Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 63, 1358-1369 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Identification of Potassium-Dependent and -Independent Components of the Apoptotic Machinery in Mouse Ovarian Germ Cells and Granulosa Cells1

Gloria I. Pereza, Daniel V. Maraveia, Alexander M. Trbovicha, John A. Cidlowskib, Jonathan L. Tillya, and Francis M. Hughes Jr2,,c

a Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114 b Laboratory of Integrative Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 c Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223

ABSTRACT

Recent studies with thymocytes have suggested a critical role for intracellular potassium in the regulation of apoptosis. In this study, we examined the pathways of K+ regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K+ during apoptosis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium-enriched medium prevented condensation, budding, and fragmentation, although it did not block DNA degradation, suggesting the existence of potassium-independent nucleases in oocytes. Culture of granulosa cells in potassium-enriched medium inhibited internucleosomal DNA cleavage, although high-molecular weight DNA cleavage was apparent, suggesting that the nuclease or nucleases responsible for generating 50-kilobase (kb) fragments in these cells is potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but not large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium regulation. In cell-free assays, processing of pro-interleukin-1ß and proteolysis of cellular actin by recombinant caspase-1 and caspase-3, respectively, were suppressed by the presence of 150 mM potassium. Other monovalent ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thus, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events including caspase activity and internucleosomal DNA cleavage. However, there also exist novel potassium-independent pathways in both ovarian germ cells and somatic cells that signal certain apoptotic events, such as large-fragment DNA cleavage.

apoptosis, follicle, granulosa cells, ovum

INTRODUCTION

Apoptosis plays a fundamental role in the development of multicellular organisms and in the maintenance of homeostasis in many tissues (reviewed in [1]). In all vertebrate species examined to date, this form of controlled cellular deletion is the underlying mechanism responsible for female germ cell depletion from the ovary under both normal and pathological conditions (reviewed in [2, 3]). For example, the tremendous levels of oogonium and oocyte attrition that occur in waves during development of the fetal ovary are a result of apoptosis, as defined by both morphological and biochemical criteria [3–5]. Furthermore, accelerated oocyte loss caused by exposure of germ cells to a variety of toxicants, including chemotherapeutic drugs and industrial chemical by-products, is also dependent upon activation of apoptosis driven by discrete signaling molecules known to regulate cell death in other model systems [6–10]. The occurrence of apoptosis in the ovary is not restricted to the germline, however, because the process of postnatal atresia of maturing follicles results from the initiation of apoptosis in the somatic (granulosa) cells that support the oocyte until ovulation [3, 11, 12]. The significance of understanding the regulation of and of manipulating apoptosis in the ovary is exemplified by recent studies demonstrating protection of the female gonads in vivo from cancer therapy-induced damage [8], as well as a dramatic prolongation of ovarian life span into advanced age [13].

It is now generally accepted that apoptosis in most species and cell types is precisely regulated by the actions of a number of intracellular molecules, derived from both active gene transcription (i.e., proteins) and various metabolic events, including mitochondrial respiration (e.g., reactive oxygen species) and release of proapoptotic factors (e.g., cytochrome c, apoptosis-inducing factor), phospholipid turnover (e.g., ceramide, sphingosine-1-phosphate, diacylglycerol), and ion fluxes (reviewed in [14–16]). Despite the diversity and complexity of the events surrounding the induction of cell death, many genetic and biochemical studies have provided evidence that there likely exists an ordered and evolutionarily conserved pathway by which cells activate, execute, and complete the process of self-destruction [1, 14–18]. One universal feature of apoptosis is a loss of cell volume leading to cytoplasmic condensation [19]. Indeed, using glucocorticoid-treated S49-Neo lymphocytes as a model, it was reported that DNA fragmentation only occurred in those cells that exhibited reductions in cell volume and that cell shrinkage in lymphocytes was necessary and sufficient for the initiation of apoptosis [20]. Although the mechanisms underlying reductions in cell volume during apoptosis remain to be elucidated, several studies have directly linked potassium ion efflux from the cell as a precipitating event. For instance, apoptosis induced by treatment of CEM-C7A lymphoblastoid cells with dexamethasone or of L cells with VP-16 is associated with a net loss of intracellular potassium and a concomitant reduction in cell volume [21, 22]. Activity of potassium ion channels appears to regulate the sensitivity of lymphoid cells to extracellular ATP-induced apoptosis [23], and cell shrinkage in eosinophils undergoing apoptosis can be inhibited in a dose-dependent manner by the presence of potassium channel blockers [24].

Until very recently, however, it was unclear whether potassium efflux directly influenced the function or activity of different effectors of apoptosis, in particular the caspase family of cystein proteases and the endonucleases that are associated with most paradigms of cell death. Using glucocorticoid-treated thymocytes, Hughes et al. [25] have reported that inhibition of potassium efflux by disruption of the normal potassium electrochemical gradient prevents caspase-3 activation as well as endogenous nuclease activity. Furthermore, caspase and nuclease activities were found to be restricted to those thymocytes exhibiting reduced intracellular potassium levels and cell shrinkage [25]. This study was shortly followed by a comprehensive evaluation of potassium efflux, caspase-3 activation, nuclease activity, and cell death in S49-Neo lymphoma cells treated with a diverse spectrum of proapoptotic agents [26], collectively supporting the hypothesis that a net loss of potassium from the cell is in fact an early and central step in a conserved cell death program.

In the present study, we have examined the efflux of potassium during the apoptotic death of female germ cells and follicular granulosa cells and explored the consequences of preventing cytosolic potassium efflux by disrupting the potassium electrochemical gradient. Additionally, on the basis of recent studies suggesting the involvement of caspases [2, 3, 27–29], serine proteases [30], and nucleases [30–32] in apoptosis in oocytes and granulosa cells, we evaluated the possibility that potassium directly modulates the activities of the enzymes required for cytoplasmic and nuclear destruction that ultimately produce the apoptotic bodies for phagocytosis. Lastly, cell-free assays were employed using purified deoxyribonuclease (DNase)-I and DNase-II, two enzymes implicated in genome fragmentation during apoptosis [32–34], as well as granulosa cell nuclear protein extracts possessing endogenous nuclease activity [30], to determine whether potassium affects the occurrence of DNA cleavage in ovarian cells by directly suppressing endonuclease activity.

