Transcription factor p53 regulates healthy human ovarian cells function.

The aim of our study was to understand the role of transcription factor p53 in the control of healthy human ovarian cell functions. Ovarian granulosa cells were transfected with a cDNA construct encoding p53. The intracellular accumulation of p53, of the apoptosis marker bax, and of the proliferation marker PCNA, as well as the release of progesterone (P4), insulin-like growth factor I (IGF-I), oxytocin (OT), and prostaglandin F (PGF) and E2 (PGE) were evaluated by quantitative immunocytochemistry and RIA/IRMA. Transfection with the p53 cDNA construct resulted in the accumulation of p53 and bax, in a reduced level of released PCNA and PGF, and in an increased PGE output. No changes in P4, IGF-I, and OT secretion were found. These observations are the first demonstration of the involvement of p53 in the control of healthy human ovarian cell functions, namely, in the downregulation of proliferation, in the upregulation of apoptosis, and in the alteration of PGF and PGE release, but not of P4, IGF-I, or OT.

Apoptosis signal-regulating kinase (ASK-1) controls ovarian cell functions.

The involvement of the apoptosis signal-regulating kinase 1 (ASK1)-related signalling pathway in the control of reproduction is unknown. This study aimed to investigate the role of ASK-1 in the control of basic ovarian functions (proliferation, apoptosis and hormone release) and its response to ovarian hormonal regulators (leptin and FSH). We compared the accumulation of ASK-1, proliferation marker proliferating cell nuclear antigen (PCNA), apoptosis marker Bax and apoptosis and proliferation regulating transcription factor p53 and the release of progesterone (P4), oxytocin (OT), insulin-like growth factor I (IGF-I) and prostaglandins F (PGF) and E (PGE) using cultured porcine ovarian granulosa cells transfected with ASK-1 cDNA and cultured with leptin or FSH. This study is the first to demonstrate that ASK-1 does not affect cell apoptosis and viability in ovarian cells, but promotes cell proliferation, suppresses p53, alters the release of ovarian hormones (P4, OT, IGF-I, PGF and PGE) and defines their response to the upstream hormonal regulators leptin and FSH. Therefore, ASK-1 can be considered a new and important regulator of multiple ovarian functions.

The involvement of the phosphorylatable and nonphosphorylatable transcription factor CREB-1 in the control of human ovarian cell functions.

The objective of our study was to elucidate the role of the transcription factor CREB-1 in controlling ovarian cell proliferation, apoptosis, and hormone release and the significance of CREB-1 phosphorylation in these processes. Human ovarian granulosa cells were transfected with a gene construct encoding wild-type CREB-1 (CREB-1 WT) or CREB-1 nonphosphorylatable mutant (CREB-1 M1). The expression of total and phosphorylated CREB-1, markers of proliferation (PCNA) and apoptosis (bax), as well as the release of progesterone, oxytocin, prostaglandin F2 alpha (PGF2), prostaglandin E2 (PGE2), and insulin-like growth factor I (IGF-I) were compared by immunocytochemistry, enzyme immunoassay (EIA), and immunoradiometric assay (IRMA). Transfection with CREB-1 WT or CREB-1 M1 increased total CREB-1 expression and proliferation and decreased the release of oxytocin, PGE2, and IGF-I by ovarian cells. CREB-1 M1, not CREB-1 WT, promoted apoptosis and inhibited progesterone output. PGF2 release was inhibited by CREB-1 WT but stimulated by CREB-1 M1 construct. Phosphorylated CREB-1 was undetected in any cell group. These observations confirm the involvement of CREB-1 in the control of ovarian cell proliferation, apoptosis, and steroid hormone release. This is the first demonstration of the involvement of CREB-1 in the regulation of the ovarian non-steroidal hormones such as oxytocin, PGF2, PGE2, and IGF-I. The absence of CREB-1 phosphorylation, similar effects exerted by CREB-1 WT and CREB-1 M1 on cell proliferation and release of oxytocin, PGE2, and IGF-I, and the influence of CREB-1 M1 on apoptosis and progesterone suggest that phosphorylation plays no role in the action of CREB-1 on the majority of analyzed functions of human ovarian cells.

