Calcium-binding proteins and calcium-release channels in human maturing oocytes, pronuclear zygotes and early preimplantation embryos

Hanna Balakier1,5, Ewa Dziak2, Agata Sojecki1, Clifford Librach1,3, Marek Michalak4 and Michal Opas2

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 Women’s 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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The study aim was to investigate the presence and localization of Ca2+-binding proteins and Ca2+-release receptor channels in human maturing oocytes, pronuclear zygotes and preimplantation embryos. METHODS: Immunocytochemical analysis, using specific antibodies against the proteins being studied, followed with confocal laser microscopy, was performed on human oocytes and embryos. RESULTS: Calreticulin and calsequestrin (the two major calcium storage proteins of somatic cells), two types of calcium release receptors, the inositol trisphosphate and ryanodine receptors (InsP3R-2, RyRs-1,2,3), and the molecular chaperone, calnexin, were identified in all investigated cell types. Calreticulin was predominant in the cell cortex and in the nuclear envelope, while calsequestrin was distributed throughout the entire cytoplasm. Generally, localization of the InsP3R-2 and RyRs was similar to that of calreticulin and calsequestrin respectively. Both types of receptor were enriched in the subplasmalemmal region of meiotic oocytes. In addition, the InsP3R was detected in the nuclear structures of oocytes and blastomeres. Calnexin distribution overlapped with that of calreticulin but appeared to be present in distinct subcompartments. CONCLUSIONS: Human oocytes and embryos express the calcium sequestration and release proteins in highly organized and developmentally regulated patterns. Fine-tuning of these proteins may play a crucial role in regulation of Ca2+ transience during oocyte maturation, fertilization and early embryo development.

Key words: calcium release receptors/calnexin/calreticulin/calsequestrin/human embryos


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Ca2+ ions regulate fundamental functions in all living cells (Berridge et al., 2000Go). The vesiculotubular network of the endoplasmic/sarcoplasmic reticulum (ER/SR) appears to be one continuous Ca2+ store, which is crucial for intracellular Ca2+ signalling (Meldolesi and Pozzan, 1998Go). To sustain the Ca2+ homeostatic machinery, this organelle system is organized into small, spatially distinct compartments that function as discrete units, whereby Ca2+ ions can act in the various contexts of space, time and amplitude (Petersen et al., 2001Go). Heterogeneity of Ca2+ storage and exchange within such separate clusters likely depends on the non-random distribution of the macromolecules governing Ca2+ uptake, release and binding. Ca2+ is released from internal stores through the inositol 1,4,5-triphosphate receptor/ryanodine receptor (InsP3R/RyR) and is taken up by sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs) (Pozzan et al., 1994Go). In the ER/SR, Ca2+ is bound to lumenal proteins, the most common of which are calreticulin and calsequestrin. Calreticulin predominates in non-muscle cells, whereas calsequestrin is more restricted to the muscle SR (Nash et al., 1994Go). Both proteins have also been identified in sea urchin and Xenopus oocytes (Henson et al., 1990Go; McPherson et al., 1992Go; Lucero et al., 1994Go; Parys et al., 1994Go).

