In order to investigate the endocrine toxicity of B(a)p to marine polychaete P erinereis aibuhitensis, vitellogenin(VTG) cDNA from the P. aibuhitensis was isolated, recombinated and expressed for the first time. The f...In order to investigate the endocrine toxicity of B(a)p to marine polychaete P erinereis 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.展开更多
Vitellogenesis is the main event of oocyte growth in oviparous animals,which is mainly manifested by the accumulation of vitellogenin(VTG).The accumulation of vitellogenin depends mainly on the absorption of exogenous...Vitellogenesis is the main event of oocyte growth in oviparous animals,which is mainly manifested by the accumulation of vitellogenin(VTG).The accumulation of vitellogenin depends mainly on the absorption of exogenous vitellogenin,which enters oocyte through endocytosis mediated by its receptor(VGR).We investigated the expression and localization of VTG and VGR during gonad development of Pampus argenteus.The qPCR results show that vtgs were not expressed in male fish,but in the ovary and liver of female fish;the expression levels went up at first and then down.The expression levels of vgr in the testis were low and only 1%-3%of that in ovary.ELISA results show that during the ovarian development of P.argenteus,VTG in liver,serum,and ovary all showed a trend from increasing to decreasing.However,VTG in liver peaked in StageⅣ,and in serum and ovary peaked in Stage V,reflecting changes in the characteristics of VTG in the liver(synthesis),blood(transport),and ovaries(accumulation).During gonad development,VGR in the ovaries first increased and then decreased,reaching a peak in Stage V,in contrast to vgr mRNA expression.The VGR content in the testis was extremely low and stable,consistent with vgr mRNA.Immunohistochemistry results show that the location and intensity of VTG and VGR positive signals were synchronized with the changes of their protein content,which revealed that VTG was mainly synthesized in the liver cytoplasm,secreted into the blood,and transported to ovary in StageⅢ.VGR is highly expressed in oocytes in StageⅡ.In StageⅢ,a large amount of VTG reaches the ovary,when VGR begins to translate and is subsequently transported to the plasma membrane of the oocyte.Therefore,the positive signal of VGR was stronger near the plasma membrane of oocytes in StagesⅠandⅡ.By using qPCR,ELISA,and immunohistochemistry,the synthesis,transport,and accumulation of vitellogenin were elucidated and the mechanism of its endocytosis on egg membrane mediated by VTG during the development of P.argenteus was revealed preliminarily.展开更多
Both pentachlorophenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） had been studied widely because of their probable anti-estrogenic activity. Sodium pentachlorophenol （PCP-Na）, as a industrial product used in ...Both pentachlorophenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） had been studied widely because of their probable anti-estrogenic activity. Sodium pentachlorophenol （PCP-Na）, as a industrial product used in many fields, usually contains a trace of TCDD. The aim of this study was to assess the anti-estrogenic effect of PCP-Na in juvenile goldfish （Gurassius auratus） hepatocyte cultures using vitellogenin （VTG） as the biomarker. The ID50 of PCP-Na was investigated and then a series of concentrations （0.001 0.5 μg/ml） of PCP-Na were evaluated to estimate the anti-estrogenic activity. Results showed that PCP-Na was cytotoxic for hepatocytes even at very low concentration 〈1.21 μg/ml, and it could not induce VTG at any concentrations tested. Since it failed to stimulate VTG production, the possibility of its anti-estrogenic effect was tested, and a well-known anti-estrogenic compound-tamoxifen was used as positive control. PCP-Na caused a reduction in VTG synthesis in juvenile goldfish （Carassius auratus） hepatocytes at concentrations 〉0.1μg/ml when co-exposure with 1μg/ml 17β-estradiol （E2）, making its anti-estrogenic activity approximately as potent as tamoxifen. Our results indicate that PCP-Na can act as negative modulators of estrogenic function in juvenile goldfish （Carassius auratus） hepatocytes.展开更多
