Impact of bisphenol-A on the spliceosome and meiosis of sperm in the testis of adolescent mice.

BACKGROUND: Bisphenol-A (BPA) has estrogenic activity and adversely affects humans and animals' reproductive systems and functions. There has been a disagreement with the safety of BPA exposure at Tolerable daily intake (TDI) (0.05 mg/kg/d) value and non-observed adverse effect level (5 mg/kg/d). The current study investigated the effects of BPA exposure at various doses starting from Tolerable daily intake (0.05 mg/kg/d) to the lowest observed adverse effect level (50 mg/kg/d) on the testis development in male mice offspring. The BPA exposure lasted for 63 days from pregnancy day 0 of the dams to post-natal day (PND) 45 of the offspring. RESULTS: The results showed that BPA exposure significantly increased testis (BPA ≥ 20 mg/kg/d) and serum (BPA ≥ 10 mg/kg/d) BPA contents of PND 45 mice. The spermatogenic cells became loose, and the lumen of seminiferous tubules enlarged when BPA exposure at 0.05 mg/kg/d TDI. BPA exposure at a low dose (0.05 mg/kg/d) significantly reduced the expression of Scp3 proteins and elevated sperm abnormality. The significant decrease in Scp3 suggested that BPA inhibits the transformation of spermatogonia into spermatozoa in the testis. The RNA-seq proved that the spliceosome was significantly inhibited in the testes of mice exposed to BPA. According to the RT-qPCR, BPA exposure significantly reduced the expression of Snrpc (BPA ≥ 20 mg/kg/d) and Hnrnpu (BPA ≥ 0.5 mg/kg/d). CONCLUSIONS: This study indicated that long-term BPA exposure at Tolerable daily intake (0.05 mg/kg/d) is not safe because low-dose long-term exposure to BPA inhibits spermatogonial meiosis in mice testis impairs reproductive function in male offspring.

Activated AMPK promoted the decrease of lactate production in rat Sertoli cells exposed to Zearalenone.

Zearalenone, which is ubiquitous in grains and animal feed, is a mycotoxin that can cause serious damage to animals and humans. Sertoli cells (SCs) can be used to study ZEA male reproductive toxicity in vitro. SCs provide energy for germ cells, where AMPK regulates intracellular energy. In order to explore the regulatory effect of AMPK on ZEA-induced lactate decline, we activated AMPK by AICAR and then inhibited AMPK by Compound C with ZEA-treated SCs for 24 h to detect intracellular lactate production-related indicators. Cell viability in the presence of 20 µmol/L ZEA and either 50 µmol/L AICAR or 5 µmol/L Compound C, respectively, did not damage SCs, and could effectively either activate or inhibit AMPK. Inhibition of AMPK promoted the production of pyruvate and lactate via increased expression of the glycolysis-related genes Pgam1 and the lactate production-related proteins GLUT1, LDHA, and MCT4. Activating AMPK inhibited the production of lactate and pyruvate by suppressing the expression of glycolysis-related genes HK1, Pgam1, and Gpi1 and that of lactate production-related proteins LDHA and MCT4. Zearalenone destroys the energy balance in SCs, activates P-AMPK, which inhibit the production of lactate and pyruvate in SCs. This also leads to the decrease of energy supply of SCs to spermatogenic cells, damages to reproductive system.

Resveratrol alleviates zea-induced decidualization disturbance in human endometrial stromal cells.

Decidualization, which endows the endometrium competency to adopt developing embryo and maintain appropriate milieu for following growth, is a pivotal process for human pregnancy. The delicate collaboration between ovarian steroid hormones estrogen and progesterone governs the process of decidualization and subsequent establishment of embryo implantation. Mycotoxin zearalenone (ZEA) is well known as endocrine disruptor due to its potent estrogenic activity. In this study, we investigated effects of ZEA on decidualization of human endometrial stromal cells. Results indicated that ZEA exhibited its inhibitory action through nuclear translocation of ERα. ZEA exposure led to dampened progress of decidualization, which could be attenuated by estrogen receptor antagonist. Notably, resveratrol (RSV) administration restored impaired decidualization process by induction of anti-oxidative gene glutathione peroxidase 3 (GPX3). This study provides novel insights into the mechanism underlying adverse effects of ZEA in human decidual stromal cells and suggests RSV a potential therapeutic candidate to alleviate ZEA-induced cytotoxicity during decidualization.