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., 1992Go). 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., 1992Go; Parys et al., 1992Go; Kume et al., 1993Go). In addition, current evidence suggests that both receptor families are present in murine (Ayabe et al., 1995Go; Mehlmann et al., 1996Go; Pesty et al., 1998Go; Fissore et al., 1999Go), bovine (Yue et al., 1995Go, 1998Go), porcine (Machaty et al., 1997Go) and human oocytes as well as in human embryos (Tesarik et al., 1995Go; Sousa et al., 1996aGo,bGo; Goud et al., 1999Go). Despite the recognition of a role for Ca2+ in oocyte maturation, fertilization and early embryo development (Jones, 1998Go; Kline, 2000Go; Carroll, 2001Go), 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., 1999Go). 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., 1997Go; Ellgaard et al., 1999Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human oocytes and embryos
Oocytes and embryos used in the present study were clinically unsuitable for uterine transfer or cryopreservation after conventional IVF or ICSI attempts. The study was approved by the University of Toronto Ethics Review Committee, and patients gave their written consent prior to participation. The study material consisted of: (i) germinal vesicle (GV) and metaphase I (MI) immature oocytes, that were not suitable for ICSI; (ii) mature metaphase II (MII) oocytes that were obtained from GV oocytes after in-vitro culture or inseminated/injected MII oocytes after failed fertilization; (iii) abnormal zygotes bearing one or three pronuclei (1PN or 3PN) from both IVF and ICSI procedures; and (iv) embryos derived from these abnormally fertilized oocytes. In addition, some embryos that originated from normal monospermic zygotes (2PN) but whose cleavage was arrested on day 3 at the 2- to 4-cell stage or on day 5 at the 5- to 8-cell stage of development were also included in this study. Oocytes and embryos exhibiting signs of degeneration or abnormal morphology were not used.

Immunofluorescence staining
Oocytes and embryos were briefly treated with acid Tyrode’s 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 45–60 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., 1991Go; Milner et al., 1991Go; Opas et al., 1991Go; Nakamura et al., 2000Go). 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 (1,2,3—respectively 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.

SDS–PAGE 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 sulphate–polyacrylamide gel (SDS–PAGE). 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Immuno-identification of calreticulin and calsequestrin in human oocytes
To determine if calreticulin and calsequestrin are expressed in human oocytes and embryos, total cell extracts were prepared from oocytes at different stages of maturation (GV and MI/MII) and early cleaved embryos (2- to 8-cell stages) followed by Western blot analysis with anti-calreticulin and anticalsequestrin antibodies. A doublet protein band at ~63 kDa and a single protein band at ~56 kDa were detected with anti-calreticulin and anti-calsequestrin antibodies respectively (Figure 1Go). The doublet seen with the anti-calreticulin antibody corresponds to Ca2+-bound and Ca2+-free calreticulin frequently seen in SDS–PAGE gels (Baksh and Michalak, 1991Go; Milner et al., 1991Go). Most importantly, the anti-calreticulin antibody used did not recognize calsequestrin, and the anti-calsequestrin antibody used did not recognize calreticulin (Michalak et al., 1991Go). In both oocytes and embryos, the intensity of the protein band immunoreactive with anticalreticulin antibodies was similar. In contrast, there was a larger quantity of immunoreactive calsequestrin in oocytes as compared with embryos. It was concluded that both calreticulin and calsequestrin are expressed in human oocytes and early embryos.



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Figure 1. Identification of calreticulin and calsequestrin in human oocytes and early cleavage embryos by Western blot analysis. The protein equivalent of ~70 oocytes or embryos was loaded per lane. The doublet seen with anti-calreticulin antibodies corresponds to Ca2+-bound and Ca2+-free calreticulin.

 
Localization of calreticulin in human oocytes and embryos
Figure 2Go shows that calreticulin is present in all specimens examined. In the immature GV oocytes (n = 19), calreticulin staining was most intense in an ~3 µm-thick layer of the oocyte cortex, immediately under the plasma membrane (Figure 2AGo). The intensity of staining decreased towards the cell centre, but it was still prominent in a subsequent layer extending for another 4–5 µm. In total, the zone of fine, bright staining was ~7–8 µm thick. The central cytoplasm of these GV oocytes had weak calreticulin staining. In addition, fluorescence signal was detectable in the perinuclear area of each GV, while the karyoplasm was not labelled. An analysis of pixel intensities along a line drawn across the oocyte (a ‘line profile’) is shown in Figure 2Go. In the GV oocyte, intense labelling of the cortex sloped down toward the cell centre and rose again at the nuclear envelope.