MATERIALS AND METHODS

Materials

Equine CG was obtained from Professional Compounding Centers of America (Houston, TX; oocyte studies), Calbiochem (La Jolla, CA; granulosa cell and follicle studies) or the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases (A.F. Parlow; granulosa cell K+ studies); hCG was obtained from Serono Laboratories (Norwell, MA). The potassium-sensitive dye PBFI (the cell-permeant acetoxylmethyl ester of potassium-binding benzofuran isophthalate) was purchased from Molecular Probes (Eugene, OR). Human recombinant caspase-1 and caspase-3 (generously provided by Dr. W.W. Wong, BASF Bioresearch Corp., Worcester, MA) were prepared as N-His affinity-tagged proteins in Escherichia coli and activated prior to each experiment by dithiothreitol treatment [28, 35]. Recombinant human pro-IL1ß and rabbit polyclonal antibodies that recognize the pro form (numbers 02–1000) and processed form (numbers 02–1100) of human IL1ß were obtained from Cistron Biotechnology (Pine Brook, NJ). Anti-actin monoclonal antibody C4 was from Boehringer-Mannheim (Indianapolis, IN). Doxorubicin (DXR) was from Alexis Biochemicals (San Diego, CA) and was prepared fresh in culture medium before each experiment. All other chemicals and reagents were obtained, unless otherwise noted, from Sigma Chemical Co. (St. Louis, MO).

Oocyte Incubations

As previously described [8, 36], virgin female B6C3F1/CrlBR mice at 7 wk of age (Charles River Laboratories, Wilmington, MA) were superovulated with 10 IU of eCG, followed by 10 IU of hCG 48 h later. Cumulus-oocyte complexes were collected from the oviducts 16 h after hCG injection. Oocytes were denuded of cumulus cells by a 1-min incubation in 80 IU/ml hyaluronidase and then washed three times with human tubal fluid (HTF) culture medium (Irvine Scientific, Santa Ana, CA) supplemented with 0.5% BSA (Fraction V, fatty-acid free). Oocytes were pooled into groups of 10, transferred into 0.1-ml drops of prewarmed culture medium (see below) under paraffin oil, and cultured for 24 h at 37°C in a humidified atmosphere of 5% CO2 and 95% air, without or with 200 nM DXR.

To examine potassium efflux, oocytes were cultured for 20 h without or with 200 nM DXR, and PBFI was added to these cultures (at final concentration of 5 µM) 1 h before analysis by fluorescence microscopy (excitation at 340 nm; emission at 505 nm). For those experiments that involved disrupting the potassium gradient, oocyte incubations were carried out in a modified ("ion-deficient") HTF medium. This medium was prepared at the National Institute of Environmental Health Sciences (NIEHS) Cell Culture Media Facility according to the composition and characteristics of HTF (Irvine Scientific), with the exception that NaCl (normally 101.6 mM) and KCl (normally 4.69 mM) were not added, so that effects of "experimental" KCl, NaCl, or LiCl (each at a final concentration of 150 mM) could be directly assessed. In those cultures lacking inclusion of exogenous KCl, NaCl, or LiCl, mannitol was added to correct for deficiencies in medium osmolality [25, 26].

At the end of the incubation period, the oocytes were either examined alive (PBFI experiments) or fixed for 30 min in neutral-buffered 1% (wt/vol) paraformaldehyde prepared in 1x Dulbecco PBS and checked by differential interference contrast (DIC) microscopy for morphological changes characteristic of apoptosis [8, 36]. The percentage of oocytes that underwent cellular fragmentation out of the total number of oocytes cultured per drop in each experiment was determined. In some experiments, oocytes were further analyzed for the occurrence of DNA cleavage using terminal transferase-mediated DNA 3'-end labeling in situ or the comet assay (see below). All studies involving mice described herein were approved by and performed in accordance with the guidelines of the Massachusetts General Hospital Institutional Animal Care And Use Committee and the NIH Guide for the Care and Use of Laboratory Animals.

In Situ DNA 3'-End-Labeling Analysis

At the end of the incubation period without or with the experimental treatments, oocytes were transferred into Tyrode solution for 30 sec at 37°C to remove the zona pellucida, washed quickly in PBS, and then immediately fixed for 30 min in neutral-buffered 1% (wt/vol) paraformaldehyde prepared in PBS containing 0.1 mg/ml polyvinyl alcohol (average molecular weight of 30 000–70 000). After fixation, oocytes were washed once more with PBS, transferred to Superfrost-Plus slides (Fisher Scientific, Pittsburgh, PA) in small drops (10 oocytes/10 µl drop), and air-dried. Slides were heated at 65°C for 4 h and then stored at 4°C until processed for in situ DNA 3'-end-labeling analysis (ISEL) as detailed elsewhere [37], with slight modifications [36]. Briefly, slide-mounted oocytes were heated at 65°C for 30 min, immediately rehydrated through a graded ethanol series (absolute, 90%, 80%, and 70% ethanol; 20 sec each) to sterile water, and then treated with proteinase K (10 g/ml) at 37°C for 30 min, followed by two washes in sterile water. To block for nonspecific binding, slide-mounted oocytes were preincubated with 3% (wt/vol) BSA for 30 min at 20°C and then pre-equilibrated with 1x terminal deoxynucleotidyl transferase (TdT) reaction buffer (Boehringer-Mannheim) for 20 min at 20°C. The TdT-mediated labeling reaction of DNA 3' ends was performed by incubating the slide-mounted oocytes in the presence of 1.25 U/µl TdT enzyme (Boehringer-Mannheim; with TdT reaction buffer and CoCl2 supplied with the enzyme) and 50 pM fluorescein-labeled dUTP (Boehringer-Mannheim) at 37°C for 15 min in the dark. After 3'-end labeling, the slides were placed in 1x-concentrated TE buffer (10 mM Tris-HCl, 100 mM EDTA, pH 8) to stop the reaction and then rinsed several times with sterile water. Excess water was blotted away, mounting medium was added, and the slides were sealed with coverslips. The occurrence of DNA cleavage was assessed by fluorescence microscopy using a fluorescein filter.

Comet Assay

The comet assay protocol developed for analysis of DNA cleavage in single human sperm [38] was followed with minor modifications. Briefly, after the 24-h incubation without or with experimental treatments, oocytes were treated with Tyrode solution for 30 sec at 20°C, followed by one wash with PBS. Immediately afterward, oocytes within each experimental treatment group were pooled (5–10 oocytes per pool) and mixed with 30 µl of 1% low-melting point agarose (Boehringer-Mannheim) previously prepared in comet assay electrophoresis buffer (45 mM Tris-HCl, 45 mM boric acid, 1.25 mM EDTA, pH 10) and maintained at 37°C. The oocytes, in agarose, were carefully pipetted onto Superfrost-Plus slides and air-dried for 2–3 days at room temperature. Slide-mounted oocytes in hardened agarose were then rehydrated and incubated in comet assay lysis buffer (2.5 M NaCl, 1% N-lauroylsarcosine, 10 mM EDTA, 10 mM Tris-HCl, 1% Triton X-100, 13.3 µg/ml proteinase K, pH 10) for 1 h at 20°C, followed by an additional 20-h incubation at 37°C. At the end of incubation, the gels, still attached to the slides and containing the oocytes, were transferred to a horizontal electrophoresis chamber containing comet assay electrophoresis buffer, equilibrated for 30 min at 20°C, and then subjected to electrophoresis at 25 V for 40 min. Afterward, slides were transferred to distilled water for 30 min, stained with ethidium bromide (50 µg/ml in PBS) for 40 min, and then gently washed once with distilled water. Excess water was blotted away, mounting medium was added, and slides were sealed with coverslips. The occurrence and pattern of DNA migration out of the permeabilized oocytes was viewed under ultraviolet (UV)-fluorescence microscopy.

