• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    The expression characteristics of vitellogenin (VTG)in response to B(a)p exposure in polychaete Perinereis aibuhitensis*

    2018-12-22 07:00:30LIWanjuan李婉娟XUEShenglun薛圣倫PANGMin龐敏YUEZonghao岳宗豪YANGDazuo楊大佐ZHOUYibing周一兵ZHAOHuan趙歡
    Journal of Oceanology and Limnology 2018年6期

    LI Wanjuan (李婉娟) , XUE Shenglun (薛圣倫), PANG Min (龐敏) , YUE Zonghao (岳宗豪) ,YANG Dazuo (楊大佐), ZHOU Yibing (周一兵) , ZHAO Huan (趙歡) ,

    1 Key Laboratory of Marine Bio-resource Restoration and Habitat Reparation in Liaoning Province, Dalian Ocean University,Dalian 116023, China

    2 Key Laboratory of Marine Ecology and Environmental Science and Engineering, First Institute of Oceanography, State Oceanic Administration, Qingdao 266061, China

    Abstract In order to investigate the endocrine toxicity of B(a)p to marine polychaete Perinereis aibuhitensis, vitellogenin (VTG) cDNA from the P. aibuhitensis was isolated, recombinated and expressed for the first time. The full length P. aibuhitensis vitellogenin gene (PaVTG) was 5 325 bp, and encoded 1 692 amino acids. It contained the vitellogenin_N domain of unknown function (DUF1943), a von Willebrand factor type D domain, as well as a conserved KALGNAG motif. The expression of VTG gene and protein were mainly up-regulated after exposed to B(a)p at transcriptional and translational levels. PaVTG gene expression did not change significantly at day 4. At day 7 PaVTG expression was up-regulated in 0.5 μg/L and 5 μg/L B(a)p group. At day 14 PaVTG was significantly up-regulated in 0.5–10 μg/L B(a)p. The protein expression of PaVTG in 0.5 μg/L and 10 μg/L B(a)p group was up-regulated with time prolonging, but the expression in 5 μg/L and 50 μg/L B(a)p group exhibited first increased and then decreased trend. With the increasing of B(a)p concentration PaVTG mRNA and protein expression both firstly increased then decreased. In contrast to B(a)p exposure, estradiol did not induce PaVTG gene and protein expression,until late times of exposure (14 d). Overall, the results in this study indicate that PaVTG could be used as a potential indicator of the effects environmental estrogenic compounds.

    Keyword: Perinereis aibuhitensis; vitellogenin; B(a)p; expression profiles

    1 INTRODUCTION

    The hormonal systems of animals are important for maintaining and regulating growth, development,reproduction, and other physiological processes.Endocrine-disrupting chemicals (EDCs) can interfere with the hormonal system, and disrupt synthesis,secretion, transport, binding, action, or elimination of natural hormones in the body (Park and Kwak, 2010).In the past few decades, increasing attention has been paid to evaluating the adverse effects of EDCs in aquatic environments. The toxic effects of EDCs on aquatic animals are diverse, and include abnormal development of the reproductive tracts and disruption of the reproductive cycle, among others.

    Benzo(a)pyrene (B(a)p) is a highly toxic polycyclic aromatic hydrocarbon (PAH). B(a)p is a representative PAH pollutant in aquatic toxicological experiments(Da Silva Rocha et al., 2012). In addition to carcinogenic characteristics, B(a)p seems to possess a negative impact on reproduction, as do other EDCs(Kanaly and Harayama, 2000; Laffon et al., 2006; Wu et al., 2017). For example, B(a)p can suppress the secretion of androstenedione and estradiol in flounderPlatichthysflesusL. (Monteiro et al., 2000). Also,Miao et al. (2009) reported that 10 μg/L B(a)p caused a significantly higher rate of DNA fracture in the gonad, a significantly higher degradation rate of oocytes, and hampered accumulation of yolk granules.

    Many researchers have documented the induction of vitellogenin (VTG) after exposure to xenoestrogens.Researchers have used VTG as a sensitive biomarker of estrogenic pollution (Marin and Matozzo, 2004;Matozzo et al., 2008). VTG is a precursor of yolk protein that is ubiquitous in oviparous animals. VTG provides nutritional and functional substances such as amino acids, fat, carbohydrates, vitamins, phosphorus,sulfur, and trace elements. These substances are essential for the development of oocytes, embryos,and early larvae, as well as to ensure survival of the young until they are self-supporting (Auttarat et al.,2006; Phiriyangkul et al., 2007; Matozzo et al., 2008).VTG is normally produced in female organisms,when exposed to exogenous estrogens, males and juveniles can also synthesize and secrete VTG (Marin and Matozzo, 2004). VTG production is regulated through the estrogen receptor pathway in vertebrates.The Organization for Economic Cooperation and Development (OECD) has developed a fish screening assay protocol that uses VTG as an endpoint for screening for EDCs with estrogenic activity (OECD,2010; Wang et al., 2015). The presence of a VTG system in aquatic oviparous invertebrates raises the possibility that invertebrates can also be used for endocrine toxicity testing (Matozzo et al., 2008).Recently, several studies have focused on the measurement of VTG/vitellins (Vn) levels after xenoestrogen exposure in groups of aquatic invertebrates, such as mollusks (Zhang et al., 2012;Ni et al., 2014; Tran et al., 2016; Liu et al., 2017) and crustaceans (Huang et al., 2006; García and Heras,2012; Jubeaux, 2012; Wen and Pan, 2015; Boulangé-Lecomte et al., 2017). Compared to the available information for mollusks and crustaceans, there is limited data about VTG induction in marine polychaetes. A significantly increase in VTG secretion was detected by enzyme linked immunosorbent assay(ELISA) in cultured eleocytes from matureNereis virensfemales when they were incubated in vitro with 1 μg/L 17β-estradiol for three d (García-Alonso et al.,2006). Zheng et al. (2010) showed that exposure of femalePerinereisnuntiato sub-lethal concentrations of cadmium caused a significant increase in the relative expression of VTG mRNA.

