Identification,Expression And Function Of Nr2f2 In Reproduction Of Nile Tilapia

Nr2f2(nuclear receptor family,subfamily 2,group F,member 2),also known as coup-tfII,plays important roles in mammalian reproduction,such as fetal sex differentiation,Leydig cell differentiation and female fertility maintenance.However,the involvement of nr2f2 in fish reproduction remains elusive.In the present study,two duplicated nr2f2 genes,nr2f2 a and nr2f2 b,were identified from the genomes of Nile tilapia(Oreochromis niloticus)and other fish species.Tissue distribution and celular localization of these two duplicates were analyzed.To explore the possible roles of nr2f2 a and nr2f2 b in Nile tilapia reproduction,CRISPR/Cas9 mediated gene knockout and specific inhibitor were used to disrupt their functions.The main results are listed as follows:(1)Isolation and sequence analyses Basing on the genome and gonadal transcriptomes,we isolated two nr2f2 genes in Nile tilapia,named as nr2f2 a and nr2f2 b.Gene specific primers were designed to amplify the open reading frame(ORF)of these two duplicates.They were 1254 bp and 1299 bp,encoding 417 and 432 amino acids,respectively.The deduced amino acid sequences of Nr2f2 s were further analyzed.Like other members of the nuclear receptors,Nile tilapia Nr2f2 a and Nr2f2 b have a highly conserved DNA binding domain(DBD)and ligand-binding domain(LBD).(2)Phylogenetic and syntenic analyses Phylogenetic analysis revealed that nr2f2 b was derived from the teleost-specific whole genome duplication,and experienced a secondary loss in some basal groups,such as Cypriniformes and Siluriformes.The duplication of nr2f2 in teleosts and secondary loss of nr2f2 b was further confirmed by synteny analysis.(3)Expression of nr2f2 s in Nile tilapia RT-PCR and Real-time PCR showed that nr2f2 a was mainly expressed in the brain,heart,spleen,ovary,testis and kidney of Nile tilapia at 300 days after hatching(dah),and nr2f2 b was only expressed in the brain and ovary.Transcriptome analysis showed that nr2f2 a has a higher expression in testes than in ovaries of Nile tilapia at 5,30,90,180,300 dah,and that nr2f2 b has a relatively higher expression in the ovaries of Nile tilapia at 300 dah than at other developmental stages.It is worthwhile to note that nr2f2 a has a higher expression than nr2f2 b in gonads of Nile tilapia at all developmental stages.(4)Celular localization of nr2f2 a and nr2f2 b in Nile tilapia gonads In situ hybridization results revealed that nr2f2 a mRNA was expressed in the oogonia and primary growth oocytes(phase I and phase II)of the ovary and both somatic and germ cells except spermatids of the testis.nr2f2 b was detected in the same cell types as nr2f2 a in ovary.The celular localization of nr2f2 a was further confirmed at protein level by immunofluorescence with the Nr2f2 a specific antibody we produced.(5)Possible roles of nr2f2 s in Nile tilapia reproduction as revealed by inhibitor treatment Specific inhibitor 4-Methoxy-1-naphthol(4-MNol)of nr2f2 was used to treat wild type XX and XY Nile tilapia from 10 to 240 days after hatching(5?M/g).Histological analysis showed that compared with control fish,the treated fish displayed degenerated oocytes in ovary and reduced spermatocytes in the testis at 240 dah.These results indicated that nr2f2 a and nr2f2 b are involved in Nile tilapia gametogenesis.(6)Roles of nr2f2 a and nr2f2 b in Nile tilapia reproduction as revealed by gene knockout nr2f2 a and nr2f2 b were mutated by CRISPR/Cas9.Sperms of nr2f2 a and nr2f2 b heterozygous males displayed normal fertilization capacity.nr2f2 a female heterozygotes showed normal spawning behaviour but reduced egg quality as indicated by the unbiased lethality of offsprings at embryonic stage.nr2f2 b female heterozygotes also showed slightly reduced egg quality.These results indicate that nr2f2 a and nr2f2 b might be involved in Nile tilapia oogenesis,which is important for the development of embryos after fertilization.The roles of nr2f2 s in Nile tilapia need further analyses.Taken together,our study firstly isolated two nr2f2 s in Nile tilapia.Phylogenetic and synteny analyses revealed that nr2f2 a is the orthologue gene of tetrapod nr2f2 and that nr2f2 b was derived from the teleost-specific whole genome duplication,and experienced a secondary loss in some basal groups,such as Cypriniformes and Siluriformes.Further functional analyses showed that nr2f2 a and nr2f2 b are important for gametogenesis in Nile tilapia.Our study provided new candidate gene involved in Nile tilapia gametogenesis which will deepen our understanding of the function of nr2f2 in fish reproduction.Our work will help to obtain high quality eggs thus increase the production of Nile tilapia and finally alleviate the food crisis caused by the growth of the world population.