Caspase-3 gene knockout defines cell lineage specificity for programmed cell death signaling in the ovary

Previous studies have proposed the involvement of caspase-3, a downstream e xecutioner enzyme common to many paradigms of programmed cell death (PCD), in mediating the apoptosis of both germ and somatic cells in the ovary. Her ein we used caspase-3 gene knockout mice to directly test for the functiona l requirement of this protease in oocyte and/or granulosa cell demise. Usin g both in vivo and in vitro approaches, we determined that oocyte death ini tiated as a result of either developmental cues or pathological insults was unaffected by the absence of caspase-3. However, granulosa cells of degene rating antral follicles in both mouse and human ovaries showed a strong imm unoreaction using an antibody raised against the cleaved (activated) form o f caspase-3. Furthermore, caspase-3 mutant female mice possessed aberrant a tretic follicles containing granulosa cells that failed to be eliminated by apoptosis, as confirmed by TUNEL (terminal deoxynucleotidyl transferase-me diated deoxy-UTP nick end labeling) analysis of DNA cleavage and 4 ' ,6-dia midino-2-phenylindole staining of nuclear morphology (pyknosis). These in v ivo results were supported by findings from in vitro cultures of wild-type and caspase-3-deficient antral follicles or isolated granulosa cells. Contr asting the serum starvation-induced occurrence of apoptosis in wild-type gr anulosa cells, caspasc-3-null granulosa cells deprived of hormonal support were TUNEL-negative, showed attenuated chromatin condensation by 4 ' ,6-dia midino-2-phenylindole staining and exhibited delayed internucleosomal DNA c leavage. Such ex vivo findings underscore the existence of a cell autonomou s (granulosa cell intrinsic) defect in apoptosis execution resulting from c aspase-3 deficiency. We conclude that caspase-3 is functionally required fo r granulosa cell apoptosis during follicular atresia, but that the enzyme i s dispensable for germ cell apoptosis in the female.