    The marine polychaetePerinereisaibuhitensisis widely distributed along the coasts of Southeast Asia in mudflats and estuarine sediments. Their life within estuarine sediments and mudflats puts them in continuous contact with sediment-associated contaminants. Furthermore, polychaetes may be important vectors for the transfer of sedimentassociated contaminants to higher trophic levels, as they are high-quality bait used for many commercial fish, for shrimp, and for crab (Lewis and Watson,2012). In addition, many kinds of polychaetes includingP.aibuhitensisare known to accumulate significant amounts of organic matter from the environment, and steady-state body burdens are a function of biotransformation and elimination processes. Due those reason, several studies have focused on toxicity assessments using polychaetes.The study of Zhang et al. (2008) showed thatP.aibuhitensisexposure to 0.15 and 0.2 mL/L of petroleum hydrocarbons could significantly inhibitP.aibuhitensisacetyl cholinesterase activity. Chen et al. (2012) found that exposure to petroleum hydrocarbons significantly induced cytochrome P450 mRNA expression inP.aibuhitensis. We also found that gene expression and enzyme activity of catalase and superoxide dismutase were positively correlated to the concentration of crude oil inP.aibuhitensis(Zhao et al., 2017). However, little information about the effect of EDCs on the endocrine system in this species has been documented.

    In this study, we isolated the full length VTG cDNA fromP.aibuhitensisand acquired recombinant protein. Then we investigated its expression after exposure to the xenoestrogen B(a)p at both transcriptional and translational levels. The results of this study will provide useful insight for the development of potential biomarkers of xenoestrogen pollutants usingP.aibuhitensis.

    2 MATERIAL AND METHOD

    2.1 B(a)p exposure

    Perinereisaibuhitensis(1.0–1.5 g wet weight) was collected from an estuary of Shuangtaizi in Panjin,Liaoning Province, in China. In preliminary experiment we found the gene expression of VTG in female worm was sensitive to B(a)p (Sigma,purity≥96%) exposure, so in this study we chose female worms as experimental animals. We could observe oocytes suspended in the coelomic cavity of sampled worms, and the development of reproductive cell in each worm was observed by microscope in order to choose the worm which was in the same stage of gonad maturation. The animals were transferred to the laboratory and acclimated in tanks (60 cm×45 cm×40 cm) containing filtered seawater (salinity 31–32, temperature 20±0.5°C) for a week before the initiation of experiments. Water was changed daily and continuously aerated. During acclimatization,P.aibuhitensisworms were fed with a powder mix containing kelp powder, gulfweed powder, fishmeal,yeast, and spirulina; worms were deprived from food during the B(a)p exposure experiment. Four concentrations of B(a)p (0.5, 5.0, 10, and 50 μg/L)were tested, according to the study of Song et al.(2011), and to our preliminary experiments. The worms were exposed to B(a)p dissolved in acetone(final acetone concentration 100 μg/L) for 14 d.Negative controls were unexposed worms, and worms exposed to acetone alone at 100 μg/L. Worms were also exposed to 50 μg/L estradiol, as an additional control group. The exposure experiment was carried out in 2-L beakers. Worms were randomly divided into seven treatment groups, and each group consisted of three replicates. Each replicate consisted of ten individuals. During the exposure experiment, seawater was renewed daily. The worms was dissected at 4, 7,and 14 d and then ground in liquid nitrogen and immediately stored at -80°C.

    Table 1 The primers used in RACE and Real-time PCR

    2.2 Cloning of P. aibuhitensis VTG full length cDNA

    The production of gametes is related to the germinal epithelia located in the body wall of this worm, so we choose the body wall ofP.aibuhitensisas sampling tissue. Total RNA was extracted from 100 mg of body wall using RNAiso Plus (TaKaRa Biotechnology Co.Ltd.) according to the manufacturer’s instructions.The quality of RNA was evaluated by electrophoresis on 1% agarose gels. Total RNA (1 μg) was reverse transcribed into cDNA for rapid amplification of cDNA ends (RACE) using Clontech SMARTTMRACE cDNA amplification kit (Clontech, Palo Alto,CA, USA). Primers for VTG were designed using Primer Premier 5.0 software, based on the partial sequence obtained from transcriptome sequencing ofP.aibuhitensis(unpublished data) (Table 1). The amplification method was conducted according to the manufacturer’s instructions. The 3′ RACE amplification protocol was as follows: 25 cycles at 94°C for 30 s, 65.2°C for 30 s and 72°C for 3 min.The 5′ RACE amplification protocol was as follows:five cycles at 94°C for 30 s, 72°C for 3 min; followed by five cycles at 94°C for 30 s, 70°C for 30 s, 72°C for 5 min; and finally 25 cycles at 94°C for 30 s, 68°C for 30 s and 72°C for 3 min. The PCR products were analyzed by electrophoresis on 1% agarose gels and then purified using an agarose gel DNA purification kit (TaKaRa Biotechnology Co. Ltd.). The purified PCR products were ligated into the pMD18-T vector,and transformed intoE.coliDH5α competent cells.Positive clones were sequenced by TaKaRa Biotechnology Co. Ltd.

    2.3 Sequence analysis

    The sequences obtained from the PCR-cloned products were analyzed for similarity with other known sequences using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). The deduced amino acid sequences were obtained via the Expert Protein Analysis System (http://www.us.expasy.org/tools). Alignment of multiple sequences was performed using CLUSTAL W software (http://www.ebi.ac.uk/clustalW). The functional sites and motifs in amino acid sequences were predicted using Motif Scan (http://www.hits.isbsib.ch/cgi-bin/PESCAN)and Expasy (http://www.au.expasy.org/prosite/)software. Phylogenetic analysis was conducted using Mega 5.0 software with the Neighbor-Joining algorithm. The tree topology was evaluated by 1 000 replication bootstraps.

