Potassium and Ovarian Somatic (Granulosa)
To explore the possibility that this novel potassium-independent pathway for nuclear destruction occurs in non-germline 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.
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).
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.
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.