Inner mitochondrial membrane potential ({Delta}{Psi}m), cytoplasmic ATP content and free Ca2+ levels in metaphase II mouse oocytes

Jonathan Van Blerkom1,2,3, Patrick Davis1,2 and Samuel Alexander2

1 Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309 and 2 Colorado Reproductive Endocrinology, Rose Medical Center, Denver, CO 80220, USA

3 To whom correspondence should be addressed. e-mail: vanblerk{at}spot.colorado.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The relative magnitude of the inner mitochondrial membrane potential ({Delta}{Psi}m) has been suggested to indicate the competence of mammalian gametes and early embryos. This study examined the response of cultured somatic cells and mouse oocytes to inhibitors and conditions that affect {Delta}{Psi}m or metabolism, or both, and measured treatment-specific changes in ATP and cytoplasmic free Ca2+. METHODS: During and after treatment, relative {Delta}{Psi}m, free Ca2+, and ATP levels and cortical granule density were determined. RESULTS: Comparable responses of somatic cells and metaphase II mouse oocytes to experimental manipulations that affect {Delta}{Psi}m and metabolism were observed and reversible loss of {Delta}{Psi}m was associated with increased intracellular free Ca2+, which in certain instances resulted in parthenogenetic activation. CONCLUSION: The findings support a mitochondrial basis for pericortical J-aggregate fluorescence but not for a direct association between high {Delta}{Psi}m and metabolism. The results extend previous findings indicating that high-polarized (high {Delta}{Psi}m, JC-1 J-aggregate-forming) mitochondria occur in pericortical domains in mouse and human oocytes and early preimplantation stage embryos and support the notion that this spatial distribution may be related to localized ionic and metabolic regulation.

Key words: {Delta}{Psi}m/intracellular free Ca2/metabolism/mitochondria/oocyte


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Interest in the role of mitochondria in early mammalian development has focused primarily on metabolic function (for review see Biggers and Borland, 1976Go; Houghton et al., 1996Go; Van Blerkom et al., 1998Go) and the extent to which respiratory activity may be related to oocyte and early embryo competence. While direct assessments of ATP content in mouse and human oocytes and preimplantation stage embryos have been reported (Ginsberg and Hillman, 1973Go; Van Blerkom et al., 1995aGo, 2000), more recent studies have used the inner mitochondrial membrane potential ({Delta}{Psi}m)-sensitive probe JC-1 (5,5'6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolycarbocyanine iodide; Reers et al., 1991Go, 1995) to determine whether mitochondrial activity and embryo performance in vitro are related (Wilding et al., 2001Go, 2002, 2003). Such a relationship has been shown in certain somatic cell lines where the spatial distribution of high-hyperpolarized, JC-1, J-aggregate-forming mitochondria (Smiley et al., 1991Go; Van Blerkom et al., 2002Go, for review) and low-polarized mitochondria has been correlated with differential metabolism, focal Ca2+ regulation and other intracellular activities that regulate cell function and behaviour (Cossarizza et al., 1996Go; Salvioli et al., 1997Go; Dedov and Roufogalis, 1999Go; Diaz et al., 1999Go).

We (Van Blerkom et al., 2002Go) and others (Ahn et al., 2002Go; Jones et al., 2002Go) have reported that JC-1, J-aggregate fluorescence in mouse and human metaphase II stage (MII) oocytes and early cleavage stage embryos is most evident in the pericortical cytoplasm and may originate from clusters of high-polarized mitochondria that may have the following functions: (i) maintenance of sufficient levels of ATP production or ion buffering capacity in the subplasmalemmal cytoplasm as other mitochondria move to perinuclear regions during stage-specific spatial cytoplasmic remodelling in the maturing oocyte and early embryo (Barnett et al., 1996Go; Van Blerkom and Runner, 1984Go; Van Blerkom, 1991Go; Van Blerkom et al., 2000Go) and (ii) reversible depolarization of high-polarized mitochondria may have a localized role in Ca2+ regulation at the earliest stages of oocyte activation (Ozil and Huneau, 2001Go).

Here, studies of cultured somatic cells and mouse oocytes exposed to conditions that differentially affect {Delta}{Psi}m and respiration provide additional support for a mitochondrial origin of J-aggregate fluorescence in the oocyte. Whether oocyte mitochondria might have regulatory functions or capacities similar to those described for somatic cells was examined by measuring levels of ATP and free Ca2+ under conditions that have transient or reversible effects on {Delta}{Psi}m. The results suggest that highly polarized pericortical mitochondria may be involved in the establishment of transient microdomains (Aw, 2000Go) of high metabolism or free Ca2+ in the mature mouse oocyte.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell and oocyte culture
Cells isolated from the cumulus oophorus of human oocytes were obtained from ICSI procedures. After dispersal in hyaluronidase, they were washed in human tubal fluid (HTF) supplemented with 10% human serum, pelleted by gentle centrifugation (300 g for 3 min), resuspended in HTF, plated onto glass coverslips and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Chemical Co., USA) supplemented with 20% fetal bovine serum (FBS; Sigma Chem. Co.). Primary mouse embryonic fibroblasts rendered mitotically inactive by X-irradiation were thawed from stock solutions (70 000 cells/ml), plated onto glass coverslips and cultured in DMEM/FBS. Representative samples were stained with mitochondria-specific fluorescent probes described below and examined by conventional or scanning laser confocal microscopy (SCLM; Van Blerkom et al., 1998Go, 2002). MII oocytes, collected from the ampulla of pregnant mare serum gonadotrophin-primed 6–8 week old ICR mice 14 h after the administration of an ovulatory dose of hCG, were denuded of cumulus and coronal cells by repeated passage through a glass micropipette, and cultured in potassium-enriched synthetic oviductal medium (KSOM) supplemented with 0.5% bovine serum albumin (BSA).