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Figure 2. Immunolocalization of calreticulin in a human immature germinal vesicle (GV) oocyte (A), a metaphase II (MII) oocyte (B), a three-pronuclear (3PN) zygote (C) and a 4-cell embryo (D). A strong fluorescent signal is detectable in the cortex of all cell types. Perinuclear labelling around the GV (A) and the blastomere nuclei (D) is clearly seen. The pronuclei of the zygote are devoid of fluorescence (C). The cytoplasm of the second polar body, attached to the zygote, shows bright labelling, while its nucleus remains unstained. In an embryo, the fourth blastomere, localized on the top of the others, can only be seen as a dark shadow (in the middle of the embryo) that obscures some blastomeres. The graph shows a distribution of intensities of fluorescence signal along a line drawn across an immature GV oocyte.

 
The distribution of calreticulin in MI and MII oocytes (n = 8 and n = 19 respectively) and in 1PN and 3PN zygotes (n = 9) was similar to that in the immature GV oocytes (Figure 2AGo) except for a patchiness of staining discernible in MI/MII oocytes (Figure 2BGo) and especially in zygotes (Figure 2CGo). A brightly labelled cortical zone was always present, and this varied in depth from 8–9 µm in MI/MII oocytes to 10–13 µm in zygotes. The pronuclei of the zygotes and their nuclear envelopes were devoid of calreticulin staining. The distribution of calreticulin staining in all embryos examined (n = 10) closely resembled that of the GV oocytes. The central cytoplasm of the blastomeres remained only faintly stained. Perinuclear labelling was readily detectable in ~80% of the blastomeres; the remaining ~20% were obscured by other blastomeres and not interpretable (Figure 2DGo). No internal nuclear labelling was observed in any of the embryos.

Localization of calnexin in human oocytes and embryos
Figure 3Go 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 3AGo). Two zones of a stronger fluorescence each 2–3 µm thick were separated by an intermediate area of distinctly less intense staining (~1–2 µm thick). In total, the cortical layer of cytoplasm displaying strong calnexin labelling occupied ~7–8 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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Figure 3. Immunolocalization of calnexin in human oocytes and embryos. Three zones of fluorescence labelling—two intense zones separated by a weaker one—are prominent in the cortex of the immature GV oocyte (A), a 3PN zygote (C) and a 4-cell embryo (D). In the MII oocyte (B), the calnexin signal is redistributed into a mosaic of fluorescent patches, which are most intense in the cell cortex. The graph shows a distribution of fluorescence signal intensities along lines drawn across an immature GV oocyte (solid line) and a MII oocyte (dotted line).

 
The distribution of calnexin changed dramatically during oocyte maturation. In MI/MII oocytes (n = 8), the three zones of cortical calnexin labelling redistributed into a single layer of patchy labelling extending 10–13 µm into the cell from the cell periphery (Figure 3BGo). After fertilization, the three zones of calnexin staining were restored in zygotes and embryos (n = 5 and n = 6 respectively; Figure 3C and DGo). Line profiles shown in Figure 3Go clearly document the tri-laminar distribution of calnexin in the oocyte cortex and the patchiness of calnexin distribution in an MII oocyte.

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 4Go). 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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Figure 4. Immunolocalization of calsequestrin in human immature GV oocyte (A), MII oocyte (B), 3PN zygote (C) and 5-cell embryo (D). Calsequestrin labelling is relatively uniformly distributed throughout the cytoplasm of all cell types, although in some cells the fluorescence signal appears to be slightly more intense in the cell cortex (in C, and in two of five blastomeres shown in D). The graph shows a distribution of fluorescence signal intensities along a line drawn across an immature GV oocyte.

