| Endocrine disrupting chemicals (EDCs) are natural organic compounds or anthropogenicchemicals discharged into the environment and disrupt the endocrine system of human andwildlife. Recent studies revealed that EDCs could affect the reproductive regulations ofneuroendocrine system. However, the mechanisms are complex and involve in all three levelsof the hypothalamic-pituitary-gonadal (HPG) axis. The HPG axis mainly be regulated bygonadotropin-releasing hormone, gonadotropin hormone and sex hormone. Rare minnow(Gobiocypris rarus) is an ideal model fish for the study of EDCs, which has been widely usedfor aquatic toxicity studies. We first selected suitable reference genes for the mRNAexpression analyses using three different statistics programs; Secondly, quantitative real-timePCR (qRT-PCR) method was applied to investigate the mRNA expressions of12differentgenes related to the hormone regulation on HPG axis after expose to BPA/EE2in the brainand gonad of rare minnow. In addition, high-throughput deep sequencing technology wasused to analyze the transcriptom of brain under EE2treatments.The major findings of present study are as follow:(1) We first cloned and sequenced five frequently used reference genes, includingelongation factor1-alpha (ef1a), glyceraldehyde-3-phosphate dehydrogenase (gapdh),glucose-6-phosphate dehydrogenase (g6pd), TATA box binding protein (tbp) and tubulin alpha1(tuba1) in G. rarus. Subsequently, mRNA expression pattern of above five genes togetherwith β-actin (actb) was tested the rare minnow in the early developmental stages (18-50dpf),and the adult and juvenile fish after the EDCs exposure. Meanwhile, three different statisticalprograms, including Bestkeeper, geNorm and NormFinder were used to investigate stabilityof these genes in different treatments. The most appropriate reference gene was selected bycomprehensively considering the results of three algorithms. Similarly, the same strategy wasapplied to select the appropriate reference genes (actb, ef1a, gapdh and tuba1) in the brainafter EDCs exposure. Last, we used above six reference genes (actb, ef1a, g6pd, gapdh, tbp,and tuba1) as house-keeping genes to investigate the mRNA expression of brain P450aromatase (cyp19a1b) in rare minnow. The results indicated that actb was most stable andg6pd was most instable in gonads of the adult fish and during rare minnow juvenile development. In the brain, the most stable reference gene was ef1a and the least stable wasgapdh. In further, we investigated the stablility of reference genes in the rare minnow underEDCs treatments. The results revealed that the expressions of actb, ef1a, gapdh and tbp werenot affected by EDCs treatment, whereas the expressions of g6pd and tuba1were variableunder EDCs treatment.(2) Eight reproductive regulation related genes, including GnRH2, GnRH3, GnRHR1A,GnRHR1B, FSHβ, LHβ, FSHR and LHR genes were first cloned and sequenced in G. rarus.The homology and phylogenetic analyses of the amino acid sequences revealed that theseeight genes share high identity in cyprinid fish. The mRNA expression analyses in differenttissues (brain, gill, eye, muscule, intestines, liver, and gonad) by qRT-PCR showed thatGnRH2, GnRH3, GnRHR1A, GnRHR1B, FSHβ and LHβ were mainly expressed in brain;while FSHR and LHR were mainly expressed in gonads.(3) Adult G. rarus (8-month old) were exposed to weak estrogen BPA (5or15μg/L) orcontrol for35days. The brain mRNA expression patterns of GnRH2, GnRH3, GnRHR1A,GnRHR1B, FSHβ and LHβ were analyzed by qRT-PCR. And mRNA expressions of ovarianP450aromatase (cyp19a1a), FSHR and LHR were analyzed in gonad.15μg/L BPA couldsuppress the ovarian development in G. rarus. And mRNA expressions of cyp19a1a in bothovaries and testes were significantly downregulated by15μg/L BPA. After35-day BPAexposure, both GnRH3and GnRHR1A were significantly upregulated by15μg/L BPA in thebrains of female.(4) Adult fish (3-month old) were exposed to EE2at1,5,25,125ng/L for3and6days.Expression of brain FSHβ, LHβ, estrogen receptors (esr1, esr2a, and esr2b) and gonadalFSHR, LHR, cyp19a1a were assessed. The brain LHβ, but not FSHβ was strongly suppressedin most EE2exposure groups of both sexes. The brain esr2b was inhibited in both sexesexposed to EE2at4concentrations for6days. And esr2a was up-regulated in the females by6-day EE2treatment at1and25ng/L. The Pearson’s correlation analysis revealed thatexpression of esr2a was significantly negatively associated with expression of LHβ in thebrain of male adult rare minnow,wheras this correlation was significantly postive in femaleadult fish. The expression level of esr2b was negatively correlated to that of LHβ. In gonads,FSHR was strongly inhibited by EE2in males, while LHR was significantly stimulated byEE2in females. And cyp19a1a was inhibited by EE2in both sexes. The positive correlationsof both FSHR and LHR with cyp19a1a responsive to EE2in testes but not in ovaries.（5）We analyzed the transcriptom of adult G. rarus (6-month old) brain under EE2(25ng/L) exposure for7d by high-throughput deep sequencing technology. A total of8DigitalGene Expression Profiling (DGE) libraries were establised and sequenced. It generated over 0.68Gb clean reads for each library. The further analysis revealed there were276genesdifferentially expressed between treatment and control group in female brain, including132up-regulated genes, and144down-regulated genes. In male brain, there were194differentially expressed genes between treatment and control groups, which include110up-regulated genes and84down-regulated genes. Gene ontology functional enrichmentanalysis of in female groups revealed that they mainly function in the metabolic process,whereas there were significantly different pattern between male groups. According to KEGGdatabase information, above differentially expressed genes can be distributed to92and85pathways, respectively. The most significantly enriched pathways were Oxidativephosphorylation, Ribosome and Metabolic pathways in female.In the present study, we investigated the impact of BPA and EE2on the endocrinedisruption of HPG axis in male and female rare minnow. Based on the current study, it notonly help us to understand the potential mechanisms underlying estrogenic effects of EDCs,but also provide a basic knowledge about adverse effects of EDCs on other vertebrates andhuman. |
PDF Full Text Request