title: Caspase-3 gene knockout defines cell lineage specificity for programmed cell death signaling in the ovary.
abstract: Previous studies have proposed the involvement of caspase-3, a downstream executioner enzyme common to many paradigms of programmed cell death (PCD), in mediating the apoptosis of both germ and somatic cells in the ovary. Herein we used caspase-3 gene knockout mice to directly test for the functional requirement of this protease in oocyte and/or granulosa cell demise. Using both in vivo and in vitro approaches, we determined that oocyte death initiated as a result of either developmental cues or pathological insults was unaffected by the absence of caspase-3. However, granulosa cells of degenerating antral follicles in both mouse and human ovaries showed a strong immunoreaction using an antibody raised against the cleaved (activated) form of caspase-3. Furthermore, caspase-3 mutant female mice possessed aberrant atretic follicles containing granulosa cells that failed to be eliminated by apoptosis, as confirmed by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling) analysis of DNA cleavage and 4',6-diamidino-2-phenylindole staining of nuclear morphology (pyknosis). These in vivo results were supported by findings from in vitro cultures of wild-type and caspase-3-deficient antral follicles or isolated granulosa cells. Contrasting the serum starvation-induced occurrence of apoptosis in wild-type granulosa cells, caspase-3-null granulosa cells deprived of hormonal support were TUNEL-negative, showed attenuated chromatin condensation by 4',6-diamidino-2-phenylindole staining and exhibited delayed internucleosomal DNA cleavage. Such ex vivo findings underscore the existence of a cell autonomous (granulosa cell intrinsic) defect in apoptosis execution resulting from caspase-3 deficiency. We conclude that caspase-3 is functionally required for granulosa cell apoptosis during follicular atresia, but that the enzyme is dispensable for germ cell apoptosis in the female.
authors.affiliation: Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston 02114, USA.
authors.forename: T
authors.initials: T
authors.lastname: Matikainen
authors.forename: G I
authors.initials: GI
authors.lastname: Perez
authors.forename: T S
authors.initials: TS
authors.lastname: Zheng
authors.forename: T R
authors.initials: TR
authors.lastname: Kluzak
authors.forename: B R
authors.initials: BR
authors.lastname: Rueda
authors.forename: R A
authors.initials: RA
authors.lastname: Flavell
authors.forename: J L
authors.initials: JL
authors.lastname: Tilly
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000032:analysis / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D046508:Culture Techniques; D053938:DNA Fragmentation; D004789:Enzyme Activation; D005260:Female; D005496:Follicular Atresia; D006107:Granulosa Cells / Q000201:enzymology; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D006080:Ovarian Follicle / Q000033:anatomy & histology; D010053:Ovary / Q000166:cytology* / Q000201:enzymology; D015398:Signal Transduction
chemical_list: C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases