ARTICLE |
Correspondence to: Dmitri V. Krysko, Dept. of Human Anatomy, Embryology, Histology and Medical Physics, Faculty of Medicine, Ghent University, Godshuizenlaan 4, B-9000 Ghent, Belgium. E-mail: Dmitri_Krysko@yahoo.com
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Summary |
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Dissipation of mitochondrial membrane potential (m) and release of cytochrome c from mitochondria appear to be key events during apoptosis. The precise relationship (cause or consequence) between both is currently unclear. We previously showed in a model of serum-free cultured granulosa explants that cytochrome c is retained in a subset of respiring mitochondria until late in the apoptotic process. In this study we further investigated the issue of heterogeneity by using the
m-sensitive probe CM-H2TMRos in combination with a DNA fluorochrome. Changes of
m were assessed qualitatively by epifluorescence microscopy and were quantified using digital imaging microscopy. This approach yielded the following results: (a) CM-H2TMRos staining is a reliable and specific procedure to detect
m changes in granulosa cells explants; (b) dissipation of transmembrane potential is an early event during apoptosis preceding nuclear changes but is confined to a subpopulation of mitochondria within an individual cell; (c) in frankly apoptotic cells a few polarized mitochondria can be detected. These findings support the hypothesis that ATP needed for completion of the apoptotic cascade can be generated during apoptosis in a subset of respiring mitochondria and is not necessarily derived from anaerobic glycolysis. (J Histochem Cytochem 49:12771284, 2001)
Key Words: mitochondrial membrane, potential, apoptosis, granulosa cells, CM-H2TMRos, cytochrome c
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Introduction |
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SINCE MITOCHONDRIA WERE DISCOVERED in 1840, they have provided fertile ground for scientific inquiry. Given the importance of mitochondria for cell life, it comes now as no surprise that mitochondrial dysfunction and failure lead to apoptotic and necrotic cell death. Apoptosis and necrosis are two forms of cell death with clearly distinguishing morphological and biochemical features (
There is general agreement that apoptosis, in contrast to necrosis, is an active, energy-requiring process. m and whether the detected heterogeneity of the mitochondrial population with respect to cytochrome c loss is paralleled by a similar heterogeneity at the level of the mitochondrial membrane potential. To measure
m we used a chloromethyl rosamine-derived probe, CM-H2TMRos, which becomes fluorescent only when it is oxidized in the cell. This dye has an alkylating chloromethyl moiety attached. Owing to their membrane potential, functional mitochondria take up the dye. Once the probe accumulates in the mitochondria, the chloromethyl group can react with accessible thiol groups of peptides and proteins to form an aldehyde-fixable conjugate (
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Materials and Methods |
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Isolation and Culture of Granulosa Cell Sheets
Granulosa cell (GC) sheets were prepared from ovarian follicles of adult regularly laying Japanese quail (Coturnix coturnix japonica). The animals were reared under continuous artificial illumination, with food (fresh lettuce and complete breeding food; Biofor AVEVE, Belgium) and water ad libitum. Animal care procedures were conducted in accordance with the guidelines set by the European Community Council Directives 86/6091 EEC. The monolayered granulosal epithelium of the largest preovulatory follicle (F1) was isolated from the surrounding thecal covering as previously described (5.8 cm2 was divided into smaller squares of
4 mm2, followed by culture in 35-mm petri dishes under serum-free conditions for up to 72 hr in humidified room air at 38C. The culture medium was M199 (Sigma; Bornem, Belgium) phenol red-free supplemented with 0.1% w/v bovine serum albumin fraction V (Sigma), 6.0 g/liter HEPES (Acros; Geel, Belgium), 1% v/v penicillinstreptomycin (Gibco BRL, Paisley, UK) at pH 7.4. To inhibit the apoptotic process, the culture medium of controls was supplemented with luteinizing hormone (LH, 100 ng/ml; Sigma) and insulin-like growth factor-I (IGF-I, 10 ng/ml; Sigma) (
Mitochondrial Staining by CM-H2TMRos