Granulosa Cell and Follicle Incubations

Immature (25-day-old) female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were housed in environmentally-controlled rooms with food and water ad libitum. Upon arrival, rats were given a single s.c. injection of 10 IU of eCG to promote growth of a cohort of healthy antral follicles over a subsequent 46-h period [11, 39]. For analysis of potassium efflux in individual granulosa cells, cells were collected by needle puncture of the largest follicles present within the gonadotropin-stimulated ovaries, washed, and cultured for 24 h at 1 x 106 cells/ml in McCoy-5a medium containing 100 U/ml penicillin and 75 U/ml streptomycin sulfate. The cells were cultured in chambers constructed by placing double-stick tape across poly-L-lysine-coated slides and suspending a coverslip between the pieces of tape. Infusions of cells, treatments, and washes were carried out by placing the solution on one side and allowing capillary action to draw the liquid in. One hour before analysis, PBFI was added to a final concentration of 5 µM. After a 1-h incubation, the cells were washed in PBS containing trypan blue to allow discrimination between cells undergoing apoptosis versus those that had undergone necrosis. Slides were then analyzed by fluorescence microscopy (excitation at 340 nm; emission at 505 nm).

To analyze apoptosis in granulosa cells of whole follicles, healthy antral follicles between 700–800 µm in diameter were isolated using nonenzymatic dissection, as detailed elsewhere [39]. Briefly, the 8–10 largest follicles in each ovary were isolated with watchmaker's forceps under a dissecting microscope and then cleaned of adherent stromal tissue and smaller follicles. Once isolated, follicles were sized for homogeneity and either snap-frozen immediately (Time 0, no incubation) or incubated under serum-free conditions for 24 h at 37°C. For these experiments, control groups of follicles were incubated in a modified ("ion-deficient") RPMI medium lacking NaCl (normally 103 mM) and KCl (normally 5.4 mM), prepared in the NIEHS Cell Culture Media Core Facility, supplemented with 0.1% BSA, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. For control cultures, mannitol was added to a final concentration of 300 mM to maintain osmolality at approximately 380 mOs. To assess the effects of the different cations on apoptosis, concentrated stock solutions of KCl, NaCl, or LiCl were added to the ion-deficient RPMI at a final concentration of 150 mM each (average osmolality of 375 ± 5 mOs). To examine the dose-response relationship of potassium concentration to DNA cleavage in granulosa cells, ion-deficient RPMI was supplemented with KCl so that final concentration of potassium reached 50, 100, or 150 mM with osmolality held constant by addition of the appropriate concentration of mannitol where needed (i.e., for 50 mM KCl, the medium also contained 200 mM mannitol; for 100 mM KCl, the medium also contained 100 mM mannitol; and for 150 mM KCl, no mannitol was added).

After incubation, follicles were collected in 12- x 75-mm polypropylene tubes, snap-frozen, and stored at -80°C until processed for analysis of low-molecular weight DNA integrity by 3'-end radiolabeling and conventional agarose gel electrophoresis (CAGE; see below) or were embedded in 0.5% agarose plugs for high-molecular weight DNA analysis after pulsed-field gel electrophoresis (PFGE; see below). For the PFGE and CAGE analyses, genomic DNA present in Time 0 follicles (no incubation) served as control data points for levels of background DNA cleavage present before the experimental manipulations in vitro. Additionally, some follicles were also fixed, embedded in paraffin, sectioned, and mounted to glass slides for fluorescence microscopy of nuclear morphology (see below). All studies involving rats described herein were approved by and performed in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees at the Massachusetts General Hospital, the University of North Carolina-Charlotte, or the NIEHS.

Conventional Agarose Gel Electrophoretic Analysis of Internucleosomal DNA Cleavage

Genomic DNA was prepared from intact follicles or isolated nuclei as described elsewhere [40]. The quantity and purity of the DNA preparations were estimated by spectrophotometric measurements of the optical density of each sample at 260 versus 280 nm. Following isolation and quantitation, DNA samples (1 µg/reaction) were 3'-end labeled with [{alpha}32P]dideoxy-ATP (3000 Ci/mmol; Amersham, Arlington Heights, IL) by using the terminal transferase reaction (Boehringer-Mannheim), fractionated through conventional 2% agarose gels, and then analyzed by autoradiography and ß-counting of low-molecular weight (<10 kb) DNA fragments, as described elsewhere [37, 40].

Pulsed-Field Gel Electrophoretic Analysis of High-Molecular Weight DNA Cleavage

As we have previously described for this model system [30], follicles were embedded in agarose plugs (50-µl total volume, 0.5% final) and immediately immersed in 10 ml of PFGE lysis buffer (100 mM EDTA, 1% N-lauroyl-sarcosine) for overnight incubation at 37°C. Plugs were then removed, immersed in 1 ml of PFGE lysis buffer containing 50 µg/ml of proteinase-K, and incubated at 50°C for an additional 12 h. After proteolytic digestion, plugs were pre-equilibrated in 0.5x TBE (0.89 M Tris-HCl, 0.89 M boric acid, 2.5 mM EDTA) for at least 3 h and then subjected to PFGE using a clamped homogeneous electric field (CHEF) pulsed-field system (Bio-Rad Laboratories, Hercules, CA) for 19 h at 14°C and 6 V/cm, with a linear-switch interval ramp from 0.5 to 45 sec. Size standards included chromosomes from S. cerevisiae and DNA size standards provided by Bio-Rad. After PFGE, DNA was visualized by ethidium bromide staining and UV transillumination.

Analysis of Granulosa Cell Nuclei in Incubated Follicles

Follicles isolated from gonadotropin-primed ovaries were either immediately fixed (4% neutral-buffered paraformaldehyde) or were fixed after incubation without or with 150 mM KCl, as described above. Individual follicles were then embedded in paraffin, sectioned (6 µm), and mounted on a glass microscope slide. Sections were then deparaffinized, rehydrated, and stained with the DNA-binding fluorescent dye, 4',6'-diamidino-2-phenylindole (DAPI), at a final concentration of 0.4 µg/ml in 1x Dulbecco PBS (pH 7.5) for 10 min at 20°C. The samples were then examined by UV-fluorescence microscopy for evidence of nuclear condensation (brightly fluorescent pyknotic nuclei) associated with apoptosis [41].