The antagonistic effect of bisphenol A and nonylphenol on liver and kidney injury in rats.

OBJECTIVE: Bisphenol A (BPA) and nonylphenol (NP) are widely distributed endocrine-disrupting compounds. We aimed to estimate the combined toxicity of BPA and NP at a clinically safe dose (100 µg/kg) in rats. MATERIALS AND METHODS: Liver and kidney functions were evaluated by detecting the relevant indicators. Hematoxylin and Eosin (HE) staining was performed to examine the injury in the tissue. TUNEL assay and Western blot were used to detect cell apoptosis and expressions of target factors, respectively. RESULTS: The body weight of rats in the BPA + NP group was lighter than that in the BPA or NP group. BPA or NP weakened liver function through increasing levels of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), cholesterol (CHOL), triglyceride TG, globulin (GLOB), treponemiapallidum (TP), and total bilirubin (TBIL). BPA and NP could induce kidney damage by elevating the levels of serum creatinine (Scr) and blood urea nitrogen (BUN). Moreover, the malondialdehyde (MDA) content was increased, whereas the activities of superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-PX), glutathione sulfotransferase (GSH-ST), catalase (CAT), and peroxidase (POD) were reduced in those groups exposed to BPA or NP. HE staining exhibited injuries of the liver and kidney. Furthermore, the apoptosis of liver and kidney cells was enhanced by exposure to BPA or NP. Additionally, the expressions of CYP2D6, CYP1A1, and CYP2E1 were triggered by the treatment of BPA or NP. The combined effect of BPA and NP seemed to be antagonistic at a low dose. CONCLUSION: BPA and NP may have potential interactions.

Permeation, stability and acute dermal irritation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica crude extract loaded transdermal gels.

In this study, permeation behaviors and chemical stability of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (PM), Thai traditional medicine, crude extract containing transdermal gels were firstly evaluated. Three different PM extract containing gels were formulated, including hydroalcoholic and microemulsion gels using carbomer, and silicone gel using silicone elastomer. In vitro permeation through porcine ear skin demonstrated that the flux and 24 h cumulative permeation of miroestrol and deoxymiroestrol were in the order of hydroalcoholic > silicone > microemulsion gels. Hydroalcoholic gel provided the highest partition coefficient from gel onto skin, and thus the skin permeability coefficient. After 24 h permeation, no miroestrol and deoxymiroestrol remained deposited in the skin. Accelerated study using heating-cooling revealed insignificant difference between the remaining percentages of miroestrol and deoxymiroestrol in aqueous and non-aqueous based gels. Long-term stability study showed that miroestrol contents remained constant for 90 d and 30 d under 5 ± 3 °C and 30 ± 2 °C, 75 ± 5%RH, respectively; whereas the percentage of deoxymiroestrol decreased significantly after 30 d storage, irrespective of storage conditions. Acute dermal irritation test on New Zealand White rabbits showed that PM hydroalcoholic gels were non-irritant, with no signs of erythema or oedema.[Figure: see text].

Targeting the Lowest Concentration of a Toxin That Induces a Detectable Metabolic Response in Living Organisms: Time-Resolved In Vivo 2D NMR during a Concentration Ramp.

In vivo nuclear magnetic resonance (NMR) is a powerful analytical tool for probing complex biological processes inside living organisms. However, due to magnetic susceptibility broadening, which produces broad lines in one-dimensional NMR, 1H-13C two-dimensional (2D) NMR is required for metabolite monitoring in vivo. As each 2D experiment is time-consuming, often hours, this limits the temporal resolution over which in vivo processes can be monitored. Furthermore, to understand concentration-dependent responses, studies are traditionally repeated using different contaminant and toxin concentrations, which can make studies prohibitively long (potentially months). In this study, time-resolved non-uniform sampling NMR is performed in the presence of a contaminant concentration sweep. The result is that the lowest concentration that elicits a metabolic response can be rapidly detected, while the metabolic pathways impacted provide information about the toxic mode of action of the toxin. The lowest concentration of bisphenol A (BPA) that induces a response was ∼0.1 mg/L (detected in just 16 min), while changes in different metabolites suggest a complex multipathway response that leads to protein degradation at higher BPA concentrations. This proof of concept shows it is possible, on the basis of "real-time" organism responses, to identify the sublethal concentration at which a toxin impacts an organism and thus represents an essential analytical tool for the next generation of toxicity-based research and monitoring.

