Expression of selected nuclear receptors in human epithelial ovarian cell line Caov3 exposed to bisphenol derivatives.

Objectives. Bisphenol A (BPA) is an indispensable industrial chemical. However, as a proven endocrine disruptor, it may be associated with several health disturbances, including the reproductive functions impairment and cancer. Due to the restriction of BPA usage, many bisphenol derivatives gradually substitute BPA. However, studies have reported adverse biological effects of BPA analogs, but the specific sites of their action remain largely unknown. Nuclear receptors (NRs) appear to play significant roles in various types of cancer. In addition, they are considered relevant targets of bisphenols. In the present study, we investigated the effects of BPA and its analogs bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF) on mRNA expression of selected NRs in the human ovarian epithelial cell line Caov3. The NRs examined included retinoic acid receptor α (RARA), retinoid X receptor α (RXRA), peroxisome proliferator activating receptor ß/δ (PPARD), chicken ovalbumin upstream promoter-transcription factor 2 (COUPTFII), and nuclear receptor-related protein 1 (NURR1). Methods. Caov3 cells were treated with the bisphenols at the concentrations of 1 nM, 100 nM, 10 µM and 100 µM. After 24 h and 72 h of incubation, cell viability was determined by the MTS assay, and the selected genes expression was analyzed using RT-qPCR. Results. Bisphenol treatment did not affect Caov3 cell viability, except the significant impairment after exposure to the highest BPAF dose (100 µM). At lower doses, neither bisphenol analog altered the expression of the NRs. However, at the highest concentration (100 µM), BPAF and BPA altered the mRNA levels of PPARD, COUPTFII, and NURR1 in a time- and receptor-specific manner. Conclusions. The effects of bisphenols on the specific NRs in the epithelial ovarian cancer cells were addressed for the first time by the present study. Although generally we did not find that bisphenols may provoke significant alterations in the expression of the selected NRs in Caov3 cells, they may alter mRNA expression of certain NRs at high concentrations.

Bisphenol analogs AF and S: Effects on cell status and production of angiogenesis-related factors by COV434 human granulosa cell line.

While Bisphenol A (BPA) has been a requisite plastic additive, as an endocrine disruptor it has been associated with adverse health effects including ovarian disorders. Following implemented restrictions on BPA usage, it is replaced by alternative bisphenols, biological effects of which have not been adequately investigated. Our study examined effects of bisphenols AF (BPAF) and S (BPS), on the human ovarian granulosa cell line COV434, and compared them with BPA, with the focus on cell viability (10-9-10-4 M) and angiogenesis-related factors (10-9-10-5 M), relevant for both the follicle development and ovarian pathologies: vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor AA (PDGF-AA), and matrix metalloproteinase 9 (MMP-9). Each bisphenol impaired cell viability and increased generation of intracellular reactive oxygen species at the highest concentration (10-4 M). While VEGF-A production in BPAF-treated groups did not differ from the control, all doses of BPS and BPA caused a marked reduction in VEGF-A output. Nevertheless, the alterations in VEGF-A production were not caused by the impact on VEGFA gene expression since there were no indications of VEGFA downregulation in the presence of either BPS or BPA. Interestingly, we observed a similar pattern of PDGF-AA output reduction in BPS- and BPA-treated groups to that of VEGF-A production. BPAF and BPS (10-5 M) increased MMP9 expression, however, this effect was not reflected by the increase in MMP-9 production. The results obtained demonstrate that the novel bisphenol analogs are not inert with respect to the ovarian cells, and their effects might contribute to dysregulation of granulosa cells functions.

Bisphenol analogs AF, S and F: Effects on functional characteristics of porcine granulosa cells.