Cell-Free Caspase Activity Assays

Using assays identical to those recently reported [27], caspase-1 activity was assessed by monitoring the conversion of pro-IL1ß (33 kDa) to the active cytokine (17 kDa) after incubation of pro-IL1ß with 1 µg of active caspase-1 in the absence or presence of 150 mM KCl, NaCl, or LiCl. Caspase-3 activity was measured by analyzing the extent of cleavage of actin present in heat-inactivated crude protein extracts (50 µg/reaction) prepared from eCG-primed rat ovarian granulosa cells after a 30-min incubation at 37°C with 1 µg of active caspase-3 in the absence or presence of 150 mM KCl, NaCl, or LiCl. The extent of cleavage of pro-IL1ß or actin was determined by immunoblot analysis by the enhanced chemiluminescence system (Amersham).

Rat Granulosa Cell Nuclear Autodigestion Assays

For each experimental replicate, nuclei were isolated as previously described [30] from approximately 1.4 x 107 granulosa cells (six eCG-primed ovaries) in 1 ml of TSN buffer (10 mM Tris-HCl, pH 7.4); 25 mM NaCl; and 0.34 M sucrose) by four strokes with a tight-fitting pestle in a Kontes glass homogenizer, followed by centrifugation at 800 x g for 10 min at 4°C. The crude cytoplasmic extract (supernatant) was removed, and the crude nuclear pellet was gently resuspended in 1.4 ml of TSN. Aliquots of the resuspended nuclei (100 µl) were dispensed into 1.5-ml tubes and then preincubated without or with 150 mM KCl, NaCl, or LiCl. An equal volume (110 µl) of 10 mM Tris-HCl (pH 7.4) or CaCl2/MgCl2 (10 mM each in 10 mM Tris-HCl, pH 7.4; 5 mM each final for the autodigestion assay) was then added to the nuclei suspensions. Incubations for granulosa cell nuclear autodigestion were carried out at 37°C for 30 min, after which nuclei were pelleted by centrifugation (5 min at 4°C; 800 x g). The supernatants were removed and discarded, and the pellets were snap-frozen for radiolabeling and CAGE. Nuclei not incubated served as Time 0 data points (i.e., background DNA cleavage) for the assays [30].

Preparation of Granulosa Cell Nuclear Protein Extracts and Plasmid DNA Degradation Assays

Granulosa cells were harvested from eCG-primed rat ovaries, and nuclei were isolated as described earlier (see Rat Granulosa Cell Nuclear Autodigestion Assays). Nuclear extracts were prepared by freeze-thawing isolated nuclei in TSN/CaCl2/MgCl2 buffer, followed by a 37°C incubation for 30 min [30]. Debris, chromatin, and membranes were pelleted by ultracentrifugation (100 000 x g) for 30 min at 4°C, and the resultant supernatant containing nuclear proteins was collected and assessed for protein content. To test for nuclease activity [42], the pUC18 plasmid (Stratagene, La Jolla, CA) was linearized with the SmaI restriction enzyme and added to tubes (1 µg/reaction) containing 3 µg of GC nuclear protein extract without or with KCl, NaCl, or LiCl (150 mM final) or 1 mM sodium aurothiomalate (SAM—a previously characterized nuclease inhibitor, [30]) in a total volume of 10 µl (in 50 mM Tris-HCl, 1 mM MgCl2, and 1 mM CaCl2). Samples were incubated for 1.5 h at 37°C, after which 20 µg of proteinase-K (1 µl of a 20 mg/ml stock) was added, followed by an additional incubation at 55°C for 1 h. Samples were then resolved through 1.0% agarose gels (1.5 h, 80 V), stained with ethidium bromide, and visualized by UV transillumination.

Plasmid DNA Degradation Assays with Purified Nucleases

To further test for effects of potassium on nuclease activity, the pBSKII plasmid (Stratagene), linearized with the EcoRI restriction enzyme (Boehringer-Mannheim), was used as a substrate for nuclease attack. The reaction buffer, consisting of 100 mM Tris-HCl (pH 7.5 for DNase-I and pH 4.6 for DNase-II), 10 mM CaCl2, and 10 mM MgCl2, was prepared without or with 0.01–0.1 U of DNase-I (Boehringer-Mannheim) or 0.01–0.1 U DNase-II (Calbiochem). The nucleases were preincubated for 5 min at 37°C in the absence or presence of KCl, NaCl, or LiCl, each at a final concentration of 150 mM. Sodium aurothiomalate (1 mM final) was also included as a positive control for these experiments because we have recently shown that this compound is a potent nuclease inhibitor [30]. Linearized plasmid was then added to the tubes (0.8 µg/reaction, 20 µl total reaction volume), and samples were incubated for 3 min at 4°C (DNase-I) or 37°C (DNase-II), mixed with 2 µl of 0.5 mM EDTA and 1.3 ml 10% sodium dodecylsulfate, and placed on ice to stop the reaction. Samples were then quickly mixed with gel-loading buffer, electrophoresed through 1% agarose gels (1 h, 100 V), stained with ethidium bromide, and visualized by UV-transillumination. A 1-kb DNA ladder (Gibco-BRL Life Technologies) was included for DNA size estimates.

Data Presentation and Analysis

All experiments were independently replicated at least three times. For qualitative analysis, a representative autoradiogram or photograph is presented where appropriate. Quantitative results represent the mean ± SEM of combined data from the replicate experiments. Statistical differences (P < 0.05) between mean values were analyzed by one-way analysis of variance, followed by Scheffe's F-test.

RESULTS

Potassium and Apoptosis in Germ Cells (Oocytes)

We have recently used in vitro culture of oocytes as a model to dissect the intracellular pathways responsible for chemotherapy-induced female germ cell apoptosis [3, 8, 9, 36]. Apoptosis is clearly apparent in these cells by observing the retraction of the oolemma away from the zona pellucida (cytoplasmic condensation), membrane budding, chromatin cleavage, and eventual fragmentation of the oocyte into apoptotic bodies of unequal sizes (Fig. 1B). Oocytes cultured in medium alone exhibited negligible levels (<3%) of apoptosis after 24 h (Figs. 1A and 2), whereas exposure of oocytes to 200 nM DXR for 24 h induced cellular fragmentation in over 60% of the oocytes (Fig. 2B). To visualize changes in intracellular potassium, the cells were loaded with a potassium-binding fluorescence indicator dye (PBFI) 1 h before microscopic analysis. Control (non-drug treated) oocytes stained brightly and uniformly with PBFI, whereas those stimulated to die by DXR treatment consistently displayed reduced fluorescence indicative of a net loss of intracellular potassium (Fig. 3). The decrease in intracellular potassium occurred coincident with a decrease in cell size and prior to cell budding (and remained low in oocytes that had undergone fragmentation into apoptotic bodies), consistent with the proposed role for the loss of this ion as an early death event mediating cell shrinkage.