Low doses of BPA induced abnormal mitochondrial fission and hypertrophy in human embryonic stem cell-derived cardiomyocytes via the calcineurin-DRP1 signaling pathway: A comparison between XX and XY cardiomyocytes.

Humans are inevitably exposed to bisphenol A (BPA) via multiple exposure ways. Thus, attention should be raised to the possible adverse effects related to low doses of BPA. Epidemiological studies have outlined BPA exposure and the increased risk of cardiovascular diseases (such as cardiac hypertrophy), which has been confirmed to be sex-specific in rodent animals and present in few in vitro studies, although the molecular mechanism is still unclear. However, whether BPA at low doses equivalent to human internal exposure level could induce cardiac hypertrophy via the calcineurin-DRP1 signaling pathway by disrupting calcium homeostasis is unknown. To address this, human embryonic stem cell (H1, XY karyotype and H9, XX karyotype)-derived cardiomyocytes (CM) were purified and applied to study the low-dose effects of BPA on cardiomyocyte hypertrophy. In our study, when H1- and H9-CM were exposed to noncytotoxic BPA (8 ng/ml), markedly elevated hypertrophic-related mRNA expression levels (such as NPPA and NPPB), enhanced cellular area and reduced ATP supplementation, demonstrated the hypertrophic cardiomyocyte phenotype in vitro. The excessive fission produced by BPA was promoted by CnAß-mediated dephosphorylation of DRP1. At the molecular level, the increase in cytosolic Ca2+ levels by low doses of BPA could discriminate between H1- and H9-CM, which may suggest a potential sex-specific hypertrophic risk in cardiomyocytes in terms of abnormal mitochondrial fission and ATP production by impairing CnAß-DRP1 signaling. In CnAß-knockdown cardiomyocytes, these changes were highly presented in XX-karyotyped cells, rather than in XY-karyotyped cells.

Inhibition of pancreatic lipase by environmental xenoestrogens.

Environmental xenoestrogens are the most accessible endocrine disrupting chemicals that have been reported with harmful effects on human health. Although the influences of xenoestrogens on the endocrine system have been extensively studied, it remains unclear whether these xenoestrogens can affect the digestive system in mammals. This study aimed to investigate the inhibitory effects and the underlying mechanism of six non-steroidal synthetic estrogens (including hexestrol, diethylstilbestrol, dienestrol, bisphenol A, bisphenol AF and bisphenol Z) on pancreatic lipase (PL), a key digestive enzyme responsible for lipid digestion and absorption in mammals. The results clearly demonstrated that hexestrol, diethylstilbestrol and dienestrol exhibited strong inhibition on PL, with the IC50 values of less than 1.0 µM. Further investigations elucidated that these three synthetic estrogens functioned as mixed inhibitors of PL, with the Ki values of less than 1 µM. Moreover, molecular dynamics simulations showed that diethylstilbestrol and its analogues might block the binding of substrate on PL via occupying the portal to the active site of PL and thereby inhibit the hydrolytic activity of this key enzyme. Collectively, these results suggested that diethylstilbestrol and its analogues were potent PL inhibitors, which might play a profound role in lipid absorption and weight gain in mammals.

Gonadoprotective ability of Vincetoxicum arnottianum extract against bisphenol A-induced testicular toxicity and hormonal imbalance in male Sprague Dawley rats.

Vincetoxicum arnottianum (Wight) of family Apocynaceae is a rich source of therapeutic alkaloids, phenolics and flavonoids. Study aims to evaluate the protective potential of methanol extract of Vincetoxicum arnottianum (VAM) on bisphenol A (BPA)-induced testicular toxicity in male Sprague Dawley rat. Quantitative analysis of VAM for total phenolic (TPC), total flavonoid (TFC) and total alkaloid content (TAC) along with HPLC analysis for polyphenolics was carried out. BPA-induced testicular toxicity was determined through analysis of antioxidant enzymes, DNA damages and testicular histopathology along with reproductive hormones in serum of rat. VAM was constituted of TFC (382.50 ± 1.67 µg GAE/mg), TPC (291.17 ± 0.82 µg RE/mg), TAC (16.5 ± 0.5%), ferulic acid (2.2433 µg/mg) and vanillic acid (2.1249 µg/mg). VAM co-administration to BPA-treated rats attenuated the toxic effects of BPA and restored the body and testis weights. Altered level of luteinizing hormone (LH), testosterone and follicle-stimulating hormone (FSH) in serum, and level of antioxidants (GSH, POD, CAT and SOD) and nitric oxide in testis tissues of BPA-induced toxicity were significantly restored by VAM. Histological and comet assay studies also sanctioned the protective potential of VAM in BPA-intoxicated rats. The presence of polyphenols and alkaloids might contribute towards the scavenging and ameliorative potential of VAM in testicular toxicity induced by BPA.

