Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells.
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-1beta 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.
Perez
G I
GI
Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114, USA.
Maravei
D V
DV
Trbovich
A M
AM
Cidlowski
J A
JA
Tilly
J L
JL
Hughes
F M
FM
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D016521:Electrophoresis, Gel, Pulsed-Field; D005260:Female; D005854:Germ Cells / Q000502:physiology*; D006107:Granulosa Cells / Q000502:physiology*; D020287:In Situ Nick-End Labeling; D051379:Mice; D009865:Oocytes / Q000502:physiology; D006080:Ovarian Follicle / Q000166:cytology; D010053:Ovary / Q000166:cytology / Q000502:physiology*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011188:Potassium / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D004247:DNA; D003850:Deoxyribonuclease I; D020169:Caspases; D011188:Potassium