (Helenius et al., 1997
; Ellgaard et al., 1999
). The molecular mechanisms responsible for the ER-dependent control of Ca2+ homeostasis and the role of Ca2+-binding chaperones in human oocyte and embryos are unknown.
In this study, the expression and localization of ER/SR Ca2+-storage and -release proteins as well as Ca2+-binding chaperones in human oocytes and embryos during meiotic maturation, fertilization and early embryogenesis were examined.
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Materials and methods |
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Immunofluorescence staining
Oocytes and embryos were briefly treated with acid Tyrodes solution (pH 2; Sigma Chemical Co., St Louis, MO, USA) to remove the zona pellucida and adhering cumulus and sperm cells. These zona-free cells were rinsed in culture medium (IVC-One; In Vitro Care Inc., San Diego, CA, USA) and incubated for 30 min at 37°C in 5% CO2. The cells were handled in phosphate-buffered saline (PBS; Somagen Diagnostic Inc., Edmonton, Alberta, Canada) supplemented with 1% human serum albumin (HSA; Somagen) to prevent adhesion of the zona-free cells to the culture dishes. PBS containing 1% HSA was also used as a working solution for all antibodies. Between each step of the procedure, specimens were washed three times in fresh PBS (5 min each). For immunolocalization of calreticulin, calsequestrin and calnexin, oocytes and embryos were fixed for 15 min in freshly prepared 3.7% paraformaldehyde (Fisher, Markham, Ontario, Canada) in PBS, washed and permeabilized for 2 min in 1% Triton X-100 (Fisher) in PBS containing 100 mmol/l PIPES (Fisher), 1 mmol/l EGTA (Sigma) and 4% polyethylene glycol 8000 (w/v) (Sigma). For immunolocalization of InsP3R-2 and RyRs, the cells were fixed in 100% methanol (Fisher) for 5 min at 20°C. While methanol treatment produced significantly better staining, shrinkage of the cells and damage to the cell membrane was occasionally observed. The specimens were then processed for labelling with the appropriate primary and secondary antibodies. Incubations with antibodies were carried out for 4560 min, either at room temperature (for calreticulin, calsequestrin and calnexin) or at 37°C (for InsP3R-2 and RyRs). To localize calreticulin, calsequestrin and calnexin, goat polyclonal anti-calreticulin (diluted 1:50), affinity-purified rabbit anti-calsequestrin (diluted 1:100) and rabbit anti-calnexin (a generous gift from Dr D.B.Williams, University of Toronto; diluted 1:30) antibodies were used respectively. The secondary antibodies were as follows: for calreticulin detection, dichloro-triazinylaminofluorescein (DTAF) conjugated donkey anti-goat IgG (H+L) antibody, diluted 1:30 (Bio/Can Scientific, Mississauga, Ontario, Canada), and for detection of calsequestrin and calnexin Texas Red-conjugated F(ab") donkey anti-rabbit IgG (H+L) antibody, diluted 1:30 (Jackson ImmunoResearch Laboratories, Inc., Bio/Can Scientific). The calreticulin, calsequestrin and calnexin antibodies have been extensively characterized (Michalak et al., 1991; Milner et al., 1991
; Opas et al., 1991
; Nakamura et al., 2000
). The expression of InsP3R-2 and RyRs was examined with affinity-purified goat polyclonal IgG antibodies: InsP3R-2 (C-20) and RyR (C-18) respectively (both antibodies diluted 1:10; Santa Cruz Biotechnology, Inc., CA, USA). The C-20 InsP3R antibodies are specific for mouse, rat and human InsP3R-2 type receptors, while the C-18 RyR antibodies recognize RyRs from brain, skeletal and cardiac muscle from mouse, rat and human, that may cross-react with three subtypes of RyR (1,2,3respectively referred to hereafter as RyRs). The secondary antibodies were the same as those used for calreticulin detection above. For simultaneous detection of DNA and InsP3R-2, cells were double-stained with the DNA dye 7-aminoactinomycin D (Sigma) at a concentration of 10 mmol/l for 30 min at 37°C. After immunostaining and final washes in PBS, the cells were mounted onto glass slides in Vinol 205S (St Lawrence Chemical, Toronto, Canada) that contained 0.25% 1,4-diazabicyclo-(2,2,2)-octane (Polysciences, Warrington, PA, USA). To prevent flattening and deformation of specimens, the glass coverslips were placed onto supports made from coverslip strips. The slides were kept at 4°C before being examined under a Bio Rad MRC-600 confocal laser scanning microscope. The images were collected at cortical, subcortical and equatorial planes.