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  		FIG. 1. Cytochemical analysis of mouse oocytes treated with DXR in the absence or presence of potassium. A–C) Representative DIC photomicrographs of oocytes after a 24-h incubation in vehicle (control, A), 200 nM DXR (B), or 200 nM DXR in the presence of 150 mM KCl (C). Note that DXR-induced oocyte fragmentation into apoptotic bodies is completely prevented by potassium. D–F) Chromatin cleavage, as assessed by the comet assay, in oocytes incubated in control medium (D) or in medium containing 200 nM DXR in the absence (E) or presence of 150 mM KCl (F). Note that although the plume of low-molecular weight DNA fragments visible in the DXR-treated oocyte (E) is absent when oocytes are coincubated with 150 mM KCl (F), some level of DNA fragmentation still occurs in oocytes treated with DXR in the presence of potassium (evidenced by the electrophoretic shift of chromatin to the oocyte plasma membrane). G–I) DNA cleavage, as assessed by ISEL, in control oocytes (G), oocytes incubated with DXR (H), or DXR with 150 mM KCl (I). Note that ISEL detects DNA cleavage in DXR-treated oocytes in the absence or presence of KCl. These data are representative of results obtained in three independent experiments



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  		FIG. 2. Quantitative analysis of the effects of potassium on the incidence of in vitro murine oocyte fragmentation induced by DXR. Pools of mature (metaphase II) oocytes harvested by superovulation from adult female mice were denuded of cumulus cells and then cultured without or with DXR (DXR, 200 nM) in the absence or presence of 150 mM KCl, NaCl, or LiCl for 24 h. After culture, the number of oocytes that underwent fragmentation out of the total number of oocytes cultured in each treatment group was determined by light-microscopic analysis. The data represent the combined results from at least four independent experiments (mean ± SEM) with the total number of oocytes analyzed per group provided over the respective bar (*P < 0.05 versus DXR alone)



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  		FIG. 3. Representative fluorescence microscopic analysis of intracellular potassium levels in control (A) and DXR-treated (B) oocytes. Oocytes were cultured without (A) or with (B) DXR (200 nM) for 20 h, after which PBFI was added to the culture at a final concentration of 5 µM. Cells were examined 1 h later using live-mount microscopy with an excitation of 340 nm and an emission of 505 nm. Arrows indicate drug-treated oocytes that have undergone fragmentation

To assess the requirement of potassium loss for the biochemical and morphological changes associated with apoptosis, oocytes were cultured in ion-deficient medium supplemented with 150 mM KCl to disrupt the electrochemical gradient of this ion. Data from other experimental models have shown that this medium will prevent potassium efflux during apoptosis and consequently will suppress the activation of apoptotic enzymes [25, 26]. Likewise, incubation of oocytes in this medium completely prevented both basal and DXR-induced oocyte fragmentation (Figs. 1C and 2). The ability of 150 mM KCl to prevent oocyte fragmentation was mimicked by 150 mM LiCl but not by 150 mM NaCl (the latter being essentially normal culture medium; Fig. 2). Interestingly, however, some level of chromatin cleavage still occurred in DXR-treated oocytes after coculture with 150 mM KCl, as revealed by both the comet assay (Fig. 1F) and ISEL analysis (Fig. 1I), despite the complete absence of cellular fragmentation (Fig. 1C). These results differ from what has previously been found in thymocytes and suggest the existence of a novel potassium-independent pathway leading to DNA degradation in oocytes.

Potassium and Ovarian Somatic (Granulosa) Cell Apoptosis

To explore the possibility that this novel potassium-independent pathway for nuclear destruction occurs in nongermline ovarian cell types as well, we next examined potassium movement during granulosa cell death induced by serum-free culture in vitro. After 24 h in culture, healthy (nonapoptotic, noncondensed) granulosa cells stained brightly with PBFI, whereas shrunken (apoptotic) or necrotic (trypan blue-positive) granulosa cells stained only lightly with PBFI (Fig. 4). To extend these observations, we next examined a follicle culture model that has been extensively used to characterize the pathways responsible for the regulation of granulosa cell death during atresia (reviewed in [3]). Healthy (nonatretic) follicles possessed intact DNA, as assessed by both CAGE (low-molecular weight DNA; Fig. 5A) and PFGE (high-molecular weight DNA; Fig. 6A). Incubation of follicles for 24 h in serum-free medium induced high- and low-molecular weight DNA cleavage indicative of apoptosis (oFigs. 5A and 6A). Inclusion of 150 mM KCl in the incubation medium completely prevented internucleosomal DNA cleavage over the 24-h culture period (Fig. 5A). The actions of KCl on suppressing internucleosomal DNA cleavage were found to be concentration dependent (Fig. 5B) and could be reproduced by using 150 mM LiCl. In contrast to the results seen with oocytes, NaCl was somewhat effective in suppressing DNA degradation but not to the extent of KCl or LiCl (Fig. 5A).



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  		FIG. 4. Representative fluorescence microscopic analysis of intracellular potassium levels in healthy and apoptotic granulosa cells. Granulosa cells were cultured 24 h without serum or other trophic support to induce apoptosis. A) A phase-contrast micrograph of the cultured cells; B is the same field viewed by fluorescence microscopy after PBFI loading. Within the population of cells, three distinct subpopulations are apparent: large healthy granulosa cells that fluoresce brightly (large arrows), shrunken apoptotic cells that weakly fluoresce (small arrows), and trypan blue-positive (necrotic) cells that also show weak fluorescence (arrowhead). Magnification x400



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  		FIG. 5. Representative autoradiograms depicting the effects of KCl, NaCl, and LiCl (A) or the concentration-dependent effects of KCl (B) on internucleosomal DNA cleavage in rat ovarian follicles incubated for 24 h without trophic hormone support. A) Follicles were snap-frozen immediately (Time 0) or were incubated in normal RPMI culture medium without added mannitol (CON/-MAN) or in "ion-deficient" RPMI supplemented with 300 mM mannitol (CON/+MAN), 150 mM KCl, 150 mM NaCl, or 150 mM LiCl. After incubation, follicles were collected and processed for autoradiographic analysis of DNA cleavage as described in Materials and Methods. B) Follicles were snap-frozen immediately (Time 0) or were incubated in "ion-deficient" RPMI supplemented with mannitol in the absence or presence of increasing concentrations of KCl (osmolality was held constant by modifying the ratio of mannitol:KCl added; see Materials and Methods for details). After incubation, follicles were collected and processed for autoradiographic analysis of DNA cleavage.



