Cong Yuan

Bisphenol A affects gene expression of gonadotropin-releasing hormones and type I GnRH receptors in brains of adult rare minnow Gobiocypris rarus.

Recent studies support the notion that endocrine disrupting chemicals (EDCs) could affect the reproductive regulations of the neuroendocrine system. The objectives of the present study were to determine whether the weak estrogenic chemical, bisphenol A (BPA), disrupts gonadotropin-releasing hormone (GnRH) system by altering the transcription of GnRHs and GnRH receptor (GnRHR) genes in adult rare minnow Gobiocypris rarus. In the present study, the histological examination of the ovary after 35-day BPA exposure at 15 μg/L demonstrated the perturbing effects of environmentally relevant BPA on the ovarian development in G. rarus. In addition mRNA expression of ovarian P450 aromatase in both ovaries and testes were significantly down-regulated by 15 μg/L BPA. GnRH2, GnRH3, GnRHR1A and GnRHR1B gene were identified in G. rarus. The expression patterns of GnRHs and GnRHR1s were analyzed in various tissues of G. rarus by quantitative real-time PCR. GnRHs and GnRHR1s were all predominantly expressed in the brains. Both GnRH3 and GnRHR1A were significantly upregulated in the brains of female exposed to 15 μg/L BPA for 35 days. It would suggest a potential negative feedback in the GnRH system in response to the disturbance of downstream of the brain-pituitary-gonadal axis. Collectively, the present findings suggest that the transcripts of some key genes in the neuroendocrine system can be used as critical biomarkers in endocrine disruption assays of teleost fish.

Oxidative stress and immunotoxic effects of bisphenol A on the larvae of rare minnow Gobiocypris rarus.

Bisphenol A (BPA), a known endocrine disrupting chemical, is ubiquitous in the aquatic environment and can pose risk to the health of aquatic organisms. Studies on immunotoxicity of BPA in aquatic organisms are limited. In this study, rare minnow (Gobiocypris rarus) larvae were exposed to 1, 225 and 1000μg/L BPA for 7 days. Inflammatory effects of BPA exposure were assessed from the increased production of nitric oxide (NO) and reactive oxygen species (ROS), the change of iNOS mRNA and other TLRs-associated immune gene expression. Our findings provide evidences that different concentrations of BPA can induce a toxic response in fish to produce reactive free radicals which can affect the function of T lymphocytes and decrease the transcription levels of cytokine genes. The excess production of H2O2, induced oxidative stress and suppressed TLR4/NF-κB signaling, leading to immunosuppressive effects in fish larvae. The present results suggest that BPA has the potential to induce oxidative stress accompanied by immunosuppression in rare minnow larvae.

Non-monotonic dose-response effect of bisphenol A on rare minnow Gobiocypris rarus ovarian development.

Bisphenol A (BPA) is widely spread in the environment, and can cause various reproductive disrupting effects on different organisms, including fish. Our previous published study showed that BPA has non-monotonic (inverted U-shaped) dose-response effect on rare minnow Gobiocypris rarus ovarian weight at different concentrations. To investigate the potential mechanism, we exposed female rare minnow to 1, 15 and 225 µg L(-1) BPA for 7 days in the present study. The levels of vitellogenin (Vtg), sex hormones, hydrogen peroxide (H2O2), glutathione (GSH) and triglyceride (TG) were measured. RNA-seq of ovary tissues was also performed. Result showed that Vtg, sex hormone and TG levels showed an inverted U-shaped increased response, while H2O2 and GSH levels showed a U-shaped inhibited response. RNA-seq data showed that many genes involved in lipid metabolism, oxidative stress, and proteolysis processes were altered. The change of Vtg, H2O2, GSH and TG levels was possibly related to the altered sex hormone levels. Sex hormones direct effect, Vtg accumulation, TG accumulation and oxidative stress induced proteolysis may contribute to the change of ovary weight.