 
Localization of InsP3R-2 in human oocytes and embryos
In the immature GV oocytes (n = 18), the most prominent InsP3R-2 labelling was localized under the plasma membrane as a thin rim of uniformly bright fluorescence, approximately 0.5 µm thick (Figure 5AGo). In addition, fine InsP3R-containing granules were seen in the cell cortex and subcortex, while the central cytoplasm showed only weak InsP3R-2 labelling. A similar distribution of InsP3R-2was observed in MI and MII oocytes (n = 26 and n = 50 respectively), 1PN and 3PN zygotes (n = 17) and in 2- to 8-cell embryos (n = 41) (Figure 5B–DGo). It should be noted that alcohol fixation had to be used for InsP3R-2 labelling; unfortunately, this causes shrinkage and distortion artefacts which may have caused the appearance of empty, unlabelled spaces under cortex (as seen in Figure 5Go).



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Figure 5. Distribution of the InsP3R-2 in a human immature GV oocyte (A), an MII oocyte (B), a 3PN zygote (C) and a 4-cell embryo (D). In all cells, a rim of intense InsP3R-2 labelling is located under the plasma membrane. The central cytoplasm of all cells shows weak InsP3R-2 labelling. InsP3R-2 labelling was also associated with nuclear structures: the arrow in (A) points to an InsP3R-positive nucleolus. While many metaphase oocytes (~30%) do not display enrichment of InsP3R-2 labelling in the spindle region (B, main panel), the majority of metaphase oocytes (~70%) do show strong InsP3R labelling localized to approximately the same area, which contained the spindle (B, insert). The position of the meiotic spindle is delineated by DNA labelling. The pronuclei of the zygote remain unstained (C). In an embryo, strong and uniform InsP3R-2 labelling is visible in all nuclei (D). In two blastomeres, the subnuclei are apparent. The graph shows a distribution of fluorescence signal intensities along a line drawn across an immature GV oocyte.

 
In addition to the cortical distribution, a prominent positive labelling with anti- InsP3R-2 antibodies was associated with nuclear structures (Figure 5Go). In GV oocytes a fluorescent signal was detected in the perinuclear area that most likely corresponded to the nuclear envelope. In addition, in ~70% of these oocytes the nucleoli displayed a distinct fluorescent signal for InsP3R-2 (Figure 5AGo, arrow). Similarly, in 70% of MI and MII oocytes, which contain meiotic spindles, large patches of bright fluorescence were prominent in the cytoplasm (Figure 5BGo). Counterstaining of the same cells with a DNA dye indicated that such patches of InsP3R-2 labelling correspond to areas occupied by MI/MII spindles (Figure 5BGo, insert). In zygotes, the pronuclei were not labelled (Figure 5CGo). Nuclear labelling became prominent in embryos, where all of the nuclei displayed a bright fluorescent signal (Figure 5DGo). Strong fluorescence was also detected within the entire cytoplasm of the first polar bodies associated with MII oocytes (not shown). The line profile in Figure 5Go clearly shows the presence of InsP3R-2 in the subplasmalemmal cortex and the nucleolus of an oocyte. The two small peaks flanking the nucleolus most likely correspond to labelling of the nuclear envelope.

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 6Go). Immunolabelling with anti-RyR antibodies revealed that RyRs localized throughout the cytoplasm, in a pattern which was reminiscent of calsequestrin localization (see Figure 4Go). 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 6Go). Subplasmalemmal enrichment is demonstrated in the line profile of a GV oocyte (solid line) and a MII oocyte (dotted line).



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Figure 6. Immunolocalization of the RyRs in a human immature GV oocyte (A), a MII oocyte (B), a 3PN zygote (C) and a 3-cell embryo (D). The fluorescence signal is distributed throughout the cytoplasm of all cell types. In metaphase oocytes, an additional relatively strong subplasmalemmal labelling can be detected. The graph shows a distribution of the intensities of the fluorescence signal along lines drawn across an immature GV oocyte (solid line) and an MII oocyte (dotted line).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, the ER/SR-associated Ca2+-binding and transport proteins in human immature and mature oocytes, pronuclear zygotes and early cleaved embryos were identified and localized. It was shown that calreticulin (an ER lumenal chaperone and a Ca2+-storage protein), calsequestrin (a Ca2+-storage protein of the SR), and both the InsP3R-2 and RyRs (the ER/SR-associated Ca2+-release channels) are present and distributed non-randomly in human oocytes and embryos at all of the developmental stages investigated. Interestingly, the distribution of calreticulin, in general, corresponded to the distribution of the InsP3R-2, while the distribution of calsequestrin corresponded to that of the RyRs. Finally, it was shown that calnexin, an integral membrane chaperone of the ER, is present in human oocytes and embryos in a pattern of distribution that overlaps with that of calreticulin, although the presence of distinct ER subcompartments was clear.