Experimental and analytical methods
Mitochondrial staining and free Ca2+ analysis
Cell cultures were stained with either rhodamine 123 (r123, 26µmol/l) or JC-1 (1.5 µmol/l) (both from Molecular Probes, USA) for 10 and 15 min respectively. Oocytes were stained with r123 as above or with JC-1 (1 µmol/l) for 30 min as previously described (Van Blerkom et al., 2002Go). The distribution of JC-1 monomer (green fluorescence detected in the fluorescein isothiocyanate channel, FITC) and J-aggregate fluorescence (orange/red J-aggregate fluorescence detected in rhodamine isothiocynate channel, RITC) was determined by conventional epifluorescence and scanning laser confocal microscopy (SLCM) as described previously (Van Blerkom et al., 2002Go). Unless indicated, all images are presented as observed. However, crossover FITC epifluorescence was digitally subtracted in some images (Van Blerkom et al., 2002Go) in order to display only the pattern of J-aggregate fluorescence in the RITC channel. The relative fluorescence intensity (RFI) of JC-1 stained oocytes was determined by SLCM (see below) in the FITC and RITC channels.

Estimation of free Ca2+ levels by SLCM involved measuring average relative fluorescence intensity (RFI) detected in individual control and experimentally treated oocytes according to the following standard protocol: oocytes were preloaded for 60 min in HEPES-buffered HTF or KSOM supplemented with 4% BSA and 5 µmol/l Fluo-4 AM (Molecular Probes), followed by a 30 min wash in normal medium. Oocytes were transferred to {Delta}T dishes containing 2 ml of HEPES-buffered medium on an inverted microscope with temperature maintained at precisely at 37°C by means of a {Delta}T controller (Bioptics, USA; Van Blerkom et al., 1995bGo) and aligned such that the polar body was evident when viewed with phase contrast optics with 1 µm scans taken through the approximate centre of the oocyte to establish baseline RFI levels. Quantification of RFI used a long path filter with emission detection >510 nm and processing of the digital images with ImageSpace software (v3.10, Molecular Dynamics, USA). Specimens were examined at the same photomultiplier detector gain and pseudo-colour images are presented as observed, with dark blue (RFI = 0) and white (RFI = 225) the lower and upper levels respectively. This standard protocol, derived from preliminary studies using different Fluo-4 AM concentrations and loading times, and SLCM scans taken at different intervals and locations, provided consistent and reproducible results both within and between cohorts with no significant inter-assay variability.

Detection of cortical granules and Ca2+ ionophore activation of oocytes
Cortical granules were visualized by SLCM in oocytes fixed in 3.7% formaldehyde and stained with biotinylated Lens culinaris agglutinin (LCA) and Texas Red–streptavidin as described by Ducibella et al. (1988Go). Stained oocytes were treated with SlowFade Light (Molecular Probes) and mounted on glass coverslips without compression. 1 µm scans were taken through each oocyte and for quantification of RFI and the section series was compressed into a single projection.

Oocytes were activated by a 2 min exposure to the calcium ionophore A23187 (Sigma Chem. Co.) at 10 µmol/l in either complete KSOM or in modified medium free of divalent cations (Ducibella et al., 1988Go), followed by culture under normal conditions. Second polar body formation, pronuclear evolution or the occurrence of cleavage stage embryos with nucleated blastomeres indicated activation.

J-Aggregate formation in A23187-activated oocytes
Oocytes were exposed to A23187 for 2 min prior to JC-1 staining (time zero), or at 5 min intervals during a 30 min exposure to JC-1. With the exception of the zero time-point, oocytes were removed from JC-1-containing medium, transferred to medium containing the ionophore, and then returned to the staining medium. The occurrence of J-aggregates was assessed at the end of the 30 min staining period. After staining, oocytes found to be J-aggregate negative were cultured under normal conditions (in the absence of JC-1) and examined at 5–10 min intervals by epifluorescence or SLCM to determine whether J-aggregates developed.

Treatment with metabolic inhibitors
Although many inhibitors that target specific steps in the mitochondrial respiratory chain are available, here we selected three inhibitors with well-known effects on metabolism or {Delta}{Psi}m, or both. In preliminary studies with cumulus cells, fibroblasts and oocytes, concentrations and exposure times with the following inhibitors were varied prior to JC-1 staining in order to derive a standard treatment consistent with a non-lethal effect: (i) oligomycin, 2.6 µmol/l for 20 min (Leist et al., 1997Go; Sigma Chem. Co), (ii) FCCP, 100 µmol/l for 20 min (carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone; Sigma Chem. Co.; Smiley et al., 1991Go), and (iii) Bongkrekic acid (BA) 60 µmol/l (Calbiochem; Zamzami et al., 1996Go; DeGiorgi et al., 2000Go). For cultured cells, the standard treatment showed no cytotoxic or lethal effects as determined by DNA staining with DAPI (4'6-diamidino-2-phenylindole; Van Blerkom et al., 1995bGo) and nuclear counts of representative colonies before and for up to 5 days after treatment. For oocytes, these treatments were found to have differential effects on {Delta}{Psi}m and metabolism consistent with the known mitochondrial specificity of these agents in cultured cells and resulted in comparable frequencies of intact or fragmented oocytes in treated and control groups (unexposed oocytes from the same cohorts) after 60 h of culture in normal medium.

Temperature
Based on preliminary experiments in which the duration of culture at each temperature was varied, cell cultures and oocytes were either (i) preincubated in 1 ml or 100 µl droplets of medium respectively, under 2 ml of paraffin oil in dishes maintained at 37, 30, 27, 26, 25, 20 and 15°C for 30 min followed by staining with JC-1 for 30 min at the same temperature(s), or (ii) stained with JC-1 for 30 min at each temperature without preincubation. After staining, oocytes and cell cultures were washed in normal medium maintained at each temperature, and examined by fluorescence microscopy, and those which showed normal levels of JC-1 fluorescence in the FITC channel but no J-aggregate fluorescence in the RITC channel were transferred to normal medium at 37°C and examined by fluorescence microscopy at 10, 20, 30 and 40 min. J-Aggregate negative cell cultures and oocytes that showed little or poor JC-1 fluorescence in the FITC channel during maintenance at relatively low temperatures were returned to 37°C, restained with JC-1, and examined during staining at 10, 15 and 30 min.

Measurement of net cytoplasmic ATP content
Measurements of net ATP content followed a previously described protocol (Van Blerkom et al., 1995aGo). Briefly, groups of 20 control or treated oocytes were rapidly frozen to –80°C in 200 µl of ultrapure water. ATP levels were quantified by measuring the luminescence (Berthold LB 9501 luminometer) generated in an ATP-dependent luciferin–luciferase bioluminescence assay (Bioluminescence Somatic Cell Assay System; Sigma Chem. Co.). A standard curve containing 14 ATP concentrations from 5 fmol to 5 pmol was generated for each series of analyses.

