Environmental polycyclic aromatic hydrocarbons mixture, in human blood levels, decreased oestradiol secretion by granulosa cells via ESR1 and GPER1 but not ESR2 receptor.

Tissue-dependent oestrogenic and anti-oestrogenic activity of polycyclic aromatic hydrocarbons (PAHs) has been suggested. In this study, the effect of two PAH mixtures, M1 composed of all 16 priority pollutants and M2 composed of five (noted in the highest levels) compounds, on follicle-stimulating hormone receptor (FSHR) expression, basal or FSH-induced oestradiol (E2) secretion and aromatase cytochrome P450 (P450arom) protein expression, by non-luteinised human granulosa cell line (HGrC1) was determined. In addition, the consequences of gene silencing of oestrogen receptor alfa (siESR1), oestrogen receptor beta (siESR2) and a G protein-coupled receptor (siGPER1) on the above parameters were described. Neither PAH mixture had an effect on basal FSHR protein expression; however, both mixtures increased FSH-induced FSHR expression. Decreased E2 secretion and P450arom expression was also demonstrated. In both basal and FSH treated cells, siESR1 and siGPER1 reversed the inhibitory effect of the mixtures on E2 secretion; however, in siESR2 cells, the inhibitory effect was still observed. This study showed that both classic ESR1 and GPER1 were involved in the inhibitory effect of both PAH mixtures on E2 secretion and confirmed that expression of P450arom could be downregulated through the aryl hydrocarbon receptor and additionally through the ESR2.

Compounds of PAH mixtures dependent interaction between multiple signaling pathways in granulosa tumour cells.

Mechanism of PAH mixtures, using granulosa tumour cells, was investigated. Cells were exposed to a mixture of all 16 priority PAHs (M1) or a mixture of five PAHs not classified as human carcinogens (M2). The effect of siAHR, siAHRR and siNFKB2 on the expression of CYP1A1, CYP1B1, GSTM1, ERα, AR and cell proliferation was described. M1 decreased AhR and CYP1A1, while increased AhRR and ARNT expression. M2 also decreased AhR and CYP1A1 but had no effect on AhRR expression. siAHRR reversed the inhibitory effect of M1 on AhR and CYP1A1,while inhibitory effect of M2 was still observed. siNFKB2 reversed inhibitory effect of both mixtures on AhR and CYP1A1 expression and stimulatory effect of M1 on AhRR expression. siAHR reversed stimulatory effect of both mixtures on ERα expression. Stimulatory effect of M1 on cell proliferation was not observed in siAHR, was still observed in siESR1 cells. M2 had no effect on cell proliferation, however stimulatory effect was appeared in siAHR and siESR1cells. In conclusion: M1 by activation of AhRR and NFkB p52, but M2 only by activation of NFκB attenuated AhR signalling and ligand-induced CYP1A1 expression. Interaction between AhR and ER following M1 and M2 exposure is primarily initiated through AhR.

Superactive human leptin antagonist (SHLA), triple Lan1 and quadruple Lan2 leptin mutein as a promising treatment for human folliculoma.

PURPOSE: There are no data showing a direct correlation between obesity and increased blood leptin levels with folliculoma. Moreover, folliculoma is not the best studied among other ovarian cancer types. We investigated whether oestradiol can modulate ObR expression in some oestrogen-responsive tissues and that leptin exerts its activity not only via the leptin receptor but also through cross talk with other signalling systems. We hypothesise that blocking ObR expression could be a novel treatment for gonadal ovarian cancer. METHODS: We evaluated the effect of SHLA, Lan1 and Lan2 blockers on cell proliferation (BrdU incorporation assay), ObR and ERα/ß gene expression (qPCR), oestradiol secretion (ELISA) and cell cycle protein expression (Western blot) in the non-cancerous cell line HGrC1 and two granulosa cancer cell lines: the juvenile form (COV434) and the adult form (KGN). RESULTS: ObR gene expression in cancer cell lines was 50% higher than in the non-cancer cells. Lan-1 and Lan-2 decreased ObR expression in COV434, while it had no effect in KGN cells. Higher ERß expression in non-cancer and higher ERα expression in both cancer cell lines was noted. SHLA and Lan-1 changed the ratio towards greater expression of ERß, characteristic of non-cancer granulosa cells. All ObR antagonists in HCrC1 and KGN but only Lan-2 in COV434 reversed leptin-stimulated proliferation. In both non-cancer and cancer granulosa cells, leptin acts as a cyclinD/cdk4, cyclin A/cdk2 and E2F inhibitor. CONCLUSION: These results indicate that SHLA and Lan2 are promising leptin receptor inhibitors that can eliminate the negative effects of leptin. These compounds should be considered in further ex vivo studies on the cancer microenvironment.