Effect of inhibitor and activator of ghrelin receptor (GHS-R1a) on porcine ovarian granulosa cell functions.

It was previously shown, that ghrelin and its agonistic analogue, ghrelin 1-18, can be a stimulator of ovarian cell functions (promoter of proliferation, inhibitor of apoptosis and stimulator of hormones release). The aim of our studies was to compare the action of two ghrelin analogues - ghrelin 1-18, activator of ghrelin receptors (GHS-R1a), and (D-Lys3)-GHRP-6, its inhibitor, on porcine ovarian granulosa cell functions. Effects of (D-Lys3)-GHRP-6 added at doses of 0, 1, 10 or 100 ng/ml on the expression of markers of proliferation (PCNA, cyclin B1, MAPK/ERK1,2), apoptosis (bax, p53, caspase 3) and release of steroid hormones (progesterone, testosterone, estradiol) were examined. In addition, some effect of ghrelin 1-8 on some of these parameters (expression of MAPK/ERK1,2, bax, p53) were verified. It was shown, that (D-Lys3)-GHRP-6 promotes all markers of granulosa cell proliferation, inhibits all markers of apoptosis and stimulates the release of all three steroid hormones. Similar effects of (D-Lys3)-GHRP-6 (inhibitor of GHS-R1a) and ghrelin 1-18 (its stimulator) suggest that the examined effects of these substances on porcine ovaries are not mediated by GHS-R1a. Both chemical analogues could be potentially useful for stimulation of reproductive processes, at least in in vitro conditions.

Lipids modulate H(2)S/HS(-) induced NO release from S-nitrosoglutathione.

Recently we observed that a gas messenger H(2)S/HS(-) released NO from S-nitrosoglutathione (Ondrias et al., Pflugers Arch. 457 (2008) 271-279). However, the effect of biological compounds on the release is not known. Measuring the NO oxidation product, which is nitrite, by the Griess reaction, we report that unsaturated fatty acid-linoleic acid and lipids having unsaturated fatty acids: asolectin, dioleoylphosphocholine and dioleoylphosphoserine depressed the H(2)S/HS(-) induced NO release from S-nitrosoglutathione. On the other hand, a depression effect of the saturated fatty acid-myristic acid and lipids having saturated fatty acids, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine was less pronounced. The inhibition effect increased with the decreasing gel-to-liquid phase transitions temperature of the fatty acids and lipids. We suggest that lipid composition of biological membranes modulates NO release from nitrosoglutathione induced by H(2)S/HS(-), assuming that a reaction of H(2)S/HS(-) with unsaturated bonds of fatty acids may be partially responsible for the effect.

Protein kinases controlling PCNA and p53 expression in human ovarian cells.

The aim of this study was to identify protein kinases (PKs) involved in the expression of proliferating cell nuclear antigen (PCNA) and p53, markers of proliferation and apoptosis in human ovarian cells. Cultured ovarian granulosa cells were subjected to transfection with 264 small-interfering RNA (siRNA) constructs from a siRNA library, which selectively blocked the expression of 88 known PKs. The efficiency of transfection and siRNA knockdown were validated by fluorescent microscopy, real-time reverse transcription polymerase chain reaction, and immunocytochemical analysis. The expression of PCNA and p53, before and after transfection with siRNA constructs, was evaluated by immunocytochemistry. The siRNA constructs suppressed the expression of their targets molecules by up to 84%. Knockdown of 32 of the 88 PKs inhibited the expression of PCNA, while the knockdown of seven of the PKs stimulated PCNA expression. Knockdown of 30 of the 88 PKs reduced the expression of p53, while knockdown of five PKs enhanced p53 expression. Our results illustrate that siRNA constructs are useful tools for understanding the role of PKs in the control of ovarian cell functions, such as proliferation and apoptosis. The specific knockdown of individual PKs has enabled the identification of a number of new PKs that control the expression of PCNA and p53 in human ovarian cells.