| Epigenetics is the study of potentially heritable changes in gene expression that does not involve changes to the underlying DNA sequence.DNA methylation is one of the most important epigenetic modification.The enzymes that catalyze DNA methylation are called DNA methyltransferases（dnmts）.Teleost-specific whole genome duplication has increased the dnmts copy number.Generally,there are 1 maintenance dnmt and 3-5 de novo dnmts in teleost.Current studies have shown that there are great differences in DNA methylation levels between the ovaries and testes.In addition,in the process of sex reversal（female to male and male to female）,the DNA methylation levels of key genes for sex determination and differentiation and the expression of some dnmts family members have changed significantly.These results suggest that DNA methylation and dnmts may play important roles in sex determination and differentiation in teleost.However,the expression analysis of dnmts in fish gonadal development and the functional studies in sex determination and differentiation are still lacking.Nile tilapia（Oreochromis niloticus）is an important cultured fish in the world.The male grows 50%faster than the female.Its sex belongs to XX/XY sex determination system,and the way of sex determination belongs to the joint determination of genetic sex determination and environmental sex determination（GSD+ESD）.The analysis of sex determination and differentiation mechanism of Nile tilapia is conducive to aquatic genetics and breeding.In this study,the expression patterns of dnmts family members in the gonads of Nile tilapia were analyzed by quantitative real-time PCR（q RT-PCR）and fluorescence in situ hybridization（FISH）.The functions of dnmts in gonadal development and sex differentiation were analyzed by using dnmts inhibitor.The construction of dnmt3aa and dnmt3ab mutant lines further revealed their roles in gonadal development.The main results were as follows:1.Expression of dnmts family members in gonadal development of Nile tilapia.The expression of six dnmts family members（including dnmt1,dnmt3aa,dnmt3ab,dnmt3ba,dnmt3bb.1 and dnmt3bb.2）in the gonads of Nile tilapia at different developmental stages（5,30,60,90,120 and 180 dah）was analyzed.q RT-PCR analysis showed that dnmts family members were sexual dimorphism during gonadal development of Nile tilapia.The expression of dnmt1 and dnmt3ba in ovaries was significantly higher than that in testes,and the expression of dnmt3aa,dnmt3ab,dnmt3bb.1 and dnmt3bb.2 in testes was higher than that in ovaries.FISH showed that dnmt1,dnmt3aa,dnmt3ba,dnmt3bb.1 and dnmt3bb.2 high expression in oogonia,phase I and phase II oocytes,granulosa cells,spermatogonia and spermatocytes,while dnmt3ab was highly expressed in granulosa cells,spermatogonia and spermatocytes.2.Effects of dnmts inhibitor 5-aza-d C treatment on gonadal development of Nile tilapia.（1）When 5-aza-d C containing different concentration gradients was fed to unisexual fry（all XX and all XY）at 5 dah,it was found that 5-aza-d C significantly increased the deformity and mortality rate of tilapia,and the weight of surviving individuals in the treatment group decreased significantly.The gonadal morphology and histological observation of tilapia at different developmental stages showed that the Gonadosomatic index（GSI）of females in the 5-aza-d C treatment group was significantly reduced,and a large number of cavities appeared in the ovaries,while there was no significant difference in gonad morphology and GSI between the male treatment group and the male control group,and only a large number of cavities were produced.The gonads apoptosis of female and male tilapia in 5-aza-d C treatment group was detected at30 and 90 dah.Compared with the control group,the number of apoptosis germ cells of5-aza-d C treatment group was significantly increased.The ovaries in treatment group still expressed the female pathway key gene cyp19a1a,the testes in treatment group still expressed the male pathway key genes amh and gsdf,but did not express cyp19a1a.These results showed that there was no sexual reversal in both male and female Nile tilapia treated with 5-aza-d C alone.（2）The 30 dah female（XX）Nile tilapia was fed with 5-aza-d C and aromatase inhibitor（AI）,histological analysis and statistics were carried out at 60and 120 dah.It was found that female fish in AI treatment group,5-aza-d C and AI treatment group had sex reversal from female to male,and there was no significant difference in sex reversal rate between AI treatment group,5-aza-d C and AI treatment group.Gonadal histology showed that spermatogenesis occurred in female fish in AI treatment group,5-aza-d C and AI treatment group at 120 dah.