CM-H2TMRos (Molecular Probes; Leiden, The Netherlands) was stored desiccated at -20C (following the instructions from the manufacturer) and dissolved in dimethylsulfoxide (DMSO; UCB, Leuven, Belgium) to give a 1 mM stock solution before use. Staining media were prepared immediately before use by adding the dye stock solution to culture medium to obtain the desired final dye concentration of 200 nM. Living GC sheets were incubated with the dye in phenol red-free medium in serum-free conditions for 15, 20, 30, and 60 min at 37C shaking in the dark. Specific mitochondrial staining was obtained after a minimum of 30 min of incubation and was totally absent at shorter incubation times; at 60 min of incubation the signal-to-noise ratio further improved. Therefore, all images shown, as well as quantitative measurements of CM-H2TMRos, are derived from experiments with a 60-min incubation period. After the incubation the GC sheets were gently rinsed in PBS at 37C before fixation in freshly prepared 4% paraformaldehyde in PBS at 37C for 15 min (following the manufacturer's instructions). Thereafter, GC sheets were rinsed in PBS and permeabilized by incubation in ice-cold acetone at -20C for 10 min (following the manufacturer's instructions). This acetone permeabilization step appeared to improve signal retention (data not shown). Finally, GC sheets were rinsed in PBS, mounted in a drop of bidest water, and allowed to air-dry. Samples were examined on a Leica DM IRB/E inverted microscope equipped with epifluorescence optics, suitable filters for FITC and TRITC detection, and an MPS-60 camera.
Double Staining (CM-H2TMRos and DAPI) and Quantification of Apoptotic Nuclei
GC sheets were stained with CM-H2TMRos according to the standard procedure (see above), fixed with paraformaldehyde, permeabilized with acetone, and rehydrated with PBS. Then GC sheets were stained with 2',6'-diamidino-2-fenylindole (DAPI; Sigma) at a concentration of 1 mg/ml in PBS at room temperature for 10 min. Then the GC sheets were mounted on slides and samples were observed under an epifluorescence microscope Leica (see above). Apoptotic cells were identified as cells showing condensed chromatin masses and/or fragmented nuclei. Small groups of apoptotic bodies were counted as remnants of one apoptotic cell. Apoptosis was expressed as the number of apoptotic nuclei per number of total nuclei counted in the same microscopic field and expressed as the percent apoptotic nuclei. This apoptotic index (AI) was averaged for 10 fields, giving a total number of about 1500 cells counted for each independent experiment.
Cytochrome c Staining
Living GC sheets were stained at 37C in culture medium, pH 7.4, for 4090 min with 2 mg/ml 3-3' diaminobenzidine-tetrahydrochloride dihydrate (DAB). For light microscopic evaluation, whole mounts of the sheets were counterstained with methyl green. The DAB technique used in the present study to localize cytochrome c in individual mitochondria was described previously by us (
Pretreatment with Mitochondrial Poisons
Valinomycin (Sigma) was dissolved in 95% ethanol to obtain a 10 mM stock solution. The stock solution was stored in small aliquots at -20C and diluted in culture medium immediately before adding to the cells to obtain a final concentration of 1 mM.
Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; Sigma) was dissolved in ethanol. The stock solution was stored in small aliquots at -20C and diluted in culture medium immediately before adding to the cells to obtain a final concentration of 50 µM.
To investigate whether mitochondrial staining with CM-H2TMRos is dependent on the m, uncultured GC sheets and GC explants cultured for 72 hr supplemented with IGF-I and LH were analyzed in which the
m was dissipated with valinomycin (5 min at 37C) and with FCCP (15 min at 37C).
Quantitative Measurements of CM-H2TMRos Fluorescence
Quantitative measurements of CM-H2TMRos fluorescence were performed by digital imaging epifluorescence microscopy. GCs were viewed with an inverted Nikon Eclipse TE 300 epifluorescence microscope using a x40 oil-immersion lens. CM-H2TMRos fluorescence images were obtained by excitation at 546 nm, reflection off a dichroic mirror with a cut-off wavelength at 564 nm, and longpass emission filtering at 590 nm. Images were captured with an intensified CCD camera (Extended Isis camera; Photonic Science, East Sussex, UK) and were stored in a PC equipped with an image acquisition and processing board (Data Translation, type DT3155; Marlboro, MA).
