Functional Study Of Dax1,Dax2,Foxj1a And Foxh1 In Gametogenesis In Tilapia

Gametogenesis is a complex progress,in which the primordial germ cells undergo mitosis and meiosis to form the mature gametes.The progress is responsible for transmitting genetic and epigenetic information across generations,ensuring the production of an offsprings generation from one generation to the next.Therefore,it is particularly important to explore the function of genes involved in the gametogenesis.In mammals,Dax1?Nr0b1?,a member of the nuclear receptor family,is expressed in the germ cells and somatic cells of both the ovary and testis.Functional study in mammals indicated that it is an important factor to regulate the spermatogenesis.However,its role in ovarian development remains unclear.Recently,two dax1 genes have been identified in the elephant shark?Callorhinchus milii?,fugu?Takifugu rubripes?and tilapia?Oreochromis niloticus?,named dax1 and dax2.However,their expression pattern and functions during gonadal development remains to be elucidated.Furthermore,Foxj1and Foxh1,two members of the Forkhead domain box family.Foxj1,the key gene for regulating the ciliogenesis,is expressed in the spermatids of the testis.Foxh1 is expressed in the oocytes,theca cells and corpora lutea of the ovary in mammals.Our previous study showed that foxj1a and foxh1 were expressed in the secondary spermatocytes and phase I,II oocytes in tilapia,respectively.Therefore,we speculate that these genes might be involved in the gametogenesis in teleosts,even in vertebrates.In the present study,the Nile tilapia was used as our experimental animal.In order to study the functions of dax1,dax2,foxj1a and foxh1 in gametogenesis,TALEN and CRISPR/Cas9 were performed and the secondary sex reversal?SSR?model was produced in Nile tilapia.The main results are as follows:1.Expression pattern analysis of dax1 and dax2.dax1 was expressed in the gonads at 5 dah?days after hatching?,without sexual dimorphism?p>0.05?.The expression of dax1 was significantly up-regulated in the ovary from 25 to 30 dah,but decreased from 75 to 180 dah,even lower than that of 5 dah?p<0.05?.The expression was significantly increased in testis from 75 to 180 dah?p<0.05?.Additionally,dax2was expressed in ovary and testis without sexual dimorphism at 5 dah?p>0.05?,but significantly up-regulated in the ovary at 90-180 dah?p<0.05?.Further,dax1 was expressed in the oocytes and interstitial cells of the ovary and the spermatocytes,Sertoli and Leydig cells of the testis.dax2 was expressed in the stage I-III oocytes of the ovary and spermatocytes,Sertoli,Leydig cells of the testis.2.Generation of dax1 homozygous mutants and analysis of gonadal phenotype.The target site of dax1 in its first exon containing Nla IV was selected for mutant analysis and screening the F0 fish.Heterozygous dax1 F1 offsprings were obtained by outcross dax1 mosaic F0 XX with WT?wild-type?XY fish.dax1^+/-F1 XX and XY fish with a 14-bp deletion were selected to breed the F2 generation.Histologically,at 30 dah,the meiosis was initiated in the control XX fish,with the cluster of pre-meiotic oocytes observed in the ovary.However,the meiosis was initiated in the dax1^-/-XX fish until 60 dah.Furthermore,the meiosis was initiated in the control XY fish at 90 dah,with the primary and secondary spermatocytes observed in the testis.However,the meiosis was initiated in the dax1^-/-XY fish until 150 dah.Taken together,the meiosis was delayed in the dax1^-/-XX and XY fish.The gonadal transcriptomics and Real-time PCR analysis of the dax1^-/-XX fish at 30 dah and dax1^-/-XY fish at 90 dah showed that the expression of RA catabolic enzyme cyp26a1 was significantly up-regulated,while synthetic enzyme aldh1a2 and meiosis related gene sycp3expression were significantly down-regulated in the dax1^-/-fish,compared with that of the control fish?p<0.01 or 0.05?.The gonadal phenotype and gene expression were rescued by administration of the exogenous RA.It was worth noting that the expression of dax2 was significantly up-regulated during the resume of the meiosis in the dax1^-/-fish?p<0.01?.Therefore,dax1 is critical for regulating the RA levels and germ cells meiosis initiation in tilapia,and its loss of function might be compensated by its duplicated gene dax2.3.Generation of dax2 heterozygous mutants and analysis of gonadal phenotype.The target site of dax2 in its first exon containing HpyCH4 III was selected for mutant analysis and screening the F0 fish.Heterozygous dax2 F1 offsprings were obtained by outcross dax2 mosaic F0 XY with WT XX fish.Histologically,the oocytes development was normal in the dax2^+/-XX fish,with the phase I and II oocytes observed in the ovary at 90 dah.The phase III and IV oocytes were observed in the ovary of the control XX fish,with the