Characterization of four nr5a genes and gene expression profiling for testicular steroidogenesis-related genes and their regulatory factors in response to bisphenol A in rare minnow Gobiocypris rarus

Bisphenol A (BPA) widely used in the manufacture of numerous products is ubiquitous in aquatic environment. To explore the mechanisms of BPA-mediated actions, male rare minnow Gobiocypris rarus were exposed to BPA at concentrations of 5, 15, and 50 μg/L for 14 and 35 days in the present study. Four subtypes of nr5a gene encoding important transcription factors for steroidogenesis were characterized, and tissue distribution analysis demonstrated distinct expression profiling of the four genes in G. rarus. BPA at environmentally relevant concentration (5 μg/L) caused increase of gonadosomatic index (GSI) of male fish. In response to BPA, no obvious changes on the testis development were observed. Modulation of vtg mRNA expression by BPA suggests estrogenic and/or anti-estrogenic effects of BPA were dependent on exposed duration (14 or 35 days). Gene expression profiling for testicular steroidogenesis-related genes, sexual steroid receptors, gonadotropin receptors, and transcription factors indicates differential regulation was dependent on exposure duration and dose of BPA. The correlation analysis at mRNA level demonstrates that the BPA-mediated actions on testicular steroidogenesis might involve sex steroid hormone receptor signaling, gonadotropin/gonadotropin receptor pathway, and transcription factors such as nuclear receptor subfamily 5, group A (Nr5a), fork head box protein L2 (Foxl2).

Effects of bisphenol A on lipid metabolism in rare minnow Gobiocypris rarus.

As one of the most abundant endocrine disrupting compounds (EDCs), bisphenol A (BPA) exists ubiquitously in an aquatic environment. Many studies on fish have focused on the reproductive toxicity effects of BPA. However, few has involved the effects of BPA on lipid metabolism. To evaluate the effects of BPA on lipid metabolism, we determine the hepato-somatic index, triglyceride contents in the liver and serum, the activities of acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), carnitine palmitoyltransferase (CPT1), and glycerol-3-phosphate acyltransferase (GPAT) enzymes and the mRNA expression of acaca, acacb, fasn, gpat1 and cpt1α in Gobiocypris rarus after exposure to BPA for 28days. BPA induced increasing tendency of triglyceride contents in male fish, possibly due to up-regulated lipid synthesis. Although in this process, fatty acid β-oxidation was up-regulated, it might be compensated by increasing lipogenesis. Our result also revealed that the GPAT enzyme might play a key role in lipid metabolism disturbance by BPA in females. Besides, the effect of BPA on the fatty acid β-oxidation pathway might be gender-dependent in G. rarus. Further studies are needed to investigate BPAs effects on the signaling pathway of lipid metabolism.

Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus

DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process.

Molecular characterization of estrogen receptor genes in loach Paramisgurnus dabryanus and their expression upon 17α-ethinylestradiol exposure in juveniles.

The full-length cDNAs for estrogen receptor 1 (esr1), esr2a and esr2b were isolated and characterized from the loach (Paramisgurnus dabryanus, Cobitidae, cypriniformes). P. dabryanus Esr1, Esr2a and Esr2b share high amino acids identities with their counterparts of cyprinid species. Quantitative real-time PCR (qRT-PCR) was used to analyze the tissue distribution of esr mRNAs in one-year-old P. dabryanus. The mRNA expression of esr1 in female liver was extremely higher than that in other tissues. esr2a mRNA expression in female intestine and in male muscle was higher than that in other tissues. esr2b mRNA expression was the highest in both male and female intestine. Two-month-old P. dabryanus were exposed to 17α-ethinylestradiol (EE2) for 3weeks and the changes of esr mRNA expression in brain, gonad and liver were analyzed by qRT-PCR. Results showed that EE2 at 1, 5 and 25 ng/L significantly suppressed testicular esr1 mRNA expression in male. The ovarian esr2a mRNA expression was significantly up-regulated at 1 ng/L EE2. In female brain, esr1 mRNA expression was significantly down-regulated at 5 ng/L EE2. Both in males and females, EE2 exposure increased the hepatic esr1 mRNA expression in a concentration-dependent manner. The present study suggests that different esrs in different tissues have differential responsiveness to EE2 and the hepatic esr1 is a sensitive biomarker to EE2 at environmental concentrations in P. dabryanus juveniles. So, the loach P. dabryanus, a typical demersal fish, is a promising ecological model organism to detect estrogenic chemicals in the sediment of aquatic environment by using molecular biomarkers.