To study the effect of mitochondrial poisons on CM-H2TMRos fluorescence, images were acquired in the two experimental groups: uncultured GC sheets and GC explants cultured for 72 hr supplemented with IGF-I and LH.
To investigate m changes during apoptosis induction by gonadotropin withdrawal, fluorescence intensity generated by CM-H2TMRos was quantified in 72-hr cultures of GCs compared to control cultures supplemented with IGF-I and LH. All images were corrected for background fluorescence (culture medium without sheets).
Statistical Analysis
Data are expressed as mean ± SEM with n denoting the number of experiments on different animals. Statistical significance was tested using a Student's t-test for unpaired observations. p values less than 0.05 were considered as statistically significant.
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Results |
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Sensitivity of CM-H2TMRos to Mitochondrial Poisons
To confirm that the mitochondrial CM-H2TMRos dye accumulation was dependent on mitochondrial transmembrane potential, uncultured (i.e., freshly isolated) GC sheets and 72-hr IGF-I- and LH-supplemented cultures were treated with the mitochondrial uncoupling agents FCCP and valinomycin. Pretreatment of GC sheets with FCCP or valinomycin before probe loading resulted in absence of punctiform mitochondrial staining and showed only diffuse cytoplasmic staining on visual inspection (Fig 1A, Fig 1E, and Fig 1F). Quantification of the CM-H2TMRos signal under these conditions showed significantly reduced fluorescence intensity with FCCP or valinomycin compared to control (Fig 2A and Fig 2B). Both mitochondrial poisons had no effect on the CM-H2TMRos fluorescence when added after loading of the mitochondrial probe.
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Subcellular Distribution of Mitochondria During Apoptosis
In uncultured GC sheets, staining of polarized mitochondria was intense and the pattern was uniform throughout the sheet. In images of GC sheets stained with CM-H2TMRos in combination with DAPI, mitochondria appeared as threadlike and granular structures homogeneously distributed throughout the GCs. The mitochondrial staining revealed by CM-H2TMRos was similar to the appearance of respiring mitochondria identified by cytochrome c staining (Fig 1A and Fig 1B).
Double staining (CM-H2TMRosDAPI) of 72-hr IGF-I- and LH-supplemented cultures, i.e., the control condition in which the apoptotic process is inhibited (AI of circa 0.64 ± 0.27% SEM; n=4), showed a homogeneous pattern in contrast to the serum-free cultures. All normal cells showed a similar number of polarized mitochondria localized in compact masses at one side of the nucleus (Fig 1C).
Seventy-two-hour cultured GC explants cultured in the absence of IGF-I and LH (AI of circa 29 ± 8.03% SEM; n=4) showed a non-uniform pattern of CM-H2TMRos staining throughout the GC explant cell layer (Fig 1D). Some cells with normal nuclei were surrounded by very few stained mitochondria, whereas other adjacent cells displayed a sizable number of polarized mitochondria localized around nuclei with normal morphology. Apoptotic cells were observed with condensed and fragmented nuclei but still containing polarized mitochondria (Fig 1G). In the majority of the apoptotic cells, however, CM-H2TMRos fluorescence was no longer detected (Fig 1G). In necrotic cells, often present at the edge of the explants and recognized by their small pyknotic nuclei, polarized mitochondria were never observed (Fig 1F).
Quantification of the CM-H2TMRos signal in the 72-hr cultured GC explants revealed that the fluorescence intensity was significantly lower (Fig 3) when IGF-I and LH were absent in the culture medium compared to the control condition (with both survival factors).
