Abstract
The role of either mTOR system/enzyme sirtuin1 (SIRT1) or transcription factor NF-κB in the direct control of ovarian function has not been estabished. The aim of our in vitro experiments was to examine the involvement of SIRT1 and the p65 and p50 subunits of NFκB in control of porcine ovarian granulosa cell functions and the interrelationships between SIRT1, NFκB (p65, p50) 30 and FSH in the ovary. Monolayers of primary granulosa cells were transfected with gene constructs encoding either SIRT1 or p65 and p50, and thereafter cultured with, or without, addition of FSH. The accumulation of markers of proliferation (cyclin B1 and cyclin-dependent protein kinase Cdc2/p34) and proteins p50, p65 and SIRT1 in the cells was detected by using SDS-PAGE/Western immunoblotting and immunocytochemistry. The secretion of progesterone (P4) and insulin-like growth factor I (IGF-I) was measured by using radioimmunoassay. It was observed that transfection of cells with a SIRT1 gene construct promoted accumulation of proliferation markers, Cdc2/p34, cyclin B1, decreased accumulation of p50 and p65 and stimulated release of P4 and IGF-I. Co-transfection of cells with cDNA p50 and cDNA p65 enhanced the accumulation of SIRT1 and the release of P4 but did not influence the release of IGF-I. Adding FSH to the culture medium stimulated accumulation of both subunits of NF-κB, as well as accumulation of Cdc2/p34, cyclin B1 and release of both P4 and IGF-I. The ability of FSH to promote NF-κB accumulation, the similarity of the main effects of FSH, SIRT1 and NF-κB, as well as the inability of NF-κB to substantially modify the the majority of FSH effects suggest that SIRT1/NF-κB system could be a mediator of FSH action on ovarian cell functions. On the other hand, SIRT1 was able to inhibit NF-κB and to change stimulatory the effect of FSH on NF-κB from stimulatory to inhibitory. This could suggest the existence of negative feedback control of FSH/NF-κB system by high amounts of SIRT1.
Our observations (1) confirm the previous data on proliferation, P4 and IGF-I release in ovarian cells and their up-regulation by FSH, (2) demonstrate the presence of SIRT1, NF-κB/p50 and NF-κB/p65 in these cells, (3) show for the first time the involvement of SIRT1 and NF-κB in direct control of proliferation and secretory activity of ovarian cells, (4) represent the first data on interrelationships between FSH, SIRT1 and NF-κB within the ovary.