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 apoptosisrelated (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. Reproduction (2008) 136 611-618

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.

The aim of the present study was to examine the role of cAMP response element-binding protein (CREB) and its phosphorylation in the regulation of ovarian cell proliferation and apoptosis, and of the response of proliferation and apoptosis to the upstream hormonal stimulators FSH and insulin-like growth factor (IGF) 1. In the first series of experiments, porcine ovarian granulosa cells, transfected or not with a gene construct encoding wild-type CREB1 (CREB1WT), were cultured with and without FSH (0, 1, 10 or 100ngmL-1). In the second series of experiments, these cells were transfected or not with CREB1WT or non-phosphorylatable mutant CREB1 (CREB1M1) and cultured with and without FSH (0, 1, 10 or 100ngmL-1) or IGF1 (0, 1, 10 and 100ngmL-1). Levels of total and phosphorylated (p-) CREB1, proliferating cell nuclear antigen (PCNA), a marker of proliferation, and BAX, a marker of apoptosis, were evaluated by western immunoblotting and immunocytochemical analysis. Transfection of cells with CREB1WT promoted accumulation of total CREB1 within cells, but p-CREB1 was not detected in any cell group. Both CREB1WT and CREB1M1 reduced cell proliferation and apoptosis. Addition of 10 and 100ngmL-1 FSH to non-transfected cells promoted CREB1 accumulation and apoptosis, whereas cell proliferation was promoted by all concentrations of FSH tested. FSH activity was not modified in cells transfected with either CREB1WT or CREB1M1. IGF1 at 100ngmL-1 promoted cell proliferation, whereas all concentrations of IGF1 tested reduced apoptosis. Transfection with either CREB1WT or CREB1M1 did not modify the effects of either FSH or IGF1, although CREB1M1 reversed the effect of IGF1 on apoptosis from inhibitory to stimulatory. These observations suggest that CREB1 is involved in the downregulation of porcine ovarian cell proliferation and apoptosis. The absence of visible CREB1 phosphorylation and the similarity between the effects of CREB1WT and CREB1M1 transfection indicate that phosphorylation is not necessary for CREB1 action on these processes. Furthermore, the observations suggest that FSH promotes both ovarian cell proliferation and apoptosis, whereas IGF1 has proliferation-promoting and antiapoptotic properties. The effect of FSH on CREB1 accumulation and the ability of CREB1M1 to reverse the effects of IGF1 on apoptosis indicate that CREB1 is a mediator of hormonal activity, but the inability of either CREB1WT or CREBM1transfection to modify the primary effects of FSH and IGF1 suggest that CREB1 and its phosphorylation do not mediate the action of these hormones on ovarian cell proliferation and apoptosis.