Expression of apelin and apelin receptor (APJ) in porcine ovarian follicles and in vitro effect of apelin on steroidogenesis and proliferation through APJ activation and different signaling pathways.

Apelin was thought to be an adipocyte-specific hormone, but recent studies indicate a link between apelin and female reproductive function. Using real-time PCR, immunoblotting, immunohistochemistry and ELISA, we demonstrated expression of apelin and its receptor (APJ) in ovarian follicles of different sizes from mature pigs. Apelin concentration in the follicular fluid, and expression of both apelin and APJ, increased with follicular growth; greatest values were found in large follicles. Immunohistochemistry revealed the positive staining for apelin and APJ in membranes of granulosa, than theca cells. Furthermore, we observed strong expression of apelin in oocytes and APJ in the zona pellucida. The effect of apelin (0.02, 0.2, 2 and 20 ng/ml) on basal and IGF1- and FSH-induced steroid hormone (progesterone [P4], and estradiol [E2]) secretion, steroidogenic enzyme (3ßHSD and CYP19A1) expression and cell proliferation (Alamar blue) was determined. Apelin was found to increase basal steroid secretion, but decrease IGF1- and FSH-induced steroid secretion, and 3ßHSD and CYP19 expression. Apelin also increased cell proliferation and the phosphorylation level of 5'-monophosphate-activated protein kinase (AMPK), phosphatidyl inositol 3' kinase/Akt (Akt) and extracellular signal-regulated kinases (ERK1/2). AMPKα was involved in the action of apelin in P4 production, and MAPK/ERK and Akt/PI3 mediated the proliferative effect of apelin. However, these effects on steroid secretion and cell proliferation were abolished when cultured in the presence of ML221, an APJ antagonist. In conclusion, apelin appears to regulate ovarian follicular functions such as steroidogenesis and proliferation via APJ activation and different signaling pathways.

Effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE47) on the enzymes of phase I (CYP2B1/2) and phase II (SULT1A and COMT) metabolism, and differences in the action of parent BDE-47 and its hydroxylated metabolites, 5-OH-BDE-47 and 6-OH-BDE47, on steroid secretion by luteal cells.

UNLABELLED: In this study we determined the effects of BDE-47 on the expression and activity of phase I (CYP2B1/2) and phase II (SULT1A and COMT) enzymes, and assessed the actions of BDE-47 and its metabolites on luteal steroidogenesis. Luteal cells collected during early (ELP), middle (MLP) and late (LLP) luteal phase were exposed to BDE-47 (0.5, 25, and 50ng/ml) or metabolites (2.5, 5 and 25ng/ml). BDE-47 decreased CYP2B1/2 activity and expression but had no effect on SULT1A or COMT. BDE-47 exerted a stimulatory action on estrogen secretion in MLP and an inhibitory in LLP, but had no effect on progesterone secretion. 5-OH-BDE-47 and 6-OH-BDE-47 decreased progesterone, but had no effect on estrogen secretion. CONCLUSIONS: The inhibitory effect of BDE-47 on CYP2B1/2 suggests the possibility of BDE-47 accumulation in the corpus luteum; by affecting steroid secretion and steroidogenesis enzymes, BDE-47 and its metabolites can be responsible for shortening luteal phase.

Superactive human leptin antagonist reverses leptin-induced excessive progesterone and testosterone secretion in porcine ovarian follicles by blocking leptin receptors.