Tributyltin（TBT）, an organometal used as an antifouling biocide, has been reported to induce masculinization of fish. Benzo [a]pyrene （BaP）, a widespread carcinogenic polycyclic aromatic hydrocarbon, has been repo...Tributyltin（TBT）, an organometal used as an antifouling biocide, has been reported to induce masculinization of fish. Benzo [a]pyrene （BaP）, a widespread carcinogenic polycyclic aromatic hydrocarbon, has been reported that its microsomal metabolites can produce an estrogenic response when tested in vitro. This study was therefore designed to examine the potential in vivo influence of TBT, BaP and their mixture on sex hormone levels in serum of Sebastiscus marmoratus, which were given 2 separate intraperitoneally （ip） injections（a single injection every 7 d） of TBT（0.5, 1, 5 and 10 mg/kg）, BaP（0.5, 1, 5 and 10 mg/kg）, or both in combination（0.5, 1, 5 and 10 mg/kg）; control fish received olive oil vehicle only. Six days after the 2nd injection, serum samples were collected and analyzed for sex hormone levels and alkali labile protein phosphorus （ALPP）, which is related to the yolk precursor protein vitellogenin. The pollutants at all doses significantly reduced serum testosterone, estradiol and ALPP content after 2 injections compared with the corresponding controls. The reduction of the estradiol levels should be response for the decrease of the vitellogenin levels. The results in the present study suggested that aromatase seems not the major target acted by TBT and BaP in fish. This study demonstrated that TBT or BaP exposure both inhibit the reproductive potential in female Sebastiscus marmoratus. Combined effect of TBT and BaP on the serum testosterone, estradiol and ALPP was not antagonism from the anticipation.展开更多
In this study, the Harmonia axyridis vitellogenin gene 2 (HmVg2) sequence was identified from transcriptomic databases of female adult H. axyridis and cloned into pMD18-T vector. The HmVg2 gene was 5 460 bp in length,...In this study, the Harmonia axyridis vitellogenin gene 2 (HmVg2) sequence was identified from transcriptomic databases of female adult H. axyridis and cloned into pMD18-T vector. The HmVg2 gene was 5 460 bp in length, and formed with an open reading frame (ORF) of 5 361 nucleotides (GenBank accession no. KY794939). The putative molecular weight of the primary HmVg2 protein was 203.459 kDa and the predicted isoelectric point (pI) was 8.59. HmVg2 contained a signal peptide, vitellogenin N-terminal (vitellogenin-N) domain, domain of unknown function 1943 (DUF1934) domain and von Willebrand factor type D (VWD) domain. The developmental expression profiling showed that HmVg2 was extremely highly expressed in female insects, but was expressed at lower levels in male insects. In female insects, HmVg2 was mainly expressed in the wing and fat body. The double-stranded RNA-HmVg2/-GFP was injected into H. axyridis, and qRT-PCR results showed that the HmVg2 gene was specifically silenced. The eggs laid during the first five days and the hatching rate of eggs was lower than controls after dsHmVg2 injection. This investigation demonstrated that the HmVg2 gene plays an important role in H. axyridis reproduction and enriches the function of the insect vitellogenin gene.展开更多
Background: Vitellogenin(VTG) is a precursor of egg yolk proteins synthesized within the liver of oviparous vertebrates. Betaine is an important methyl donor that is reported to improve egg production of laying hens w...Background: Vitellogenin(VTG) is a precursor of egg yolk proteins synthesized within the liver of oviparous vertebrates. Betaine is an important methyl donor that is reported to improve egg production of laying hens with an unclear mechanism. In the present study, we fed betaine-supplemented diet(0.5%) to laying hens for 4 wk and investigated its effect on VTGII expression in the liver.Results: Betaine did not affect chicken weight, but significantly(P < 0.05) increased egg laying rate accompanied with a significant(P < 0.05) increase in hepatic concentration and plasma level of VTGI. Plasma estrogen level did not change,but the hepatic expression of estrogen receptor α(ERα) mR NA was significantly(P < 0.05) up-regulated. Betaine did not affect the protein content of ERα, but significantly(P < 0.05) increased hepatic expression of glucocorticoid receptor(GR)at both mR NA and protein levels. Also, ERα/GR interaction tended to be enhanced in the liver nuclear lysates of betainesupplemented hens as determined by co-immunoprecipitation. Furthermore, dietary betaine supplementation significantly increased(P < 0.05) the hepatic expression of methyl-transfer enzymes, such as BHMT, GNMT, and DNMT1,which was associated with higher SAM/SAH ratio and hypomethylation of GR promoter regions.Conclusions: Betaine activates hepatic VTGII expression in association with modified DNA methylation of GR gene promoter, GR expression and ERα/GR interaction. Activation of hepatic VTGII expression may contribute, at least partly, to improved egg production in betaine-supplemented hens.展开更多
基金Supported by the National Natural Science Foundation of China(No.41306138)the National Marine Public Welfare Research Project(Nos.201305002,201305043)the Foundation of Education Department of Liaoning Province(No.L201609)
文摘In order to investigate the endocrine toxicity of B(a)p to marine polychaete P erinereis 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.