In order to replace industrial functions of the restricted endocrine disruptor bisphenol A (BPA), its structural analogs are increasingly employed without adequate assessment of their biological actions. Our study examined effects of the bisphenols AF (BPAF), S (BPS) and F (BPF), on functions of porcine ovarian granulosa cells (GCs) with the focus on viability, steroid production (10-9-10-4M), and expression of factors (10-9-10-5M) important for the follicle development: vascular endothelial growth factor A (VEGFA), matrix metalloproteinase 9 (MMP9), forkhead box O1 (FOXO1), and aryl hydrocarbon receptor (AHR). Cell viability was not impaired by the bisphenol analogs, except for the highest BPAF concentration (10-4M). While the lower concentrations of the bisphenols were without effect, each of them reduced follicle-stimulating hormone (FSH)-induced progesterone synthesis at the highest dose. Estradiol synthesis was sensitive to BPS, inhibitory effects of which were manifested from the concentration of 10-6M. Treatment of GCs with the selected bisphenol concentrations did not result in marked alterations in steroidogenic enzyme expression. Bisphenols did not significantly modulate VEGFA mRNA expression or output either under basal or FSH-stimulated conditions. BPF at 10-5M increased MMP9 expression in FSH-stimulated cells. FSH upregulated FOXO1 expression, however, none of the bisphenols significantly affected FOXO1 levels either in basal or in FSH-stimulated conditions. AHR mRNA expression remained unchanged after bisphenol treatment. Although the significant effects of BPAF, BPS and BPF appeared only at supraphysiological doses, the results obtained indicate that BPA analogs are not inert with regard to ovarian physiology.

The use of ex vivo ovary culture for assessment of alterations in steroidogenesis following neonatal exposure to poly(ethylene glycol)-block-polylactide methyl ether or titanium dioxide nanoparticles in Wistar rats.

OBJECTIVES: Rapid development and widespread application of different types of nanoparticles (NPs) may result in increased exposure of humans and animals to NPs. Recently, reproductive toxicity due to NP exposure has become a major component of risk assessment. Current data have suggested that NPs may pose adverse effects on male and female reproductive health by altering normal testis and ovarian structure, and sex hormone levels. To detect possible alterations in steroidogenesis in adult and infantile rats following neonatal exposure to polymeric poly(ethylene glycol)-block-polylactide methyl ether (PEG-b-PLA) or titanium dioxide (TiO2) NPs, whole ovary cultures were used. METHODS: Newborn female Wistar rats were intraperitoneally (i.p.) injected daily with two different doses of PEG-b-PLA NPs (20 and 40 mg/kg body weight, b.w.) or TiO2 NPs (1% LD50 TiO2=59.2 µg/kg b.w. and 10% LD50 TiO2=592 µg/kg b.w.) from postnatal day 4 (PND 4) to PND 7. The ovaries were collected on PND73 and PND15 of PEG-b-PLA- and TiO2 NP-treated rats, respectively, and their corresponding control animals. Minced ovaries were cultured in vitro in the absence (basal conditions) or presence of gonadotropins (follicle-stimulating hormone, FSH and luteinizing hormone, LH) and insulin-like growth factor-1 (IGF-1) (stimulated conditions) for 6 days. At indicated time intervals, culture media were collected for steroid hormone (progesterone, estradiol) analysis by specific radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Basal progesterone and estradiol secretion by ovaries from adult rats (PND73) were significantly decreased (p<0.01) in both PEG-b-PLA-treated groups after 3 days and 1 day of ex vivo ovary culture, respectively, compared with control group. With the presence of FSH/LH and IGF-1 in the culture medium, progesterone and estradiol production significantly increased (p<0.001) compared to basal levels. Stimulated progesterone production was significantly decreased (p<0.05) in PEG-b-PLA40-treated group after 3 days of culture compared with controls. After ex vivo culture of rat ovaries collected on PND15, basal progesterone and estradiol levels measured in the culture media did not differ between control and both TiO2 NP-treated groups. The ovaries from rats neonatally exposed to both doses of TiO2 NPs failed to respond to FSH/IGF stimulation in progesterone secretion at all time intervals. CONCLUSIONS: The obtained results indicate that neonatal exposure to NPs in female rats may alter ovarian steroidogenic output (steroid hormone secretion) and thereby might subsequently induce perturbation of mammalian reproductive functions. Possible mechanisms (induction of oxidative stress, inflammation) of adverse effects of NPs on ovarian function should be further elucidated.

Effect of selected bisphenol derivatives on nuclear receptor expression in ovarian cell line COV434.

