The Role Of 11β-hydroxylase (Cyp11b2) On Gametogenesis In Tilapia

Most vertebrates have both male and female sex,the ovary for the female,and the testis for the male.Gonads have the function of gametogenesis and sex steroid hormones synthesis.Gametogenesis is a series of complex processes from germ cells to mature gametes,that is,the differentiation of eggs and sperm.Steroid hormones play an important role on gametogenesis,maturation and fertility in teleosts,even the whole vertebrates.11β-hydroxylase is a steroidogenic enzyme,encoding by cyp11b2(Cytochrome P450,family 11,subfamily B,polypeptide 2),which belongs to the B subfamily of the cytochrome P450 superfamily and is named cyp11c1 in zebrafish.In human and mammals,the gene catalyzes the synthesis of glucocorticoid aldosterone,and mutation of cyp11b2 resulted in a variety of disease symptoms.In teleosts,cyp11b2 involves in the synthesis of specific androgen 11-ketotestosterone(11-KT)and endogenous glucocorticoid cortisol,which expresses in the testis and head kidney,respectively.Different from mammals,the primary androgen of teleosts is 11-KT,but not testosterone(T).Androgen promotes the testicular development and plays an essential role in spermatogenesis,the development of secondary sexual characteristics and reproductive behavior.However,in fish,most studies of androgen are concentrated in hormone treatment and knockout of androgen receptor(AR),and no study about the effect on testicular development,spermatogenesis,maturation and fertility by knockout androgen synthetase.Even more,whether there is a direct effect for 11-KT on the oogenesis and maturation in females needs to be further studied.Cortisol is the primary glucocorticoid in fish and previous studies about its roles mainly focused on stress response and energy metabolism,as well as the adverse effects of elevated cortisol level on oogenesis,while the effects of cortisol insufficiency or deficiency on oogenesis are rarely studied.In our previous study,we cloned cyp11b2 gene and produced polyclonal antibody.We also have been demonstrated that its expression was detected in testicular Leydig cells,head kidney and kidney,which began at 30 dah and continued to rise until 180 dah.To further elucidate the exact role of 11-KT and cortisol on gametogenesis in teleosts,we took the Nile tilapia(Oreochromis niloticus)as the experimental animals and successfully generated cyp11b2 mutants by CRISPR/Cas9 to analyze the effects on gonadal phenotype and gametogenesis.The main results are as follows:1、Generation of cyp11b2 homozygous mutant fish.Cyp11b2 mutants were successfully generated by CRISPR/Cas9 and the target site was located on the first exon and cytochrome P450 superfamily domain.The restriction enzyme Mlu CI located on the target site was selected to screen the F0 fish.Heterozygous mutants were obtained by outcross cyp11b2 mosaic F0 XY with wild type XX fish.XX and XY heterozygous fish with a 7-bp deletion were selected to breed homozygous mutants.2 、 Analyses of gonadal phenotype and spermatogenesis.We found that heterozygous and homozygous mutant males had the same phenotype,and the phenotype of homozygous mutants was more serious through the analysis of gonadal phenotype,spermatogenesis and sperm characteristics.Histologically,the meiosis process of germ cells had been initiated and spermatogonia,primary spermatocytes and secondary spermatocytes were observed in the control testis,while only spermatogonia were detected in the homozygous mutant testis at 90 days after hatching(dah),a critical period of spermatogenesis in wild type tilapia.Moreover,the serum T level of homozygous mutants was significantly higher than the wild type males.However,at 180 dah,histological observation showed that,like the wild type male fish,various stages spermatogenic cells could be detected in the testis of mutants,including spermatocyte and spermatid,indicating that spermatogenesis has been recovered partially or completely.Meanwhile,significantly higher serum T level was detected in homozygous mutant males compared with the control male fish.In addition,11-KT was not detected in the serum of homozygous mutant male fish at 90 dah and 180 dah.T,11-KT and Cortisol were used to rescue treatment male homozygous mutants from 30 dah to 90 dah.Histological observation found that spermatogonia,primary spermatocytes and secondary spermatocytes were detected in the 11-KT treatment group,which was consistent with the phenotype of wild type males.Only spermatogonia,a small number of primary