Function Of Foxl2/Cyp19a1 In Sex Determination And Fertility Of Nile Tilapia

Many aquacultural fishes,such as Nile tilapia,Oreochromis niloticus,show significant sexual dimorphism in growth rates.Research on the sex determination mechanism of tilapia has been a difficult and hot topic in breeding research,driven by an aquacultural interest in stocking of all-male fingerlings,which is not only because males grow faster that greatly improve growth rates and overall production but also to avoid uncontrolled reproduction before harvest.To date,several candidate genes,involved in the testicular development,have been recognized as the sex determining genes in fishes.However,there were few reports on female sex-determining genes in fishes.Foxl2,a member of the Fox gene family,was involved in ovarian differentiation and oogenesis in vertebrates.Knockouts of Foxl2 in XX individuals triggered partial or complete sex reversal in mammals,demonstrating that Foxl2 is a key ovarian-determining gene.foxl2 genes have been cloned from numerous fishes and showed sexually dimorphic expression in the gonads,with higher expression in ovaries compared with testes.Previous studies showed that either overexpression of the Foxl2dominant-negative mutant or knockdown Foxl2 in XX tilapia caused sex reversal,and recently,foxl2 mutation also resulted in female-to-male sex reversal in zebrafish,suggesting that Foxl2 is a key gene in female sex determination of fish.However,the mechanism of Foxl2 on the sex determination remains unclear.Foxl2 was reported to be a regulator of cytochrome P450 aromatase（encoded by cyp19a1a）,the key enzyme responsible for estrogens synthesis.It was reported previously by our group that knockdown Cyp19a1a in XX tilapia resulted in female-to-male sex reversal.Recently,sex reversal was also observed in cyp19a1a mutant zebrafish and medaka.In addition,the roles of estrogens in controlling male fertility are well established in mammals.Blockage of estrogen signal in mouse caused male infertility due to efferent duct obstruction.However,no obvious reproductive defects were observed in cyp19a1a and cyp19a1b mutant zebrafish.Thus,the role of estrogen in male reproduction of fish needs more investigations.The previous study by our group showed that it was Cyp19a1b,not Cyp19a1a,expressed in XY tilapia testes,indicating that the estrogen synthesis in XY male was catalyzed by Cyp19a1b.To further demonstrate the function of Foxl2/Cyp19a1a in female sex determination and Cyp19a1b in male reproduction,in the present study,we obtained and analyzed the foxl2,cyp19a1a,cyp19a1b mutant fish in Nile tilapia.The main results are listed as follows:1.The foxl2（single-exon gene）mutant tilapia with a 2-bp deletion in the exon was obtained.When examined at 90 days after hatching（dah）,the foxl2^-/-XX fish developed as male.When compared with the XX gonads,immunohistochemical and Western blot analyses revealed that male pathway genes（Sf1,Dmrt1 and Gsdf）were significantly up-regulated in the mutant XX gonads.Real-time PCR showed that the expressions of female pathway genes（β-cat1,β-cat2 and figla）were significantly down-regulated in the mutant XX gonads.No significant differences in fertilization rate were observed between the mutant XX and the control XY male fish when mated with normal XX females.By IHC,the expression of Dmrt1 was detected in the mutant XX gonads at 5 and 30 dah,similar to expression the pattern in the control XY gonads.Cyp19a1a was expressed in the control XX gonads but not in the mutant XX and control XY gonads at 5,30 and 90 dah.These results indicated that the mutant XX fish shifted directly to the male pathway without any female differentiation.Consistently,serum E2（17β-estradiol）concentration was significantly decreased in the mutant XX fish.The sex reversal caused by Foxl2 mutation could be rescued by E2 treatment.Cyp11b2（the enzyme responsible for the synthesis of 11-KT,11-ketotestosterone,the main androgen of fish）)expression and serum 11-KT concentration in the mutant XX fish were up-regulated.Star1,controlling the rate-limiting step in steroidogenesis,was reported to be expressed in XY gonads but not in the XX gonads and involved in androgen synthesis of tilapia.In foxl2 mutant fish,we found that Star1 was expressed in the XX gonads at 30 and 90 dah.Promoter analyses revealed that Foxl2 alone can activate star1 transcription in a dose-dependent manner.Therefore,loss of Foxl2 led to Star1-dependent androgen production,which,in turn,promoted testicular development and spermatogenesis in the sex-reversed gonad.It has been reported in tilapia,as well as in other vertebrates,that