    2.4 Quantitative real-time PCR

    SYBR Green quantitative real-time PCR was used to investigate the expression of the VTG gene inP.aibuhitensis. Gene-specific primers for PaVTG(Table 1) were designed according to the full length PaVTG cDNA sequence.β-actinwas chosen as reference gene according to the previous studies(Chen et al., 2012). Standard curve testing was performed using serial 10-fold samples dilutions.The slopes of standard curve and the PCR efficiency were calculated to confirm the accuracy of real-time PCR data. PCR amplification was done in a 20-μL reaction mixture containing 10 μL of SYBR?Premix Ex TaqTMII (TaKaRa Biotechnology Co. Ltd.),0.8 μL of each primer (10 mmol/L), 0.4 μL of Rox Reference Dye, 2.0 μL of cDNA, and 6 μL of diluted water. The amplification protocol was carried out at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s,and 60°C for 34 s. Melting curve analysis of the amplification products was done at the end of each PCR assay to confirm the specificity of the PCR products. Each product generated a single discrete peak in the melting curve analysis, which demonstrated the specificity of the PCR products.The PCR efficiency of PaVTG andβ-actinwere 100.076% and 99.959%.

    2.5 Expression and purification of recombinant PaVTG

    The conserved domain of full length PaVTG cDNA(30-740 aa) was amplified by PCR, and the PCR product was purified and digested with Nde I and Hind III to generate fragments with overhanging ends that could be ligated into the multiple cloning site of the expression vector PET30a. The recombinant plasmid was transformed intoE.coliBL21(DE3)cells, and PaVTG positive clones were confirmed by PCR, restriction enzyme digestion, and sequence analysis. The expression of PaVTG was induced by 1 mmol/L Isopropyl β-D-Thiogalactoside (IPTG) at 37°C for 4 h. Bacteria were suspended in phosphatebuffered saline (PBS) and lysed by sonication on ice,until the bacterial suspension was no longer viscous.Bacteria were then centrifuged at 12 000 r/min for 30 min. The expressed PaVTG protein accumulated in inclusion bodies, according to the analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The protein was collected by centrifugation at 13 000 r/min for 30 min and by disruption of the inclusion bodies in Tris-urea buffer(50 mmol/L Tris, 8 mol/L urea, pH 8.0). The resulting solution was centrifuged, and the supernatant was purified by using a Ni-NTA column. The purified protein was sent to GenScript (China, Nanjing) to prepare PaVTG-specific antibodies.

    2.6 Western-blotting analysis

    Purified PaVTG protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), and then transferred onto a Polyvinylidene Fluoride (PVDF) membrane at 120 V at 4°C for 1 h. PVDF membranes protein were blocked with 5% nonfat dry milk in Tris-Buffered Saline and Tween 20 (TBST) at 4°C overnight. The membranes were washed three times with TBS for 5 min, and incubated with anti-PaVTG rabbit serum(1:4 000 dilution in TBST) for 1 h at 37°C. The membranes were washed three times, and incubated with goat anti-rabbit IgG (H+L) conjugated to horseradish peroxidase (HRP) (1:4 000 dilution,Proteintech, Chicago, IL, USA) for 1 h at 37°C. After three washes with TBST, the immunoreative bands were visualized using DAB substrate solution(OriGene, Wuxi, China).

    2.7 Indirect Enzyme-linked immunosorbent assay

    The expression of PaVTG protein in femaleP.aibuhitensisunder B(a)p exposure was detected by ELISA. The protein of each sample was extracted using lysates (Beyotime, Shanghai, China), and diluted to a concentration of 0.2 μg/mL with PBST.Ninety-six well microtiter plates (Corning, Shanghai,China) were coated overnight with 100 μL of the sampling protein, plates were then blocked with 3%(w/v) BSA in PBS for 2 h at 37°C. After incubation,the plates were washed three times with PBST for 5 min. Anti-PaVTG rabbit serum (100 μL, 1:2 000 dilution) was added to each well and incubated at 37°C for 1 h. After incubation, the plates were washed with PBST three times for 5 min. HRP Affini Pure Goat Anti-Rabbit IgG (H+L) (Proteintech) was diluted 1:4 000 (v/v) in PBST, and 100 μL of the dilution were added to each well. After incubation at 37°C for 1 h, 100 μL of TMB substrate solution(Solarbio, Beijing, China) were added to each well.The plates were incubated at room temperature for 10 min in the dark. The reaction was stopped by adding 50 μL of ELISA terminator (Solarbio).Absorbance values at 450 nm were measured using a microplate reader (Perlong, DNM-9602A).

    2.8 Statistical analysis

    The 2-ΔΔCtmethod was used to analyze the relative expression level of PaVTG mRNA. Data are expressed as mean±standard deviation (S.D.). The differences between samples exposed to different concentrations of B(a)p at each sampling time, were determined using one-way analysis of variance(ANOVA) followed by Tukey’s test, using SPSS19.0 software.Pvalues ≤ 0.05 were considered statistically significant.

    3 RESULT

    3.1 Molecular characterization of PaVTG

    The full length cDNA of PaVTG has a length of 5 325 bp, including a 199-bp 3′ untranslated region(UTR), a 47-bp 5′ UTR, and a 5 079-bp open-reading frame. The open-reading frame predicted a protein of 1 692 amino acids (aa) with a predicted molecular mass of 185.27 kDa, and a theoretical isoelectric point of 8.66 (Fig.1). The 3′ UTR of PaVTG contains a putative polyadenylation consensus signal(AATAAA). The cDNA sequence of the PaVTG gene has been deposited in NCBI with accession number KF212194.1. The predicted amino acid sequence of VTG contained various functional motifs, such as the lipoprotein amino-terminal region (vitellogenin_N domain) (27–643 aa), a domain of unknown function(DUF domain) (681–974 aa), and a von Willebrand and factor type D domain (1 394–1 553 aa). A conserved amino acid motif KALGNAG in the vitellogenin-domain (544–550 aa), and a consensus RXXR cleavage sequence motifs (RKQR and RASR)were also found. SignalP analysis indicated that the signal peptide was positioned at the first 23 aa. The sequence of theP.aibuhitensisVTG protein had no phosvitin or polyserine domains, which are involved in receptor binding, and in phosphate and metal ion transport, in insects and vertebrates. Clustal W analysis revealed that the predicted amino acid sequence of PaVTG shared 59% identity with VTG protein from polychaetePlatynereisdumeriliiand had 21% to 27% homology with VTG protein from other species. And the sequence matched with 2.28%VTG sequence ofperinereissp. acquiring by pyrosequencing method (Phoonsamran et al., 2017).Figure 2 also showed that the PaVTG sequence had the closest relationship to VTG fromP.dumerilii.The two sequences were located in the same branch,and then clustered together with mollusks. The VTG sequences from lophotrochozoan were located in the same branch, and clustered with tardigratal sequence.

