PARENT SESSION
SLIDE SESSION 8. SIGNAL TRANSDUCTION
Chairs: Teresa Woodruff, Mary Hunziker-Dunn, Chun-Ying Ku (Trainee)
Univ Ottawa-Morisset 218
2:30 PM-4:30 PM

ROLE OF NFB IN GONADOTROPIC REGULATION OF X-LINKED INHIBITOR OF APOPTOSIS PROTEIN (XIAP) EXPRESSION IN RAT OVARIAN GRANULOSA CELL IN VITRO.

Wang, Yifang^1,2, Chan, Simon¹, Tsang, Benjamin^1,2, ^{1 2}

ABSTRACT- The fate of the developing ovarian follicles (continual growth/ovulation verses atresia) is determined by the fate of their granulosa cells (proliferation, differentiation verses apoptosis). It has been demonstrated that XIAP plays an important role in the maintenance of granulosa cell survival by FSH during follicular development in vitro. However, the cellular mechanism(s) by which FSH induces XIAP expression is unknown. The nuclear factor (NF) B family of transcription factors are important intracellular mediators in transcriptional regulation in a number of biological systems. The objective of the current study was to examine the role of NFB in the gonadotropic regulation of granulosa cell XIAP expression in vitro. Granulosa cells harvested from eCG-primed immature rats were cultured in RPMI 1640 medium in the absence or presence of FSH (100ng/ml) ± SN50 (a cell permeable inhibitory peptide of NFB translocation) or SM50 (its inactive analogue) [50-200 ng/ml]. XIAP, IB and phospho-IB protein contents were measured by Western blot. NFB binding activity was assessed by electrophoretic mobility shift assays, using granulosa cell nuclear protein extracts and a probe containing a consensus B enhancer motif. Whereas FSH failed to elicit a significant change in granulosa cell phospho-IB and total IB contents in vitro, it increased NFB binding activity and XIAP protein content in a concentration-dependent manner. These latter responses were also time-dependent, with a significant increase at 10 min and 24 h after FSH stimulation, respectively. Immunocytochemical studies showed clear translocation of NFB containing p65 subunit from the granulosa cell cytoplasm to nucleus 15 minutes after gonadotropin challenge. Addition of SN50, but not of SM50, to granulosa cell incubations not only suppressed FSH-stimulated NFB binding but also attenuated XIAP expression induced by the gonadotrophin. In conclusion, these studies demonstrated for the first time, NFB activation plays an important role in the regulation of granulosa cell XIAP expression by FSH, a process independent of IB phosphorylation. [Supported by Canadian Institutes of Health Research (MOP-10369) and the Natural Science and Engineering Research Council of Canada (PGSB-222413-1999)].

KEY WORDS: FSH, granulosa cell , NFB, XIAP