| Glucocorticoids are important endocrine regulatory factors in vertebrates,widely involved in physiological processes such as energy metabolism,immune regulation,inflammation and stress response,as well as reproductive regulation.Glucocorticoids act mainly through glucocorticoids receptor(GR).Most teleost fishes have two GRs due to the third round of whole genome duplication,however,the naming of the two GRs is still controversial.Cortisol is the main glucocorticoids in fish.Whether there is a difference in the responsiveness of the two GR to cortisol remains unclear? To date,the role of glucocorticoids in reproduction has been widely investigated,while the role of GRs in reproduction is rarely reported.Nile tilapia is a worldwide-farmed fish with XX/XY sex determination system.The high quality genome and transcriptome,as well as wellestablished monosex fish breeding systems and molecular biology techniques including gene editing,make tilapia an excellent model for the study of GRs function.In this study,we first isolated two GR in Nile tilapia and named them GR1 and GR2 according to amino acid sequence similarity with tetrapods.Then,we compared the transcriptional regulatory ability and ligand affinity of GR1 and GR2 through Dual-Luciferase Reporter Assay.Subsequently,we analyzed the expression level of gr1 and gr2 in different tissues of tilapia by RT-q PCR and transcriptome analysis,and the cellular location in gonads by immunohistochemistry.Finally,we investigated the role of gr1 and gr2 in gametogenesis by mutation analysis.The results are listed as follows:1)Naming of GR1 and GR2.The GR of Homo sapiens was isolated and used as a query to identify GRs of Mus musculus,Gallus gallus,Alligator mississippiensis,Xenopus tropicalis,Latimeria chalumnae,Lepidosteus oculatus,Danio rerio,Oryzias latipes,Takifugu rubripes,Oncorhynchus mykiss,Ictalurus punctatus,Maylandia zebra,Neolamprologus brichardi,Atractosteus tropicus and Pundamilia nyererei using blastp.One GR was isolated in Homo sapiens,Mus musculus,Gallus gallus,Alligator mississippiensis,Xenopus tropicalis,while two GRs were isolated in fishes except Latimeria chalumnae,Lepidosteus oculatus,Danio rerio.The amino acid sequence alignment revealed that even though vetebrate GRs show high similarty,one of the fish GR possess nine amino acids insertion "WRARQWTDG" in the DNA binding domain compared with tetrapods GRs.In fish,the GR with nine amino acids insertion was named as GR2 and the other was named as GR1.Phylogenetic analysis showed that the tetrapod GRs were clustered into one clade,and the bony fish GRs were clustered into another,which could be further divided into two clades,corresponds to GR1 and GR2.Syntenic analysis showed that teleost fishes GR1 were more conserved with tetrapod GR.2)Analysis of the transcriptional regulatory ability and ligand affinity of tilapia GR1 and GR2In order to compare the transcriptional regulatory ability of GR1 and GR2,we constructed the overexpression plasmids of GR1 and GR2,p CDNA3.1-gr1 and p CDNA3.1-gr2,and promoter plasmid containing glucocorticoids response element,p GL3-GRE.The Dual-Luciferase Reporter Assay results revealed higher transcriptional regulatory ability of GR2 than GR1 as indicated by the higher luciferase activity after GR2 overexpression at the existence of same concentration of cortisol,the main glucocorticoid in most of mammals and fishes.Corticosterone is the main glucocorticoid in rodents,birds,reptiles and amphibians,and also present in fishes,so we investigated the affinity of GR1 and GR2 to cortisol and corticosterone as well.The Dual-Luciferase Reporter Assay results showed that corticosterone could also bind to GR1 and GR2 and activate luciferase activity,but its activation ability was significantly lower than that of cortisol.In addition,at the same corticosterone concentration,the activation ability of GR2 overexpression was significantly higher than that of GR1.These results indicated that GR2 has a stronger transcriptional regulation ability than GR1,and cortisol is the most efficient ligand for GR1 and GR2 in tilapia.In order to verify whether the nine amino acids insertion