    3.2 PaVTG gene expression in female P. aibuhitensis after exposure to B(a)p

    The expression pattern of PaVTG in femaleP.aibuhitensisin response to B(a)p exposure is shown in Fig.3. The expression of PaVTG in acetone group did not have significant difference with that in control group, which indicated the solvent used to dissolve B(a)p had no effect in these processes.Estradiol failed to induce PaVTG gene expression at day 4 and 7, but it enhanced the expression of PaVTG at day 14 (P<0.05). With the increase of B(a)p concentration PaVTG mRNA expression was firstly increased then decreased. B(a)p induced PaVTG gene expression in a time-dependent manner. At day 4 of exposure, PaVTG gene expression in B(a)p-exposed worms did not change significantly, relative to the unexposed controls (P>0.05). On day 7 of exposure to 0.5 μg/L and 5 μg/L B(a)p, PaVTG expression was up-regulated in worms , but only the gene expression in 0.5 μg/L B(a)p group had significant difference with control group (P<0.05). At day 14 of exposure,PaVTG was significantly up-regulated in worms exposed to 0.5–10 μg/L B(a)p (P<0.05), but not in worms exposed to 50 μg/L B(a)p.

    3.3 Expression and purification of the recombinant protein

    The PaVTG recombinant protein was expressed successfully as a Histidine-tag fusion protein with an expected molecular mass of 79.5 kDa. The recombinant protein was expressed at a relatively higher level when bacteria were incubated at 37°C for 4 h with 1 mmol/L IPTG (Fig.4). Most of the recombinant protein existed in inclusion bodies, and was extracted with urea. The protein was purified by Ni-NTA resin column and refolded. Only a single band at the appropriate position corresponding to the molecular mass was showed in Fig.5, and it was indicated the specificity of antibody.

    3.4 VTG protein expression in female P. aibuhitensis exposed to B(a)p

    Fig.1 The full length cDNA and deduced amino acid sequence of PaVTG

    Fig.2 Phylogenetic neighbor-joining tree of VTG

    Fig.3 PaVTG expression in female P. aibuhitensis under B(a)p exposure

    An indirect ELISA method was used to detect the protein expression of PaVTG in femaleP.aibuhitensisexposed to B(a)p (Fig.6). The expression of PaVTG protein in acetone and estradiol group did not have significant difference with that in control group.Expression of the PaVTG protein was slightly upregulated by B(a)p at day 4, but there were no significant differences between groups (P>0.05).Compared to the protein expression in day 4, the concentration of the PaVTG protein in day 7 in each B(a)p concentration group was increased especially in 5 μg/L B(a)p group (P<0.05). On day 14 of exposure, the concentration of the PaVTG protein was decreased significantly in worms exposed to 5 μg/L and 50 μg/L B(a)p, but the concentration of the PaVTG protein was sightly increased in 0.5 μg/L and 10 μg/L B(a)p.

    4 DISCUSSION

    Fig.4 Expression analysis of the recombinant protein using SDS-PAGE

    In order to investigate the estrogenic effect of B(a)p toP.aibuhitensis, the full length cDNA of VTG inP.aibuhitensiswas identified and characterized for the first time. The sequence conserved domains predicted PaVTG protein was identified as a member of the lipid transport protein family (Zheng et al.,2012). In our study, a DUF domain was also found in PaVTG, which has been rarely detected in insects and vertebrates (Smolenaars et al., 2007). This domain family possesses a structure consisting of several large open beta sheets (Thompson and Banaszak,2002). The function of this domain has not been identified yet. The predicted sequence of PaVTG had no phosvitin or polyserine domains, which are involved in receptor binding, and in phosphate and metal ion transport in insects and vertebrates. This phenomenon has also been found in most crustaceans(Hwang et al., 2010). The absence of phosvitin and polyserine domains indicates that a different mechanism for VTG receptor binding may exist in the marine polychaeteP.aibuhitensis. Moreover, absence of phosvitin in aquatic invertebrates makes the egg yolk protein more susceptible to oxidative damage produced by pollutants (Matozzo et al., 2008).

    Fig.5 Western blotting analysis of the antigenicity of polyclonal antibody

    Fig.6 Levels of vitellogenin in body wall determined by indirect ELISA in adult female P. aibuhitensis samples

    Most studies concerning VTG induction in aquatic invertebrates have been conducted in economically important species, such as mollusks and crustaceans.Little information about changes in VTG levels in polychaetes (which form the dominant sediment dwelling fauna of most mud flat and estuaries) has been documented. In order to evaluate the possible estrogenic effect of B(a)p onP.aibuhitensis, we included an estradiol group in this study for comparison. Figures 4 and 6 show that estradiol induced PaVTG gene and protein expression on day 14 of exposure. These results contradict previous studies on the marine polychaeteN.virens. García-Alonso et al. (2006) showed that VTG secretion was increased in the presence of 17β-estradiol in cultured eleocytes from mature female (but not immature)worms ofN.virens. Wu et al. (2017) investigated the VTG gene expression level in femalePerinereis nuntia, the results showed that VTG mRNA level increased significantly in 2.5 and 25 μg/L B(a)p groups after inducing for 60 d. Conflicting reports about VTG expression after estradiol exposure have also been reported in bivalves. Gagné et al. (2001)showed enhanced levels of a VTG-like protein in the haemolymph of musselsElliptiocomplanataafter they were injected with estradiol. However, Puinean et al. (2006) did not observe any significant change in VTG expression in gonad homogenates from musselsMytilusedulisafter a 10-day exposure to estradiol.Similarly, exposure to estradiol failed to induce any significant change in VTG levels inE.complannatamussels (Won et al., 2005). These conflicting reports in bivalves suggest the presence of a steroid regulatory mechanism that may be different in distinct bivalve species, or distinct stages of gamete development.Alternatively, these conflicting results may indicate that VTG in bivalves is under the control of other hormones (Matozzo et al., 2008).P.aibuhitensisandN.virensbelong to the same family (Nereidae), the reproductive control strategy that has been most studied. There is consensus in that there is single hormonal system inNereidae, the supra-oesophageal ganglion that secretes a juvenile hormone (nereidine)performing a range of functions (Lewis and Watson,2012). The process of oogenesis and growth inNereidaeis related to the contents of nereidine, so the titre of nereidine may be different at different gamete developmental stages. So the difference between our study and García-alonso’s study may be due to the difference in developmental stage.