Accumulating evidence indicates that leptin plays an important role in controlling reproductive function. At physiological levels, leptin stimulates steroidogenesis and follicle maturation, whereas supraphysiological concentrations of leptin have been suggested to be an independent risk factor for cyst formation. Since the discovery of the link between leptin and obesity, which is frequently associated with polycystic ovarian syndrome (PCOS), a number of leptin mutants exhibiting antagonistic properties have been developed, opening new avenues for leptin-related research. Here, using a superactive human leptin antagonist (SHLA), we sought to determine whether blocking leptin receptors can reverse the actions of leptin in ovarian follicles. Antral porcine ovarian follicles, collected from prepubertal and mature animals, were exposed to 100, 250 and 500 ng/ml SHLA for 24 hours, after which leptin receptor (ObR), leptin, CYP11A1 and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) levels in follicles were evaluated by Western blotting. Levels of secreted leptin, progesterone (P4), and testosterone (T) in the follicle incubation medium were measured using enzyme-linked immunosorbent assays. The effects of SHLA on leptin-stimulated P4 and T secretion were also tested by exposing follicles to 40 ng/ml leptin in the presence and absence of SHLA. These experiments revealed that SHLA acted through inhibition of ObR expression and leptin expression and secretion decreased P4 and T secretion by ovarian follicles from both prepubertal and mature animals. Our data further suggest that the mechanism underlying this action of SHLA involves inhibition of CYP11A1 and 17ß-HSD protein expression. Importantly, SHLA reversed leptin-induced increases in P4 and T secretion. Collectively, these data indicate that, in addition to their potential application in novel therapeutic strategies in oncology, which has received considerable recent research attention, leptin receptors antagonists might also be beneficial in controlling reproduction.

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 (3ßHSD, CYP17A1, 17ßHSD, 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 3ßHSD, CYP17A1, and 17ßHSD, suggesting an autocrine and/or paracrine regulatory role in the porcine ovary during the estrous cycle.

Hexachlorobenzene and pentachlorobenzene accumulation, metabolism and effect on steroid secretion and on CYP11A1 and CYP19 expression in cultured human placental tissue.

Hexachlorobenzene and pentachlorobenzene accumulation and the effect on CYP1A1, SULT1A, COMT and steroid secretion in term placental tissue were determined. Explants of placental tissue were exposed to between 0.02 and 2 ng/ml HCBz or PeCBz for 6-72 h. Accumulation was measured by capillary gas chromatography and quadrupole mass spectrometry. CYP1A1, SULT1A, COMT activity and progesterone secretion were analysed by EIA. Protein expression was quantified by Western blot; 6% HCBz and 7% PeCBz were detected in the tissue. Fast induction of CYP1A1 activity and protein expression in the presence of HCBz were observed. HCBz increased, while PeCBz decreased COMT protein expression. The stimulatory effect of HCBz, and the inhibitory of PeCBz on progesterone secretion and CYP11A1 protein expression were noted. Later activation of CYP1A1, inhibition of COMT protein expression and progesterone secretion by PeCBz suggest greater exposure to PeCBz and pointing at PeCBz as the main factor responsible for the disruption of placental function.

Activation of the enzymes of phase I (CYP2B1/2) and phase II (SULT1A and COMT) metabolism by 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in the pig ovary.

The aim of the current study was to determine metabolism of polybrominated diphenyl ether (BDE-47) in the porcine ovary. We analyzed the activity and expression of enzymes involved in phase I (CYP1A1 and CYP2B1/2) and phase II (SULT1A and COMT) of BDE-47 metabolism. Basal CYP1A1 and CYP2B1/2 activity increased during culture. BDE-47 had no effect on CYP1A1, however increased CYP2B1/2 activity after exposure for 6h. Basal SULT1A activity was 2.5 fold lower than that of COMT, and both proteins were stable during culture. BDE-47 increased SULT1A after exposure for 6 h, and COMT activity after exposure for 24 and 48 h. BDE-47 had no effect on the expression of all investigated enzymes. In conclusion, fast activation of CYP2B1/2 and late activation of COMT (with a very low basal SULT1A activity) indicates a possible action of locally produced hydroxylated metabolites prior to their detoxification.

Expression of ghrelin and its receptor in porcine ovarian follicles collected from prepubertal and estrous cycle animals.

Ghrelin, a hormone predominantly found in the stomach, was described as a factor that controls female reproductive function. Using real time PCR and western blot, we measured gene and protein expression of ghrelin and its receptor. Enzyme-like immunoassay (ELISA) was used to measure the concentration of acylated (Ac) and unacylated (UnAc) forms of ghrelin as well as levels of estradiol (E2) in follicular fluid. For all analyses, we compared small, medium and large ovarian follicles collected from ovaries of prepubertal and estrous cycling pigs. We demonstrated that the gene expression levels of ghrelin significantly increased in ovarian follicles from cycling animals, with the maximum expression in large follicles, without any change in prepubertal. However, the protein expression of ghrelin and its concentration was increased with increasing follicle size both in prepubertal and cycling animals and it was positively correlated with E2 levels in follicular fluid. In addition, both receptor growth hormone secretagogue receptor (GHSR) and GHSR type GHSR-1a expression were significantly higher in ovarian follicles from cycling animals than prepubertal. Results of this study suggest the possibility of local synthesis of ghrelin in the ovarian follicles and point to important modulatory functions for ghrelin before puberty and during the estrous cycle.