accumulation of yolk granule and lipid drops at180 dah.However,the failure of the lipid drops and vitellogenin accumulation was observed in the dax2^+/-fish,compared with that of the control XX fish,leading to the vacuolation of the stage IV oocytes and female infertility.Therefore,we failed to obtain the dax2 homozygous mutant lines.Taken together,dax2 is involved in the accumulation of yolk granule and lipid drops in oocytes at the vitellogenesis stage.4.Generation of foxj1a homozygous mutants and phenotype analysis.foxj1a was expressed in the secondary spermatocytes of the testis,the renal tubule and glomerulus of the kidney.Furthermore,the target site of foxj1a containing BsrB I adjacent to protospacer adjacent motif?PAM?in its second exon,was selected for mutant analysis and screening the F0 fish.Heterozygous foxj1a F1 offsprings were obtained by outcross foxj1a mosaic F0 XY with WT XX fish.Foxj1a^+/-F1 XX and XY fish with a 5-bp deletion were selected to breed the F2 generation.We found that the foxj1a^-/-fish exhibited deaths at 8 dah,due to the body curve,kidney cysts and swimming defects,while WT fish were normal and viable.The foxj1a^-/-fish were survived until 45 dah under the recycling water,which helped them to swim and obtain the foods.Of the survived foxj1a^-/-fish,the cystic index in the kidney and kidney/body ratio was significantly up-regulated?p<0.01?,compared with that of the control fish.Histologically,the spermatogenesis was initiated in the control XY fish at 90 dah,with the primary and secondary spermatocytes observed.Nevertheless,the spermatogenesis was delayed in the foxj1a^-/-XY fish.The spermatogenesis was resumed in the foxj1a^-/-XY fish at 210 dah.The matured spermatoza were observed in the spermatic fluid of the foxj1a^-/-XY fish at 300 dah.However,the morphological abnormality of the sperm was observed,with abnormal flagella shortening and folding,which caused sperm motility defects and male infertility?p<0.01?.Therefore,foxj1a is critical for the kidney development,spermatogenesis initiation and sperm flagellation.5.Generation of foxh1 homozygous mutants and ovarian phenotype analysis.The target site of the foxh1 containing Hae III adjacent to protospacer adjacent motif?PAM?in its second exon,was selected for mutant analysis and screening the F0 fish.Heterozygous foxh1 F1 offsprings was obtained by outcross foxh1 mosaic F0 XY with WT XX fish.foxh1^+/-F1 XX and XY fish with a 4-bp insertion were selected to breed the F2 generation.Histologically,the development of the oocytes was normal in the WT and foxh1^-/-XX fish,with the phase I and II oocytes appeared at 90 dah.In control XX fish,the phase III and IV oocytes were surrounded by the granulosa and theca cells at180 dah,while the oocytes development was arrested at stage I and II oocytes,with the surrounded monolayer pre-granulosa cells.Therefore,foxh1 is critical for the oocytes development at the folliculogenesis stage.6.Expression pattern analysis of dax1,dax2,foxj1a and foxh1 during SSR in XY fish.The gonadal differentiated XY tilapia were treated with TR?Trilostane,inhibitor of 3?-HSD?,MN?Metopirone,inhibitor of 11?-HSD?and GA?Glycyrrhetinic acid,inhibitor of Cyp11b2?alone,or in combination with E2?17b-estrodiol?from 30 to90 dah.Only blockage of androgen synthesis and simultaneous administration of E2 can successfully induced SSR in XY fish,which was rescued by administration of 11-KT or MT.While in E2,TR,MN and GA XY fish,the spermatogenesis was disrupted,due to the inhibition of the androgens production.Consistantly,the expression of dax1 and foxj1a was significantly down-regulated in the XY treatment fish?p<0.05?,while foxh1and dax2 showed no significant difference?p>0.05?,compared with that of the control fish.In TR+E2,MN+E2 and GA+E2-SSR XY fish,the ovarian tissue differentiation with the ectopic oocytes was observed in the testis.Consistantly,the expression of dax1and foxj1a was further significantly down-regulated,while the expression of foxh1 and dax2 was significantly up-regulated in SSR XY fish?p<0.05?,compared with that of other groups.These results showed that dax1,foxj1a and dax2,foxh1 might be involved in the development of the testis and ovary.In summary,the dax1?dax2?foxj1a and foxh1 mutants were obtained by TALEN and CRISPR/Cas 9.We found dax1 is involved in meiosis initiation in tilapia,and loss of its function might be compensated by dax2.However,dax2 has an effect on the accumulation of yolk granules and lipid drops in the oocytes at vitellogenic stage.Furthermore,foxj1a is essential for both the kidney development,spermatogenesis and sperm flagellation in testis,while foxh1 is involved in the oocytes development at folliculogenesis stage.Therefore,dax1?dax2?foxj1a and foxh1 played the critical roles in gametogenesis at different stages.