Objectives. Bisphenol A (BPA), as an indispensable plastic additive, has also been proven as an endocrine disruptor associated with adverse health effects including impaired ovarian function and cancer. Due to the restrictions of its usage, several analogs have been employed to replace BPA. Although many studies revealed a harmfulness in the biological effects of BPA analogs, their specific targets remain largely unknown. Nuclear receptors (NRs) may be one of the most important targets of bisphenols. Therefore, in this study, our attention was directed to explore the effect of BPA and its analogs, AF and S, on the mRNA expression of selected NRs involved in the steroidogenic and carcinogenic pathways in the human granulosa cell line COV434. The NRs investigated included: thyroid hormone receptor α (THRA), peroxisome proliferator activating receptor ß/δ (PPARD), retinoid X receptor α (RXRA), chicken ovalbumin upstream promoter-transcription factor II (COUPTFII), nuclear receptor-related protein 1 (NURR1), and liver receptor homolog-1 (LRH1).Methods. COV434 cells were treated with the bisphenols at the concentrations of 10-9 M, 10-7 M, and 10-5 M, and after 24 and 48 h, cell viability was monitored by the MTS assay and gene expressions were analyzed using RT-qPCR.Results. Bisphenol treatment did not alter the COV434 cell viability. After 24 h, the expression of neither of the NRs was changed. Likewise, after 48 h, the expression of the selected genes was not altered. However, both BPAF and BPS increased, at the highest concentration (10-5 M) used, the mRNA levels of both PPARD and NURR1 NRs after 48 h of the treatment. In the BPA-treated groups, no significant upregulation was observed.Conclusions. In the present study, the effect of bisphenols on COUP-TFII, Nurr1, and LRH-1 NRs was investigated for the first time. Although generally we did not observe that BPs provoked any alterations in the expression of the selected NRs in COV434 cells, at specific concentrations and time points they might alter mRNA expression of certain NRs (NURR1, PPARD).

Simultaneous effects of endocrine disruptor bisphenol A and flavonoid fisetin on progesterone production by granulosa cells.

In the present study, we aimed to examine effects of different concentrations of the endocrine disruptor Bisphenol A (BPA; 1 nM, 1 µM, 100 µM) and the flavonoid fisetin (1, 10, 25, 50 µM), individually and in combinations, on steroidogenic function of porcine ovarian granulosa cells (GCs) represented by progesterone production. We confirmed that BPA inhibited progesterone production by GCs at the highest concentration. Fisetin reduced gonadotropin-stimulated progesterone synthesis dose-dependently, and in this manner, fisetin impaired progesterone production when added to BPA-treated GCs. The mechanisms of the inhibitory effects of the combinations included a significant down-regulation of the key steroidogenesis-related genes (STAR, CYP11A1, HSD3B). Our findings suggest for the first time that fisetin might interfere with ovarian steroidogenesis, and might not have beneficial but rather aggravating effects in terms of modulating progesterone synthesis altered by high concentrations of BPA.

Effect of Neonatal Exposure to Poly(Ethylene Glycol)-block-Poly(Lactic Acid) Nanoparticles on Oxidative State in Infantile and Adult Female Rats.

Our goal was to evaluate the potential health risk of the polymeric NP, poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA), from the view of redox imbalance of the organism in two different life stages. Female Wistar rats were neonatally administered intraperitoneally with PEG-b-PLA NPs [20 mg/kg of b.w. (PEG20) or 40 (PEG40) mg/kg of b.w.] from postnatal day 4 (PND4) to PND7. We measured antioxidant capacity (TEAC), level of protein carbonyls and lipoperoxides in plasma, activities of catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD) in hemolysates of infantile (sacrificed on PND17) and adult (sacrificed after PND176) rats. Compared to controls, neonatal PEG40 exposure induced a significant TEAC reduction in the infantile rats. Protein carbonyls and lipoperoxide levels were not affected after any dose of PEG-b-PLA NP administration. In adult rats, PEG20 administration caused a significant decrease of protein carbonyl levels compared to controls. In infantile rats, both doses of PEG-b-PLA NP administration increased catalase, Gpx, and SOD activities compared to controls. Surprisingly, in adult rats, the activities of Gpx and SOD decreased significantly after administration of both doses of PEG-b-PLA NPs. Obtained data indicate a possible age-related association between the oxidative status and neonatal PEG-b-PLA NP administration in female rats.

Impact of endocrine disrupting chemicals on onset and development of female reproductive disorders and hormone-related cancer.

A growing body of evidence suggests that exposure to chemical substances designated as endocrine disrupting chemicals (EDCs) due to their ability to disturb endocrine (hormonal) activity in humans and animals, may contribute to problems with fertility, pregnancy, and other aspects of reproduction. The presence of EDCs has already been associated with reproductive malfunction in wildlife species, but it remains difficult to prove causal relationships between the presence of EDCs and specific reproductive problems in vivo, especially in females. On the other hand, the increasing number of experiments with laboratory animals and in vitro research indicate the ability of different EDCs to influence the normal function of female reproductive system, and even their association with cancer development or progression. Research shows that EDCs may pose the greatest risk during prenatal and early postnatal development when organ and neural systems are forming. In this review article, we aim to point out a possible contribution of EDCs to the onset and development of female reproductive disorders and endocrine-related cancers with regard to the period of exposure to EDCs and affected endpoints (organs or processes).

Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic-pituitary-ovarian axis development and function in Wistar rats.

We studied delayed effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether (PEG-b-PLA) on the endpoints related to pubertal development and reproductive function in female Wistar rats from postnatal day 4 (PND4) to PND 176. Female pups were injected intraperitoneally, daily, from PND4 to PND7 with PEG-b-PLA (20 or 40mg/kg b.w.). Both doses of PEG-b-PLA accelerated the onset of vaginal opening compared with the control group. In the low-dose PEG-b-PLA-treated group, a significantly reduced number of regular estrous cycles, increased pituitary weight due to hyperemia, vascular dilatation and congestion, altered course of hypothalamic gonadotropin-releasing hormone-stimulated luteinizing hormone secretion, and increased progesterone serum levels were observed. The obtained data indicate that neonatal exposure to PEG-b-PLA might affect the development and function of hypothalamic-pituitary-ovarian axis (HPO), and thereby alter functions of the reproductive system in adult female rats. Our study indicates a possible neuroendocrine disrupting effect of PEG-b-PLA nanoparticles.

Effect of polymeric nanoparticle poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) on in vitro luteinizing hormone release from anterior pituitary cells of infantile and adult female rats.

OBJECTIVES: Polymeric PEG-b-PLA nanoparticles (NPs) were developed for delivery of poorly water-soluble drugs via blood brain barrier into brain parenchyma. We analyzed neuroendocrine disrupting effects of neonatal exposure of female rats to PEG-b-PLA NPs and diethylstilbestrol (DES) on the function of adenohypophyseal gonadotrophs of infantile or adult rats by examining in vitro luteinizing hormone releasing hormone (LHRH)-induced luteinizing hormone (LH) release. METHODS: Neonatal female Wistar rats were injected intraperitoneally, daily, from postnatal day (PND) 4 to PND7 with PEG-b-PLA NPs (20 mg.kg b.w.(-1)), DES (4 µg.kg b.w.(-1)) or vehicle. At the necropsy day (PND15 in infantile and the first estrus day after PND176 in adult rats), adenohypophyseal cells were isolated by enzymatic digestion, plated in 96-well plates (5×10(4) cells.well(-1)) in serum-supplemented medium and left to recover for 96 h. LHRH (10-7 mol.L(-1)) treatment was performed in serum-free medium for 60 min and LH levels in culture media were determined by radioimmunoassay. RESULTS: In all experimental groups, in vitro LHRH treatment significantly stimulated LH release from pituitary cells of infantile but not adult female rats. Neonatal DES treatment increased basal LH secretion from cultured pituitary cells of adult but not infantile rats. In both, infantile and adult rats, neonatal treatment with PEG-b-PLA significantly increased basal and LHRH-induced LH release from pituitary cells compared to corresponding controls and DES-treated group. CONCLUSIONS: Data indicate that neonatal exposure to PEG-b-PLA NPs may alter pituitary LH release, and thereby modify reproductive system development in infantile female rats leading to reproductive dysfunctions in adult age.

Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium.

OBJECTIVE: To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC). DESIGN: Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling. SETTING: A reproductive biology laboratory. PATIENT(S): Not applicable. INTERVENTION(S): Porcine OCC were cultured in vitro in a medium with FSH/LH in the presence/absence of lapatinib. MAIN OUTCOME MEASURE(S): Methods performed: real-time reverse transcriptase-polymerase chain reaction (PCR), immunofluorescence, RIA. RESULT(S): In FSH/LH-stimulated and expanded cumulus oophorus extracellular matrix, HA was detected with biotinylated HA-binding proteins. However, weaker HA- and weaker cytoplasmic TNFAIP6 were detected were detected in lapatinib-pretreated OCC. The expression of the two cumulus expansion-associated gene transcripts was significantly decreased and synthesis of HA by cumulus cells was reduced. Lapatinib (10 µM) inhibited FSH/LH-induced oocyte meiotic maturation. Progesterone production increased after OCC stimulation with FSH/LH and was significantly decreased by lapatinib (10 µM). CONCLUSION(S): Lapatinib inhibits oocyte maturation and reduces expression of cumulus expansion-associated transcripts, and synthesis of HA and P in OCC cultured in vitro in FSH/LH-supplemented medium.

