| Estradiol is an essential sex steroid involved in the regulation of animal growth,reproduction,and metabolism,while the specific mechanism of regulating crustacean ovarian development and lipid metabolism remains obscure.In vertebrates,estrogen normally performs its function by binding to estrogen receptor(ER).In invertebrates,however,ER gene has not been functionally identified to date.Instead of ER,another nuclear receptor,the estrogen related receptor(ERR),has been found with high sequence homology to vertebrate ER.ERR has common signaling pathways with ER and functions in multiple biological processes including reproduction and growth,but its role in crustacean reproduction is still unclear.In this paper,the swimming crab Portunus trituberculatus was studied to confirm the regulatory effects of estradiol and ERR on ovarian development and lipid metabolism,and explore its possible mechanism.The results are as follows:1)Throughout the ovarian development,the PtVg-mRNA expression level was the highest in ovary and hepatopancreas of stage Ⅳ,and the PtVg-mRNA expression level in hepatopancreas was much higher than that of ovary during stages Ⅱ to Ⅳ;PtVgmRNA positive signals was mainly detected in follicle cells(FC),the cytoplasm of previtellogenic oocytes(PRO)and endogenously vitellogenic oocytes(EN)of ovary,as well as the nuclei of fibrillar cell(F-cell)and resorptive cell(R-cell)of hepatopancreas;At endogenously vitellogenic stage,the positive signal of PtVg-mRNA in ovary was stronger than that in hepatopancreas,while at exogenously vitellogenic stage,the positive signal in ovary was much weaker than that in hepatopancreas.Estradiol of 10-9 mol/L could significantly increase the PtVg-mRNA expression level in ovary at endogenously vitellogenic stage,and only high concentration of estradiol could lead to a significant increase in PtVg-mRNA expression level of hepatopancreas at exogenously vitellogenic stage.In summary,the vitellogensis of P.trituberculatus was stage-specific,and most vitellogenin was mainly produced by hepatopancreas.Moreover,exogenous estradiol had significant effects on the vitellogenesis in the ovary of endogenously vitellogenic stage and the hepatopancreas of exogenously vitellogenic stage,which showed dose-dependence and stage-specificity.2)In this study,only one PtERR cDNA sequence was cloned,and analysis showed that it had characteristics unique to nuclear receptors.PtERR had no signal peptide sequence or transmembrane domain.Phylogenetic analysis showed that PtERR and crustacean ERR clustered together.qPCR results showed that PtERR-mRNA expressed in various tissues,of which the PtERR-mRNA expression level in ovary was the highest;PtERR-mRNA had a higher expression level in ovary during the ovarian development,and the peak was reached at stage III(exogenous vitellogenic stage).For the hepatopancreas,and the highest PtERR-mRNA expression level was detected at stage II.The results of in situ hybridization showed that the positive signal of PtERR-mRNA was found in follicular cells and oocytes of the ovary.35 days after PtERR-dsRNA injection,both the gonadosomatic index and the expression levels of PtERR and PtVg showed a significant downward trend,and a stagnation was also observed in the oocyte development.The fluorescent ligand-binding trial showed that PtERR lacked the ability to bind estradiol.The in vitro culture results showed that high concentration of estradiol treatment(10-7/10-6 mol/L)induced the expression of PtERR-mRNA and PtVg-mRNA at exogenously vitellogenic stage.The analysis of transcription factor binding site revealed that the potential 1/2 estrogen response element(1/2 ERE)sequences were found at-1968 bp,-1271 bp,-499 bp,and-359 bp of the PtVg promoter sequence.On the whole,PtERR had a positive regulatory effect on ovarian development,which might regulate the expression of Pt Vg by binding to response elements on the PtVg promoter.3)The combined analysis of transcriptome and proteome was performed in the ovary and hepatopancreas of P.trituberculatus after PtERR-dsRNA(ERRi)and GFPdsRNA(GFP)injection.Results showed that the number of genes that were significantly up-regulated and down-regulated at the transcription level and protein level in ovary was 0,and the number of genes that were significantly down-regulated was only one.But in the hepatopancreas,the number of genes significantly up-regulated and down-regulated at the transcription level and protein level were 20 and 49,respectively.Enrichment analysis showed that the most significantly enriched KEGG pathways of genes with consistent expression changing pattern in hepatopancreas were glycolysis/gluconeogenesis(ko00010),histidine metabolism(ko00340),and fatty acid metabolism(ko01212).Compared with the GFP-treated group,the differential genes related to reproduction in the ERRi-treated group mainly include estrogen sulfotransferase and juvenile hormone epoxide hydrolase-like protein,and each differential gene was significantly down-regulated at both the transcription and protein levels.In addition,the differential genes related to nutrient metabolism in the ERRi treatment group mainly include acyl-CoA delta-9 desaturase and very long-chain fattyacid-CoA ligase,acetyl-CoA carboxylase,medium-chain specific acyl-CoA dehydrogenase,fatty acid transport protein,fatty acid binding protein 3,long-chain specific acyl-CoA dehydrogenase,phosphoglycerate kinase and fructose-1,6bisphosphatase.The results showed that ERR possibly influenced ovarian development of P.trituberculatus by regulating its nutrient metabolism in vivo.4)Estradiol and its antagonist(tamoxifen)could significantly change the expression level of the lipid metabolism-related genes,lipid composition and tissue structure in the hepatopancreas of P.trituberculatus.In particular,the estradiol treatment altered the expression of fatty acid β-oxidation and fat production-related genes,and reduced the content of polar lipids and total monounsaturated fatty acids,but increased the content of total polyunsaturated fatty acids and the volume of B cells in the hepatopancreas.Tamoxifen down-regulated the expression levels of lipid breakdown-related genes acyl-CoA oxidase(ACOX)and fatty acid transport protein(FATP),but significantly increased the mRNA level and enzyme activity of CPT-1,which led to the brittle wall of tubules and more blister-like cells.The above results indicated that estradiol promoted the lipid catabolism in hepatopancreas,while tamoxifen damaged the hepatopancreas and caused a decrease in its lipid deposition in P.trituberculatus. |
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