Statistical analysis
ATP levels (fmol/oocyte) and RFI values for JC-1, Texas Red and Fluo-4 were analysed statistically by the {chi}2-test and unless indicated were considered significant at P < 0.01.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Cellular controls
Ionophores and metabolic inhibitors
The effects of A23187, mitochondrial inhibitors and temperature on J-aggregate fluorescence were examined in triplicate in human cumulus and irradiated mouse fibroblast cultures. Figure 1A–C (arrows) shows the typical patterns of mitochondria-specific fluorescence in mouse fibroblasts stained with r123 (Figure 1A) or JC-1 (Figure 1B, C). High/hyperpolarized J-aggregate-forming mitochondria are readily detectable in both the FITC (arrows, Figure 1B) and RITC channels (arrow, Figure 1C) as intensely fluorescent rod-shaped structures. Exposure of either cell type to A23187 for up to 15 min had no obvious effect on the occurrence or apparent intensity of J-aggregate fluorescence (10 min, arrow, Figure 1D).



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Figure 1. Fluorescent microscopic images of cultured mouse fibroblasts and human cumulus cells stained with the mitochondria-specific probes rhodamine 123 (r123) (A) and JC-1 (BR) under normal and experimental conditions (see text for details). For the metaphase II oocyte, the characteristic pericortical distribution of JC-1 J-aggregate fluorescence in normal and treated oocytes is shown in U and W. Changes in intracellular free Ca2+ signified by elevated intensities of Fluo-4 fluorescence at 0, 10, 30 and 80 s after exposure to A23187 are shown in T14. Corresponding effects of A23187 treatment on pericortical J-aggregate fluorescence (V, W) and cortical granule density at 0, 15 and 30 min (XZ) are discussed in the text.

 
FCCP is a proton ionophore that uncouples oxidation from phosphorylation by dissipating the chemiosmotic gradient ({Delta}{Psi}m: Mitchell and Moyle, 1967Go) while leaving the electron transport system functional. Exposure of fibroblasts or cumulus cells to FCCP followed by JC-1 staining was associated with a uniform rather than punctate pattern of cytoplasmic JC-1 fluorescence (Figure 1E) and virtually no detectable J-aggregate fluorescence (5 min exposure, Figure 1F). The uniformity of JC-1 monomeric fluorescence is characteristic of cytoplasmic but not mitochondrial uptake of the stain. The same result occurred with cells exposed to JC-1 and FCCP simultaneously.

Oligomycin inhibits oxidative phosphorylation by binding ATP synthase and blocking the mitochondrial proton channel, but has no short-term effect on electron transport or the chemiosmotic gradient associated with the formation of J-aggregates. This activity was confirmed by detection of JC-1 fluorescence in the green channel (Figure 1G) and mitochondrial J-aggregate fluorescence in the red channel (Figure 1H) after treatment of cumulus cells at concentrations between 2.6 and 26 µmol/l. A similar finding was obtained with mouse fibroblasts exposed to oligomycin for 1–1.5 h at concentrations up to 52 µmol/l (RITC channel, arrow, Figure 1I). In a second series of experiments, cells were exposed to oligomycin (20–30 min), washed in normal medium, treated with FCCP (10 min) and stained with JC-1 (15 min). Similar to exposure to FCCP alone, no detectable J-aggregates formed during FCCP treatment (Figure 1L), demonstrating the specificity of these agents on the ability of mitochondria to maintain a {Delta}{Psi} consistent with JC-1 multimerization in oligomycin but not FCCP.

BA is a potent inhibitor of the mitochondrial permeability transition pore (PTP, i.e. prevents or retards {Delta}{Psi}m collapse, see below) and also inhibits the mitochondrial adenine nucleotide translocator (transporter) situated on the inner aspect of the inner mitochondrial membrane where ADP is delivered into the matrix. Figure 1J and K shows the typical pattern of J-aggregate fluorescence in mouse fibroblast in the FITC (Figure 1J) and RITC channels (Figure 1K) after a 30 min pre-exposure to BA followed by JC-1 staining for 30 min in the presence of the inhibitor. The detection of J-aggregate fluorescence is consistent with the ability of BA to prevent collapse of {Delta}{Psi}m. In a second series of experiments, cultures were exposed to BA for up to 60 min, washed extensively for 10 min, exposed to FCCP (in the absence of BA), and then stained with JC-1 in normal medium as described above. Although normal intensities of JC-1 were detected in the FITC channel, no J-aggregate fluorescence was observed (comparable to Figure 1L). Similar to the oligomycin results, this finding indicates that under the conditions used, mitochondria remain hyperpolarized in BA and collapse {Delta}{Psi} in response to FCCP.

Effects of reduced temperature
J-Aggregate formation under conditions of reduced temperatures was examined in mouse fibroblasts to determine whether the apparent {Delta}{Psi}m could be manipulated without the use of chemical inhibitors. Based on findings from each temperature point, 25°C was the critical temperature for detectable J-aggregate formation. At 25°C the relative intensity (RFI) of JC-1 staining in the FITC channel remained largely unchanged from levels seen between 30 and 37°C (comparable to Figure 1G), and the distinct green mitochondrial fluorescence detectable at higher temperatures was still evident (Figure 1M). However, J-aggregate fluorescence was virtually undetectable in the RITC channel (Figure 1N). The intense foci of fluorescence indicated by arrows in Figure 1M and N is autofluorescence most likely associated with lipid droplets observed in some fibroblasts. When these stained cultures were washed and incubated in normal medium at 37°C, the typical pattern and intensity of J-aggregate fluorescence occurred within 15 min without restaining (Figure 1O).

At 20°C, the JC-1 RFI in the FITC channel was significantly lower than observed at higher temperatures and no J-aggregate positive mitochondria were detected. At 15°C, JC-1 RIF was of low intensity and showed uniform rather than punctate pattern, indicating cytoplasmic uptake but no apparent mitochondrial incorporation (data not shown). However, in cultures maintained at <=20°C for up to 60 min, J-aggregate fluorescence returned to normal levels within 15 min after restaining with JC-1 at 37°C (initial culture at 15°C; arrow, Figure 1P).