The immunofluorescent staining procedure for each cell type (oocytes, zygotes, embryos) was repeated at least three times with comparable results. As no differences were observed in the distribution of antigens between the cells obtained from conventional IVF and ICSI procedures, the results were pooled. Control staining using only secondary antibodies was always performed during each experiment (two to three cells per staining). In each case only faint cytoplasmic staining was detected (not shown). Image analysis was carried out using Image J software downloadable from http://rsb.info.nih.gov/ij/index.html.
SDSPAGE and Western blotting
For Western blotting, about 70 cells per lane were loaded. The zona-free oocytes (GV and MI/MII stages) and embryos were collected in 5 µl aliquots of lysis buffer (150 mmol/l Tris-HCl, 120 mmol/l NaCl and 0.5% NP-40, pH 8.0). Samples were snap-frozen in liquid nitrogen and stored at 80°C. Proteins were separated electrophoretically on a 7% one-dimensional sodium dodecyl sulphatepolyacrylamide gel (SDSPAGE). Subsequent transfer of proteins from SDS gels to nitrocellulose paper (Millipore, Bedford, MA, USA) was carried out during overnight incubation in blocking solution [5% milk powder (Nestlé) at 4°C, followed by washing in PBS with 0.05% Tween 20. (Bio-Rad, Hercules, USA)]. Western blots were then probed for 1 h at room temperature with the primary polyclonal antibodies against calreticulin or calsequestrin (diluted 1:300 in PBS), followed by incubation with HRP-conjugated donkey anti-goat or HRP-conjugated donkey anti-rabbit IgG (H+L) secondary antibodies (diluted 1:10 000). Immunoreactive bands were detected with an enhanced chemiluminescence kit (ECL; Amersham, Buckinghamshire, UK). Crude extracts of human skin cells and isolated cardiac calsequestrin were used as positive controls for calreticulin and calsequestrin respectively (data not shown).
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Results |
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Localization of calnexin in human oocytes and embryos
Figure 3 shows that in all human oocytes and embryos examined, calnexin localized primarily to the cell cortex. In GV oocytes (n = 5), fine staining formed three distinct zones localized under the plasma membrane (Figure 3A
). Two zones of a stronger fluorescence each 23 µm thick were separated by an intermediate area of distinctly less intense staining (
12 µm thick). In total, the cortical layer of cytoplasm displaying strong calnexin labelling occupied
78 µm. From that point, the fluorescent signal decayed gradually and almost disappeared in the central cytoplasm. Occasionally, a weak fluorescent signal could be detected around the perinuclear area.
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Localization of calsequestrin in human oocytes and embryos
In contrast to the predominantly cortical localization of calreticulin and calnexin, calsequestrin was distributed throughout the entire cytoplasm of oocytes (22 GV and 26 MI/MII), zygotes (n = 18) and 2- to 8-cell embryos (n = 15) (Figure 4). Calsequestrin was excluded from the nuclear and perinuclear areas. Occasionally, calsequestrin labelling appeared to be slightly enriched in cortex and subcortex and less intense in central ooplasm; however, it was difficult to attribute any specificity to such increase in fluorescence as it could have been due to the geometry of the cell.
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Localization of RyRs in human oocytes and embryos
All oocytes (20 GVs and 33MI/MII), zygotes (n = 6) and 2- to 8-cell embryos (n = 22) examined were positively labelled for RyRs (Figure 6). Immunolabelling with anti-RyR antibodies revealed that RyRs localized throughout the cytoplasm, in a pattern which was reminiscent of calsequestrin localization (see Figure 4
). An intense labelling was observed in the cortex and subcortex that gradually diminished toward the cell centre. In MI/MII oocytes an additional thin subplasmalemmal rim of bright fluorescence could be detected. As alcohol fixation was also used for RyRs labelling, possible artefact-related, empty, unstained spaces under the cell cortex could be seen (Figure 6
). Subplasmalemmal enrichment is demonstrated in the line profile of a GV oocyte (solid line) and a MII oocyte (dotted line).
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Discussion |
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Calreticulin and calsequestrin are Ca2+-binding proteins which play important roles in Ca2+ storage in the lumen of the ER and SR respectively. With respect to somatic cells, calreticulin is abundant in non-muscle and smooth muscle cells; however, it is expressed at very low levels in skeletal and cardiac muscle (Michalak et al., 1999). Calsequestrin expression is restricted to the muscle SR, and it has not been identified in any non-muscle mammalian cells except for avian Purkinje neurones, where it co-exists with calreticulin (Michalak et al., 1991
; Villa et al., 1992
; Meldolesi and Pozzan, 1998
). Calreticulin and calsequestrin have also been detected in Xenopus and sea urchin oocytes (Henson et al., 1990
; Lucero et al., 1994
; Parys et al., 1994
). Here, the identification of these two proteins in human oocytes and embryonic cells is reported. The presence of both calreticulin and calsequestrin in oocytes from such diverse species suggests that oocytes may represent a unique cell type, which requires both types of these high-capacity Ca2+-binding proteins for correct function of their complex Ca2+ homeostasis and signalling system.