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  		FIG. 6. Representative analysis of high-molecular weight DNA integrity and nuclear morphology in cells of rat ovarian follicles incubated without trophic hormone support in the absence or presence of potassium. A) Pulsed-field gel electrophoretic analysis of high-molecular weight DNA cleavage in rat ovarian follicles prior to incubation (Time 0) and after a 24-h incubation in normal RPMI culture medium (CON/-MAN) or in "ion-deficient" RPMI supplemented with 300 mM mannitol (CON/+MAN), 150 mM KCl, 150 mM NaCl, or 150 mM LiCl. A DNA size standard (MARKER) is included on the left side of the gel for estimations of DNA fragment size (note that the fastest migrating standard is approximately 48 kb). B–D) Fluorescence microscopic analysis of nuclear morphology (using 4',6'-diamidino-2-phenylindole [DAPI]) in granulosa (gc) and theca-interstitial (tic) cells of rat ovarian follicles before culture (B) or after a 24-h incubation in the absence (C) or presence of 150 mM KCl (D). Note the widespread cell death and nuclear pyknosis in the KCl-treated follicle, despite the complete absence of internucleosomal DNA cleavage (Fig. 5).

However, PFGE analysis revealed that none of the ions tested prevented high molecular weight DNA fragmentation in cultured follicles, although an attenuation of 50-kb fragment accumulation was consistently noted in KCl- and LiCl-treated follicles compared with the case of controls (Fig. 6A). These results suggest that a potassium-independent pathway or pathways of DNA degradation is also present in ovarian somatic (granulosa) cells, findings confirmed and extended by fluorescence analysis of nuclear morphology that revealed that pyknosis (brightly fluorescent, condensed nuclei) remained widespread in the granulosa and theca-interstitial cell layers of follicles incubated with 150 mM KCl (Fig. 6D).

Effects of KCl on DNA Cleavage in Isolated Granulosa Cell Nuclei

The above results suggest the presence of a potassium-dependent pathway leading to internucleosomal DNA cleavage and a potassium-independent pathway that catalyzes large DNA fragment generation. To examine the mechanism by which intracellular potassium levels suppress internucleosomal DNA cleavage, we switched to a cell-free nuclear autodigestion assay. In these experiments, nuclei were prepared from nonapoptotic granulosa cells; incubated with calcium and magnesium in the absence or presence of KCl, NaCl, or LiCl; and then assayed for both low- (internucleosomal; CAGE) and high- (PFGE) molecular weight DNA cleavage. In the absence of divalent (and monovalent) cations, no evidence of DNA cleavage was detected (Fig. 7). Addition of calcium and magnesium resulted in extensive high- and low-molecular weight DNA cleavage, whereas inclusion of KCl, NaCl, or LiCl completely prevented the internucleosomal DNA cleavage activated by calcium and magnesium (Fig. 7A). However, high-molecular weight DNA fragments remained detectable (Fig. 7B), further supporting the potassium-independent nature of large DNA fragment generation in granulosa cells.



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  		FIG. 7. Representative conventional agarose and pulsed-field gel electrophoretic analyses of the effects of potassium on DNA cleavage in isolated rat granulosa cell nuclei. Nuclei prepared from nonapoptotic granulosa cells were incubated without calcium and magnesium (-Ca2+/Mg2+) or with calcium and magnesium (+Ca2+/Mg2+) in the absence or presence of 150 mM KCl, NaCl, or LiCl. After incubation, nuclei were processed for CAGE (A) or PFGE (B) to assess the occurrence and extent of low- and high-molecular weight DNA cleavage, respectively. B) A DNA size standard (MARKER) is included on the left side of the gel for estimations of DNA fragment size (note that the fastest migrating standard is approximately 48 kb). Integrity of DNA in nuclei before incubation (Time 0) is shown in both panels for comparative purposes.

Direct Inhibition of Nuclease Activity by KCl

Because it was possible that the potassium-mediated inhibition of internucleosomal DNA cleavage in isolated granulosa cell nuclei could be indirect via a suppression of nuclear protease activity, two final experiments were conducted to further examine the possible direct actions of KCl on nuclease activity. Consistent with data recently reported using a cell-free assay to monitor granulosa cell nuclease activity [30], nuclear protein extracts prepared from nonapoptotic granulosa cells catalyzed cleavage of linearized plasmid DNA (Fig. 8). However, inclusion of 150 mM KCl, NaCl, or LiCl in the reaction mixture completely suppressed nuclease activity present in the granulosa cell nuclear protein extracts (Fig. 8). Because all nuclease activity in this assay was suppressed by potassium, it may be that the large-fragment cleavage enzyme was not efficiently extracted from the granulosa cell nuclei or that the large-fragment cleaving nuclease requires DNA to be in a chromatin conformation in order to be active.



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  		FIG. 8. Potassium-mediated suppression of plasmid DNA degradation catalyzed by granulosa cell nuclear protein extracts. Representative photograph depicting a conventional agarose gel electrophoretic analysis of linearized plasmid DNA (intact plasmid is indicated by the arrow) after incubation with vehicle (control, CON) or with granulosa cell nuclear protein extracts in the absence or presence of 150 mM KCl, NaCl, or LiCl. Note the smear of DNA degradation products generated by the nuclear protein extract nuclease or nucleases and the inhibition of plasmid degradation by the presence of KCl, NaCl, and LiCl.

These data were reinforced by the results of the second set of experiments, in which the actions of KCl, NaCl, and LiCl were assessed in a simple system containing only ions, purified nucleases, and naked linearized plasmid. Incubation of plasmid DNA with either DNase-I (Fig. 9, A and B) or DNase-II (Fig. 9, C and D) resulted in a rapid degradation of the plasmid, with the extent of degradation dependent upon amount of enzyme used (Fig. 9, B and D) and the length of reaction (data not shown). At low levels of added nuclease (0.01 U/reaction), inclusion of 150 mM KCl, NaCl, or LiCl in the reaction completely suppressed plasmid DNA degradation (Fig. 9A; data not shown for 0.01 U DNase-II), and these effects were mimicked by the general nuclease inhibitor SAM (Fig. 9A). The direct suppressive effects of these ions could be overcome, however, because all three ions tested were unable to suppress plasmid DNA degradation catalyzed by high amounts of nuclease (0.1 U/reaction; Fig. 9C; data not shown for 0.1 U DNase-I) whereas SAM remained an effective inhibitor of nuclease activity (Fig. 9C). The ability of 150 mM KCl to inhibit plasmid DNA degradation catalyzed by increasing amounts of DNase-I or DNase-II is shown in Figure 9, B and D, respectively.