Counts of DAPI fluorescent nuclei taken in the same regions of irradiated (non-dividing) fibroblast cultures before and after each type of inhibitor or temperature treatment used in this study showed no significant cell loss, with >90% of the cells intact at 5 days post exposure treatment (data not shown).

Stability of J-aggregate fluorescence in inhibitor- and temperature-treated fibroblasts
If cell cultures were first stained with JC-1 and then treated with FCCP or exposed to temperatures at or below critical levels, J-aggregate fluorescence was clearly detectable after treatment (e.g. FCCP, arrow, Figure 1Q). A similar finding was obtained with cells fixed in formaldehyde (Figure 1R) or glutaraldehyde after JC-1 staining under standard conditions. Figure 1S shows the absence of fluorescence in unstained cultures fixed with formaldehyde. These results indicate that, once formed, J-aggregates may remain relatively stable when followed by treatments that collapse {Delta}{Psi}m.

Metaphase II oocytes
For each time-point or experimental treatment in which free Ca2+ or ATP levels were measured, a minimum of 150 or 200 MII mouse oocytes respectively were used, with each experiment performed in duplicate. A minimum of 100 and 40 oocytes were used in each experiment that involved detection of JC-1 and cortical granule fluorescence respectively. ATP levels shown indicate the average net ATP content/oocyte, ± 70 fmol. For measurements of intracellular Ca2+ levels, the relative average maximum fluorescence intensity (RFI ± 15%) was determined by SLCM using pseudocolour values between 0 and 225 as shown on the scale in Figure 1T1 (Van Blerkom et al., 2000Go). Approximately 4% of the MII oocytes exposed to A23187 or FCCP showed RFI measurements that were completely off scale (>>225). As described below, a similar percentage of control oocytes showed atypical patterns of punctate cytoplasmic J-aggregate fluorescence that was unusually intense. Approximately 0.5% of the several hundred oocytes examined showed no cytoplasmic increase in intracellular free Ca2+ in response to A23187 or FCCP. Oocytes that were non-responsive to these agents or where RFI levels were off scale were considered abnormal and not included in the values shown.

Effects of A23187 on intracellular free Ca2+ levels, J-aggregate formation, cortical granule exocytosis and net cytoplasmic ATP content
MII mouse oocytes were exposed to the Ca2+ ionophore A23187 under conditions known to induce cortical granule exocytosis and parthenogenetic activation (Ducibella et al., 1988Go). Changes in the RFI of Fluo-4 AM were recorded at 5 s intervals after the addition of A23187 and quantified for individual sections and for the entire cytoplasm from compiled images (Figure 2). This treatment was associated with a pronounced increase in intracellular free Ca2+ initially detected at the portion of the oocyte plasma membrane first exposed to the ionophore (time = 10 s; Figure 1T2), which then progressed across the cell to involve the entire cytoplasm and first polar body within 30 s (time = 30 s; Figure 1T3; column B, Figure 2. By 80 s (Figure 1T4) the RFI returned to a level only slightly higher than the initial background (time = 0; Figure 1T1; column A, Figure 2). There was no significant difference in RFI between oocytes exposed to A23187 in either normal or modified medium.



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Figure 2. Histographic representation of the maximum average cytoplasmic fluorescent intensity (RFI) of Fluo-4-AM stained metaphase II (MII) mouse oocytes determined by quantitative analysis of pseudocolour images of compiled sections obtained by scanning laser confocal microscopy. The increased RFI signifies changes in intracellular free Ca2+ levels in control (column A) and treated (columns B–I) MII mouse oocytes exposed to metabolic inhibitors [A23187, FCCP (carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone, oligomycin], reduced temperature (25°C, column H) or a combination of treatments (columns D, E, I) according to times and conditions described in the text. P <= 0.01 are denoted by an asterisk with the corresponding RFI range indicated by a bar at the top of each column.

 
The typical distribution of punctate pericortical J-aggregate fluorescence detected in the RITC channel in untreated oocytes is shown in Figure 1U (arrows; with FITC crossover fluorescence subtracted). A marked reduction in the density of pericortical J-aggregate fluorescence was detected after the standard 30 min JC-1 exposure if treatment with A23187 occurred during the first 5–10 min of staining (10 min, arrow, Figure 1V). In contrast, the RFI of JC-1 monomeric staining detected in the FITC channel in control and ionophore-treated oocytes after 30 min was similar (compare with Figure 3H).



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Figure 3. (A, D, GN) Fluorescent microscopic images of JC-1-stained mouse oocytes in the fluorescein isothiocynate (H, I) and rhodamine isothiocynate (A, D, G, J, KN) channels showing the presence (arrows) or absence of pericortical J-aggregate fluorescence after exposure to metabolic inhibitors or reduced temperature (see text for details). (B1B3) The rise in intracellular free Ca2+ reported by the intensity of Fluo-4 fluorescence after treatment with FCCP (carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone) (B2) followed by exposure to A23187 (B3) (untreated control, B1). (C, E and F) Fluorescent patterns and intensities of Texas Red-tagged cortical granules in control and experimentally treated oocytes as described in the text.

 
In order to determine whether J-aggregates could develop after ionophore treatment, J-aggregate negative oocytes exposed to A23187 during the first 10 min of JC-1 staining were placed in normal medium at the end of the 30 min staining period. The characteristic pericortical distribution of punctate J-aggregate fluorescence occurred within 30 min of culture and in the absence of additional staining (arrows, Figure 1W, with FITC subtraction; asterisk indicates position of first polar body). This finding indicates that A23187 may be associated with a transient reduction in the hyperpolarized state of pericortical mitochondria.

Cortical granule exocytosis was confirmed with representative untreated (time = 0 min; Figure 1X) and ionophore-exposed oocytes examined at 5 min intervals (e.g. 15 min, Figure 1Y and 30 min, Figure 1Z). Similar to the findings of Ducibella et al. (1988Go), cortical granule-associated fluorescence was largely absent by 30 min, although in some oocytes, small domains of intense fluorescence were evident (arrow, Figure 1Z). Compared with untreated oocytes, the average RFI of the cortical granule complement decreased by ~45% at 15 min and >90% at 30 min.

