Adult Rare Minnow Gobiocypris Rarus Research Articles

Adverse effects of bisphenol A on Sertoli cell blood-testis barrier in rare minnow Gobiocypris rarus

Bisphenol A (BPA), an environmental contaminant, has been shown to disturb the dynamics of Sertoli cell blood-testis barrier (BTB) in mammal testis. However, the effects of BPA on Sertoli cell barrier (SC barrier) were little known in fish to date. To evaluate the potential mechanism of reproductive toxicity of BPA, we studied the damage of SC barrier using in vivo models. In this study, male adult rare minnow Gobiocypris rarus were exposed to 15 μg/L BPA for 7–35 days. Gonadal histology and the integrity of SC barrier were analyzed. Meanwhile, the expressions of SC barrier -associated proteins, tumor necrosis factor (TNFα) content, and the mRNA expressions of genes in the mitogen activated protein kinase (MAPK) pathway were detected. Histological analysis demonstrated 15 μg/L BPA promoted the infiltration of inflammatory cells in fish testes after 7-days exposure. The biotin tracer assay showed that 7-days BPA exposure increased permeability for spermatid cysts. In addition, the BPA treatment caused increased TNFα in testis, which was reportedly related to SC barrier impairment. The expressions of Occludin and β-Catenin protein were significantly decreased in the testes after 7- and 21-days exposure. BPA also altered the mRNA expressions of occludin, β-catenin, p38 MAPK and JNK. Therefore, the detrimental effects of BPA on reproduction of male fish may attribute to the disturbed expressions of SC junction proteins.

Responses of gonadal transcriptome and physiological analysis following exposure to 17α-ethynylestradiol in adult rare minnow Gobiocypris rarus

17α-ethynylestradiol (EE2), a synthetic estrogen commonly used in the oral contraceptive pills, disrupts the sexual differentiation, gonadal development and reproduction in aquatic species. Nowadays aquatic species and even humans still have the potential risks of exposure to EE2. However, the mechanism of EE2 endocrine disruption is still unclear. Aiming to elucidate molecular mechanisms, we analyzed transcriptome profiling of gonads, gonadal histology and the sex steroid hormones in response to EE2 in G. rarus. Through this study, we obtained eight RNA-Seq libraries upon EE2 exposure, and found some key genes and pathways in correlation with the disruption effects of EE2. We found EE2 could disrupt oocyte development and spermatogenesis in adult G. rarus, and EE2 has more obvious disruption effects on male G. rarus than females. Interestingly, EE2 was indicated to be an exogenous DPC-inducing agent and ppp2r3b was suggested to be a spermatogenesis candidate gene in rare minnow. The differential gene expressions of rps30, samp9, ppp2r3b and spartan upon EE2 exposure suggest EE2's disruption effects on gonads could attribute to altered pathways of translation, ribosome biogenesis and cell division.

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.

Effect of low-dose malathion on the gonadal development of adult rare minnow Gobiocypris rarus

Malathion is an organophosphorus pesticide that extensively used in agriculture and veterinary practices. To investigate the effects of low dose malathion on rare minnow Gobiocypris rarus gonadal development, we exposed adult rare minnow to environmentally relevant concentration malathion (2 and 20μg/L) for 21 days. Gonadal histology, sex hormone levels and mRNA expressions of steroidogenic genes were investigated. Malathion at both 2 and 20μg/L significantly up-regulated rare minnow testicular weight and promoted the progression of spermatogenesis. Neither ovarian weight nor process of ovary development was markedly changed. In testis, 2μg/L malathion significantly down-regulated testosterone and 11-ketotestosterone levels, and up-regulated mRNA expression of steroidogenic genes. In ovaries, 2 and 20μg/L malathion significantly inhibited estradiol17β levels and induced testosterone levels, both in concentration dependent manners; mRNA expressions of almost all the detected ovarian steroidogenic genes were up-regulated. The present result suggested that malathion even at low dose could pose a potential threat to adult rare minnow gonadal development.

