Agnieszka Rak-Mardyła

Congener-specific action of PBDEs on steroid secretion, CYP17, 17β-HSD and CYP19 activity and protein expression in porcine ovarian follicles.

The available data on reproductive toxicity of PBDEs are limited. In the present study we evaluated the direct effects of BDE-47, -99 and -100 on porcine ovarian follicular steroid secretions and the activity and expression of enzymes involved in its synthesis. Follicles were exposed to BDE-47 (0.5, 25 and 50 ng/ml), BDE-99 (0.25, 10 and 17.5 ng/ml), or BDE-100 (0.1, 4 and 12.5 ng/ml) for 24h. Progesterone (P4), androstenedione (A4), testosterone (T) and estradiol (E2) levels in the media were determined by EIA. CYP17, 17β-hydroxysteroid dehydrogenase (17β-HSD) and CYP19 activity was measured by conversion of P4>A4, A4>T and T to E2, respectively. Protein expression of CYP17, 17β-HSD and CYP19 was measured by western blot. All of the congeners explored in this study increase testosterone secretion. However, in the case of BDE-47 due to activation of 17 β-HSD and BDE-100 due to activation of CYP17, a corresponding failure to activate CYP19 expression and inhibition of CYP19 activity was seen. The lack of an effect of BDE-99 on the expression and activity of all of the investigated enzymes indicates action on enzymes before progesterone secretion, i.e., STAR or 3β-HSD activity.

Effect of ghrelin on proliferation, apoptosis and secretion of progesterone and hCG in the placental JEG-3 cell line

To determine the effect of ghrelin on placental cell proliferation, apoptosis and hormone secretion we cultured human JEG-3 cells with 100, 250, 500 or 1000 pg/ml of ghrelin for 48 hours. Ghrelin stimulated cell proliferation and decreased caspase-3 activity. All of the investigated ghrelin concentrations decreased progesterone (P(3)) but had no effect on human chorionic gonadotrophin (hCG) secretion. Stimulatory effects on cell proliferation paralleled inhibitory effects on cell apoptosis suggesting a possible role for ghrelin in placental formation or remodeling.

Cooperation of bisphenol A and leptin in inhibition of caspase-3 expression and activity in OVCAR-3 ovarian cancer cells

This study was designed to investigate the effect of bisphenol A and leptin on caspase-3 expression and activity in OVCAR-3 ovarian cancer cells. Caspase-3 and survivin expression was measured at the transcript level by real-time PCR and at the protein level by Western blotting. In addition, caspase-3 activity was measured, using a fluorometric assay, upon exposure to bisphenol A (40 nM) alone, leptin (2.5 nM) alone, and the combination of both agents. 17β-estradiol (40 nM) was used as a positive control for estrogenic properties of bisphenol A. Results showed that the interaction between bisphenol A and leptin, which was similar to that observed between 17β-estradiol and leptin, led to the inhibition of caspase-3 expression and activity in OVCAR-3 cells. Surprisingly, survivin was found to not be involved in the anti-apoptotic activity of either agent. Also, results showed that leptin inhibits caspase-3 activity by acting on the signal transducers and activators of transcription 3 (STAT3) pathway, but bisphenol A and 17β-estradiol by the extracellular-signal-regulated kinases 1/2 (ERK1/2) pathway. In conclusion, the study reveals that bisphenol A and leptin interact to inhibit caspase-3 expression and activity by modulating STAT3 and ERK1/2 signaling pathways in OVCAR-3 cells.

Expression of ghrelin and the ghrelin receptor in different stages of porcine corpus luteum development and the inhibitory effects of ghrelin on progesterone secretion, 3β-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity and protein expression

Recent studies have suggested that ghrelin plays a direct role in controlling female reproduction. The aim of the present study was to investigate the mRNA and protein expression of ghrelin and its receptor (via real time PCR, Western blot and immunohistochemistry analysis, respectively) in porcine corpora lutea (CL) collected during early (CL1: 1-2 days after ovulation), middle (CL2: 7-10 after ovulation), and late luteal phase (CL3: 13-15 after ovulation). Ghrelin expression and concentration of both acylated and unacylated forms of ghrelin significantly increased during CL development. Immunohistochemistry analysis shown localization of ghrelin protein in the cytoplasm of large luteal cells. No changes in the expression of the ghrelin receptor were observed. Direct in vitro effects of ghrelin on progesterone (P4) secretion and 3-beta-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3β-HSD protein expression were then analyzed. To assess 3β-HSD activities, mature luteal cells were first cultured for 24 h with ghrelin at 100, 250, 500 and 1000 pg/mL with P5, or with aminoglutethimide (AMG). AMG is an inhibitor of CYP11A1-mediated hydroxylation; an addition of AMG and P5 enabled P4 production to serve as an index of 3β-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3β-HSD activity and protein expression were observed. In conclusion, the presence of ghrelin and its receptor in porcine corpora lutea and the direct inhibitory effects of ghrelin on luteal P4 secretion and 3β-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.