As shown in Figure 4, no significant change in the average net cytoplasmic ATP content of MII oocytes was detected during or after ionophore treatment, and levels measured at 30 min persisted for 24 h in culture in normal medium (data not shown). Ionophore-induced activation was confirmed by pronuclear formation or the first cleavage division in ~80% and ~73% of treated oocytes at 18 h (24/30) and 32 h (56/77) respectively after exposure.



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Figure 4. Net average cytoplasmic ATP contents (± 70 fmol/oocyte, >=400 oocytes/time-point) of untreated (control) metaphase II mouse oocytes, and oocytes exposed to the Ca2+ ionophore A23187, the metabolic inhibitors oligomycin (oligo), FCCP (carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone), Bongkrekic acid (BA), or reduced temperature (25°C) as described in the text. Treatments that resulted in J-aggregate (JA) positive (JA+) or negative (JA–) oocytes are indicated. *P <= 0.01 refers to each time-point in the corresponding series of treatments.

 
Effects FCCP on pericortical J-aggregate fluorescence, intracellular free Ca2+ and cytoplasmic ATP
We have previously reported that exposure of MII oocytes to FCCP precluded formation of J-aggregate fluorescence in oocytes (Van Blerkom et al., 2002Go), as demonstrated here, an identical affect was observed with cultured cells. However, the present study shows that the characteristic pattern of punctate pericortical J-aggregate fluorescence developed in treated oocytes (10 min in FCCP) ~30 min after placement in normal medium and without restaining in JC-1 (arrows, Figure 3A, image shown with FITC fluorescence subtracted). Comparable responses of somatic cells and oocytes to FCCP under the conditions used here indicate that collapse of {Delta}{Psi}m is reversible.

In order to determine whether mitochondrial depolarization affected levels of cytoplasmic free Ca2+, the average maximum RFI of oocytes preloaded with Fluo-4 was measured at timed intervals following exposure to FCCP. In the presence of FCCP, a progressive rise in intracellular free Ca2+ levels occurred (untreated, Figure 3B1) with maximum Fluo-4 RFI after 10 min (Figure 3B2) ~50% (column C, Figure 3) of the level observed with A23187 alone (column B, Figure 3).

The potential mitochondrial contribution to intracellular free Ca2+ was estimated by varying the order in which oocytes were exposed to A23187 and FCCP and the time of free Ca2+ analysis. In the first series of experiments, A23187 was added when the FCCP associated Fluo-4 RFI reached a maximum (10 min). The addition of A23187 was accompanied by a further increase in Fluo-4 RFI (column D, Figure 3) within the first 10 s of treatment (Figure 3B3). In a second experiment, FCCP was added after peak Fluo-4 RFI induced by A23187 occurred (30–45 s). In this instance, no significant increase in Fluo-4 RFI was observed (comparable to levels shown in column B, Figure 3). In a third series of experiments, FCCP was added after A23187-associated Fluo-4 RFI returned to and stabilized at normal baseline levels (5 min in ionophore-free, normal medium; levels comparable to value shown in Figure 1T4 and in column A, Figure 3), and at 5 min intervals for 30 min, and at 10 min intervals between 30 and 60 min. The first significant increase in the level of intracellular free Ca2+ was detected ~20 min following baseline stabilization (column E, Figure 3), and by 30 min, the magnitude of the increase was comparable to levels seen with FCCP alone (e.g. column C, Figure 3). Similar maximum average RFI levels were measured when FCCP was added at 40, 50 and 60 min after A23187 treatment. These findings suggest that mitochondria may contribute to intracellular free Ca2+ after ionophore exposure, but may require a period of time to repolarize (and resequester this ion) in order to discharge Ca2+ in response to FCCP-induced {Delta}{Psi}m collapse.

Exposure to FCCP was accompanied by a progressive decline in cytoplasmic ATP content from normal levels of ~700 fmol/oocyte to ~150 fmol/oocyte after 10 min of treatment (Figure 4). As previously reported (Van Blerkom et al., 1995aGo), average cytoplasmic ATP contents returned to near normal levels (600 fmol/oocyte) after 20 min of culture in normal medium and the oocytes remained intact after 24 h in normal medium.

Although a morphometric analysis of cortical granules was beyond the scope of this study, exposure to FCCP for 10–15 min was associated with an average reduction in the RFI of LCA–Texas Red-tagged oocytes (Figure 3C) of ~40% when compared with control RFI values (e.g. Figure 1X). The increase in cytoplasmic free Ca2+ and partial loss of the cortical granule complement resulting from FCCP exposure was not accompanied by parthenogenetic activation during the subsequent 28 h of culture in normal medium. This finding suggests that the magnitude of the Ca2+ may be subthreshold with respect to activation.

Oligomycin
Oligomycin is a classic inhibitor of oxidative phosphorylation that is widely used to experimentally modulate ATP levels in somatic cells (Leist et al., 1997Go). As shown in Figure 4, exposure of MII mouse oocytes to this inhibitor at 2.6 µmol/l reduced the average net cytoplasmic ATP concentration from 700 to 200 fmol/oocyte after 10 min. At 60 min, the average ATP content was 120 fmol/oocyte (data not shown). However, similar to the findings described above for somatic cells, exposure to oligomycin did not inhibit the development of pericortical J-aggregate fluorescence in oocytes exposed to the inhibitor for 20–30 min in the presence of JC-1 (Figure 3D), during which cytoplasmic ATP content declined markedly (Figure 4).

No significant increase in Fluo-4 RFI was observed in oocytes exposed to oligomycin for up to 30 min (Figure 2, column F) and there was no evident effect on the distribution or RFI of lectin-tagged cortical granules after treatment or during subsequent culture in normal medium (e.g. 6 h, Figure 3E). Approximately 10% of the oocytes exposed to this inhibitor showed cortical granule clustering in the form of highly fluorescent patches (Figure 3F). However, the relative RFI of lectin-tagged cortical granules was comparable to values obtained from untreated controls or other oligomycin-treated oocytes, indicating a redistribution rather than exocytosis in these instances.