Global and cyp19a1a gene specific DNA methylation in gonads of adult rare minnow Gobiocypris rarus under bisphenol A exposure

As a weak estrogenic chemical, bisphenol A (BPA) has been extensively studied for reproductive toxicity and the effects on the steroidogenesis. In the present study, we aim to explore the effects of BPA on epigenetic modification in rare minnow Gobiocypris rarus. We have detected the global and cyp19a1a gene specific DNA methylation in gonads of adult G. rarus under BPA exposure. The global DNA methylation level was significantly increased in testis of the male fish exposed to BPA for 7 days, and it was significantly increased in the ovary following 35-days exposure. DNA methyltransferases (DNMTs) catalyze the transfer of a methyl moiety from S-adenosyl-l-methionine to the cytosine of a CpG dinucleotide. The alteration of the detected dnmts mRNA expression could affect the global DNA methylation levels following 15μg/L BPA exposure. Cytochrome P450 aromatase (CYP19A1A), is responsible for the conversion of androgens into estrogens, which plays a vital role in estrogen synthesis in gonads. In the present study, the methylation level of ovarian cyp19a1a gene was significantly suppressed and stimulated by 7- and 35-day BPA exposure, respectively. There was a significant negative correlation between cyp19a1a mRNA expression and methylation levels of the four CpGs at the 5′ flanking region in the ovary of adult G. rarus following BPA exposure. So we hypothesize that there are some association between the reproductive toxicity of BPA and the global DNA methylation under BPA exposure. And the alteration of cyp19a1a expression in female G. rarus by BPA might attribute to the change of its DNA methylation status.

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.

DNA methylation in the 5′ flanking region of cytochrome P450 17 in adult rare minnow Gobiocypris rarus — Tissue difference and effects of 17α-ethinylestradiol and 17α-methyltestoterone exposures

Cytochrome P450 17 (CYP17) plays a vital role in hormone production in the body. In our previous study, mRNA expression of cyp17a1 was regulated by endocrine disrupting chemicals in rare minnow Gobiocypris rarus. However, the mechanism underlying the regulation is unclear. In the present study, we aim to explore whether the differential expression of cyp17a1 in distinct tissues and the modulation of its expression upon 17α-ethinylestradiol (EE2) and 17α-methyltestoterone (MT) are related to the DNA methylation status in G. rarus. The 732-bp fragment of 5′ flanking region of cyp17a1 gene was isolated in G. rarus. The bisulfite sequencing PCR result showed that DNA methylation levels in 5′ flanking of cyp17a1 in the gonads were significantly lower than those in the brains, which is negatively related to its mRNA expression in the 2 tissues in the previous study. The 7-day EE2 exposure of 25ng/L caused a significant increase of methylation levels of cyp17a1 gene and a significant decrease of its transcript in testis. While 100ng/L MT exposure for 7days caused a significant decrease of methylation levels of cyp17a1 gene and a significant increase of its transcript in the ovary. The present findings indicate that the methylation status of cyp17a1 gene is negatively correlated with its mRNA expression in response to EE2 and MT in G. rarus. We hypothesize that the regulation of cyp17a1 expression by EE2 and MT might attribute to the change of its DNA methylation status in G. rarus.

Responsiveness of four gender-specific genes, figla, foxl2, scp3 and sox9a to 17α-ethinylestradiol in adult rare minnow Gobiocypris rarus

Proteins encoded by figla, foxl2, scp3 and sox9a play important roles in gonad differentiation and reproduction. In the present study, we aimed to determine the responsiveness of figla, foxl2, scp3 and sox9a to 17α-ethinylestradiol (EE2) in the gonads of adult Gobiocypris rarus. Full-length cDNAs of figla, scp3 and sox9a were cloned and characterized by RT-PCR and RACE methods. Expression patterns in adult tissues were investigated. Results indicated that figla was predominantly expressed in adult ovaries and scp3 was restrictively expressed in the male testes and sox9a was principally expressed in the brains of both genders and the testes of males. Gene expression profiles of figla, foxl2, scp3 and sox9a were analyzed in the gonads of adult G. rarus exposed to EE2 at 1, 5, 25, and 125ng/L for 3 and 6days. Three-day EE2 treatment at 1–125ng/L all caused a significant increase of figla transcript in testes and foxl2 transcript in ovaries. However, six-day EE2 exposure at 1–125ng/L repressed figla and scp3 transcript in testes and foxl2 transcript in ovaries. The present study indicates that the testicular transcripts of figla and scp3 in males and the ovarian foxl2 transcript in females have high responsiveness to EE2 and they can be used as sensitive molecular biomarkers for early warning to monitor the environmental estrogenic chemicals in fresh water environment. The present study also suggests that the effective EE2 dosage for feminization in male G. rarus might be at least 25ng/L.