Expression of ghrelin and the ghrelin receptor in different stages of porcine corpus luteum development and the inhibitory effects of ghrelin on progesterone secretion, 3ß-hydroxysteroid dehydrogenase (3ß-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 (3ß-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3ß-HSD protein expression were then analyzed. To assess 3ß-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 3ß-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3ß-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 3ß-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.

Hydroxylated estrogens (2-OH-E2 AND 4-OH-E2) do not activate cAMP/PKA and ERK1/2 pathways activation in a breast cancer MCF-7 cell line.

OBJECTIVE: The current study was undertaken to determine the involvement of cAMP/PKA and MAPK-mediated signalling pathways in the regulation of cell proliferation by hydroxylated metabolites of 17ß-estradiol (E2). METHODS: MCF-7, human breast cancer cells, were cultured in phenol red-free DMEM and treated with 1 nM 2-OH-E2 or 4-OH-E2. E2 was used as a positive control. Cell proliferation was measured using the BrdU incorporation assay. Cellular levels of cAMP and PKA were determined using ELISA kits. ERK1/2 protein expression was evaluated by Western Blot analysis. To determine the involvement of different intracellular pathways in the regulation of cell proliferation appropriate activators or inhibitors were used. RESULTS: Hydroxylated estrogens, as E2, exhibited no influence on cAMP accumulation and PKA activation. In concomitant treatments with forskolin, cell proliferation decreased to the amount noted under the influence of forskolin alone. A PKA inhibitor (PKI) had no statistically significant effect on proliferation stimulated by E2 and its hydroxylated metabolites. Phospho-ERK1/2 protein expression in cells stimulated with E2, 2-OH-E2 and 4-OH-E2 was not significantly different from the control. However, co-treatment with both PD98059 and E2 or its hydroxylated metabolites reversed the effect of tested compounds on cell proliferation. CONCLUSIONS: We have shown that E2 hydroxylated metabolites do not activate cAMP/PKA in breast cancer cells and confirm previously published data, which showed a lack of ERK1/2 activation in a breast cancer cell line. The observed reversible action of PD98059 on cell proliferation can be explained by the fact that hydroxylated estrogens, as E2, stimulate secretion of a number of growth factors, which affect MAPK activity, suggested by Lobenhofer et al. (2000).

Congener-specific action of PBDEs on steroid secretion, CYP17, 17ß-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, 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and CYP19 activity was measured by conversion of P4>A4, A4>T and T to E2, respectively. Protein expression of CYP17, 17ß-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 ß-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 3ß-HSD activity.

The ability of hydroxylated estrogens (2-OH-E2 and 4-OH-E2) to increase of SHBG gene, protein expression and intracellular levels in MCF-7 cells line.

OBJECTIVE: Sex Hormone-Binding Globulin (SHBG) - specific carrier for sex steroids - regulates hormone bioavailable fraction and estrogen signaling system in breast cancer cells. This study was conducted to elucidate the effects of hydroxylated estrogen (E2) metabolites (2-OH-E2 and 4-OH-E2) on sex hormone binding globulin (SHBG) mRNA and protein expression as well as on intracellular and extracellular SHBG levels. METHODS: MCF-7 human breast cancer cells were cultured with 2-OH-E2 or 4-OH-E2 in concentration of 1, 10 and 100 nM for 24 h, 17ß-estradiol being used as a positive control. SHBG levels were measured in medium and cells by ELISA, SHBG mRNA expression was evaluated by real-time-PCR and protein expression by Western blot analysis. RESULTS: 4-OH-E2 in high doses and 2-OH-E2 in the highest dose, while 17ß-estradiol in all doses used increased intracellular but not extracellular SHBG levels. Both metabolites increased SHBG mRNA expression, the rank order of potency being E2 > 4-OH-E2 > 2-OH-E2. Both E2 and its metabolites increased SHBG protein expression. CONCLUSION: Although the metabolites showed a lower potency than 17ß-estradiol, further studies are needed to clarify whether hydroxylated metabolites of E2 are potent ligands for SHBG.

ERK 1/2 and PI-3 kinase pathways as a potential mechanism of ghrelin action on cell proliferation and apoptosis in the porcine ovarian follicular cells.

