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.
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).
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.