Bongkrekic acid
Exposure to BA for 60 min at concentrations up to 150 µmol/l prior to JC-1 staining had no obvious effect on the intensity or pattern of pericortical the J-aggregate fluorescence (arrows, Figure 3G). The average net ATP content/oocyte declined progressively during a 60 min exposure (60 µmol/l; 450 fmol/oocyte at 30 min Figure 4; 400 fmol/oocyte, 60 min, data not shown) but returned to control levels within 15 min of culture in normal medium. Intracellular free Ca2+ levels indicated by the maximum average RFI of Fluo-4 measured at 10 min intervals during 60 min of culture in BA at 60 µmol/l remained unchanged (Figure 2, column G). No change in cortical granule distribution or density (RFI) was observed (after 60 min in BA) and no evidence of parthenogenetic activation was detected during a subsequent 28 h culture in normal medium.

Exposure of oocytes to reduced temperature
Effects on JC-1, J-aggregate formation
The association between J-aggregate fluorescence and temperature detected in cultured cells was examined as follows: oocytes were (i) preloaded with JC-1 at 37°C for 30 min and then transferred to medium maintained at the same temperatures used for somatic cells (15–37°C) or (ii) stained and examined at each temperature and, if J-aggregate negative, returned to 37°C and re-examined as described below.

Similar to findings obtained with somatic cells, 25°C was also found to be the critical temperature for detectable pericortical J-aggregate formation in MII oocytes under the conditions used in this study. At 25°C, the relative intensity of JC-1 fluorescence was largely unchanged (Figure 3H) from levels seen at higher temperatures (e.g. 37°C, Figure 3I), but pericortical J-aggregate fluorescence (37°C, Figure 3J) was undetectable (Figure 3K), with the exception of scant fluorescence in the first polar body (PB1) of some oocytes (arrow, Figure 3K). When returned to normal culture conditions, oocytes that were J-aggregate negative at 25°C exhibited normal punctate pericortical fluorescence within 20 min of culture at 37°C without restaining (arrows, Figure 3L). Oocytes cultured at 15°C showed very low intensity JC-1 fluorescence in the FITC channel and no J-aggregate signal. When returned to 37°C and restained, normal pericortical J-aggregate fluorescence was detectable within 20 min of staining (20 min, arrows, Figure 3M).

After JC-1 exposure for 30 min at 37°C, oocytes were cultured in the absence of JC-1 for up to 60 min at 25, 20 and 15°C. The persistence of pericortical J-aggregate fluorescence (similar to image shown in Figure 3J) indicates that, once formed, mitochondria J-aggregates may remain relatively stable at temperatures that appear to be inconsistent with nascent JC-1 multimerization.

Effects on intracellular free calcium and ATP levels
Oocytes were preloaded with Fluo-4 at 37°C and then transferred to medium maintained at 30, 25, 20 and 15°C as described above. The RFI was measured within 1 min of transfer and at 2 min intervals during the first 20 min of culture, and at 10 min intervals between 20 and 60 min. Temperatures at these levels were not associated with detectable changes in free Ca2+ levels (e.g. 30 min 20°C; column H, Figure 2). An elevation of intracellular Ca2+ to levels seen in untreated oocytes (column B, Figure 2) occurred when cooled oocytes were incubated at 37°C for 15–20 min and then exposed to A23187 without additional Fluo-4 staining (e.g. 60 min at 25°C; column I, Figure 2). This finding suggests that a normal elevation in intracellular Ca2+ levels was temporally associated with the occurrence of normal pericortical J-aggregate fluorescence.

In order to determine whether J-aggregate fluorescence, temperature and ATP content were related, ATP levels were measured at 25°C during 60 min of culture. In parallel cultures containing JC-1, representative oocytes were examined at 30 min and all were J-aggregate negative. The slight decline in the average ATP content during the first 30 min at 25°C (Figure 4) was not significant and remained at this level for an additional 30 min (data not shown). No significant change in cortical granule distribution or density determined empirically by SLCM and quantitatively by measurement of the LCA–Texas Red–streptavidin RFI was observed during 60 min of culture at 25°C (comparable to image shown in Figure 3E). In this instance, the absence of J-aggregate fluorescence was unrelated to net ATP content.

Atypical patterns of J-aggregate fluorescence
Approximately 4% of the several hundred control and experimentally treated JC-1-stained oocytes (BA, oligomycin) examined in this study exhibited an atypical pattern of J-aggregate fluorescence characterized by intense punctate cytoplasmic fluorescence (Figure 3N, oligomycin-treated) after a standard 30 min exposure to JC-1. This pattern was not observed in any of the several hundred oocytes exposed to FCCP or cultured at <=25°C. The potential correspondence between oocytes with atypical J-aggregate fluorescence and those producing atypical intensities of Fluo-4 fluorescence is discussed below.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fertilization and early developmental failure in the human have been suggested to involve inadequate levels of metabolism associated with mitochondrial genetic defects (Keefe et al., 1995Go; Brenner et al., 1998Go; Barritt et al., 1999Go, 2000), functional disorders (Muller-Hocker et al., 1996Go; Wilding et al., 2001, 2003), mtDNA copy number (Chen et al., 1995Go; Steuerwald et al., 2000Go; Reynier et al., 2001Go) and abnormal segregation patterns that may lead to unequal inheritance between blastomeres during cleavage (Van Blerkom et al., 2000Go). Recent non-invasive assessments of sperm, oocyte and embryo mitochondrial activity (Garner and Thomas, 1999Go; Wilding et al., 2001Go, 2002, 2003; Marchetti et al., 2002Go) that may be indicative of competence are founded on the relationship between {Delta}{Psi}m and oxidative metabolism (Mitchell and Moyle, 1967Go) observed in some somatic cell cultures stained with the {Delta}{Psi}m-sensitive cationic lipophilic carbocyanine dye JC-1 (Salvioli et al., 1997Go).