An important finding of the present study was that calsequestrin and calreticulin are distributed differentially in human oocytes and early embryos. In oocytes, zygotes and blastomeres, calsequestrin is uniformly present throughout the entire cytoplasm of the cells, whilst calreticulin is always concentrated in the cell cortex. Such uniform localization of calsequestrin resembles its distribution pattern in Xenopus and sea urchin oocytes. However, the cortical accumulation of calreticulin observed herein was different from the uniform distribution reported for these other species (Parys et al., 1994). Calsequestrin has been localized to the nuclear envelope of sea urchin oocytes (Henson et al., 1990
), but this was not the case in the present study. Rather, it was calreticulin that was localized to the perinuclear regions of GVs of immature oocytes and of blastomere nuclei. The calreticulin-rich perinuclear regions observed here likely correspond to the nuclear envelopes. Such calreticulin localization has already been described in numerous somatic cell types (Michalak et al., 1999
). Since calreticulin and calsequestrin are ER/SR lumenal resident proteins, their differential distribution observed in the present study, and the changes in this distribution during development, suggest that these cells have uniquely specialized Ca2+ storage compartments. Therefore, they may play different roles during human oocyte maturation, fertilization and subsequent embryo development.
Like Ca2+-binding proteins, Ca2+-release channels in human oocytes and embryos are also distributed differentially. Importantly, a relationship was observed between the distribution of Ca2+-binding and -release proteins. The InsP3R-2 and calreticulin both localize to similar subcellular regions, which are different but overlapping with areas in which the RyRs and calsequestrin localize. The InsP3R-2 localizes to the subplasmalemmal region and the cell cortex, and it is scarce in the central cytoplasm. This pattern is in agreement with the cortical localization of the InsP3R type 1 reported for human oocytes and embryos (Goud et al., 1999), as well as for mouse oocytes (Mehlmann et al., 1996
; Pesty et al., 1998
; Fissore et al., 1999
). In Xenopus oocytes, which lack the RyR, the InsP3R localizes to regions that contain both calreticulin and calsequestrin (Kume et al., 1993
, 1997
; Parys et al., 1994
). The RyRs are present throughout the cytoplasm of both human oocytes and embryos. In addition, an enrichment of the RyRs was detected in the peripheral cytoplasm of MI/MII oocytes. Cortical accumulation of the RyR in a thin rim was also observed in sea urchin, mouse and bovine oocytes (McPherson et al., 1992
; Ayabe et al., 1995
; Yue et al., 1998
). However, central cytoplasmic localization of the RyR has not been reported in any other species. Functional studies on Ca2+ dynamics, using ryanodine as an activating agent, have shown that the Ry-sensitive stores operate in the central cytoplasm but not in the cell cortex and subcortex in human oocytes (Tesarik et al., 1995
; Sousa et al., 1996a
,b
). In contrast, in sea urchin, mouse and bovine oocytes the cortical presence of RyR has been associated with cortical granule release (McPherson et al., 1992
; Ayabe et al., 1995
; Yue et al., 1995
, 1998
). Interestingly, a recent report on hamster oocytes attributes cortical localization of calreticulin to its presence in cortical granules (Munoz-Gotera et al., 2001
). In this study, oocyte activation with a Ca2+ ionophore resulted in calreticulin exocytosis to the perivitelline space. These observations suggest that there is a spatial and functional dichotomy of the RyR, which may be important in Ca2+ homeostasis and signalling. In spite of this unsolved issue of physiological significance of the receptors, the present study provides direct evidence for the dual presence of InsP3R and RyR in human oocytes and embryos at distinct subcellular compartments, where one is enriched in calreticulin and the InsP3R-2, and the other contains calsequestrin and RyRs. Due to such compartmentalization, the ER Ca2+ stores may generate spatially and temporally distinct Ca2+ signals to control individual Ca2+-dependent processes or to activate processes at a global level during oocyte and embryo development. Therefore, our findings could be related to the previously proposed unique patterns of Ca2+ oscillations in human oocytes and early embryos; for example, cortical InsP3R activity, subcortical and central oscillator activity of RyR and cyclic exchanges of Ca2+ ions between InsP3-sensitive and Ry-sensitive Ca2+ stores (Sousa et al., 1996a
,b
; Tesarik and Sousa, 1996
).
