ABSTRACT
Extensive apoptotic oocyte reduction occurs during fetal ovarian development. The regulatory pathways responsible for oocyte selection to programmed cell death are, however, poorly understood. The aim of this study was to investigate the potential involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 and decoy receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in the apoptotic process characterizing human fetal and adult ovaries. For this purpose, in situ hybridization and immunohistochemistry were applied to human fetal and adult ovarian samples to study the mRNA and protein expression of TRAIL pathway components, and a human granulosa cell tumor-derived cell line (KGN) was used to elucidate functional effects of TRAIL on apoptosis. TRAIL was expressed in human fetal ovary from the 11th week until term. The pro-apoptotic TRAIL-R2/DR5 and the anti-apoptotic TRAIL-R4/DcR2 were also expressed in human ovaries throughout the fetal period. Among the different ovarian cell types, these TRAIL pathway components were mainly localized in the oocytes, and their expression increased towards term. Expression of TRAIL-R1/DR4 and TRAIL-R3/DcR1 was negligible in all of the fetal ovaries studied. Adult ovaries expressed TRAIL, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in granulosa cells and oocytes of small primary/secondary follicles as well as in granulosa and theca cells of more developed antral follicles. In KGN cells, TRAIL efficiently induced apoptosis in a dose-dependent manner, and this was blocked by a caspase inhibitor. The results indicate a role of the TRAIL pathway components in the regulation of granulosa cell apoptosis in in vitro and suggest that these factors may have a role in regulating ovarian apoptosis also in vivo.
Subject(s)
Apoptosis , Granulosa Cells/metabolism , Ovary/cytology , Ovary/physiology , TNF-Related Apoptosis-Inducing Ligand/physiology , Female , Fetus/cytology , Fetus/physiology , Gene Expression Regulation , Granulosa Cells/cytology , Granulosa Cells/drug effects , Humans , Immunohistochemistry , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacologyABSTRACT
Part of heterodimeric inhibin, inhibin-alpha is crucial for mammalian ovarian function. Regulation of inhibin-alpha expression in granulosa cells is both endocrine, primarily by follicle-stimulating hormone (FSH), and paracrine, primarily by members of the transforming growth factor beta (TGF-beta) superfamily. Smad proteins transmit TGF-beta signals to the nucleus, but the cooperating transcription factors involved in inhibin-alpha promoter activation remain unknown. Transcription factor GATA-4 regulates inhibin-alpha in gonadal cells, and the FSH cascade activates GATA-4. We hypothesized that the TGF-beta signalling cascade and GATA-4 also cooperate to regulate inhibin-alpha expression. In KK-1 granulosa tumour cells, which resemble normal granulosa cells and express inhibin-alpha, we found that TGF-beta upregulated GATA-4 expression. Transient transfection experiments in KK-1 cells demonstrated that dominant negative GATA-4 variants or mutations of GATA-binding sites in the inhibin-alpha promoter attenuated TGF-beta-induced gene activation. In GATA-4-deficient COS-7 cells, TGF-beta enhanced the expression of the inhibin-alpha promoter only in the presence of exogenous GATA-4. Smad3, but not Smad2, cooperated with GATA-4 in the transcriptional activation of the inhibin-alpha promoter, and immunoprecipitation experiments in KK-1 cells revealed a physical Smad3:GATA-4 interaction. Our data suggest that GATA-4, interacting with Smad3, is a cofactor for TGF-beta signalling to activate inhibin-alpha in granulosa cells.