JC-1 accumulates in monomeric form within the mitochondrial matrix and its fluorescent emission characteristics are a function of the magnitude of {Delta}{Psi}m (Reers et al., 1991Go, 1995; Cossarizza et al., 1996Go). Low {Delta}{Psi}m (low polarized) organelles fluoresce green, while higher polarized organelles fluorescence orange–red owing to multimerization of JC-1 and the formation of ‘J-aggregates’ (Jelly, 1937Go), which shifts the emission spectrum to longer wavelengths (Reers et al., 1995Go). For human oocytes and blastomeres, ratiometric analysis of JC-1 emissions has been suggested to specify levels of oxidative metabolism (Wilding et al., 2002Go, 2003). Other studies have reported patterns of JC-1 staining in which J-aggregate fluorescence is largely localized to the pericortical cytoplasm of oocyte and blastomeres (Ahn et al., 2002Go; Jones et al., 2002Go; Van Blerkom et al., 2002Go), suggesting that high-polarized mitochondria may have unique or location-dependent functions at these stages.

Unlike the fully developed mitochondria within highly flattened cultured cells commonly used to study {Delta}{Psi}m and organelle function (Smiley et al., 1991Go; Diaz et al., 1999Go), the comparatively undeveloped mitochondria of mammalian oocytes and early embryos are too numerous (~85 000 in mouse, Piko and Matsumoto, 1976Go; ~150 000 in human; Cummins, 2002Go) and too small (<0.4 µm, both species; Dvorak et al., 1987Go) to permit their individual detection with fluorescent probes such as r123, MitoTracker, or JC-1 (Van Blerkom et al., 1998, 2002Go). Consequently, functional correlations between mitochondrial activities and patterns of J-aggregate fluorescence in the oocyte are inferred rather than direct as in somatic cells.

Here, we compared the response of JC-1-stained MII mouse oocytes and somatic cells (human cumulus cells and mouse fibroblasts) to culture conditions and inhibitors with known effects on respiration or {Delta}{Psi}m, or both. We also addressed an issue raised in a previous study (Van Blerkom et al., 2002Go), which proposed that location-associated differences in {Delta}{Psi}m may be associated with the regulation of intracellular free Ca2+ levels in oocytes and blastomeres. With respect to pericortical J-aggregate fluorescence in the oocyte, the following are the major findings of this study: (i) comparable responses of oocytes and somatic cells to agents that affect metabolism or {Delta}{Psi}m, or both, support a mitochondrial origin as previously suggested (Van Blerkom et al., 2002Go), (ii) high {Delta}{Psi}m can occur despite reduced mitochondrial respiration, and (iii) transient loss of high {Delta}{Psi}m in the pericortical cytoplasm of MII mouse oocytes is accompanied by increased levels of intracellular free Ca2+.

J-Aggregate fluorescence, mitochondrial metabolism and intracellular free Ca2+
The present findings indicate that a high {Delta}{Psi}m can be maintained under conditions that reversibly reduce the capacity of mitochondria to generate ATP. In these instances, the absence of an effect on {Delta}{Psi}m may be explained by the relationship between {Delta}{Psi}m, ion transport and the mitochondrial permeability transition pore (PTP), a non-selective channel of the inner mitochondrial membrane. Opening the PTP can collapse {Delta}{Psi}m and, if persistent, can result in a Ca2+ efflux that for fully developed mitochondria may occur focally or involve the entire organelle (Ichas et al., 1997Go). As noted above, oligomycin reduces metabolic activity without collapsing {Delta}{Psi}m, and BA is a specific PTP inhibitor (Fiore et al., 1998Go) that maintains polarity by preventing PTP opening and Ca2+ efflux (Rottenberg and Marbach, 1990Go; Zamzami et al., 1996Go). Adverse metabolic effects of prolonged BA exposure are associated with the inhibition of the adenine nucleotide transporter. Despite declining ATP levels, no increase in intracellular Ca2+ was detected in oocytes that retained the capacity to form J-aggregates during metabolic inhibition, which is consistent with PTP function in the presence of these inhibitors under the conditions used in this study. In contrast, FCCP rapidly abolishes {Delta}{Psi}m in oocytes and somatic cells thus preventing J-aggregate formation. Respiratory effects of this inhibitor (Van Blerkom et al., 1995aGo) were confirmed by a marked reduction in oocyte ATP content. If mitochondria discharge Ca2+ in response to a sudden collapse in {Delta}{Psi}m and retain the PTP in an open state, a progressive rise in levels of intracellular free Ca2+ should occur. The effects of FCCP on mouse oocytes is consistent with such a response and suggests that mitochondria in general, and high-polarized mitochondria in particular, could contribute to the rise in intracellular free Ca2+ detected with this inhibitor. After transfer to normal medium, the development of pericortical J-aggregate fluorescence indicates that FCCP-induced mitochondrial depolarization was reversible and, as discussed below, may be associated with resequestration of this cation.

A different relationship between J-aggregate fluorescence, metabolism and intracellular free Ca2+ levels was observed with A23187 or during culture at reduced temperatures. For both cultured cells and oocytes, J-aggregate fluorescence was undetectable at 25°C. For the oocyte, the absence of J-aggregate fluorescence occurred against a background of apparently normal levels of JC-1 uptake, ATP contents and intracellular free Ca2+. One explanation for this finding is that at the critical temperature, {Delta}{Psi}m may have declined below a threshold required for intramitochondrial JC-1 multimerization (Reers et al., 1995Go). This notion is supported by the occurrence of J-aggregate fluorescence in cooled oocytes after return to 37°C (without re-exposure to JC-1). These findings do not preclude a temperature effect on metabolism such that a normal ATP content at 25°C could reflect a change in the equilibrium between synthesis and utilization. Treatment with A23187 was accompanied by a reversible loss of the ability to develop J-aggregate fluorescence, an immediate and significant increase in intracellular free Ca2+, but no detectable change in cytoplasmic ATP content. In this case, loss of high {Delta}{Psi}m has no metabolic consequences but may be associated with a mitochondrial Ca2+ efflux as discussed below.

