Preliminary Study On The Function Of Foxo3 In Oogenesis Of Cultured Gobiocypris Rarus

Fish can economically provide high-quality protein,which has significant social and economic value.How to obtain high quantity and high quality fish eggs has become one of the main objectives of aquaculture.Foxo3 is a member of Fox O family and plays an important role in cell cycle,DNA damage repair,apoptosis,oxidative stress and energy metabolism.During oogenesis,the gene expression in oocytes will change dynamically.These changes are regulated by well coordinated transcription factors in germ line and cells.Foxo3,a transcription regulator expressed in germ cells and somatic cells of gonads,plays a potential key role in reproductive development.In mammalian ovaries,Foxo3 inhibited the activation of primordial follicles and regulated the growth and development of follicles by inducing apoptosis of oocytes and granulosa cells.However,there are few studies related to Foxo3 in fish ovaries.In this study,we cloned two duplicated foxo3 genes(foxo3a and foxo3 b,due to teleost specific 3rd genome duplication)and preliminarily analyzed their functions in the ovary of Gobiocypris rarus.The results are as follows:1.The full-length cDNA sequences of foxo3 a and foxo3 b were cloned using RACE.We obtained a 2562-bp cDNA of foxo3 a gene,containing 1953-bp open reading frame(ORF)encoding a protein of 650 amino acids,185-bp 5’ untranslated terminal region(UTR),231-bp 3’ UTR;and a 2804-bp cDNA of foxo3 b gene,containing a 1959-bp ORF encoding a protein of 652 amino acids,470-bp 5’ UTR,375-bp 3’ UTR.Sequence alignment displayed that the similarity were 85.90% and85.62% among vertebrate Foxo3 a amino acids and vertebrate Foxo3 b amino acid,respectively.Both two genes contained forkhead(FH),FOXO-TAD(unique TAD domain of FOXO protein)and FOXO＿KIX＿bdg(forkhead box o,KIX binding domain of CR2)domain.Phylogenetic and syntenic analyses indicated more conserved of foxo3 b with tetrapod foxo3 than foxo3 a.2.RT-PCR and qRT-PCR showed that foxo3 a was only expressed in brains and ovaries with a high level among all detected tissues.foxo3 b was highly expressed in brains and ovaries,and also found to be expressed in all other detected tissues.foxo3 a m RNA was detected in the ovaries with high levels from 2 month onward and slightly increased from 4 to 5 month.There was no significance of foxo3 b expression in ovaries at all developing stages.In situ hybridization results showed that both foxo3 a and foxo3 b were expressed in the germ cell and follicular cell of II、III、IV stage oocytes.3.Rapamycin,one of the mTOR pathway inhibitor,was used to treat3-month-old female Gobiocypris rarus to 4 months old,then normal feeding to 5months old.Histologically,stage IV and earlier oocytes in 3,4,5-month-old control groups and 3-month-old treatment groups,stage II and earlier oocytes in 4-month-old treatment groups,stage III and earlier oocytes in 5-month-old treatment groups,could be observed.GSI and gene expressions of foxl2、fshr、cyp11a1、cyp19a1a,bmp15 were significantly lower in treatment groups than control groups,while expression of figlα was significantly higher in treatment groups than control groups.Rapamycin treatment resulted in oogenesis arrested at stage II oocytes in G.rarus.foxo3 a expression was affected,but foxo3 b not,after the treatment by rapamycin.In conclusion,These findings indicate that foxo3 a and foxo3 b may play some roles in the oogenesis of G.rarus.We suspected that mTOR pathway possibly promotes oocytes development and maturation by regulating steroidgenesis related genes and TGFβ pathway genes.This study can provide reference for efficient artificial reproduction of economic fish.