MNU-induced mammary gland carcinogenesis: chemopreventive and therapeutic effects of vitamin D and Seocalcitol on selected regulatory vitamin D receptor pathways.

The effects of administration of vitamin D3 and Seocalcitol on MNU-induced carcinogenesis of mammary gland in Sprague-Dawley rats have been investigated. Administration of both substances in a weekly dose of 7 µg/kg caused prolonged latency of mammary gland tumors. The latency of tumors was markedly prolonged for 30-40 days by Seocalcitol. Using PET analysis, reduction in [¹8F]2-fluoro-2-deoxy-d-glucose (FDG) uptake or tumor volume in tumors chemopreventively treated with vitamin D3 were detected in MNU-induced tumors, vitamin D3 reduced expression of 25-hydroxylase (25OHase) (p<0.01) and 24-hydroxylase (24OHase) (p<0.01) and Seocalcitol 24OHase. Positive regulation of 25OHase mRNA level after the treatment with vitamin D3 was observed in liver, while in kidney, vitamin D3 and Seocalcitol induced expression of 24OHase was significant. Our observations indicate a cross talk between respective pathways of VDR, RARs/RXRs, TRs and ERs in carcinogenesis process.

Activation of cumulus cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-cumulus cell expansion and steroidogenesis.

Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine cumulus cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH-EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine cumulus cells and is involved in the control of both cumulus expansion and steroidogenesis.

Development of functional LH Receptors on pig cumulus-oocyte complexes cultured in vitro by a novel two-step culture system.

We show in the present study that freshly isolated pig cumulus-oocyte complexes (COCs) display a limited response to LH, as assessed by the expression of hyaluronan synthase 2 (Has2) mRNA, activation of protein kinase A (PKA), production of hyaluronic acid (HA) and progesterone, cumulus cell expansion and resumption of meiosis. These data indicate that freshly isolated COCs do not possess a sufficient number of functional LH receptors (LHR). However, the expression of Lhr significantly increased during the culture of COCs in vitro in a medium supplemented with FSH. Assuming that the effect of FSH on LHR induction is mediated via cAMP signaling pathways, we developed a new culture system, in which the COCs were pre-cultured for 72 hr in a medium supplemented with dbcAMP. The pre-cultured COCs remained in the germinal vesicle stage, their cumulus investment underwent a dramatic increase in size and gap junctions between the cumulus cells were preserved. The stimulation of such COCs with either FSH or LH led to the resumption and completion of meiosis, activation of PKA, expression of Has2, synthesis of large amounts of HA and progesterone, and extensive expansion of cumulus cells. We conclude that the formation of functional LHR is stimulated in cumulus cells during the culture in vitro in a cAMP-dependent pathway. The dbcAMP-treated COCs thus represent a new model in which the resumption of meiosis and cumulus expansion can be induced exclusively by the action of recombinant LH.

Effects of selected endocrine disruptors on meiotic maturation, cumulus expansion, synthesis of hyaluronan and progesterone by porcine oocyte-cumulus complexes.

In most mammals, before ovulation, cumulus cells synthesize a large amount of hyaluronan (HA) that is organized into an extracellular matrix (ECM), which provides an essential microenvironment for in vivo oocyte fertilization. This process is called cumulus expansion. The present study assessed effects of selected endocrine disruptors (bisphenol A, BPA; 4-chloro-3-methyl phenol, CMP; di(2-ethylhexyl) phthalate, DEHP; and benzyl butyl phthalate, BBP) in a range of 100pM-100microM, on follicle-stimulating hormone (FSH)-induced meiotic maturation and cumulus expansion of porcine oocyte-cumulus complexes (OCC) cultured in vitro. Moreover, FSH-stimulated production of hyaluronic acid (HA) and progesterone by cumulus cells was measured. Both phenols, BPA and CMP (100microM), significantly affected meiotic maturation of oocytes. The number of oocytes that underwent germinal vesicle breakdown (GVBD) (78.7% and 72.4%, respectively) as well as the rate of oocytes that reached metaphase II stage (MII) (50% and 53.6%, respectively) after 44h culture were decreased compared to control (89.6% for GVBD and 81.5% for MII). FSH-stimulated expansion of cumulus was altered by the highest concentration of BPA and CMP (70% and 64%, respectively vs. 80.3% in control). Although BPA did not alter FSH-stimulated HA synthesis by cumulus cells, its incorporation within the complex was reduced to a half of control value. Progesterone production by OCC was significantly changed in the presence of BPA or DEHP. Finally, our results provide valuable information that oocyte meiotic progression was adversely affected during in vitro culture with endocrine disruptors.