（3）Male（XY）Nile tilapia was fed with 5-aza-d C and E2（17β-estradiol,E2）at the same time for 60 days.At 75 dah,histological analysis showed that a small number of oocytes were produced in the 5-aza-d C and E2 simultaneous treatment group.Immunofluorescence showed that the gonads of the 5-aza-d C and E2 simultaneous treatment group expressed cyp19a1a and oocyte specific marker gene 42sp50,as well as the key genes of male pathway amh and gsdf.At120 dah,statistical analysis showed that the sex reversal rate of 5-aza-d C and E2 treatment group was significantly higher than that of E2 treatment group.The methylation level of cyp19a1a promoter in gonads of 5-aza-d C and E2 simultaneous treatment group and control group was analyzed at 75 and 120 dah.It was found that the methylation level of cyp19a1a promoter in 5-aza-d C and E2 simultaneous treatment group was significantly decreased than that in male control group.3.Effects of dnmt3aa and dnmt3ab mutation on gonadal development of Nile tilapia.In order to further study the function of dnmts family members in gonadal development of tilapia,the key dnmts family members dnmt3aa and dnmt3ab were mutated by CRISPR/Cas9 in Nile tilapia.Dnmt3aa-/-and dnmt3ab-/-mutants were identified,and their gonads were analyzed.The results showed that at 60,120 and 240dah,the ovaries of dnmt3aa-/-XX fish were atrophied and degenerated,and the GSI decreased significantly,and the number of oocytes decreased significantly compared with WT（wild type）,while the ovaries and GSI of dnmt3ab-/-XX fish had no significant difference compared with WT.At 60 dah,there was no significant difference in GSI and the number of spermatogonia between dnmt3aa-/-and WT testes.At 120 and 240 dah,the testes of dnmt3aa-/-were more transparent,GSI was significantly reduced,and there were a large number of cavities in the testes,spermatocytes were significantly decreased,while there was no significant difference between dnmt3ab-/-and WT testes.At 60 dah,dnmt3aa-/-ovaries were still expressed cyp19a1a.In addition,a significant reduction of germ cells was also observed in female ovaries of F0 generation,another target of dnmt3aa.Apoptosis detection showed that compared with dnmt3ab-/-and WT ovaries,the apoptosis of germ cells in dnmt3aa-/-ovaries at 60 dah was significantly increased,and the apoptosis of spermatocytes in dnmt3aa-/-testes at 120 dah was significantly increased compared with dnmt3ab-/-and WT testes.q RT-PCR results showed that the expressions of apoptoses-related genes baxa,baxb,caspase3a,caspase3b and caspase8 were significantly increased in dnmt3aa-/-ovaries at 60 dah.The expressions of apoptosis-related genes baxa,baxb,caspase3b and caspase8 were significantly increased in the testes of dnmt3aa-/-at 120 dah.Interestingly,in dnmt3ab-/-ovaries compensatory increase expression of dnmt3aa was observed at 60 dah.Compensatory increase expression of dnmt3ab and dnmt3aa was observed in dnmt3aa-/-and dnmt3ab-/-testes at 120 dah.The semen of dnmt3aa-/-,dnmt3ab-/-and WT were collected and analyzed at 240 dah.Compared with dnmt3ab-/-and WT,the sperm concentration and the progressive sperm in dnmt3aa-/-mutants decreased significantly and the immotile sperm increased significantly.In addition,the VSL（straight linear velocity）,VCL（curvilinear velocity）and BCF（beat frequency of sperm flagella）of sperm from the dnmt3aa-/-fish were significantly lower than those of the WT and dnmt3ab-/-fish.Morphologically,similar to the WT sperms,the sperms from the dnmt3ab-/-mutants were characterized with straight and long tail,while the sperms from dnmt3aa-/-mutants consisted of some abnormal sperms with curly and short tail from Papanicolaou staining and scanning electron microscope analysis.5-methylcytosine（5-m C）antibody was used to detect the methylation levels of dnmt3aa-/-,dnmt3ab-/-and WT ovaries and testes at 120 dah.It was found that the global 5-m C levels of dnmt3aa-/-ovaries and testes were significantly lower than dnmt3ab-/-and WT.In addition,the 5-m C levels of dnmt3aa-/-granulosa cells,spermatogonia,spermatocytes and spermatids were significantly lower than dnmt3ab-/-and WT.These results suggest that dnmt3aa plays important roles in Nile tilapia gonadal development.In summary,dnmts family members were expressed in sexual dimorphism in Nile tilapia gonads.The expression of dnmt1 and dnmt3ba in ovaries was significantly higher than that in testes,and the expression of dnmt3aa,dnmt3ab,dnmt3bb.1 and dnmt3bb.2 in ovaries was higher than that in ovaries.These dnmts family members highly expressed in ovarian oocytes,granulosa cells and testicular spermatogenic cells.Inhibition of dnmts alone could not lead to sex reversal of tilapia,but increased apoptosis of ovarian and testicular germ cells.The simultaneous treatment of E2 and dnmts inhibitor can significantly reduce the methylation level of cyp19a1a and increase the secondary reversal rate of XY fish.Mutation dnmt3aa or dnmt3ab did not change the sex of tilapia,but in dnmt3aa-/-mutants,apoptosis of germ cells in testes and ovaries increased,sperm quality decreased,and 5-m C level in testes and ovaries decreased significantly.Mutation dnmt3ab has no obvious gonadal phenotype,which is likely due to the compensation of dnmt3aa.These results suggest that dnmt3aa but not dnmt3ab plays important roles in gonadal genes de novo methylation,sex differentiation and gonadal development of Nile tilapia.It enriches our understanding of epigenetic modification in the gonadal development of teleost. |
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