基金Supported by the National Natural Science Foundation of China(Nos.31872586,42076118)the Major Project of Science,Technology and Innovation 2025 in Ningbo City(No.2021Z003)the K.C.Wong Magna Fund in Ningbo University。
文摘Vitellogenesis is the main event of oocyte growth in oviparous animals,which is mainly manifested by the accumulation of vitellogenin(VTG).The accumulation of vitellogenin depends mainly on the absorption of exogenous vitellogenin,which enters oocyte through endocytosis mediated by its receptor(VGR).We investigated the expression and localization of VTG and VGR during gonad development of Pampus argenteus.The qPCR results show that vtgs were not expressed in male fish,but in the ovary and liver of female fish;the expression levels went up at first and then down.The expression levels of vgr in the testis were low and only 1%-3%of that in ovary.ELISA results show that during the ovarian development of P.argenteus,VTG in liver,serum,and ovary all showed a trend from increasing to decreasing.However,VTG in liver peaked in StageⅣ,and in serum and ovary peaked in Stage V,reflecting changes in the characteristics of VTG in the liver(synthesis),blood(transport),and ovaries(accumulation).During gonad development,VGR in the ovaries first increased and then decreased,reaching a peak in Stage V,in contrast to vgr mRNA expression.The VGR content in the testis was extremely low and stable,consistent with vgr mRNA.Immunohistochemistry results show that the location and intensity of VTG and VGR positive signals were synchronized with the changes of their protein content,which revealed that VTG was mainly synthesized in the liver cytoplasm,secreted into the blood,and transported to ovary in StageⅢ.VGR is highly expressed in oocytes in StageⅡ.In StageⅢ,a large amount of VTG reaches the ovary,when VGR begins to translate and is subsequently transported to the plasma membrane of the oocyte.Therefore,the positive signal of VGR was stronger near the plasma membrane of oocytes in StagesⅠandⅡ.By using qPCR,ELISA,and immunohistochemistry,the synthesis,transport,and accumulation of vitellogenin were elucidated and the mechanism of its endocytosis on egg membrane mediated by VTG during the development of P.argenteus was revealed preliminarily.
基金The National Natural Science Foundation of China (No. 40332023)
文摘Both pentachlorophenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） had been studied widely because of their probable anti-estrogenic activity. Sodium pentachlorophenol （PCP-Na）, as a industrial product used in many fields, usually contains a trace of TCDD. The aim of this study was to assess the anti-estrogenic effect of PCP-Na in juvenile goldfish （Gurassius auratus） hepatocyte cultures using vitellogenin （VTG） as the biomarker. The ID50 of PCP-Na was investigated and then a series of concentrations （0.001 0.5 μg/ml） of PCP-Na were evaluated to estimate the anti-estrogenic activity. Results showed that PCP-Na was cytotoxic for hepatocytes even at very low concentration 〈1.21 μg/ml, and it could not induce VTG at any concentrations tested. Since it failed to stimulate VTG production, the possibility of its anti-estrogenic effect was tested, and a well-known anti-estrogenic compound-tamoxifen was used as positive control. PCP-Na caused a reduction in VTG synthesis in juvenile goldfish （Carassius auratus） hepatocytes at concentrations 〉0.1μg/ml when co-exposure with 1μg/ml 17β-estradiol （E2）, making its anti-estrogenic activity approximately as potent as tamoxifen. Our results indicate that PCP-Na can act as negative modulators of estrogenic function in juvenile goldfish （Carassius auratus） hepatocytes.