    There was a small but not statistically significant increase in PaVGT expression at the gene and protein levels on day 4 of B(a)p exposure. Gene and protein expression of PaVTG increased over time during B(a)p exposure, reaching a maximum at day 14. The lack of PaVTG induction at the beginning of B(a)p exposure may reflect potential cross talk between biological pathways, such as the detoxification pathway (Zhang et al., 2012). Previous studies have shown that B(a)p can be transformed into more hydrophilic intermediate metabolites through the reaction of cytochrome P450 detoxification enzymes.The existence of this mechanism has been confirmed in marine polychaetes. Polychaetes are known to accumulate significant amounts of PAH from the environment. Steady-state body PAH burdens are a function of biotransfomation and elimination processes (J?rgensen et al., 2008). Chen et al. (2012)identified a full-length cDNA coding for Cytochrome P450 subfamily 4 (CYP4) inP.aibuhitensis, and found that PAH could induce its expression on day 4 of exposure, in a time and dose-dependence manner.Enhanced CYP4 expression during B(a)p exposure may explain why PaVTG expression is not influenced by B(a)p inP.aibuhitensisat early time points. The possible relationship between the expression of CYP4 and PaVTG needs to be confirmed. Besides, why PaVTG expression is not influenced by B(a)p may be related to the estrogenic effect of intermediate metabolites of B(a)p. B(a)p can produce 1-,3-,7- and 9-OH-B(a)p,4,5-7,8- and 9,10-diOH-B(a)p and other intermediate metabolites with the participation of CYP450 enzymes (Fertuck et al., 2001). B(a)p intermediate metabolites, such as 3- and 9-OH-B(a)p,have been proved to possess estrogenic effects, but hydroxyl group is not necessary for exhibiting estrogenic or antiestrogenic activity (Charles et al.,2000; Hayakawa et al., 2007).

    According to our preliminary experiments, females were more sensitive to B(a)p than males. Therefore,in this study only the female worms were investigated for gene and protein expression of PaVTG. The higher expression of PaVTG in femaleP.aibuhitensismay be related to the existence of mechanisms for reducing the toxicity of pollutants. Volz and Chandler (2004)reported that the VTG concentration in female copepodsAmphiascustenuiremiswas significantly induced by a 12-d exposure to phenylpyrazole insecticide, but no significant induction was observed in males. The authors suggested that lipovitellin induction in females may be a resistance mechanism for reducing the toxicity of lipophilic environmental contaminants by sequestration into lipoproteins.Besides, the absence of a phosvitin domain in the PaVTG sequence may also indicate that the egg yolk protein in this species is more susceptible to oxidative damage produced by pollutants. Further studies are needed to confirm this issue.

    5 CONCLUSION

    In conclusion, this is the first report regarding VTG gene expression profiles after exposure ofP.aibuhitensisto EDCs, such as B(a)p. The B(a)p induced up-regulation of PaVTG gene and protein expression suggests that VTG could be used as an potential indicator of the effects of estrogenic compounds on polychaete reproduction.