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Discussion |
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Cationic lipophilic fluorochromes such as 3,3'-dihexiloxocarbocyanine iodide (DiOC6), rhodamine 123 (R123), and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) have been widely used to assess the functionality of mitochondria in diverse biological scenarios, including differentiation (
Compared to DiOC6 and R123, JC-1 is a reliable and sensitive probe, as shown in several studies. Its loading is impaired by several drugs able to collapse m, which is not the case for DiOC6 and R123 owing to a high sensitivity to changes of plasma membrane potential for DiOC6 and energy-independent binding sites for R123 (
m. We analyzed fluorescence intensity after impairing mitochondrial function with commonly used, mechanism-specific mitochondrial poisons (FCCP, a proton translocator, and valinomycin, a potassium ionophore) in uncultured GC sheets and in 72-hr cultures supplemented with IGF-I and LH. Pretreatment with FCCP and valinomycin before probe loading gave no punctiform mitochondrial staining (Fig 1E) and significantly reduced the intensity of fluorescence (Fig 2A and Fig 2B). A possible explanation for the presence of the diffuse cytoplasmic staining could be that cells still oxidized the probe in their cytoplasm but, as mitochondrial respiration is already uncoupled, the dye is not sequestered in the mitochondria but is distributed in the cytoplasm.
The second argument to support the mitochondrial specificity of the probe can be found in the similarity of the m staining and cytochrome c localization in respiring mitochondria (Fig 1A and Fig 1B). Our data are in agreement with results of
m after fixation.
The second part of our work was to study m changes in the GC explants cultured up to 72 hr under serum-free conditions, which elicits apoptosis. Both the images and the fluorescence measurements show that, in our model system, dissipation of transmembrane potential in individual mitochondria is an early event in GCs not displaying nuclear manifestations of apoptosis. Moreover, we have shown the presence of a subset of polarized mitochondria that exhibited still normal
m until late in the apoptotic process (Fig 1G). These data confirm our previous conclusions on mitochondrial heterogeneity during apoptosis with a subset of respiring mitochondria retaining cytochrome c function until the stage of chromatin condensation and nuclear fragmentation (
treatment in rat hepatocytes expressing an IkB super-repessor that a gradual onset of mitochondrial depolarization in a subpopulation of mitochondria occurred using the tetramethylrhodamine methyl ester (TMRM) probe, in conjunction with calcein, a probe marking MPT in individual mitochondria. The authors concluded that, for several hours during the apoptotic response, hepatocytes contain both polarized and depolarized mitochondria, the latter being the presumptive source of released cytochrome c. These results are perfectly in line with our observations. However, because these authors used a non-fixable mitochondrion-selective probe, TMRM, it was impossible to check whether vital cells with apoptotic nuclear morphology still contain polarized mitochondria as was done in the present study by using the fixable CM-H2TMRos probe and DAPI staining.
Recently, m is a heterogeneous phenomenon both at the single organelle level and at the cellular level, and therefore is not unequivocally related to the execution of the death process but rather is an ancillary event. Several reports described an early
m decrease before the exposure of phosphatidylserine on the cell surface or DNA fragmentation (
m was found in several other models of apoptosis (
In several experimental models, cell stress is accompanied by an early hyperpolarization of mitochondria (
Several authors reported mitochondrial clustering in various apoptosis paradigms as an early event, independent of m changes or cytochrome c release (
In this study we have shown that (a) CM-H2TMRos staining evaluated after aldehyde fixation is a reliable and specific probe to detect m changes in the granulosa explant system, (b) dissipation of transmembrane potential is an early event during apoptosis, preceding nuclear changes, and is again confined to a subpopulation of mitochondria within an individual cell, and (c) the majority of frankly apoptotic cells are devoid of polarized mitochondria, but in some apoptotic cells a few polarized mitochondria can be detected. Necrotic cells do not display polarized mitochondria.
The present findings support the hypothesis that ATP needed for completion of the apoptotic cascade can be generated during apoptosis in a subset of respiring mitochondria and is not necessarily derived from anaerobic glycolysis.
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Acknowledgments |
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Supported by the BOF (Bijzonder Onderzoeksfonds) to KD 01115099. DVK is a recipient of a predoctoral student grant from Universiteitsvermogen R.U.G. P 0211 (FR). LL was supported by the Fund for Scientific Research, Flanders, Belgium (3G023599 and G.0012.01), the Belgian Society for Scientific Research in Multiple Sclerosis (WOMS) (grant no. 51F06700), and Ghent University (grant no. 01115099).
We thank Prof Dr M. Espeel, Ms S. Van Hulle, and Mr H. Stevens for excellent photographic assistance, and Ms B. De Prest for compiling the bibliography and for the cytochrome c staining.
Received for publication January 24, 2001; accepted May 16, 2001.
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