Involvement of transcription factor p53 and leptin in control of porcine ovarian granulosa cell functions.

The aim of our in vitro experiments was to examine the role of transcription factor p53 and the metabolic hormone leptin, in controlling basic functions (proliferation, apoptosis and secretory activity) of ovarian cells, as well as involvement of p53 in mediating or modulating actions of leptin, on ovarian cells. Porcine ovarian granulosa cells, transfected and non-transfected with a gene construct encoding p53, were cultured with leptin (at concentrations of 0, 1, 10 or 100 ng/ml). Accumulation of p53 and of apoptosis-related (bax) and proliferation-related (PCNA, cyclin B1) substances was evaluated by SDS-PAGE-western blotting. Secretion of progesterone (P4) was measured by RIA. Transfection with the p53 gene construct promoted accumulation of this transcription factor within cells. It also stimulated expression of bax (which can be thought of as a marker of apoptosis), and reduced accumulation of proliferation-related substances PCNA and cyclin B1. Overexpression of p53 resulted in reduced P4 secretion. Leptin, when added alone, increased accumulation of p53, bax and PCNA, decreased accumulation of cyclin B1 and had no effect on P4 secretion. Transfection of cells with p53 gene construct reversed effects of leptin on cyclin B1 and induced stimulatory effects of leptin on P4 release, but did not modify leptin action on p53, bax and PCNA. These multiple effects of the p53 gene construct on granulosa cells, cultured with and without leptin, (i) demonstrate that leptin can be involved in control of porcine ovarian cell proliferation, apoptosis and expression of p53, but not on P4 release; and (ii) confirm involvement of p53 in promoting apoptosis and suppression of proliferation and P4 secretion in these cells. (iii) The similarity of p53 and leptin's actions on bax and cyclin B1, and inability of p53 to further promote leptin action on this parameter suggest that p53 can be a mediator of leptin's action on ovarian cell apoptosis. (iv) On the other hand, p53 can modulate, but probably not mediate the effects of leptin on ovarian cell proliferation and P4 release.

Involvement of the transcription factor STAT1 in the regulation of porcine ovarian granulosa cell functions treated and not treated with ghrelin.

The aim of our in vitro experiments was to study the role of the transcription factor STAT1 and the hormone ghrelin in controlling porcine ovarian function. The effects of treatment with ghrelin (0, 1, 10, 100 ng/ml), transfection-induced overexpression of transcription factor STAT1, and their combination on apoptosis (expression of apoptosis-related peptides caspase-3, BAX and anti-apoptotic peptide BCL2), proliferation (expression of proliferating cell nuclear antigene PCNA, proliferation-associated protein kinase MAPK/ERK1,2) and release of the hormones progesterone (P(4)), prostaglandin F (PGF) and oxytocin (OXT) in cultured porcine ovarian granulosa cells was evaluated using RIA, immunocytochemistry and SDS-PAGE-western immunoblotting. It was found that ghrelin, when given alone, increased the expression of proliferation-associated PCNA and MAPK/ERK1,2, decreased the accumulation of apoptosis-related substances caspase-3, BAX, BCL2, decreased P(4), and increased PGF and OXT release. Ghrelin tended to promote accumulation of STAT1 in both control and transfected cells, although in transfected cells ghrelin at 1 ng/ml decreased STAT1 accumulation. Transfection of porcine granulosa cells by a gene construct encoding STAT1 promoted the expression of STAT1 and apoptosis-related-BAX but the expression of BCL2 did not, and decreased the accumulation of proliferation-associated MAPK/ERK1,2 but not that of PCNA. It also promoted PGF and OXT but not P(4) release. Overexpression of STAT1 reversed the effect of ghrelin on STAT1, PCNA, PGF, OXT (from stimulatory to inhibitory), BCL2, P(4) (from inhibitory to stimulatory), prevented ghrelin effect on caspase-3 and BAX, but did not affect ghrelin's effect on MAPK/ERK1,2 expression. These results suggest that ghrelin directly affects porcine ovarian cells function - stimulates proliferation, inhibits apoptosis and affects secretory activity. Furthermore, they demonstrated the involvement of the transcription factor STAT1 in controlling these functions, the promotion of some markers of apoptosis (BAX), inhibition of some markers of proliferation (MAPK/ERK1,2) and stimulation of PGF release. Finally, the obtained data failed to demonstrate that STAT1 is involved in mediating the action of ghrelin on ovarian cell functions.

Transcription factor p53 can regulate proliferation, apoptosis and secretory activity of luteinizing porcine ovarian granulosa cell cultured with and without ghrelin and FSH.

The aim of our in vitro experiments was to examine the role of transcription factor p53 in controlling the basic functions of ovarian cells and their response to hormonal treatments. Porcine ovarian granulosa cells, transfected and non-transfected with a gene construct encoding p53, were cultured with ghrelin and FSH (all at concentrations of 0, 1, 10, or 100 ng/ml). Accumulation of p53, of apoptosis-related (MAP3K5) and proliferation-related (cyclin B1) substances was evaluated by immunocytochemistry. The secretion of progesterone (P(4)), oxytocin (OT), prostaglandin F (PGF), and E (PGE) was measured by RIA. Transfection with the p53 gene construct promoted accumulation of this transcription factor within cells. It also stimulated the expression of a marker of apoptosis (MAP3K5). Over-expression of p53 resulted in reduced accumulation of a marker of proliferation (cyclin B1), P(4), and PGF secretion and increased OT and PGE secretion. Ghrelin, when added alone, did not affect p53 or P(4), but reduced MAP3K5 and increased PGF and PGE secretion. Over-expression of p53 reversed the effect of ghrelin on OT, caused it to be inhibitory to P(4) secretion, but did not modify its action on MAP3K5, PGF, or PGE. FSH promoted the accumulation of p53, MAP3K5, and cyclin B1; these effects were unaffected by p53 transfection. These multiple effects of the p53 gene construct on luteinizing granulosa cells, cultured with and without hormones 1) demonstrate the effects of ghrelin and FSH on porcine ovarian cell apoptosis and secretory activity, 2) confirm the involvement of p53 in promoting apoptosis and inhibiting P(4) secretion in these cells, 3) provide the first evidence that p53 suppress proliferation of ovarian cells, 4) provide the first evidence that p53 is involved in the control of ovarian peptide hormone (OT) and prostaglandin (PGF and PGE) secretion, and 5) suggest that p53 can modulate, but probably not mediate, the effects of ghrelin and FSH on the ovary.