Halowax 1051 affects steroidogenesis, 17β-hydroxysteroid dehydrogenase (17β-HSD) and cytochrome P450arom (CYP19) activity, and protein expression in porcine ovarian follicles.

We evaluated the effect of Halowax 1051 on ovarian follicular testosterone (T) and estradiol (E2) secretion determined by EIA and on the expression of 17β-HSD and CYP19 analyzed by Western blot. 17β-HSD and CYP19 activity were determined indirectly by measuring the conversion of androstenedione (A4) to T and T to E2, respectively. Additionally, CYP19 activity by dibenzylfluorescein assay. Follicles were exposed to 1-1000pg/ml of Halowax 1051 for 24, 48 or 72h. It was found that Halowax 1051, in all doses used, increased basal T secretion concomitant with a decrease in basal E2 secretion. Inhibitory action on 17β-HSD and stimulatory action on CYP19 (activity and protein expression) under the influence of 1pg/ml, and opposite effect under the influence of 10-1000pg/ml was noted. In conclusion, we suggest non-linear dose-response effect of Halowax 1051 on steroidogenesis and steroidogenic enzymes activity and protein expression.

A Role for Resistin in the Ovary During the Estrous Cycle

In a previous study, we showed that resistin expression increased during ovarian follicle development in prepubertal pigs and had direct effects on steroidogenesis, suggesting an important role for resistin in the ovary during puberty. To determine its potential regulatory role in the ovary during the estrous cycle, using real-time PCR, immunoblotting, immunohistochemistry, and ELISA methods, we quantified the expression, immunolocalization and concentration of resistin in different sized ovarian follicles (small, 2-4 mm; medium, 4-6 mm; and large, 8-12 mm) in mature pigs. We then determined the effects of recombinant resistin (0.1, 1, and 10 ng/ml) on steroid hormone (progesterone-P4, androstendione-A4, testosterone-T, and estradiol-E2) secretion and steroidogenic enzyme (3βHSD, CYP17A1, 17βHSD, and CYP19A1) gene and protein expression in ovarian follicles. We found no differences in the resistin expression between all of the examined follicles. Immunostaining analysis also showed resistin expression in the cytoplasm of both granulosa and theca cells, where it was localized more abundantly in the granulosa cells compared to the theca cells. Recombinant resistin direct stimulated P4, A4, and T secretion via increased expression of 3βHSD, CYP17A1, and 17βHSD, suggesting an autocrine and/or paracrine regulatory role in the porcine ovary during the estrous cycle.

Supraphysiological leptin levels shift the profile of steroidogenesis in porcine ovarian follicles toward progesterone and testosterone secretion through increased expressions of CYP11A1 and 17β-HSD: a tissue culture approach

Evidence from both clinical and animal studies suggests that exposure to excess androgens results in cyst formation. The present in vitro study assessed the effects of supraphysiological concentrations of leptin (20 and 40 ng/ml) on progesterone (P(4)), androstenedione androstendione (A(4)), testosterone and estradiol (E(2)) secretion by ELISA and the expression of CYP11A1, CYP17, 17b-hydroxysteroid dehydrogenase (17b-HSD) and CYP19 by western blot to answer the question of whether leptin could be independent risk factor for cystformation in pigs. Small- and medium-sized ovarian follicles were collected from prepubertal and cycling pigs. Increased P(4) and testosterone secretions were observed in both small- and medium-sized follicles in prepubertal and cycling animals whereas there was no change in E2 secretion. Leptin treatment resulted in an increase in CYP11A1 and 17b-HSD protein expression but had no effect on CYP17 and CYP19 expression in follicles of either size from prepubertal and cycling pigs. Results of presented data suggest that leptin in elevated doses, by stimulatory effect on CYP11A1 and 17b-HSD protein expression resulting in elevated P(4) and testosterone secretions could be an independent risk factor for cyst formation in both prepubertal and cycling pigs.