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., 1999Go). 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., 1991Go; Villa et al., 1992Go; Meldolesi and Pozzan, 1998Go). Calreticulin and calsequestrin have also been detected in Xenopus and sea urchin oocytes (Henson et al., 1990Go; Lucero et al., 1994Go; Parys et al., 1994Go). 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., 1994Go). Calsequestrin has been localized to the nuclear envelope of sea urchin oocytes (Henson et al., 1990Go), 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., 1999Go). 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., 1999Go), as well as for mouse oocytes (Mehlmann et al., 1996Go; Pesty et al., 1998Go; Fissore et al., 1999Go). In Xenopus oocytes, which lack the RyR, the InsP3R localizes to regions that contain both calreticulin and calsequestrin (Kume et al., 1993Go, 1997Go; Parys et al., 1994Go). 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., 1992Go; Ayabe et al., 1995Go; Yue et al., 1998Go). 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., 1995Go; Sousa et al., 1996aGo,bGo). In contrast, in sea urchin, mouse and bovine oocytes the cortical presence of RyR has been associated with cortical granule release (McPherson et al., 1992Go; Ayabe et al., 1995Go; Yue et al., 1995Go, 1998Go). Interestingly, a recent report on hamster oocytes attributes cortical localization of calreticulin to its presence in cortical granules (Munoz-Gotera et al., 2001Go). 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., 1996aGo,bGo; Tesarik and Sousa, 1996Go).

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., 1999Go), 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, 1997Go; Pesty et al., 1998Go; Fissore et al., 1999Go), and has been investigated extensively in Xenopus eggs (Parys et al., 1992Go, 1994Go; Kume et al., 1993Go; Stehno-Bittel et al., 1995Go). 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 5CGoGo), 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., 1996aGo, 1997Go; Payne et al., 1997Go). 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, 1995Go; Santella and Carafoli, 1997Go; Petersen et al., 1998Go; Hardingham and Bading, 1999Go; Berridge et al., 2000Go; Ashby and Tepikin, 2001Go). 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., 1997Go; Ellgaard et al., 1999Go) as well as non-glycosylated substrates (Saito et al., 1999Go). 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 3Go). 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 2Go). 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 cell’s 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., 1997Go). 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., 1997Go). 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., 1990Go; Parys et al., 1994Go; Shiraishi et al., 1995Go; Mehlmann et al., 1996Go).

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The material for this study was collected in the START IVF clinic, and the authors thank the staff for their kind assistance. These studies were supported by grants from the Canadian Institutes of Health Research (to M.M. and M.O.), from the Heart and Stroke Foundations of Alberta (to M.M.) and Ontario (to M.O.). M.M. is a Canadian Institutes of Health Research Senior Investigator and a Medical Scientist of the Alberta Heritage Foundation for Medical Research.


    Notes
 
5 To whom correspondence should be addressed at: CReATe Program Inc., 790 Bay Street, Suite 1020, Toronto, Ontario M5G 1N8, Canada. E-mail: hbalakier{at}sympatico.ca Back

The results of this study were presented at the 57th Annual Meeting of the American Society for Reproductive Medicine, Orlando, Florida, USA, October 20–25, 2001


    References
 Top
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 Introduction
 Materials and methods
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Submitted on March 13, 2002; resubmitted on June 20, 2002; accepted on July 26, 2002.