spermatocytes and a very few of secondary spermatocytes were observed in the T treatment group.In Cortisol treatment group,only spermatogonia,no spermatocytes were observed as the homozygous mutant male fish,.Our results suggest that 11-KT is a more efficient androgen than T and plays an important role in the initiation of spermatogenesis in fish.In addition,the deficiency of androgens,not cortisol,is the major cause of delayed spermatogenesis.Histological examination and quantitative analysis of spermatogenic cells indicated that there is an irregular arrangement of spermatogenic cysts in the testes of homozygous mutants,and the number of spermatid and Leydig cells significantly decreased at 360 dah.Additionally,GSI and semen volume were lower in the testes of homozygous mutants than the control.IHC showed that the positive signal of Leydig cells marker gene(Cyp17a2)was significantly lower in the cyp11b2 deficient testis than that of the control testis.Nevertheless,it was unexpectedly found that sperm characters such as the instantaneous movement trajectories,concentration,motility,morphplogical abnormality of sperm,beat frequency of flagellum,curvilinear velocity(VCL),straight line velocity(VSL),sperm quality and fertility in homozygous mutant fish were normal,compared with the wild type males by computer assisted sperm analysis system.It demonstrated that 11-KT affected the testicular structure and semen volume,but not affected the sperm quality and fertility.3 、 Analyses of gonadal phenotype and oogenesis.We analyzed gonadal phenotype and oogenesis of heterozygous and homozygous female fish,respectively.Histological observation demonstrated that oogenesis and maturation were normal and various stages oocytes were present in the heterozygous mutant ovaries at 360 dah.Compared with wild type females,there was no difference in the number of oocytes and GSI,and heterozygous mutants had a smaller diameter of eggs in despite natural ovulation and normal fertilization rate.However,either wild type or mutant males were crossed with heterozygous mutant females,the development of embryos was arrested at 2 days post fertilization(dpf).The development of embryos can be successfully rescued by cortisol alone administration with heterozygous mutant females,not 11-KT.Transcriptome analysis of mature eggs(phase V)of heterozygous female fish revealed that insufficient cortisol may affect the expression of a large number of genes,and significant variations were observed in the expression of maternal genes,such as zar1,zar1 l,tdrd5.It indicated that the quality of eggs was affected in heterozygous mutant females,and maternal cortisol insufficient is a vital factor.Histological analysis demonstrated that various stages oocytes were already present in the wild type ovaries,including phase I,II,III and IV oocytes at 360 dah.In contrast,only phase I,II and a few III oocytes were detected in the homozygous mutant ovaries,no phase IV oocytes.GSI of homozygous mutant females was significantly lower than that of wild type females.Additionally,TUNEL detection revealed that the positive signal(red fluorescence)in the homozygous mutant ovaries was significantly higher than that of wild type females.Real-time PCR showed that the mRNA expressions of cyp19a1 a,foxl2,esr1,esr2 b,lhr,fshr,amh,vtg2,vtg3 were significantly down-regulated,while bmp15,figlα,gdf9,42sp50,zar1,zar1 l were evidently up-regulated in the homozygous mutant ovaries.Consistently,compared with the control females,the serum E2,Cortisol and Vtg levels and the expression of Vtg in liver of homozygous mutant females were significantly decreased.E2 was used to rescue homozygous mutant females.Phase IV oocytes were found in homozygous mutant females rescued by E2,suggesting that E2 can partially rescue gonadal phenotypes.In summary,we generated cyp11b2 mutants by CRISPR/Cas9 in tilapia.We found that cyp11b2 mutation resulted in delayed spermatogenesis and a low volume of semen in male fish,while it had no effects on sperm motility and fertility due to the compensation effects of precursor androgen T,indicating that 11-KT is a more efficient androgen than T in fish.In addition,insufficient cortisol affected egg quality and deficiency cortisol resulted in oogenesis arrest.The study not only enriches the effects of 11-KT on spermatogenesis in teleosts,but also innovatively finds out the new effects of cortisol on oogenesis and preliminarily clarify its possible molecular mechanism,indicating that cortisol plays an important role on oogenessis.