Analysis Of Thyroid Hormone Receptor Mediating Thyroid Hormone Regulation Of Metamorphosis In Japanese Flounder(Paralichthys Olivaceus)

Thyroid hormone(TH)is a class of amino acid derivatives that have a wide range of effects on individual development,maintenance of homeostasis,cell proliferation,and differentiation.Paralichthys olivaceus is an important marine economically cultured fish in China.Its transition from larvae to juveniles undergoes a severe metamorphosis development process,accompanied by the loss of body symmetry and the migration of the right eye.This process is regulated by thyroid hormones.It has been confirmed that thyroid hormones accelerate the metamorphosis of P.olivaceus larvae through thyroid hormone receptors.But so far,little is known about the mechanisms by which thyroid hormones regulate larval metamorphosis.This article starts from three aspects and the main conclusions are as follows:1 Related expression and regulation of deiodinases and thyroid hormone receptors during metamorphosis in the Japanese flounder(Paralichthys olivaceus)To study the effect of exogenous TH on deiodinase and TRs during metamorphosis of P.olivaceus,we randomly divided 15 dph larvae into 3 groups: normal control(NC),TH-treated,and TU-treated,and reared until 41 dph.To assess whether TU(thiourea)treated P.olivaceus can rescue their metamorphosis by exogenous TH,we designed a rescue experiment at 35 dph while the NC group larvae had achieved metamorphosis,dividing the TU larvae into 3 groups: TU group continue feeding in seawater containing thiourea.TU + NC group was transferred to natural seawater.TU + TH group was transferred to seawater containing exogenous thyroxine.The expression of three thyroxine receptors TR?A,TR?B,TR? and three deiodinase D1,D2,and D3 m RNAs in each experimental group was detected by real-time quantitative PCR(RT-q PCR),enzyme-linked immunosorbent assay(ELISA)was utilized to detect the overall level of thyroid hormones T4 and T3 in P.olivaceus larvae of each experimental group.The results showed that the expression of D1,D2,TR?A,TR?B and TR? began to increase as the P.olivaceus larvae began to undergo metamorphosis(16-21 dph),as did the level of thyroid hormone in the body;all other analyzed elements except D3 in this study all peaked at the peak of metamorphosis(25-31 dph),after which the expression levels all declined in a similar manner until the end of metamorphosis(35-41 dph).In contrast,TR? transcription levels after metamorphosis are still high.D3 shows irregular wavy expression changes,which was different from other elements analyzed during metamorphosis.Exogenous TH treatment significantly promoted the metamorphosis of P.olivaceus larvae.The results of ELISA showed that exogenous TH treatment increased the level of TH in P.olivaceus in the early stage of metamorphosis,but there was no significant effect afterwards.q RT-PCR showed that D1 expression was down-regulated by exogenous TH during the entire metamorphosis.D2,TR?A,and TR?B were up-regulated by TH treatment early in the metamorphosis and were subsequently down-regulated.In the early and peak stages of metamorphosis,TH treatment up-regulated the expression of D3 and TR?.In contrast to TH treatment,TU treatment inhibited the metamorphosis of larval larvae.Our results show that TU treatment reduces the level of TH in P.olivaceus body during the entire metamorphosis.D1 was up-regulated by exogenous TU treatment in the peak stage of metamorphosis and late stage metamorphosis;D2 and TR?B were down-regulated at peak stage and up-regulated after peak stage;D3 and TR? were down-regulated by TU during peak stage and late metamorphosis stage.In this experiment,we demonstrated that deiodinase and TRs have diverse responses to TH and TU treatment,indicating that they may have complementary roles in the metamorphosis of P.olivaceus.One of the interesting results we found was the time-dependent difference in the response of deiodinases and TRs to TH and TU treatment.In the rescue experiment,D1 and D2 expression was significantly reduced compared to the TU group,while D3 expression showed a significant increase,especially in the TU + TH group.These results further support the opinion that D3 protects tissues against excessive TH and complements D1 and D2.For TRs,only TR? showed a significant increase in rescue experiments.Combined with the results of exogenous TH and TU treatment experiments,we believe that TR? and TR? have important but different roles in the process of P.olivaceus metamorphosis,and TR? may play a major role.2 Thyroid hormone receptor-interacting proteins and their regulation by thyroid hormoneIn this study,we selected thyroid hormone receptor ? that we believe to play a leading role in mediating thyroid hormone-induced P.olivaceus metamorphosis.We developed polyclonal antibodies and used immunoprecipitation to explore TR?-interacting proteins in TR?-overexpressed P.olivaceus embryonic cells(FEC).We identified 22 reliable P.olivaceus proteins that reached this mass spectrometry identification threshold score from immunoprecipitated complexes of TR? polyclonal antibodies and TR?