the number of germ cells showed significant difference between testes and ovaries at early stages.In mutant XX gonads at 10 dah,the number of germ cells was significantly reduced,indicating that germ cell proliferation was suppressed in the mutants.2.The cyp19a1a mutant tilapia with a 7-bp deletion in the first exon was obtained.When examined at 90 dah,the cyp19a1a^-/-XX fish developed as male.When compared with control XX gonads,Dmrt1 was up-regulated in the mutant XX gonads at 5,30 and 90 dah.Significantly decreased serum E2 level was found in the mutant XX fish.Up-regulation of Sf1,Dmrt1,Gsdf,Cyp11b2 and down-regulation ofβ-cat1,β-cat2,figla were observed in the mutant XX gonads.No significant differences in fertilization rate were observed between the mutant XX and the control XY male when mated with the normal XX females.The sex reversal caused by cyp19a1a mutation could be rescued by constant E2 treatment,however,different from foxl2^-/-XX fish,once the treatment was stopped,the cyp19a1a^-/-XX gonads reversed to testes.Similar to the situation of foxl2^-/-XX fish,the number of germ cells in the cyp19a1a^-/-XX gonads was significantly reduced at 10 dah,indicating that germ cell proliferation may be regulated by estrogens.3.The cyp19a1b mutant tilapia with a 8-bp deletion in the second exon was obtained.When examined at 240 dah,serum E2 level was significantly decreased in the cyp19a1b^-/-XY fish.The mutant fish failed to obtained any offsprings when mating with normal XX females.No semen was obtained from the mutant fish by in vitro extrusion.Gonadal hypertrophy was observed in the mutant fish.Consistently,GSI of the mutant fish was significantly up-regulated.When compared with the control XY fish,histological observation showed smaller efferent ducts in the mutant testes.Efferent duct area index of the mutant fish was significantly decreased.The semen collected from testes of the mutant fish by gonadal autopsy contained normal sperms,showing no significant differences in morphology,sperm motility,average curvilinear velocity,flagellum beat frequency,average straightness and fertilization rate when compared with control sperms.Consistently,real-time PCR analyses showed that germ cell markers（vasa,nanos2,piwil1,dazl）and spermatogenesis markers（fshr,lhr,sycp3,spo11,aspm,suz12a,cdk16,dusp26）displayed no significant difference in mRNA levels between the mutant and control testes.Additionally,low convolution and less branching in the efferent duct and no blood vessel inside the efferent duct wall were observed in the mutant fish.Real-time PCR analyses showed that mRNA levels of ion exchange-related genes,including aqp1ab,slc12a7b,slc20a1a,slc26a1,slc26a3,slc9a6b,were down-regulated in the mutant testes.Cyp19a1b mutation led to an increase of positive TUNEL staining in the epithelial cells of efferent ducts and some other somatic cells of the mutant testes.Meanwhile,expression of apoptosis markers,including tnfsf10,bcl2l13,bnip3la,tradd,frzd and col1a1,were up-regulated in the mutant testes.At 360 dah,degenerative testes with short gonads and fibrotic efferent duct were observed in the mutant fish.Severe degeneration with few sperms in the posterior testes and slight degeneration with abundant sperms in the anterior testes were observed in the mutant fish.The GSI of the mutant fish was significantly down-regulated.These results indicated that long-term obstruction of the efferent duct resulted in testicular atrophy and tubular fibrosis.The sperms collected from the mutant testes showed no significant differences in fertilization rate when compared with the sperms collected from control XY fish.In summary,we obtained foxl2,cyp19a1a,cyp19a1b mutant Nile tilapia.Both the foxl2^-/-XX fish and the cyp19a1a^-/-XX fish displayed female-to-male sex reversal.The mutant phenotype could be rescued by E2 treatment.Our results demonstrated that Foxl2 promotes ovarian differentiation and development by upregulating Cyp19a1a expression,increasing germ cell number,and repressing male pathway gene expression.These data enrich the understanding of mechanism of sex determination in vertebrates and provide the theoretical basis for sex control in aquaculture.Estrogen deficiency by Cyp19a1b mutation resulted in male sterility due to efferent duct obstruction in tilapia.Spermatogenesis appeared normal and the sperms were fertile in the pathological testes.Long-term obstruction of the efferent duct led to testicular atrophy and tubular fibrosis.Our findings provide the genetic evidence,similar with mammals,that estrogens are required to achieve and maintain normal fertility in XY tilapia.