Mitochondria and Ca2+ regulation
Results from varying the timing and order of exposure to FCCP and A23187 suggest the possibility that Ca2+ storage sites may be differentially targeted by each ionophore, with FCCP-associated release involving mitochondria, while A23187 directly induces release from inositol 1,4,5-triphosphate (IP3)-responsive elements such as the smooth endoplasmic reticulum (SER) or subplasmalemmal storage granules (Sousa et al., 1996Go, 1997). If high-polarized oocyte mitochondria have an ability similar to their somatic cell counterparts to release Ca2+ in response to electrical and ionic fluxes (Babcock et al., 1997Go; Ichas et al., 1997Go; Diaz et al., 1999Go), the transient effect of A23187 on mitochondrial polarity may be an indirect one initiated by a Ca2+-induced Ca2+ release (CICR) pathway (Loew et al., 1994Go) originating from A23187-responsive Ca2+ storage elements. In this respect, the delayed response to FCCP after A23187 exposure may be related to the time required for mitochondrial repolarization and Ca2+ resequestration.

The present findings demonstrate that pericortical J-aggregate fluorescence and free Ca2+ levels are related, but while relevance to developmental processes in the oocyte or embryo has been suggested (Ahn et al., 2002Go; Jones et al., 2002Go; Van Blerkom et al., 2002Go), a direct association remains to be established. Whether oocytes that show intense cytoplasmic mitochondrial hyperpolarization (J-aggregate fluorescence) also have abnormally high ATP contents (Van Blerkom et al., 1995aGo) is under investigation. However, this phenotype could reflect abnormal conditions (e.g. Ca2+ overload; Berridge et al., 1998Go) that result in an atypical complement of hyperpolarized mitochondria. When combined with mobilization from other sites of sequestration (FitzHarris et al., 2003Go), Ca2+ efflux from these mitochondria could have contributed to cytoplasmic levels that were off scale after A23187 (but not FCCP) exposure. In this regard, lethality of the tw32/tw32 mutation in the early mouse embryo may be relevant to this atypical wild type phenotype as it is characterized by abnormally high rates of ATP synthesis and the presence of intramitochondrial inclusions containing unusually high levels of Ca2+ (Hillman and Hillman, 1975Go). Developmental significance would be demonstrated if such oocytes were unfertilizable or unable to develop normally through the preimplantation stages, and clinical significance suggested if a similar phenotype occurs in human oocytes (Van Blerkom et al., 2002Go).

Whether J-aggregate-forming pericortical mitochondria have metabolic rates, levels of Ca2+ sequestration /discharge, or response times (e.g. to electrical fluxes or CIRC) that are significantly different from their apparently lower polarized counterparts, remain to be determined. If confirmed, the unique location of domains of high-polarized mitochondria may have important functions in the oocyte and early embryo. For example, if pericortical J-aggregate fluorescence is associated with comparatively high metabolic activity, the presence of clusters of mitochondria with high {Delta}{Psi} may compensate for the loss of other mitochondria from cortical regions as these organelles aggregate in the perinuclear cytoplasm of the maturing oocyte and pronuclear and cleavage stage embryos (for review, see Van Blerkom et al., 2002Go).

Ca2+ is a universal signalling agent and regulatory cues can be encoded in both the frequency and the amplitude of acute or periodic waves (transients or oscillations). These transients can effect the activity of organelles such as mitochondria and initiate differential gene activation through Ca2+-dependent signal transduction pathways that regulate cellular development, differentiation, proliferation, and molecular events related to life or death (Berridge et al., 1998Go). In somatic cells, transient changes in {Delta}{Psi}m have been correlated with metabolism and free Ca2+ levels, and can occur throughout the cytoplasm or act focally, such as in regions subjacent to the plasma membrane (Ichas et al., 1997Go), where transient opening and closing of the PTP and corresponding changes in {Delta}{Psi}m (so-called mitochondrial ‘flickering’; DeGiorgi et al., 2000Go) have been suggested to contribute to the amplification and propagation of Ca2+ signals (Ichas et al., 1997Go).

The role of Ca2+ as a critical epigenetic factor in mammalian development has been reported by Ozil and Huneau (2001Go), who demonstrated that reducing the amplitude of the first Ca2+ transient within the first seconds of rabbit oocyte activation had significant and adverse developmental consequences that were not evident until well after implantation. However, it is an intriguing possibility that transient depolarization(s) of high-polarized mitochondria in the mature oocyte could act locally to increase the amplitude or rate of propagation, or both (DeGiorgi et al., 2000Go), of Ca2+ transients in the maturing oocyte (FitzHarris et al., 2003Go), as well as the initial transient(s) associated with activation. Whether an association between high-polarized mitochondria and elements of the SER detected in the pericortical cytoplasm (Van Blerkom et al., 2002Go) represent focal sites Ca2+ of regulation responsive to electrical and ionic fluxes (Duchen, 2000Go; Pozzan et al., 2000Go) is under investigation.

Gianaroli et al. (1994Go) reported that the ‘first event’ of sperm activation after penetration in the human oocyte was the generation of an outward fertilization current which induces a hyperpolarization of the plasma membrane over a 60–120 min period. However, a different and smaller inward current was detected within minutes after sperm contact with the oolemma. The progressive increase in the amplitude of the secondary outward fertilization current was demonstrated to be Ca2+-dependent with this cation mobilized from stores responsive to sperm-derived IP3. Although speculative at present, it is an interesting possibility that the early inward currents effect a transient depolarization of high-polarized subplasmalemmal mitochondrial clusters with a resulting focal elevation of free Ca2+ (as observed in cultured cells; Ichas et al., 1997Go), such that high-polarized pericortical mitochondria may first release Ca2+ in response to an electric current and later, after repolarization, in response to IP3-induced CIRC (Abbott et al., 1999Go; Tang et al., 2000Go). If the findings of Ozil and Huneau (2001Go) are relevant to other mammals, including the human, it may be this first release that activates developmentally significant signalling pathways (Berridge et al., 1998Go).

Here, we show that reversible changes in intracellular free Ca2+ and high {Delta}{Psi}m can occur in mature mouse oocytes without a corresponding reduction in ATP levels. If an epigenetic role for Ca2+ is confirmed, it could offer a potentially new perspective on developmental forces in early mammalian embryogenesis and for clinical IVF, a different context in which normal, abnormal and failed development may be investigated with regard to mitochondrial DNA copy number (Reynier et al., 2001Go), complement size and inheritance patterns during cleavage (Van Blerkom et al., 2000Go), and certain structural defects described for the oocytes of women of advanced reproductive age (Muller-Hocker et al., 1996Go).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 31, 2003; resubmitted on June 24, 2003; accepted on July 29, 2003.