    一级毛片精品| 午夜老司机福利片| 两人在一起打扑克的视频| 日本在线视频免费播放| 色播在线永久视频| 九色亚洲精品在线播放| 欧美亚洲日本最大视频资源| 午夜福利欧美成人| 19禁男女啪啪无遮挡网站| 午夜免费成人在线视频| 欧美在线黄色| 亚洲,欧美精品.| 久久中文字幕人妻熟女| 狠狠狠狠99中文字幕| 好男人在线观看高清免费视频 | 午夜免费成人在线视频| 国产亚洲精品综合一区在线观看 | 18禁美女被吸乳视频| 亚洲第一青青草原| 精品久久久久久成人av| 在线av久久热| 国产人伦9x9x在线观看| 电影成人av| 亚洲成人国产一区在线观看| 久久久久亚洲av毛片大全| tocl精华| av视频在线观看入口| 欧美一区二区精品小视频在线| 亚洲欧美日韩另类电影网站| 精品日产1卡2卡| 好男人在线观看高清免费视频 | 久久久国产精品麻豆| 黄片小视频在线播放| 国产精品综合久久久久久久免费 | 国产精华一区二区三区| 嫩草影院精品99| 日日干狠狠操夜夜爽| 成在线人永久免费视频| 午夜福利免费观看在线| 亚洲精品中文字幕在线视频| 国产成+人综合+亚洲专区| 老司机在亚洲福利影院| 亚洲色图av天堂| 天天一区二区日本电影三级 | 中文字幕另类日韩欧美亚洲嫩草| 亚洲伊人色综图| 成人国产一区最新在线观看| 欧美精品亚洲一区二区| 亚洲成人免费电影在线观看| 亚洲色图综合在线观看| 三级毛片av免费| 亚洲色图 男人天堂 中文字幕| 日韩三级视频一区二区三区| 亚洲av美国av| 日韩视频一区二区在线观看| 久久久久国产精品人妻aⅴ院| 性少妇av在线| 日本撒尿小便嘘嘘汇集6| 国产精品 国内视频| 久久国产精品影院| 九色亚洲精品在线播放| 欧美一级a爱片免费观看看 | 久久精品国产清高在天天线| 色播在线永久视频| 午夜免费鲁丝| 男男h啪啪无遮挡| 亚洲成a人片在线一区二区| 久久久国产精品麻豆| 动漫黄色视频在线观看| 在线观看舔阴道视频| www.精华液| 日本vs欧美在线观看视频| 亚洲熟妇中文字幕五十中出| 美国免费a级毛片| 婷婷丁香在线五月| 黄频高清免费视频| 老汉色av国产亚洲站长工具| 国产精品综合久久久久久久免费 | 88av欧美| 黄色女人牲交| 国产精品98久久久久久宅男小说| 大型黄色视频在线免费观看| 国产欧美日韩精品亚洲av| 少妇裸体淫交视频免费看高清 | 极品教师在线免费播放| 欧美乱妇无乱码| 国产熟女午夜一区二区三区| 国产aⅴ精品一区二区三区波| 一边摸一边做爽爽视频免费| 在线天堂中文资源库| 十八禁人妻一区二区| 欧美激情高清一区二区三区| 久久久国产成人免费| 自拍欧美九色日韩亚洲蝌蚪91| 香蕉久久夜色| 老汉色∧v一级毛片| 欧美av亚洲av综合av国产av| 露出奶头的视频| 免费搜索国产男女视频| 一本久久中文字幕| 久久香蕉精品热| 亚洲欧洲精品一区二区精品久久久| 真人一进一出gif抽搐免费| 午夜福利在线观看吧| 19禁男女啪啪无遮挡网站| 欧美激情极品国产一区二区三区| 宅男免费午夜| 美女午夜性视频免费| 男女午夜视频在线观看| 精品久久久久久,| 亚洲va日本ⅴa欧美va伊人久久| 校园春色视频在线观看| 性欧美人与动物交配| 久久亚洲真实| 日韩高清综合在线| 久久中文看片网| 午夜福利免费观看在线| a级毛片在线看网站| 亚洲一卡2卡3卡4卡5卡精品中文| 真人做人爱边吃奶动态| 悠悠久久av| av欧美777| 亚洲性夜色夜夜综合| 好男人在线观看高清免费视频 | 国产一级毛片七仙女欲春2 | 久久精品国产亚洲av高清一级| 成人手机av| 日日摸夜夜添夜夜添小说| 精品电影一区二区在线| 视频区欧美日本亚洲| 操出白浆在线播放| 看免费av毛片| 搡老岳熟女国产| 麻豆久久精品国产亚洲av| 啦啦啦免费观看视频1| 搡老岳熟女国产| 久久中文字幕人妻熟女| 91麻豆av在线| 美国免费a级毛片| 国产欧美日韩一区二区三区在线| 淫秽高清视频在线观看| 日韩有码中文字幕| 看黄色毛片网站| 侵犯人妻中文字幕一二三四区| 精品国产一区二区三区四区第35| 夜夜爽天天搞| 黄频高清免费视频| 免费av毛片视频| 久久久久久久久中文| 男男h啪啪无遮挡| 多毛熟女@视频| 伦理电影免费视频| 又大又爽又粗| АⅤ资源中文在线天堂| 亚洲熟妇熟女久久| 制服丝袜大香蕉在线| 国产精品av久久久久免费| 久久精品影院6| 亚洲精品在线观看二区| 久久精品亚洲熟妇少妇任你| 十八禁人妻一区二区| 国产精品免费一区二区三区在线| 大陆偷拍与自拍| 亚洲性夜色夜夜综合| 一区在线观看完整版| 18禁黄网站禁片午夜丰满| 两性夫妻黄色片| 丝袜在线中文字幕| 亚洲国产中文字幕在线视频| 欧美另类亚洲清纯唯美| 亚洲精品久久国产高清桃花| 精品电影一区二区在线| 亚洲中文字幕日韩| ponron亚洲| 91大片在线观看| 亚洲中文日韩欧美视频| 亚洲精品av麻豆狂野| 高潮久久久久久久久久久不卡| 国产单亲对白刺激| 男女之事视频高清在线观看| 黑人巨大精品欧美一区二区蜜桃| 国产99久久九九免费精品| 亚洲精品粉嫩美女一区| 亚洲国产精品sss在线观看| 亚洲精品中文字幕一二三四区| av网站免费在线观看视频| 后天国语完整版免费观看| 色精品久久人妻99蜜桃| 中文字幕人妻熟女乱码| 最近最新中文字幕大全电影3 | 99在线人妻在线中文字幕| 国产成人精品在线电影| 日本精品一区二区三区蜜桃| 变态另类成人亚洲欧美熟女 | 一区二区三区精品91| 黄色视频,在线免费观看| 男人操女人黄网站| 黄色片一级片一级黄色片| 国产亚洲精品综合一区在线观看 | 成熟少妇高潮喷水视频| 亚洲成人国产一区在线观看| 欧美成人免费av一区二区三区| 国产精品爽爽va在线观看网站 | 国产一区二区三区综合在线观看| 成在线人永久免费视频| 亚洲精品美女久久久久99蜜臀| 女人爽到高潮嗷嗷叫在线视频| 身体一侧抽搐| 亚洲第一电影网av| 桃色一区二区三区在线观看| 亚洲免费av在线视频| 在线永久观看黄色视频| 露出奶头的视频| 他把我摸到了高潮在线观看| 久久精品aⅴ一区二区三区四区| 变态另类丝袜制服| 免费人成视频x8x8入口观看| 亚洲色图av天堂| 19禁男女啪啪无遮挡网站| 国产亚洲精品久久久久久毛片| 99国产精品一区二区蜜桃av| 18禁观看日本| 欧美另类亚洲清纯唯美| 国产在线精品亚洲第一网站| 国产高清有码在线观看视频 | 欧美日韩福利视频一区二区| 婷婷六月久久综合丁香| 亚洲三区欧美一区| 久久性视频一级片| 淫秽高清视频在线观看| 老司机福利观看| 日本a在线网址| 久久久久久亚洲精品国产蜜桃av| www.