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  		FIG. 9. Effects of potassium on plasmid DNA degradation catalyzed by purified DNase-I or DNase-II. A) Linearized plasmid DNA was incubated without nuclease (-DNase I) or with 0.01 U of DNase-I in the absence (CON, control) or presence of 150 mM KCl, NaCl, or LiCl or with 1 mM SAM. The reactions were then subjected to CAGE, and gels were stained with ethidium bromide to visualize the DNA. Note that the linearized plasmid (indicated by the arrow) is cleaved by DNase-I and that this is prevented by inclusion of KCl, NaCl, LiCl, or SAM in the reaction. Similar data were obtained with 0.01 U of DNase-II (not shown; see D for KCl data). B) Reactions were prepared as described in A, with the exception that increasing amounts of DNase-I were mixed with plasmid DNA in the absence (CON) or presence of a fixed concentration of KCl (150 mM). The ability of KCl to suppress nuclease activity was lost with increasing amounts of nuclease added. C) Plasmid DNA degradation reactions were prepared as described in A using 0.1 U of DNase-II in place of DNase-I. Note that despite the fact that SAM maintains the ability to inhibit plasmid DNA degradation catalyzed by this level of DNase-II activity, KCl, NaCl, and LiCl are without effect. Similar data were obtained with 0.1 U of DNase-I (not shown; see B for KCl data). D) Linearized plasmid was incubated with increasing amounts of DNase-II, as described for DNase-I in B, in the absence or presence of 150 mM KCl. Similar to the case of results obtained with DNase-I (B), the ability of KCl to suppress DNase-II activity was lost with higher amounts of the nuclease

Modulation of Caspase Activity by KCl

Caspases are believed to be central to the execution of apoptosis in a wide variety of cell types (reviewed in [43]), including germ cells and granulosa cells of the ovary [2, 3]. Moreover, these enzymes are believed to function upstream of DNA cleavage, with release of caspase-activated DNase from its inhibitor protein offered as an example [44]. Therefore, we next analyzed the sensitivity of recombinant caspases to inhibition by potassium and other ions using cell-free proteolysis assays, as previously described [27]. Cleavage of pro-interleukin (IL)-1ß to its mature form is a major function for caspase-1 and a clear indicator of its activity. As shown in Figure 10A, incubation of pro-IL1ß with 1 µg of recombinant caspase-1 resulted in rapid cleavage of the protein to the mature cytokine. The presence of 150 mM KCl, NaCl, or LiCl in the assay mixture inhibited caspase-1-mediated cleavage of pro-IL1ß, although under these experimental conditions, the extent of inhibition was not complete because first-site cleavage was observed in all cases (Fig. 10A). However, cleavage of pro-IL1ß by lower concentrations of caspase-1 (100 ng) was completely suppressed by all of the ions tested (data not shown). Similarly, we observed that the ability of 1 µg of recombinant caspase-3 to catalyze cleavage of cytoplasmic actin was inhibited by the presence of 150 mM KCl, NaCl, or LiCl (Fig. 10B).



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  		FIG. 10. Potassium-mediated suppression of caspase activity in cell-free substrate cleavage assays. A) Inhibitory effects of 150 mM KCl, NaCl, or LiCl on the sequential processing of recombinant pro-IL1ß (33 kDa) to the mature cytokine (17 kDa) catalyzed by 1 µg of recombinant caspase-1. B) Inhibition of caspase-3-mediated cleavage of actin (intact protein, 42 kDa; first cleavage product, 41 kDa), present in crude cytoplasmic extracts prepared from rat granulosa cells, by 150 mM KCl, NaCl, or LiCl

DISCUSSION

Recent evidence, derived primarily from studies of cultured thymocytes, suggests that a major efflux of intracellular potassium occurs in the early stages of apoptosis and that this efflux is required for the activation of key components of the cell death machinery [25, 26, 45, 46]. In the present study, we undertook a series of experiments to assess potassium levels during apoptosis of ovarian germ cells and granulosa cells and the role of this ion in controlling several morphological and biochemical characteristics of cell death. Using anti-cancer drug-treated murine oocytes as a model for female germ cell apoptosis [3, 8, 9, 36], we observed a consistent decrease in intracellular levels of potassium as the oocytes died. As recently shown in thymocytes [25, 26], placing cells in medium containing a high level of K+ prevented K+ efflux during apoptosis and blocked many features of apoptosis, such as cellular shrinkage and fragmentation [25, 26]. This high-K+ medium was also effective in blocking many aspects of apoptosis in oocytes and granulosa cells, suggesting similar K+-dependent apoptotic pathways in these cells. Unlike thymocytes, however, some apoptotic events in oocytes, such as DNA degradation, were not completely blocked, suggesting that both potassium-dependent and potassium-independent pathways function in germ cells to mediate different apoptotic events.

Interestingly, suppression of oocyte condensation and fragmentation could also be reproduced with LiCl, but not with NaCl, indicating some level of ion selectivity for these effects. Although previous in vitro studies with thymocytes indicated that the cell death endpoints studied were equally affected by all monovalent cations [25, 26], the general similarity in the results across these two studies suggest that ionic strength, as opposed to ionic specificity, is the main modifier. The importance of potassium in vivo is therefore probably due to its high ionic concentration, as opposed to the presence of potassium per se, in the cytoplasm of viable cells [46]. The finding that LiCl was as effective as KCl in preventing some of the biochemical and morphological events associated with apoptosis may be related to the fact that lithium can pass through some types of potassium channels [47], resulting in an exchange of potassium for lithium inside the cell. Furthermore, recent studies have shown that LiCl protects rat cerebellar granule cells from apoptosis induced by a number of external stimuli [48], collectively suggesting that lithium may indeed have pharmacologic similarity to potassium action in this regard. The relative ineffectiveness of NaCl in suppressing apoptotic effects is not surprising because the NaCl medium is essentially normal medium, and the cell expends considerable energy to maintain the Na+ gradient across the membrane. The mechanisms employed are not circumvented in this study, and thus NaCl in the medium would not be expected to affect apoptosis. It should be noted that a small effect of NaCl on DNA degradation was detected in rat follicles, although it was not as strong or complete as seen with K+ or Li+. The ability of Na+ to inhibit apoptotic enzymes in vitro further suggests that ionic strength, as opposed to ionic specificity, is the main modifier of the apoptotic program.