Effects of 17 α-methyltestosterone on transcriptome, gonadal histology and sex steroid hormones in rare minnow Gobiocypris rarus

The 17α-methyltestosterone (MT), a synthetic androgen, is known for its interference effects on the endocrine system. Aiming to investigate the transcriptome profiling of gonads induced by MT and to understand the molecular mechanism by which MT causes adverse effects in fish, transcriptome profiling of gonads, gonadal histology and the sex steroid hormones in response to MT were analyzed in Gobiocypris rarus. Eight libraries, 4 from the ovary and 4 from the testis, were constructed and sequenced and then a total number of clean reads per sample ranging from 7.03 to 9.99 million were obtained. In females, a total of 191 transcripts were differentially regulated by MT, consisting of 102 up-regulated transcripts and 89 down-regulated transcripts. In males, 268 differentially expressed genes with 108 up-regulated and 160 down-regulated were detected upon MT exposure. Testosterone serves as the major sex steroid hormone content in G. rarus of both sexes. The concentrations of 17β-estradiol, testosterone and 11-ketotestosterone were significantly increased in females and decreased in males after MT exposure. Interestingly, MT caused a decreased number of vitellogenic oocytes in the ovary and spermatozoa in the testis. After MT exposure, four differentially expressed genes (ndufa4, slc1a3a, caskin-2 and rpt3) were found in G. rarus of both sexes. Overall, we suggest that MT seemed to affect genes involved in pathways related to physiological processes in the gonads of G. rarus. These processes include the electron transfer of Complex IV, endothelial cell activation, axon growth and guidance, and proteasome assembly and glutamate transport metabolic.

Gonadal development and transcript profiling of steroidogenic enzymes in response to 17α-methyltestosterone in the rare minnow Gobiocypris rarus

It is well known that natural and anthropogenic chemicals interfere with the hormonal system of vertebrate and invertebrate organisms. How these chemicals regulate gonadal steroidogenesis remains to be determined. The main objective of this study was to evaluate the effects of 17α-methyltestosterone (MT), a synthetic model androgen, on gene expression profiles of six key steroidogenic genes in adult rare minnow. The full-length cDNA encoding 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) was firstly isolated and characterized by RT-PCR and RACE methods. The gonadal transcript changes of StAR, cyp11a1, 3β-HSD, cyp17a1, 11β-HSD2 and cyp19a1a in 6-month adult Gobiocypris rarus exposed to MT and 17α-ethinylestradiol (EE2) for 7, 14 and 21 days were detected by qRT-PCR. To make an effort to connect the transcriptional changes of steroidogenic enzymes with effects on higher levels of biological organization and on VTG, one remarkable sensitive target of steroids, body and gonad weights, histology of gonads, and hepatic vtg mRNA level were measured. MT caused varying degree of abnormalities in ovaries and testes. The hepatic vtg mRNA level was highly inhibited in females and slightly altered in males by MT. Transcripts of several steroidogenic genes including StAR, cyp17a1, and cyp11a1 showed high responsiveness to MT exposure in G. rarus. The gene expression profiles of these steroidogenic genes in MT-treated groups were much distinct with the EE2-treated group.

Hepatic expression patterns of aryl hydrocarbon receptor, pregnane X receptor, two cytochrome P450s and five phase II metabolism genes responsive to 17alpha-methyltestosterone in rare minnow Gobiocypris rarus

17Alpha-methyltestosterone (MT), a synthetic androgen, is widely used in aquaculture. Aquatic organisms can receive continuous exposure to residual MT throughout their lives. Aiming to evaluate the effects of MT on genes involved in biotransformation pathway, meanwhile attempting to unravel the MT metabolic pathway at the transcriptional level in fish, here we isolated the cDNAs of previously unreported AHR2, Sult1 st1, Ugt2a1 and Ugt2b6 in rare minnow, and predominantly investigated the hepatic transcriptional patterns of AHR2, PXR and five biotransformation genes after MT exposure in both genders adult rare minnow Gobiocypris rarus. The present findings suggest that AHR2 and PXR should play important roles in regulating biotransformation enzymes related to MT catabolism, moreover, CYP1A, CYP3A, SULT1 ST4, SULT1 ST6 and UGT2A1 may play certain roles in catabolism of MT in adult G. rarus. Additionally, UGT2A1 may make greater contribution than SULT1 ST4 and SULT1 ST6 in MT catabolism in males.