To assess the requirement of potassium loss for the biochemical and morphological changes associated with apo-ptosis, 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. Likewise, incubation of oocytes in this medium completely prevented both basal and DXR-induced oocyte fragmentation (Figs. 1C and 2).
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. 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 apo-ptotic 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.
Plasmid DNA Degradation Assays with Purified Nucleases
To further test for effects of potassium on nuclease activity, the pBSKII plasmid (Stratagene), linearized with the £coRI 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 (Calbi-ochem). 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.
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. 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.
Cell-Free Caspase Activity Assays
Using assays identical to those recently reported, caspase-1 activity was assessed by monitoring the conversion of pro-IL1p (33 kDa) to the active cytokine (17 kDa) after incubation of pro-IL1p with 1 ^g 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 ^g/reaction) prepared from eCG-primed rat ovarian granulosa cells after a 30-min incubation at 37°C with 1 ^g of active caspase-3 in the absence or presence of 150 mM KCl, NaCl, or LiCl. The extent of cleavage of pro-IL1p or actin was determined by immunoblot analysis by the enhanced chemiluminescence system (Amersham).
Pulsed-Field Gel Electrophoretic Analysis of High-Molecular Weight DNA Cleavage
As we have previously described for this model system, follicles were embedded in agarose plugs (50-^l total volume, 0.5% final) and immediately immersed in 10 ml of PFGE lysis buffer (100 mM EDTA, 1% N-lauroyl-sar-cosine) for overnight incubation at 37°C. Plugs were then removed, immersed in 1 ml of PFGE lysis buffer containing 50 ^g/ml of proteinase-K, and incubated at 50°C for an additional 12 h. After proteolytic digestion, plugs were preequilibrated in 0.5 X 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.
After incubation, follicles were collected in 12- X 75mm 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.
To analyze apoptosis in granulosa cells of whole follicles, healthy antral follicles between 700-800 ^m in diameter were isolated using nonenzymatic dissection, as detailed elsewhere. 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 ^g/ml streptomycin sulfate.
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. 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 comet assay protocol developed for analysis of DNA cleavage in single human sperm 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 (510 oocytes per pool) and mixed with 30 ^l 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.