The effects of selected phenol and phthalate derivatives on steroid hormone production by cultured porcine granulosa cells.

We have investigated the effects of several phenols (octylphenol [OP], nonylphenol [NP], tert-octylphenol [tOP]) and phthalates (dioctylphthalate [DOP], diisodecylphthalate [DiDP], diisononylphthalate [DiNP]) on steroid hormone production by porcine ovarian granulosa cells after a 72-hour incubation. These chemicals are widely used as plasticisers and are suspected to possess endocrine disrupting properties. No changes were exhibited in basal progesterone production after treatment with NP or tOP, or with the tested phthalates. However, OP tended to decrease progesterone levels, while DOP and DiDP, at the lowest concentration used (10(-8)M), increased progesterone levels in the culture media. Neither of the tested phenols affected follicle stimulating hormone (FSH)-stimulated progesterone production, except for OP and NP at 10(-4)M, which decreased progesterone levels. The phthalates, tested at higher concentrations, were able to amplify FSH-stimulated progesterone release into the culture medium. An inhibitory action on oestradiol production by porcine granulosa cells was observed after the treatment with both groups of test chemicals. The results obtained in the experiments on primary granulosa cell cultures indicate that ovarian steroidogenesis might be one of the possible sites affected by the endocrine disrupting actions of phenols and phthalates.

Alterations in steroid hormone production by porcine ovarian granulosa cells caused by bisphenol A and bisphenol A dimethacrylate.

We have investigated the effects of bisphenol A (BPA) and BPA-dimethacrylate (BPA-DMA), endocrine disruptors used as plasticizers, on steroid hormone production by porcine ovarian granulosa cells after 72 h incubation. BPA at 10(-8) M to 10(-5) M increased basal progesterone levels, while the same concentration range of BPA-DMA did not cause any changes. After FSH-stimulation of the cells, BPA-DMA showed a tendency to inhibit progesterone production. BPA, however, at 10(-7) M and 10(-6) M concentrations was even able to amplify FSH-stimulated progesterone synthesis. BPA as well as BPA-DMA inhibited FSH-induced estradiol production in the whole concentration range. LH-stimulated progesterone production was not altered by BPA in 10(-8) M to 10(-5) M, while BPA-DMA decreased progesterone levels in the cultured media. Significant inhibitory effect of both tested agents at 10(-4) M concentrations was observed specifically on progesterone production, basal as well as gonadotropin-stimulated. The results indicate that ovarian steroidogenesis might be one of the possible sites afflicted by the endocrine disrupting action of BPA and BPA-DMA.

Ovarian intrafollicular processes as a target for cigarette smoke components and selected environmental reproductive disruptors.

Steroidogenesis, expansion of oocyte-cumulus complex, and meiotic maturation of the oocyte represent intrafollicular processes taking important part in the background of successful fertilisation. The reproductive health of female could be affected by a number of endogenous as well as exogenous factors, such as exposure to agents from specific lifestyle habits, environmental pollutants with endocrine disrupting properties, or heavy metals. Published data indicate that exposure to chemicals may cause alterations in reproductive behavior and contribute to sub-fecundity, infertility, or ovarian failure. Female reproductive functions can be compromised by exposure to toxic chemicals at a variety of sites, including ovary or reproductive tract. Substantial harmful effects of cigarette smoke on fecundity and reproduction have become apparent but are not generally appreciated. The effects of cigarette smoke components (cadmium, nicotine, cotinine) absorbed into the organism on intrafollicular processes may thus in part explain the negative impact of smoking on female fertility. Moreover, it is now evident that a variety of man-made pollutants present in the environment are capable to disrupt normal endocrine function in many species. Examples of these "endocrine disrupters" include plasticizers, such as phthalates and bisphenol A. The effects of selected environmental chemicals on the processes of steroidogenesis, expansion of oocyte-cumulus complex, and meiotic maturation of the oocyte are summarized in the present paper and possible mechanisms of action of these agents are suggested. However, for complete understanding the mechanisms by which chemical agents from the environment can affect the intrafollicular processes, a lot of further investigation is needed.