InsP3R-2 and calreticulin are clearly present in the nuclear structures throughout early human embryonic development. InsP3R-2 was found in the nucleoli of the GVs, then in association with MI/MII chromatin, and finally throughout the entire nuclei of embryos. Such developmental redistribution is in partial agreement with the findings of others (Goud et al., 1999), though these authors did not detect InsP3R-1 in the nuclei of blastomeres. An association of the InsP3R-1 with the nuclear envelope and/or chromatin has also been noted in starfish and mouse oocytes (Santella and Kyozuka, 1997
; Pesty et al., 1998
; Fissore et al., 1999
), and has been investigated extensively in Xenopus eggs (Parys et al., 1992
, 1994
; Kume et al., 1993
; Stehno-Bittel et al., 1995
). In the present study it was shown that, in contrast to the InsP3R, calreticulin is not present intranuclearly but instead appears to be enriched in the nuclear envelope of human oocytes and embryos. Exceptionally, in the pronuclei of 1-cell stage zygotes, neither the InsP3R nor calreticulin were observed (Figures 2C and 5C
), which might be related to the reorganization of their Ca2+ homeostasis machinery and specific Ca2+ oscillation patterns. This notion derives support from reports showing that, in human zygotes, the redistribution of cytoplasmic organelles (the ER, mitochondria) coincides with the reversal of direction of Ca2+ waves from centripetal (just after fertilization) to centrifugal (at pronuclear stage) (Sousa et al., 1996a
, 1997
; Payne et al., 1997
). Importantly, the nuclear envelope functions as a Ca2+ store continuous with the ER, and Ca2+-mediated events have been implicated in a variety of important nuclear activities including modulation of chromatin structure and function, gene expression, DNA synthesis, nucleocytoplasmic transport and changes in nuclear architecture (Bachs and Agell, 1995
; Santella and Carafoli, 1997
; Petersen et al., 1998
; Hardingham and Bading, 1999
; Berridge et al., 2000
; Ashby and Tepikin, 2001
). Therefore, the presence of the InsP3R-2 and Ca2+-binding chaperones (including calnexin, see Results) within nuclear structures of human oocytes and embryos might also be crucial for similar nuclear activities.
A novel observation of the present work is the identification and intracellular localization of calnexin, an integral ER membrane chaperone in human oocytes and embryos. This protein, together with calreticulin, plays an essential role in the folding of glycoproteins (Helenius et al., 1997; Ellgaard et al., 1999
) as well as non-glycosylated substrates (Saito et al., 1999
). In all mammalian somatic cells examined, calnexin and calreticulin are always confined to overlapping ER compartments. In human oocytes and embryos, calnexin also overlaps with calreticulin but displays an unusual tri-laminar cortical localization (outer and inner strong layer of fluorescence, but central weak layer; see Figure 3
). Most importantly, the protein appears to undergo a dramatic rearrangement in MI/MII oocytes, that has not been shown in any morphological studies performed to date. This is in sharp contrast to a uni-laminar distribution of calreticulin found in similar regions of all human oocytes and embryos examined by us (outer layer strong, inner weaker; see Figure 2
). It should be stressed that the ER localization of calnexin and calreticulin is so well established that the proteins are considered to be the best markers of the ER membrane. Therefore, it is possible that such distinct intracellular localization of both proteins may reflect their functional differences. Calnexin is an important chaperone, whereas calreticulin plays a dual role as a chaperone and Ca2+-storage protein. Functionally, certain cell regions which are enriched in calnexin may be involved in the protein synthesis required for oocyte maturation and embryo development, while other calreticulin-enriched compartments may play an essential role in generating Ca2+ signals necessary for the cells fundamental functions. In the other subcompartments, both proteins might be associated in mutual chaperoning activity. Further studies, however, are required to determine the exact functions and interactions of these Ca2+-binding chaperones in human oocytes and embryos.
The developmental alterations of Ca2+-storage and -release protein distribution observed in the present study might reflect developmental rearrangement of the ER structures. It has been shown that in human oocytes and embryos, smooth ER occurs in different forms of small/large vesicles as well as tubular aggregates which undergo dynamic morphological transitions and rearrangements (Sousa et al., 1997). Furthermore, the search for loosely bonded, mobilized calcium using the pyroantum staining technique has revealed that in addition to different forms of smooth ER, mitochondria may also act as a source of intracellular calcium (Sousa et al., 1997
). Indeed, a correlation between reorganization of the ER network and redistribution of calreticulin, calsequestrin and InsP3R has been reported in Xenopus, sea urchin, hamster and mouse oocytes (Henson et al., 1990
; Parys et al., 1994
; Shiraishi et al., 1995
; Mehlmann et al., 1996
).
In summary, human oocytes and embryos possess highly organized and diversified calcium sequestration and release mechanisms. Studies of the proteins controlling Ca2+ homeostasis, the evaluation of their response to various stimuli, and investigation of their developmental roles remain an intriguing challenge for future research.
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Acknowledgements |
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Notes |
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The results of this study were presented at the 57th Annual Meeting of the American Society for Reproductive Medicine, Orlando, Florida, USA, October 2025, 2001
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References |
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Submitted on March 13, 2002; resubmitted on June 20, 2002; accepted on July 26, 2002.