Molecular Aspects of Mycotoxins-A Serious Problem for Human Health.

Mycotoxins are toxic fungal secondary metabolities formed by a variety of fungi (moulds) species. Hundreds of potentially toxic mycotoxins have been already identified and are considered a serious problem in agriculture, animal husbandry, and public health. A large number of food-related products and beverages are yearly contaminated by mycotoxins, resulting in economic welfare losses. Mycotoxin indoor environment contamination is a global problem especially in less technologically developed countries. There is an ongoing effort in prevention of mould growth in the field and decontamination of contaminated food and feed in order to protect human and animal health. It should be emphasized that the mycotoxins production by fungi (moulds) species is unavoidable and that they are more toxic than pesticides. Human and animals are exposed to mycotoxin via food, inhalation, or contact which can result in many building-related illnesses including kidney and neurological diseases and cancer. In this review, we described in detail the molecular aspects of main representatives of mycotoxins, which are serious problems for global health, such as aflatoxins, ochratoxin A, T-2 toxin, deoxynivalenol, patulin, and zearalenone.

Perinatal High-Fat Diet and Bisphenol A: Effects on Behavior and Gene Expression in the Medial Prefrontal Cortex.

Both high-fat diets (HFD) and bisphenol A (BPA), an environmental endocrine disruptor, are prevalent in industrialized societies. Previous studies have detected separate effects of BPA and HFD; however, none have assessed possible interactive effects. Here, pregnant dams consumed 0, 40, or 400 µg BPA/kg/day and were fed either a control (CON; 15.8% kcal fat) or HFD (45% kcal fat) from gestational day 2 through parturition. The pups were individually dosed with BPA from postnatal days (P) 1-10, while the dams continued to consume one of the two diets. Maternal behavior increased with the HFD while the offspring's periadolescent social play decreased with BPA, but no interactive effects were observed. Neither HFD nor BPA exposure changed performance on a social recognition task, and only BPA had an effect on the elevated plus maze. BPA increased several cytokines in the medial prefrontal cortex (mPFC) of P10 males but not females. Expression of several genes related to hormone synthesis and receptors, inflammation, oxidative stress, and apoptosis in the mPFC on P10 and P90 were altered due to BPA and/or HFD exposure with rare interactive effects. BPA resulted in an increase in the gene expression of Esr1 in the mPFC of females on both P10 and P90. Epigenetic analysis on P90 did not show a change in methylation or in the levels of pre-mRNA or microRNA. Thus, perinatal BPA and HFD have separate effects but rarely interact.

Mice lacking membrane estrogen receptor 1 are protected from reproductive pathologies resulting from developmental estrogen exposure†.

Both membrane and nuclear fractions of estrogen receptor 1 (ESR1) mediate 17ß-estradiol (E2) actions. Mice expressing nuclear (n)ESR1 but lacking membrane (m)ESR1 (nuclear-only estrogen receptor 1 [NOER] mice) show reduced E2 responsivity and reproductive abnormalities culminating in adult male and female infertility. Using this model, we investigated whether reproductive pathologies caused by the synthetic estrogen diethylstilbestrol (DES) are mitigated by mESR1 ablation. Homozygous and heterozygous wild-type (WT and HET, respectively) and NOER male and female mice were subcutaneously injected with DES (1 mg/kg body weight [BW]) or vehicle daily from postnatal day (PND) 1-5. Uterine histology was assessed in select DES-treated females at PND 5, whereas others were ovariectomized at PND 60 and treated with E2 (10 µg/kg BW) or vehicle 2 weeks later. Neonatal DES exposure resulted in ovary-independent epithelial proliferation in the vagina and uterus of WT but not NOER females. Neonatal DES treatment also induced ovary-independent adult expression of classical E2-induced transcripts (e.g., lactoferrin [Ltf] and enhancer of zeste homolog 2 [Ezh2]) in WT but not NOER mice. At PND 90, DES-treated WT and HET males showed smaller testes and a high incidence of bacterial pyogranulomatous inflammation encompassing the testes, epididymis and occasionally the ductus deferens with spread to lumbar lymph nodes; such changes were largely absent in NOER males. Results indicate that male and female NOER mice are protected from deleterious effects of neonatal DES, and thus mESR1 signaling is required for adult manifestation of DES-induced reproductive pathologies in both sexes.

Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERß-dependent) at higher concentrations. BPC induced zfERß-mediated luciferase expression in vitro, and the zfERß agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

Quantitative proteomic analyses of mammary organoids reveals distinct signatures after exposure to environmental chemicals.

Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation.

Nonylphenol causes shifts in microbial communities and nitrogen mineralization in soil microcosms.

The aims of this work was to investigate, in soil microcosms, the effects on soil microbial community structure and function of increasing concentrations of 4-Nonylphenol (NP). The lasts is a product of degradation of NPEOs (Nonylphenol polyethoxylates) with a known toxic and estrogenic capacity able to disrupt animal's hormonal systems. The effect of increasing concentrations of NP (0, 10, 30, 90, and 270 mg NP kg-1 of dry soil) in soil microcosms in three sampling dates (28, 56, and 112 days) over soil microbial activity and function were assessed. Soil microbial activity was estimated by microbial ATP content, and both bacterial and fungal communities composition were estimated using the terminal restriction fragment length polymorphism technique (T-RFLP). Abundance of ammonia-oxidizing bacteria (AOB) was estimated by qPCR of gene encoding for the bacterial ammonia-monoxygenase (amoA). Changes in biologically mediated soil properties were also assessed, namely water-soluble NH+4, NO-2 and NO-3 content, the two last allowing the assessment of mineralization rates. NP-spiking had some unexpected impacts on microbial community structure and functions, since (i) impacted both bacterial and fungal communities structure at the highest NP concentration tested, bacterial communities were resistant to lower concentrations, while fungal communities were increasingly impacted until the end of the incubation at day 112; (ii) no community structure resilience was observed in bacteria at the highest NP concentration nor for fungi at any concentration; (iii) microbial activity decreased with NP after 28 and 56 d, but increased in the last sampling at the highest concentrations tests, coupled to an enrichment in AOB taxa after 56 and 112 days, that at least partly explain also explain the observed speed up of nitrification rates.

Modulation of brain kisspeptin expression after bisphenol-A exposure in a teleost fish, Catla catla.

Endocrine-disrupting chemicals (EDCs) affect the neuroendocrine system which in turn influences the reproductive regulation. Neuronal genes disrupted by EDCs are the gonadotropin-releasing hormone (gnrh2), the Kiss/GPR54 system that regulates gonadotropin release and cyp19b gene encoding brain aromatase. In the present study, pubertal Catla catla expected to spawn for first the time in the coming season were exposed to graded concentration of bisphenol-A (10, 100, 1000 µg/l) for 14 days. Messenger RNA (mRNA) levels of neuroendocrine genes, i.e., kisspeptins and their receptors, gonadotropin-releasing hormone type II and brain aromatase were studied after 14 days exposure. Results showed that bisphenol-A (BPA) strongly upregulated expression of kiss1, kiss2, gpr54a, and gnrh2 in fish exposed to 10 µg/l BPA. Fish exposed to 1000 µg/l BPA, expression of kiss1 and gnrh2 were comparable to control while kiss2 mRNA increased compared to controls. Brain aromatase (cyp19b) mRNA expression increased in fish exposed to both 10 and 1000 µg/l BPA. These results indicate that BPA exposure can disrupt organization of the kisspeptin signaling pathways. This neuroendocrine disruption may be the underlying mechanism by which a suite of reproductive abnormalities are induced.

In utero exposure to both high- and low-dose diethylstilbestrol disrupts mouse genital tubercle development.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development. Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra. This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol, at a high, clinically relevant dose, and a low, previously untested dose, administered via water. The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency. Females exhibited hypospadias and urethral-vaginal fistula. These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

Differentiation of sow and mouse ovarian granulosa cells exposed to zearalenone in vitro using RNA-seq gene expression.