Expression of two zona pellucida genes is regulated by 17α-ethinylestradiol in adult rare minnow Gobiocypris rarus

Zona pellucida (ZP) proteins are glycoproteins synthesized in liver, ovary or in both tissues in fish. In the present study, we aimed to determine the responsiveness of ZP2 and ZP3 to 17α-ethinylestradiol (EE2) in adult rare minnow Gobiocypris rarus. The full length of ZP3 cDNA was firstly characterized and its tissue distribution revealed that ZP3 mRNA was predominantly expressed in ovary of G. rarus. The gene expression profiles of ZP2, ZP3 and vitellogenin (VTG) were analyzed in gonad and liver of adult G. rarus exposed to EE2 at 1, 5, 25, and 125ng/L for 3 and 6days. The results show that ZP2 is more sensitive than ZP3 in gonads of both genders, and VTG in liver is extremely sensitive to EE2 in male fish. However, at lower concentrations (1 and 5ng/L), the ZP2 in testes shows higher responsiveness to EE2 compared with VTG in rare minnow. The 5′ flanking regions of ZP2 and ZP3 were isolated and the comparison of transcription factors in the regions of ZP2 and ZP3 suggested that the disparity for the responsiveness of ZP2 and ZP3 to EE2 could partly be a result of differential cis-elements such as oocyte-specific protein (Osp1) binding sites or/and sex-determining region Y (SRY) binding site.

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 15μg/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 15μg/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 15μg/L BPA for 35days. 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.

Effects of 17α-ethinylestradiol and bisphenol A on steroidogenic messenger ribonucleic acid levels in the rare minnow gonads

Previous studies showed that the endocrine disrupting chemicals (EDCs) affect reproductive physiology in teleosts. How the EDCs regulate gonadal steroidogenesis remains to be determined. The gonadal transcript changes of steroidogenic enzyme genes in adult rare minnow Gobiocypris rarus exposed to 17α-ethinylestradiol (EE2) and bisphenol A (BPA) were detected in the present study. The full-length cDNAs encoding steroidogenic enzymes, including steroidogenic acute regulatory protein (StAR), cytochrome P450-mediated side-chain cleavage enzyme (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 17 A1 (CYP17A1) were isolated and characterized by RT-PCR and RACE methods. The homology and phylogenetic analyses of the amino acid sequences confirmed that the nucleotide sequences of these steroidogenic genes were correct. The mRNA tissue distribution results indicated that StAR, cyp11a1, and cyp17a1 mRNAs were mainly expressed in the gonads and 3β-HSD was mainly expressed in both the gonads and the brains. The 233 dpf adult G. rarus were exposed to EE2 (25ng/L) and BPA (5, 15, and 50μg/L) dissolved in dimethyl sulfoxide (DMSO) or control for 7 days. The gonadal mRNA levels of StAR, cyp11a1, 3β-HSD, cyp17a1 and ovarian cytochrome P450 aromatase (cyp19a1a) were quantified by qRT-PCR. Our data indicated that 25ng/L EE2 had different degrees of inhibitory effects on the expression of steroidogenic genes in the gonads. BPA at different levels caused concentration-specific effects on the mRNA expression of the steroidogenic genes. The transcripts of several ovarian steroidogenic genes were more sensitive to 15μg/L BPA than that at other two levels. These findings suggest that EE2 could impair gonadal steroidogenesis by suppressing mRNA expression of steroidogenic genes and BPA could cause variations in gonadal steroidogenesis modulation with a potential consequence of compensation for the disturbance.