The analysis of the potassium-independent chromatin cleavage patterns in oocytes proved intriguing. Although potassium apparently did not completely prevent DNA degradation, it did alter its pattern of electrophoretic movement as assessed by the comet assay. On the basis of the results of the ISEL analysis, we conclude that potassium prevents activation and/or activity of the endonuclease or endonucleases responsible for final degradation of oocyte chromatin to low-molecular weight fragments (as revealed by the absence of a plume of DNA outside of the oocyte when assessed by the comet assay) but that higher order DNA cleavage still occurs (as assessed by ISEL). The latter would only permit accumulation of the cleaved high-molecular weight chromatin against the oocyte plasma membrane. This proposal is supported by our findings from analysis of DNA cleavage in granulosa cells of incubated rat ovarian follicles. In these experiments, internucleosomal, but not high-molecular weight, DNA cleavage was inhibited by KCl, NaCl, and LiCl. Furthermore, the occurrence of high-molecular DNA breaks in cells of KCl-treated follicles was sufficient for nuclear pyknosis to proceed. These findings are similar to those obtained in previous studies of rat ovarian follicles cultured with SAM, in which morphological criteria of apoptosis (e.g., nuclear pyknosis) were preserved in granulosa cells because of high-molecular weight DNA degradation, despite the complete absence of internucleosomal DNA cleavage [27, 30].

To determine whether the effect of potassium on internucleosomal DNA cleavage in ovarian cells occurs at the level of a direct suppression of endonucleolytic activity, we used a number of previously characterized cell-free assays to monitor activity of nuclear extract-derived or purified nucleases. The results from these experiments demonstrated that potassium directly suppresses activity of the nuclease(s) responsible for internucleosomal DNA cleavage that is present in granulosa cell nuclei, a finding consistent with data derived from comparable analyses of potassium effects on internucleosomal DNA-cleaving nucleases involved in thymocyte or S49-Neo lymphocyte apoptosis [25, 26]. Furthermore, the present results extend these previous reports by showing that the activities of two purified nucleases in particular, DNase-I and DNase-II, both of which have been implicated in chromatin degradation during apoptosis [32–34], are directly inhibited by potassium. The present results differ, however, from data derived using thymocytes in which large-fragment DNA generation is blocked by potassium with a similar IC50 as that for the inhibition of internucleosomal DNA cleavage (unpublished results). In the present studies, the generation of 50-kb chromatin fragments was not affected by 150 mM potassium either in the culture medium (intact cells) or in the autodigestion reaction (cell-free assays). In thymocytes, a potential large-fragment cleavage enzyme has been identified as NUC18 (cyclophilin), but in vitro studies have shown this nuclease to be sensitive to potassium-mediated inhibition [49]. The present results therefore suggest that oocytes and granulosa cells possess a potassium-independent pathway leading to the activation of a large DNA fragment-generating nuclease or nucleases distinct from NUC18/cyclophilin.

Because of the fact that changes in cellular morphology and internucleosomal DNA cleavage associated with apoptosis are events that reportedly depend upon the activity of caspases [43, 50], we next employed substrate cleavage assays to assess a role for potassium in the regulation of caspases during apoptosis of oocytes and granulosa cells. We observed that KCl, NaCl, and LiCl effectively blocked caspase-1-mediated processing of pro-IL1ß and caspase-3-catalyzed cleavage of cellular actin. These data differ from those of Hughes et al. [25] using thymocytes and those of Bortner et al. [26] using S49-Neo lymphoma cells; those studies showed that potassium prevented procaspase processing (i.e., generation of the active enzyme) but did not inhibit active caspases, as assessed by cleavage of a tetrapeptide (DEVD) known to be a substrate for several members of this family of enzymes. Although the reasons for this discrepancy remain unclear, it may be that not all caspases are inhibited by potassium. If this is the case, monitoring cleavage of a caspase substrate in cell lysates presumably containing many caspases [25, 26], as opposed to the cleavage of caspase substrates by individual recombinant caspases (present study), would be predicted to generate different results. Nonetheless, the present data extend this earlier work by showing that in addition to procaspase processing [25, 26], potassium directly inhibits active caspase-1 and caspase-3.

In summary, the results presented underscore the importance of intracellular potassium homeostasis in suppressing activity of effector molecules required for the execution phase of apoptosis (i.e., caspases and nucleases) and suggest that loss of potassium from oocytes and granulosa cells may be involved in generating the cascade of events leading to their demise. Moreover, these data, taken with numerous studies published to date regarding the molecular biology of apoptosis in the ovary (reviewed in [2, 3]), further support the hypothesis that a conserved program of events responsible for the regulation and execution of apoptosis in diverse species and cell types functions to coordinate normal and pathologic cell death in the female gonad. However, the differences noted regarding the impact of potassium on various intracellular events associated with apoptosis in thymocytes ([25, 26] and unpublished results) versus ovarian cells (present study) indicate that although potassium efflux may be a conserved feature of apoptosis [46], cell lineage specificity will determine the final cascade of events set in motion by a net loss of intracellular potassium.

ACKNOWLEDGMENTS

We would like to thank Ms. Kim I. Tilly (Vincent Center for Reproductive Biology, Massachusetts General Hospital) and Mr. Joshua M. Newton (Department of Biology, University of North Carolina at Charlotte) for outstanding technical assistance and Dr. Winnie W. Wong (Department of Biochemistry, BASF Bioresearch Corp., Worcester, MA) for recombinant caspases. Portions of this study are in partial fulfillment of the requirements for the doctoral studies of D.V.M and A.M.T. in the Cell and Molecular Biology Graduate Program at the Boston University School of Medicine, Department of Pathology and Laboratory Medicine.

FOOTNOTES

First decision: 29 March 2000.

1 This study was supported by NIH grants R01-HD34226, R01-AG12279, and R01-ES08430 (J.L.T.), by Vincent Memorial Research Funds (J.L.T.), by NIEHS intramural funds (J.A.C.), and by internal research funds from the University of North Carolina at Charlotte (F.M.H.). Back

2 Correspondence: Francis M. Hughes, Jr., Department of Biology, UNCC, 9201 University City Blvd., Charlotte, NC 28223. FAX: 704 547 3128; mhughes{at}email.uncc.edu Back

Accepted: June 8, 2000.

Received: March 8, 2000.

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Apoptosis in the ovary: molecular mechanisms
Hum. Reprod. Update, March 1, 2005; 11(2): 162 - 178.
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 Am. J. Physiol. Cell Physiol.Home page
D. S. Kempe, P. A. Lang, K. Eisele, B. A. Klarl, T. Wieder, S. M. Huber, C. Duranton, and F. Lang
Stimulation of erythrocyte phosphatidylserine exposure by lead ions
Am J Physiol Cell Physiol, February 1, 2005; 288(2): C396 - C402.