Transcription factor NF-κB (p50/p50, p65/p65) controls porcine ovarian cells functions

Abstract

The aim of these in vitro studies was to examine the involvement of transcription factor NF-κB (p50/p50, p65/p65) and FSH in control of porcine ovarian granulosa cells functions and the possible role of dimers p50/p50, p65/p65 in mediating FSH actions on these cells. Monolayer of primary granulosa cells was transfected with plasmids encoding human p50 cDNA and p65 cDNA, and cultured with or without addition of FSH (0, 1, 10 or 100 ng/ml). The accumulation of proteins p50 and p65, as well as of proliferation markers (PCNA and MAPK/ERK1,2) and marker of apoptosis (Bax) in cells was detected by using SDS-PAGE-Western immunoblotting and immunocytochemistry. DNA fragmentation was evaluated by TUNEL assay. Release of hormones insulin-like growth factor I (IGF-I), progesterone (P 4), oxytocin (OT), prostaglandins E 2 (PGE 2) and F 2α (PGF 2α) was measured by using RIA. We observed, that p50/p50 promoted the accumulation of PCNA, MAPK/ERK1,2, the release of OT, PGF 2α; inhibited the occurrence of TdT-positive cells, the release of IGF-I and P 4, and did not influence the accumulation of Bax and the release of PGE 2. p65/p65 enhanced the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, PGE 2 and PGF 2α; decreased the percentage of cell containing TdT and did not affect the release of P 4. FSH stimulated the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, P 4, PGE 2; but reduced the proportion of TdT-positive cells and the release of PGF 2α. These observations suggest (1) the involvement of NF-κB (p50/p50) in stimulation of proliferation, inhibition of apoptosis and in either stimulation (OT, PGE 2) or inhibition (IGF-I, P 4, but not PGF 2) of hormones release by porcine ovarian granulosa cells; (2) the involvement of NF-κB (p65/p65) in stimulation of proliferation and mitochondrial/Bax-related apoptosis, inhibition of nuclear/TdT-related apoptosis and in stimulation of ovarian hormones (IGF-I, OT, PGE 2, PGF 2α, but not P 4) release; (3) the role of FSH in up-regulation of both ovarian cell proliferation and mitochondrial/Bax-related apoptosis, in inhibition of nuclear/TdT-related apoptosis, in promotion of IGF-I, P 4, OT, PGE 2 and suppression of PGF 2α release by porcine ovarian cells. The majority of results demonstrates the involvement of NF-κB (p50/p50 and p65/p65) and FSH in control of basic ovarian functions (proliferation, apoptosis, and secretory activity), but not the functional interrelationships between these regulators.