Recently, we reported the stimulatory effect of ghrelin on ovarian cell proliferation in parallel with the inhibitory action of ghrelin on cell apoptosis. The aim of the presented data propose local activation of extracellular signal-regulated protein kinase 1 and 2 (ERK 1/2) and phosphoinositide-3 (PI-3) kinase pathways as a mechanism of ghrelin effect in the porcine ovary. To test this hypothesis, action of ghrelin on levels of ERK 1/2 with PI-3 kinase activity and protein expression using ELISA and western blot analysis, respectively, was examined. Additionally, to determine which pathways (ERK 1/2 or PI-3 kinase) are the potential signals of ghrelin-mediated cell proliferation and apoptosis in ovarian cells, we used PD098059 (50 microM) and wortmannin (200 microM), well-known inhibitors of these kinases. Treatment of ovarian coculture cells with ghrelin (100, 250, 500 and 1000 pg/ml) showed stimulation of phospho-ERK 1/2 levels and PI-3 kinase activity, with the maximum effect observed after 15 min of cell incubation. Additionally, western blot analysis indicated that ghrelin increased expression of both kinases. Moreover, ghrelin used in combination with PD098059 or wortmannin significantly decreased cell proliferation, which was measured by the Alamar Blue assay and increased apoptosis, which was measured by caspase - 3 activity and DNA fragmentation. In conclusion, these results suggest that the ERK 1/2 and PI-3 kinase pathways may be potential signals of ghrelin mediate the cell proliferation and apoptosis of ovary cells.

Mechanisms of action of two different natural mixtures of persistent organic pollutants (POPs) in ovarian follicles.

The present work investigated the effects of two different natural mixtures on aryl hydrocarbon receptor (AhR) and oestrogen receptor (ER)beta protein levels, as well as on the activity of cytochrome P450 (CYP) 1A1 and CYP2B. Consequently, the authors observed the effects of these mixtures on gonadotropine-stimulated steroid secretion by ovarian follicles. The natural mixtures that were studied were 'Mjosa' extracted from burbot liver, which contains a high level of PBDEs, and 'Marine mix', extracted from Atlantic cod liver, which contains a high level of polychlorinated biphenyls (PCBs). Follicular cells were exposed in vitro to 'Marine mix' and 'Mjosa mix' at doses of 3.6 and 1.4 microg ml(-1), respectively. Media were collected and used for steroid analysis and cell viability assays. Cells were used to estimate aromatase activity (CYP19), AhR and ER protein levels, and CYP1A1 and CYP2B1 activity. Western blot analysis indicated down-regulation of AhR by 'Marine mix' and down-regulation of ERbeta by Mjosa mix. Up-regulation of CYP1A1 expression and activity were seen following treatment with Marine mix, but not Mjosa mix. Increased CYP2B1 activity was noted after treatment with both 'Marine mix' and Mjosa mix. Both mixtures increased luteinizing hormone (LH)-stimulated progesterone and testosterone secretion, follicle-stimulating hormone (FSH)-stimulated oestradiol secretion, and CYP19 activity. These results suggest that: (1) 'Marine mix' is a mixed-type CYP inducer; (2) 'Mjosa mix' is an inducer of ERbeta and CYP2B; and (3) both 'Marine mix' and 'Mjosa mix' stimulate aromatase activity as a consequence of oestradiol secretion through activation of CYP19.

A low halogenated biphenyl (PCB3) increases CYP1A1 expression and activity via the estrogen receptor beta in the porcine ovary.

Polychlorinated biphenyls (PCBs) have been detected at high levels, up to hundreds of pg/ml, in human ovarian follicle fluid. The effect of PCBs on the ovary and the consequences of exposure are largely unknown. We have previously shown that PCB3 (4-chlorobiphenyl) increases the secretion of estradiol and the activity of cytochrome P450s (CYPs) in ovarian follicle cells. Our goal here is to elucidate the mechanism of CYP induction by this congener. Exposure of porcine follicle cells, a co-culture of theca and granulosa cells, to 6 ng/ml of PCB3 caused an increase in CYP1A1 protein and enzymatic activity, in the same manner as exposure to exogenous 17beta-estradiol. No changes were seen in the protein level of the aryl hydrocarbon receptor (AhR), which mediates the first step in the signaling pathway of CYP1A1 induction. However, a strong reduction was seen in the protein level of estrogen receptor beta (ERbeta), while no effect was seen on ERalpha protein levels. These result suggest that: 1) PCB3 acts as an agonist of ERbeta but not the Ah receptor in the ovarian follicles, 2) PCB3 is not only an efficacious inducer of CYP1A1 expression and activity, but also a substrate for this enzyme. Changes in the expression level of CYP1A1 not only alter the intensity of the activity of PCB3, but also the activity of estrogen in the ovary.