Zearalenone (ZEA), a natural contaminant found in feed, has been shown to have a negative impact on domestic animal reproduction, particularly in pigs. There are species-specific differences in the ZEA-induced toxicity pattern. Here, we investigated the different biological effects of ZEA exposure on porcine and mouse granulosa cells, using RNA-seq analysis. We treated murine and porcine granulosa cells with 10 µM and 30 µM ZEA during 72 h of culturing, in vitro. The results showed that 10 µM ZEA exposure significantly altered mitosis associated genes in porcine granulosa cells, while the same treatment significantly altered the steroidogenesis associated genes in mouse granulosa cells. Exposure to 30 µM ZEA resulted in significantly up-regulated expression of inflammatory related genes in porcine granulosa cells as well as the cancer related genes in mouse granulosa cells. Similarly, 30 µM ZEA exposure significantly decreased the expression of tumor suppressor factors in the mouse granulosa cells. Furthermore, immunofluorescence, RT-qPCR as well as western-blot analysis verified the different expression of related genes in ZEA exposed porcine and mouse granulosa cells. Collectively, these results illustrate the presence of species differences with regards to ZEA effects between porcine and mouse ovarian granulosa cells, in vitro.

Bisphenol A alters oocyte maturation by prematurely closing gap junctions in the cumulus cell-oocyte complex.

In ovarian follicles, cumulus cells communicate with the oocyte through gap junction intercellular communication (GJIC), to nurture the oocyte and control its meiosis arrest and division. Bisphenol A (BPA) is a monomer found in polycarbonate-made containers that can induce functional alterations, including impaired oocyte meiotic division and reduced molecule transfer in GJIC. However, how BPA alters oocyte meiotic division is unclear. We investigated whether BPA effects on oocyte meiotic division were correlated with reduced transfer in GJIC. Cumulus cell-oocyte complexes (COCs) isolated from mouse preovulatory follicles were cultured with 0, 0.22, 2.2, 22, 220, and 2200 nM BPA for 2 h. An additional 16-h incubation with epidermal growth factor (EGF) was performed to promote the occurrence of meiotic resumption and progression to metaphase II. Without EGF stimulus, BPA treatment increased the percentage of oocytes undergoing meiotic resumption, decreased GJIC in the COCs, and did not modify GJIC gene (Cx43 and Cx37) and protein (CX43) expression. Following EGF stimulus, BPA increased the percentage of oocytes that remained at the anaphase and telophase stages, and decreased the percentage of oocytes reaching the metaphase II stage. Concomitantly, BPA reduced the expansion of cumulus cells. Carbenoxolone (a GJIC inhibitor) and 6-diazo-5-oxo-l-norleucine (a cumulus cell-expansion inhibitor) exerted effects on meiotic division similar to those exerted by BPA. These data suggest that BPA accelerates meiotic progression, leading to impaired prophase I-to-metaphase II transition, and that this adverse effect is correlated with reduced bidirectional communication in the COC.

Effects of bisphenol A on ovarian follicular development and female germline stem cells.

Bisphenol A (BPA), one of the most frequently detected emerging pollutants in the environment, has been implicated in adverse effects in male and female reproduction at extremely low concentrations. This study aimed to investigate the effects and potential mechanism of BPA on mouse ovarian follicular development and female germline stem cells (FGSCs). Female CD-1 adult mice were administered gradient concentrations of BPA (12.5, 25, and 50 mg/kg/day) by intraperitoneal injection. We found that the number of atretic ovarian follicles was significantly increased at high BPA concentrations. Additionally, the numbers of primordial follicles, primary follicles, and corpus luteum (CL) were significantly reduced at high BPA concentrations. Interestingly, the number of FGSCs was remarkably reduced in BPA-treated ovaries. Furthermore, the increased apoptotic rate of FGSCs in vitro was triggered by BPA accompanied by increased BPA concentrations. To investigate the mechanism of BPA in ovarian follicular development, 193 differentially expressed proteins were identified in BPA-treated ovaries by the isobaric tags for relative and absolute quantification-coupled 2D liquid chromatography-mass spectrometry technique. A total of 106 proteins were downregulated and 85 proteins were upregulated. Among these proteins, the apoptosis-related protein SAFB-like transcriptional modulator (SLTM) was remarkably upregulated, and this result was consistent with western blotting. Taken together, our results suggest that an ovarian follicular development, especially, the development of primordial follicles, primary follicles, and the CL, is inhibited by high BPA concentrations, and the ovarian follicle atresia is initiated by BPA through upregulated expression of SLTM. Furthermore, BPA induces apoptosis of cultured FGSCs. The effect of BPA on ovarian follicular development and FGSCs, especially the effect on FGSCs, suggests a novel mechanism of how BPA causes female infertility.