-overexpressed FEC total protein.GO enrichment analysis showed that the identified proteins were mainly distributed in the ribosome,cytoplasm,nucleus,inner mitochondrial membrane,chromosome and extracellular region.Most of the proteins were concentrated in mitochondria and cytoplasm;their molecular functions including ATP binding and unfolded protein binding,ribosome structural composition,kinase activity,NAD-dependent histone deacetylase activity,transmembrane transporter activity,GTPase activity,translation elongation factor activity,peroxiredoxin reductase activity,hydrolase activity,actin filament binding,RNA binding,DNA binding,chemokine activity and extracellular matrix structure components.And they involved in these biological processes: translation,regulation of transcription,protein folding and refolding,cellular redox homeostasis of cell division,nucleosome assembly,estrogen responses,stress responses,and immune responses.At the same time,the results of the KEGG pathway analysis showed that the identified proteins involved in the ribosome pathway,cell cycle,endoplasmic reticulum protein processing,MAPK signal transduction pathways,thyroid hormone signaling pathway,spliceosomes,focal adhesions,oxidative phosphorylation,endocytosis,RNA degradation,amino acid metabolism,bile secretory,messenger RNA surveillance pathway,salmonella infection,herpes simplex infection,and cytokine receptor interaction pathways.8 identified proteins with the highest confidence and comprehensive score were selected for further analysis.They were: beta-F1-ATPase,heat shock protein 90 beta(Hsp90?),chaperone subunit 6A containing TCP1(Cct6A),40 S ribosomal protein S15A(RPS15A),creatine kinase 1(CK1),solute carrier family 25? member 5(Slc25a5),elongation factor 1(EF1a)and histone deacetylase 1-B(HDAC1-B).These proteins play important roles in the vital activity of cells.Among them,histone deacetylases(HDACs)have been found to regulate downstream gene transcription in anuran by binding to unliganded thyroid hormone receptors.Further analysis of m RNA expression of the genes encoding them shows that their expressions show significant changes after exogenous TH treatment,among which the expression of beta-F1-ATPase,Hsp90?,Cct6 A,RPS15A,Slc25a5,EF1 a and HDAC1-B were significantly downregulated by exogenous TH treatment.CK1 was diametrically opposite,its expression was significantly up-regulated by TH treatment.Treatment with TU almost had no significant effect on the analyzed genes.These results indirectly support the relationship between the analyzed credible proteins and P.olivaceus thyroid hormone receptor beta.3 Proteomic variation in metamorphosing Paralichthys olivaceus induced by exogenous thyroid hormoneIn this study,the varieties and expression levels of differentially expressed proteins in the TH-treated and untreated larvae of P.olivaceus during metamorphosis were analyzed by TMT-coupled 2D LC-MS/MS.Real-time quantitative PCR(RT-q PCR)and Western blotting(WB)were used to verify changes in protein expression levels.The possible biological significances of these differentially expressed proteins in the response of metamorphosing P.olivaceus larvae to exogenous TH treatment were further evaluated by a series of bioinformatics methods.We identified 815 proteins that showed significant changes in expression response to exogenous thyroid hormone stimulation,with 412 proteins upregulated and 403 downregulated.These proteins,which were significantly affected by thyroid hormone,have a variety of biological functions.It is worth noting that here we found five groups of proteins that have strong interactions and are significantly regulated by thyroid hormone,including hbbe2-hbae1-pah,tnnt3a-zgc:92233-tpm1-myhz2-smyhc2-myh11a-tnnc2-ttnb-speg,fabp6-fabp3-apoba-apoa1 a,rlbp1b-opn1mw4-grk7 a and gapdh-tktaldoaa-aldob-gpib-ldha-pkmb-idh1.Quantitative proteomic analysis showed that except for one unknown protein(zgc:92233),two of the five interaction groups(hbbe2-hbae1-pah and tnnt3a-zgc:92233-tpm1-myhz2-smyhc2-myh11a-tnnc2-ttnb-speg)were significantly up-regulated by TH treatment,and the other three groups(fabp6-fabp3-apoba-apoa1 a,rlbp1bopn1mw4-grk7 a and gapdh-tkt-aldoaa-aldob-gpib-ldha-pkmb-idh1)were significantly down-regulated.Further studies have shown that proteins of these two up-regulated protein interaction groups are known to be involved in blood cell formation and function and muscle development,respectively.Proteins of the other three down-regulated protein interaction groups are known to play an important role in the process of bioenergy metabolism,development of the visual system and metabolism regulation.In general,we confirmed that thyroid hormone enhance the maturation of blood cells during the metamorphosis of P.olivaceus,strengthen the muscle development of P.olivaceus larvae,weaken the visual system and the energy metabolism system,from the perspective of proteomics.