Modulatory effect of ghrelin in prepubertal porcine ovarian follicles.

Ghrelin is a novel growth hormone-releasing peptide, originally identified in rat stomach as an endogenous ligand of the growth hormone secretagogue receptor. Ghrelin is an important regulator of growth hormone secretion, food intake, and reproductive function. This study investigates whether or not ghrelin can modulate prepubertal pig ovary function, which was measured as ovarian estradiol secretion, aromatase activity, cell proliferation, and apoptosis. To investigate this, ovarian cells were co-cultured with four different doses of ghrelin (100, 250, 500, and 1000 pg/ml) for 48 h. Culture media samples were collected, and estradiol levels were determined, while aromatase expression was measured in the cultured cells. Cell apoptosis was measured by determination of caspase-3 activity, DNA fragmentation and TUNEL assay. Ghrelin in 250 and 500 pg/ml doses stimulated estradiol secretion. At all doses ghrelin stimulated aromatase activity and protein expression. Moreover, ghrelin increased cell proliferation and decreased apoptosis. This study provides novel evidence that ghrelin has a modulatory effect in the ovary. We suggest two mechanisms that explain how ghrelin acts on estradiol secretion: 1) ghrelin directly influences aromatase activity and protein expression; 2) ghrelin stimulates cell proliferation and antiapoptotic actions.

DDT- and DDE-induced disruption of ovarian steroidogenesis in prepubertal porcine ovarian follicles: a possible interaction with the main steroidogenic enzymes and estrogen receptor beta.

We evaluated impact of DDT isomers, o, p'- DDT [1, 1-dichloro-2, 2-bis (p, p'-chlorophenyl) ethylene] and p, p'-DDT [1, 1, 1-trichloro-2, 2-bis (p-chlorophenyl) ethane], and their metabolites, o, p'-DDE and p, p'-DDE, on ovarian steroidogenesis. All these compounds, except for p, p'-DDT, demonstrated estrogenic effects on steroid secretion in co-cultures of porcine prepubertal granulosa and theca cells. p,p'-DDT decreased progesterone and estradiol release, which was reversed by the addition of testosterone. In contrast, o, p'-DDT inhibited progesterone secretion with parallel stimulation of basal and testosterone-stimulated estradiol release. DDEs stimulated progesterone and estradiol secretion. The fluorometric assay confirmed that p,p'-DDE, o,p'-DDT, and o,p'-DDE stimulated aromatase activity. Western blots indicated that o,p-DDT and o,p'-DDE diminished the expression of estrogen receptor beta (ERbeta). This study demonstrated the isomer-dependent action of DDT in pig ovarian cells. We propose that DDT could disrupt ovarian steroidogenesis either by interfering with main steroidogenic enzymes or affecting ERbeta.

Differential accumulation of low-chlorinated (Delor 103) and high-chlorinated (Delor 106) biphenyls in human placental tissue and opposite effects on conversion of DHEA to E2.

OBJECTIVE: Polychlorinated biphenyls (PCBs) and related compounds elicit a diverse spectrum of toxic responses. Additionally, they are able to pass through the human placenta. The aim of the presented data was to compare the action of low-chlorinated (Delor 103) and (Delor 106) high-chlorinated biphenyls on placental steroidogenesis. METHODS: Explants of human placental tissue were used to test differences in PCBs accumulation and influence on placental steroidogenesis. Delor 103 or 106, were added daily for six days at a dose of 200 pg from day 0 to day 6 of culture. The media in the control and experimental groups were changed every day, and collected and frozen for steroid analysis by RIA. Determinations of PCBs of tissue and medium were analysed by GC/MS/MS. RESULTS: Delor 103 was found at a higher level in the tissue than Delor 106. The first day of exposure to Delor 103 had no effect on the conversion of dehydroepiandrosterone (DHEA) to estradiol (E2) while there was a 2-fold decrease in E2 secretion from days 3 to 6. Conversely, Delor 106 caused an immediate increase in E2 secretion, which was maintained at higher levels throughout the exposure period. CONCLUSION: Differences between the accumulation of lower chlorinated and higher chlorinated biphenyls in human placental tissue and in the properties of the congeners can have multiple effects that may intensify or counteract the effects on uterine contraction by PCBs.