熟女人妻精品国产| 一区二区三区国产精品乱码| 亚洲人成伊人成综合网2020| 欧美色视频一区免费| 香蕉丝袜av| 母亲3免费完整高清在线观看| 可以在线观看的亚洲视频| 啪啪无遮挡十八禁网站| 亚洲精品久久国产高清桃花| 香蕉国产在线看| 午夜福利一区二区在线看| 精品不卡国产一区二区三区| 日韩成人在线观看一区二区三区| 久久婷婷人人爽人人干人人爱 | 12—13女人毛片做爰片一| 女人爽到高潮嗷嗷叫在线视频| 国产精品一区二区免费欧美| 精品高清国产在线一区| 亚洲最大成人中文| 欧美日韩精品网址| 老汉色∧v一级毛片| 国产亚洲精品第一综合不卡| 午夜免费观看网址| www.999成人在线观看| 性色av乱码一区二区三区2| 给我免费播放毛片高清在线观看| 国产97色在线日韩免费| 亚洲avbb在线观看| 国产99久久九九免费精品| 日本撒尿小便嘘嘘汇集6| 国产片内射在线| 久久精品亚洲精品国产色婷小说| 国产精品秋霞免费鲁丝片| 久久精品国产综合久久久| 精品免费久久久久久久清纯| 成人手机av| 天天一区二区日本电影三级 | 亚洲欧美精品综合久久99| 午夜福利高清视频| 真人一进一出gif抽搐免费| 757午夜福利合集在线观看| 久久久久久久午夜电影| 免费观看精品视频网站| 99久久久亚洲精品蜜臀av| 亚洲av电影在线进入| 非洲黑人性xxxx精品又粗又长| 久久精品亚洲精品国产色婷小说| 日韩欧美一区二区三区在线观看| 别揉我奶头~嗯~啊~动态视频| 无限看片的www在线观看| 日本欧美视频一区| 欧美日韩一级在线毛片| 亚洲三区欧美一区| 狠狠狠狠99中文字幕| 国产高清videossex| 亚洲国产欧美日韩在线播放| 亚洲五月婷婷丁香| 欧美日韩瑟瑟在线播放| 欧美日本中文国产一区发布| 国产精品一区二区在线不卡| 丁香欧美五月| 亚洲黑人精品在线| 精品久久久精品久久久| 亚洲精品美女久久av网站| 亚洲欧美精品综合久久99| 国产成人免费无遮挡视频| 少妇粗大呻吟视频| 制服诱惑二区| 国产1区2区3区精品| 国产不卡一卡二| 成人手机av| 国产99久久九九免费精品| 99久久99久久久精品蜜桃| 久热爱精品视频在线9| 成人三级黄色视频| 久久香蕉国产精品| 国产成人影院久久av| 中出人妻视频一区二区| 亚洲午夜理论影院| 精品电影一区二区在线| 十八禁网站免费在线| tocl精华| 久久影院123| 久久国产精品影院| 亚洲片人在线观看| 高清黄色对白视频在线免费看| 热99re8久久精品国产| 国产精品 欧美亚洲| 国产精品野战在线观看| 久久久久久人人人人人| cao死你这个sao货| 精品久久久精品久久久| 久久久久久国产a免费观看| 中文字幕最新亚洲高清| 一a级毛片在线观看| 无人区码免费观看不卡| 老汉色∧v一级毛片| 亚洲精品中文字幕一二三四区| www.熟女人妻精品国产| 精品少妇一区二区三区视频日本电影| 午夜福利一区二区在线看| 精品国产乱码久久久久久男人| 日日夜夜操网爽| 亚洲人成网站在线播放欧美日韩| 一区福利在线观看| www.999成人在线观看| a级毛片在线看网站| 精品熟女少妇八av免费久了| 国产成人欧美在线观看| 一本综合久久免费| 国产三级在线视频| 欧美日韩亚洲综合一区二区三区_| 欧美日韩福利视频一区二区| 国产成人免费无遮挡视频| 亚洲专区中文字幕在线| 亚洲无线在线观看| 亚洲免费av在线视频| 久热爱精品视频在线9| 久久久久久久午夜电影| 欧美成人免费av一区二区三区| 一区二区三区激情视频| 亚洲av日韩精品久久久久久密| 人妻久久中文字幕网| 99久久久亚洲精品蜜臀av| 国产精品精品国产色婷婷| 级片在线观看| 日韩中文字幕欧美一区二区| 午夜福利免费观看在线| 在线播放国产精品三级| 天天躁夜夜躁狠狠躁躁| 国产伦人伦偷精品视频| 多毛熟女@视频| 久久久国产欧美日韩av| 国产欧美日韩一区二区精品| 欧美激情久久久久久爽电影 | 精品熟女少妇八av免费久了| 美女国产高潮福利片在线看| 999久久久国产精品视频| 日本免费a在线| 国产激情久久老熟女| 亚洲成人免费电影在线观看| 国产精品香港三级国产av潘金莲| 国产午夜福利久久久久久| 无遮挡黄片免费观看| videosex国产| 非洲黑人性xxxx精品又粗又长| 搡老熟女国产l中国老女人| 国产精品99久久99久久久不卡| 日韩欧美国产一区二区入口| av视频免费观看在线观看| 满18在线观看网站| 人妻久久中文字幕网| 侵犯人妻中文字幕一二三四区| 久久中文看片网| 欧美乱妇无乱码| 91在线观看av| 日韩 欧美 亚洲 中文字幕| 视频在线观看一区二区三区| 亚洲欧美日韩高清在线视频| 日本免费一区二区三区高清不卡 | 日本 欧美在线| 久久影院123| 男人舔女人的私密视频| 国产精品av久久久久免费| 久久久久久久久免费视频了| 老熟妇乱子伦视频在线观看| 欧美黑人欧美精品刺激| 正在播放国产对白刺激| 亚洲av成人不卡在线观看播放网| 99精品久久久久人妻精品| 在线观看www视频免费| 国产精品 国内视频| 国产精品1区2区在线观看.