can affect the transcriptional regulation ability of GR2,we constructed a mutated GR2 overexpression plasmid without the nine amino acids insertion,p CDNA3.1-mutgr2.The Dual-Luciferase Reporter Assay results showed that the activation ability of GR2 without nine amino acids was significantly lower than that of the wildtype GR2,demonstrating that the nine amino acids insertion in the DNA binding domain enhanced the transcriptional regulation ability of GR2 to its target genes.3)Expression analysis of gr1 and gr2 in tilapia.RT-q PCR results showed that gr1 was mainly expressed in liver,intestine,ovary,testis,kidney and muscle,while gr2 was mainly expressed in brain,pituitary gland,gill,liver,intestine,ovary,testis,kidney and headkidney.The expression of gr1 and gr2 in testis was significantly higher than that in ovary.Gonadal transcriptome analysis at 5,30,90,180,300 dah(days after hatching)revealed that gr1 expressed higher in testis than in ovary at 30,90,180,300 dah and gr2 expressed higher in testis than in ovary at all these developmental stages.gr1 showed higher expression than gr2 in gonads at all developmental stages.Immunohistochemistry results showed that GR1 and GR2 were expressed in both germ cells and somatic cells in ovary and testis.These results suggested that GR1 and GR2 may be involved in the reproduction of tilapia.4)Effects of gr1 and gr2 mutations on oogenesis of tilapia.The H&E staining results showed that compared with the control fish,the oocyte number in the ovaries of gr1 or gr2 single mutant fish at 60 dah was significantly reduced and the somatic cells were significantly increased.The oocyte number in the ovaries of gr1 and gr2 double mutant fish was further reduced and the somatic cell number was further increased,and the ovarian wall of gr1 and gr2 double mutant fish was significantly thickened.No obvious phenotype was found in the testes of gr1,gr2 single or double mutant fish.Immunohistochemistry results showed that the expression of Cyp19a1a(key gene involved in estrogen synthetase)in the ovaries of gr1,gr2 single or double mutation fish was significantly decreased compared with the wild type female fish,indicating that gr1 and gr2 involved in tilapia oogenesis.The expression of Cyp11c1(key gene involved in androgen synthetase)in the testis of mutant fish was not influenced compared with the wild type male fish.To figure out how the gr1 and gr2 invovled in oogenesis,the expressions of cyp19a1 a,cyp11c1,estrogen receptors esr1,esr2 a,esr2b,androgen receptors ar1,ar2,and amh the key gene in oogenesis,in the gonads of gr1 and gr2 mutant fish at 60 dah were analyzed by RT-q PCR.The results showed that compared with the wild type female fish,the expression of cyp19a1 a and amh in the ovary of gr1 mutant fish was significantly decreased,and the expressions of cyp19a1 a,amh,esr1 and esr2 a in the ovary of gr2 mutant fish was significantly decreased.However,compared with wild type male fish,the expression of cyp11c1,amh,ar1 and ar2 in the testis of mutant fish was not influenced.To investigate the possible reason for the decreased cyp19a1 a and amh expression in gr1 and gr2 mutant fish,we predicted the GR binding sites in the promoter region of cyp19a1 a and amh using JASPAR and verified it by Dual-Luciferase Reporter Assay.The results showed that gr1 overexpression significantly up-regulated the transcriptional activity of the amh promoter,and gr2 overexpression significantly upregulated the transcriptional activity of cyp19a1 a and amh promoter.The results demonstrated that gr1 and gr2 can regulate oogenesis by influencing the expression of amh and estrogen pathway genes.In summary,there are two GRs in Nile tilapia,GR1 and GR2.Compared with GR1,GR2 has nine amino acid insertions in the DNA binding domain,and its transcriptional regulation ability is stronger than GR1.GR1 and GR2 are expressed in different tissues,as well as germ cells and somatic cells in gonad.GR1 and GR2 can regulate oogenesis through the amh and the estrogen signaling pathway.This study laid a foundation for the further analysis of GR function in gametogenesis and the functional divergence of GR1 and GR2. |