基金TheN ationalNaturalScienceFoundationofChina (No.40476049)andtheStateKeyLaboratoryofM arineEnvironm entalScience(X iam enU niversity),China(No.M EL201)
文摘Tributyltin（TBT）, an organometal used as an antifouling biocide, has been reported to induce masculinization of fish. Benzo [a]pyrene （BaP）, a widespread carcinogenic polycyclic aromatic hydrocarbon, has been reported that its microsomal metabolites can produce an estrogenic response when tested in vitro. This study was therefore designed to examine the potential in vivo influence of TBT, BaP and their mixture on sex hormone levels in serum of Sebastiscus marmoratus, which were given 2 separate intraperitoneally （ip） injections（a single injection every 7 d） of TBT（0.5, 1, 5 and 10 mg/kg）, BaP（0.5, 1, 5 and 10 mg/kg）, or both in combination（0.5, 1, 5 and 10 mg/kg）; control fish received olive oil vehicle only. Six days after the 2nd injection, serum samples were collected and analyzed for sex hormone levels and alkali labile protein phosphorus （ALPP）, which is related to the yolk precursor protein vitellogenin. The pollutants at all doses significantly reduced serum testosterone, estradiol and ALPP content after 2 injections compared with the corresponding controls. The reduction of the estradiol levels should be response for the decrease of the vitellogenin levels. The results in the present study suggested that aromatase seems not the major target acted by TBT and BaP in fish. This study demonstrated that TBT or BaP exposure both inhibit the reproductive potential in female Sebastiscus marmoratus. Combined effect of TBT and BaP on the serum testosterone, estradiol and ALPP was not antagonism from the anticipation.
基金supported by the National Natural Science Foundation of China (31272095)the National Key Research and Development Program of China (2017YFD0201004)the Hebei Modern Agriculture Industry Technology System, China (HBCT2018060204)
文摘In this study, the Harmonia axyridis vitellogenin gene 2 (HmVg2) sequence was identified from transcriptomic databases of female adult H. axyridis and cloned into pMD18-T vector. The HmVg2 gene was 5 460 bp in length, and formed with an open reading frame (ORF) of 5 361 nucleotides (GenBank accession no. KY794939). The putative molecular weight of the primary HmVg2 protein was 203.459 kDa and the predicted isoelectric point (pI) was 8.59. HmVg2 contained a signal peptide, vitellogenin N-terminal (vitellogenin-N) domain, domain of unknown function 1943 (DUF1934) domain and von Willebrand factor type D (VWD) domain. The developmental expression profiling showed that HmVg2 was extremely highly expressed in female insects, but was expressed at lower levels in male insects. In female insects, HmVg2 was mainly expressed in the wing and fat body. The double-stranded RNA-HmVg2/-GFP was injected into H. axyridis, and qRT-PCR results showed that the HmVg2 gene was specifically silenced. The eggs laid during the first five days and the hatching rate of eggs was lower than controls after dsHmVg2 injection. This investigation demonstrated that the HmVg2 gene plays an important role in H. axyridis reproduction and enriches the function of the insect vitellogenin gene.
基金supported by the National Natural Science Foundation of China(31672512)Special Fund for Agro-scientific Research in the Public Interest(201003011)+1 种基金the Fundamental Research Funds for the Central Universities(KYZ201212)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)
文摘Background: Vitellogenin(VTG) is a precursor of egg yolk proteins synthesized within the liver of oviparous vertebrates. Betaine is an important methyl donor that is reported to improve egg production of laying hens with an unclear mechanism. In the present study, we fed betaine-supplemented diet(0.5%) to laying hens for 4 wk and investigated its effect on VTGII expression in the liver.Results: Betaine did not affect chicken weight, but significantly(P < 0.05) increased egg laying rate accompanied with a significant(P < 0.05) increase in hepatic concentration and plasma level of VTGI. Plasma estrogen level did not change,but the hepatic expression of estrogen receptor α(ERα) mR NA was significantly(P < 0.05) up-regulated. Betaine did not affect the protein content of ERα, but significantly(P < 0.05) increased hepatic expression of glucocorticoid receptor(GR)at both mR NA and protein levels. Also, ERα/GR interaction tended to be enhanced in the liver nuclear lysates of betainesupplemented hens as determined by co-immunoprecipitation. Furthermore, dietary betaine supplementation significantly increased(P < 0.05) the hepatic expression of methyl-transfer enzymes, such as BHMT, GNMT, and DNMT1,which was associated with higher SAM/SAH ratio and hypomethylation of GR promoter regions.Conclusions: Betaine activates hepatic VTGII expression in association with modified DNA methylation of GR gene promoter, GR expression and ERα/GR interaction. Activation of hepatic VTGII expression may contribute, at least partly, to improved egg production in betaine-supplemented hens.