| 99国产精品一区二区蜜桃av| 熟妇人妻久久中文字幕3abv| av在线天堂中文字幕| 国内精品久久久久精免费| 亚洲精品国产精品久久久不卡| 中文字幕色久视频| 欧美日本亚洲视频在线播放| e午夜精品久久久久久久| 午夜视频精品福利| 精品无人区乱码1区二区| 日本免费一区二区三区高清不卡 | 午夜视频精品福利| 好男人在线观看高清免费视频 | 亚洲五月色婷婷综合| 午夜福利,免费看| 欧美另类亚洲清纯唯美| 欧美在线黄色| 热99re8久久精品国产| 涩涩av久久男人的天堂| 国产精品亚洲av一区麻豆| 婷婷精品国产亚洲av在线| 别揉我奶头~嗯~啊~动态视频| 久久人人97超碰香蕉20202| 后天国语完整版免费观看| 动漫黄色视频在线观看| av网站免费在线观看视频| www日本在线高清视频| 日本免费a在线| av电影中文网址| 亚洲精品中文字幕一二三四区| 国产伦一二天堂av在线观看| 亚洲熟妇熟女久久| 国产精品亚洲av一区麻豆| 一边摸一边做爽爽视频免费| 18美女黄网站色大片免费观看| 激情在线观看视频在线高清| 天堂动漫精品| 99久久综合精品五月天人人| 999久久久精品免费观看国产| 精品久久久久久久毛片微露脸| 大码成人一级视频| 精品不卡国产一区二区三区| 亚洲精品国产精品久久久不卡| 桃色一区二区三区在线观看| 亚洲欧美激情在线| 琪琪午夜伦伦电影理论片6080| 麻豆一二三区av精品| 天天躁夜夜躁狠狠躁躁| 国产av精品麻豆| 久久精品国产99精品国产亚洲性色 | 99国产综合亚洲精品| 免费一级毛片在线播放高清视频 | xxx96com| 99精品欧美一区二区三区四区| 欧美日韩亚洲国产一区二区在线观看| 国产男靠女视频免费网站| 热99re8久久精品国产| 韩国精品一区二区三区| 热99re8久久精品国产| 久久久国产欧美日韩av| 亚洲欧美激情综合另类| 国产男靠女视频免费网站| 精品久久久久久成人av| 色综合亚洲欧美另类图片| 国产人伦9x9x在线观看| 国产欧美日韩一区二区三| 色av中文字幕| 乱人伦中国视频| 一级,二级,三级黄色视频| 成人精品一区二区免费| 亚洲视频免费观看视频| 三级毛片av免费| 色老头精品视频在线观看| 精品人妻在线不人妻| 十八禁人妻一区二区| 亚洲午夜理论影院| 在线观看一区二区三区| 后天国语完整版免费观看| 欧美精品亚洲一区二区| 90打野战视频偷拍视频| 熟女少妇亚洲综合色aaa.| 自线自在国产av| 嫁个100分男人电影在线观看| 老司机深夜福利视频在线观看| 国产精品,欧美在线| 亚洲成国产人片在线观看| 欧美+亚洲+日韩+国产| 极品人妻少妇av视频| 午夜福利18| www国产在线视频色| 他把我摸到了高潮在线观看| 精品久久久久久久久久免费视频| 精品国产美女av久久久久小说| 两人在一起打扑克的视频| 国产一级毛片七仙女欲春2 | 性少妇av在线| 日本欧美视频一区| 色尼玛亚洲综合影院| 长腿黑丝高跟| 精品国产国语对白av| 日本免费a在线| 久久精品亚洲熟妇少妇任你| 久久性视频一级片| 99国产精品一区二区三区| 欧美日韩中文字幕国产精品一区二区三区 | 国产精品 国内视频| 精品卡一卡二卡四卡免费| 欧美黄色片欧美黄色片| 欧美中文日本在线观看视频| 久久精品91蜜桃| av视频在线观看入口| 亚洲国产欧美网| 法律面前人人平等表现在哪些方面| 午夜影院日韩av| 桃色一区二区三区在线观看| 男女午夜视频在线观看| www国产在线视频色| 日韩欧美一区视频在线观看| 国内久久婷婷六月综合欲色啪| 男女做爰动态图高潮gif福利片 | 色播在线永久视频| 国产视频一区二区在线看| 久久久久九九精品影院| 久久香蕉精品热| 亚洲精品久久国产高清桃花| 国产精品乱码一区二三区的特点 | 淫秽高清视频在线观看| 亚洲色图综合在线观看| 美女大奶头视频| 最新在线观看一区二区三区| 91字幕亚洲| 亚洲黑人精品在线| 国产精品久久电影中文字幕| 无遮挡黄片免费观看| 国产麻豆69| 国产精品久久久久久亚洲av鲁大| 久久这里只有精品19| 日韩精品青青久久久久久| 国产av又大| 亚洲成人久久性| 成人手机av| 激情在线观看视频在线高清| 免费搜索国产男女视频| 老司机在亚洲福利影院| 亚洲黑人精品在线| 国产精品国产高清国产av| 人成视频在线观看免费观看| 少妇熟女aⅴ在线视频| a级毛片在线看网站| 激情视频va一区二区三区| 久99久视频精品免费| 日本 欧美在线| 国产激情欧美一区二区| 国产成人精品在线电影| 一本综合久久免费| 免费搜索国产男女视频| 麻豆久久精品国产亚洲av| 日韩一卡2卡3卡4卡2021年| 欧美av亚洲av综合av国产av| 亚洲avbb在线观看| 免费在线观看完整版高清| 亚洲 欧美一区二区三区| 90打野战视频偷拍视频| 亚洲一区二区三区不卡视频| 嫩草影院精品99| 欧美 亚洲 国产 日韩一| 亚洲精华国产精华精| 久久久久久免费高清国产稀缺| 免费在线观看黄色视频的| 亚洲少妇的诱惑av| 成熟少妇高潮喷水视频| 国产私拍福利视频在线观看| 人妻丰满熟妇av一区二区三区| 久久久久久大精品| www日本在线高清视频| 久久久国产成人免费| 一区二区日韩欧美中文字幕| 亚洲av片天天在线观看| 青草久久国产| 男女下面进入的视频免费午夜 | 法律面前人人平等表现在哪些方面| 午夜免费观看网址| 看免费av毛片| 国产精品精品国产色婷婷| 欧美精品啪啪一区二区三区| 69av精品久久久久久| 午夜激情av网站| 大香蕉久久成人网| 国产99白浆流出| 欧美色视频一区免费| 免费看a级黄色片| 大码成人一级视频| 亚洲视频免费观看视频| 一级毛片精品| 免费看美女性在线毛片视频| 亚洲欧美精品综合一区二区三区| 午夜福利视频1000在线观看 | 国产精品美女特级片免费视频播放器 | 一个人免费在线观看的高清视频| 亚洲三区欧美一区| 色av中文字幕| 大陆偷拍与自拍| 麻豆久久精品国产亚洲av| 亚洲国产精品久久男人天堂| 精品国产乱码久久久久久男人| 国产高清视频在线播放一区| 伦理电影免费视频| 少妇裸体淫交视频免费看高清 | 精品高清国产在线一区|