Differential accumulation of HCBz and PeCBz in porcine ovarian follicles and their opposing actions on steroid secretion and CYP11, CYP17, 17β-HSD and CYP19 protein expression. A tissue culture approach

In the current study, we determined in vitro accumulation of hexachlorobenzene (HCBz) and pentachlorobenzene (PeCBz) in porcine ovarian follicles, the effect on steroidogenesis and the expression of enzymes responsible for steroid synthesis. Sixty percent of the HCBz and almost 100% of the PeCBz that was added to the culture medium accumulated in ovarian tissue, and only 1% of each was found in the medium. An inhibitory HCBz effect and stimulatory PeCBz effect on testosterone and estradiol secretion were noted. Immunoblot analyses showed an inhibitory effect of HCBz on CYP17, 17β-HSD and CYP19, a stimulatory effect of PeCBz on CYP17 and CYP19 and no effect on 17β-HSD protein expression. In conclusion, the greater exposure to an estrogenic action of PeCBz than anti-estrogenic HCBz would be a consequence of the preferential accumulation of PeCBz in the ovarian follicles. As one of the mechanisms of action, we propose modulation of steroidogenic enzymes expression.

Ghrelin Negatively Affects the Function of Ovarian Follicles in Mature Pigs by Direct Action on Basal and Gonadotropin-Stimulated Steroidogenesis:

We previously showed that expression of ghrelin messenger RNA is significantly increased in the ovaries of cycling pigs but not in prepubertal animals and that ghrelin stimulates estradiol (E2) secretion by ovarian follicles in prepubertal animals. The present study investigated in vitro the role of ghrelin in regulating the ovarian steroidogenesis during estrus cycle in mature pigs. Small (SFs), medium (MFs), and large (LFs) ovarian follicles were collected on days 4 to 6, 10 to 12, and 16 to 18 of the estrous cycle from cycling pigs and exposed to 20, 100, and 500 pg/mL ghrelin for 24 hours. In additional experiments, MFs were exposed to ghrelin plus 100 ng/mL follicle-stimulating hormone (FSH) or luteinizing hormone (LH). Levels of progesterone (P4), testosterone (T), and E2 in culture medium were determined by enzyme-linked immunosorbent assay, and the expression of the steroid pathway enzymes 3β hydroxysteroid dehydrogenase (HSD), 17β-HSD, and cytochrome P450 aromatase (CYP19) was evaluated by Western blotting. Ghrelin had no effect on steroid secretion when present at 20 pg/mL, its concentration in follicular fluid, whereas at 100 pg/mL and 500 pg/mL, its concentration in serum, ghrelin significantly decreased secretion of P4, T, and E2. Moreover, all concentrations of ghrelin decreased steroid secretion in FSH- and LH-stimulated follicles. Western blot analysis showed that ghrelin inhibited expression of 3β-HSD, 17β-HSD, and CYP19 proteins. These results suggest that ghrelin, by direct inhibition of 3β-HSD, 17β-HSD, and CYP19 protein expression, inhibits LH- and FSH-stimulated steroid secretion by ovarian follicles, thus negatively affecting ovarian steroidogenesis in mature pigs.

In vitro interaction between resistin and peroxisome proliferator-activated receptor γ in porcine ovarian follicles

In the present study, using real-time polymerase chain reaction and immunoblotting methods, we quantified the expression of peroxisome proliferator-activated receptor (PPAR) γ, PPARα and PPARβ in different sized ovarian follicles (small (SF), medium (MF) and large (LF) follicles) in prepubertal and adult pigs. In prepubertal pigs, PPARγ and PPARα expression was highest in LF; however, PPARβ expression did not differ among SF, MF and LF. In mature pigs, only protein expression of PPARγ and PPARα increased during ovarian follicle development. Following identification of very high levels of PPARγ expression in LF in prepubertal and adult pigs, using in vitro culture of ovarian follicles, we determined the effect of resistin at 0.1, 1 and 10ngmL(-1) on PPARγ mRNA and protein expression and the effect of rosiglitazone at 25 and 50µM (a PPARγ agonist) on resistin mRNA and protein expression. Resistin increased PPARγ expression in ovarian follicles in both prepubertal and adult pigs, whereas rosiglitazone had an inhibitory effect on resistin expression. The role of PPARγ in regulating the effects of resistin on ovarian steroidogenesis was investigated using GW9662 (a PPARγ antagonist at dose of 1μM). In these studies, GW9662 reversed the effect of resistin on steroid hormone secretion. The data suggest that there is local cooperation between resistin and PPARγ expression in the porcine ovary. Resistin significantly increased the expression of PPARγ, whereas PPARγ decreased resistin expression; thus, PPARγ is a new key regulator of resistin expression and function.