Steroidogenic Factor 1(SF-1) Is Essential For Steroidogenesis, Gonadal Development And Sex Differentiation In Nile Tilapia

Orphan nuclear receptor steroidogenic factor 1(SF-1/NR5A1) is a master regulator of steroidogenesis and reproduction. In mammals, SF-1 is highly expressed in steroidogenic tissues. Its targets include genes at every level of the hypothalamic-pituitary-gonadal axis, as well as many genes involved in adrenal steroidogenesis. Animals with SF-1 deficiency failed to form adrenal glands and gonads, displayed male to female sex reversal, exhibited impaired gonadotrope function, and lacked the ventromedial hypothalamic nucleus. Thus SF-1 is essential for adrenal and gonadal development, and sexual determination/differentiation. Due to the lack of specific antibody and other technological limitations, the study of sf-1 in fish has been limited to molecular cloning, mRNA expression and in vitro experiments. There has been no report on loss of function study of sf-1 in teleosts. CRISPR/Cas9 targeted gene knockout technology is one of the most dominant genome editing techniques in recent years. The Nile tilapia, Oreochromis niloticus, is a world wide aquaculture fish with male growing 50% faster than females. Culture of monosex fish has high economic value. In the present study, in association with Foxl2, we analyzed the expression pattern of Sf-1 in Nile tilapia gonads by immunohistochemistry(IHC) using a Sf-1 specific antibody. We mutated sf-1 and analyzed the gonadal phenotype, gene expression, serum E2(estradiol-17β) and 11-KT(11-ketotestosterone) level of the F0 and F1 mutant fish. The main results are as follows: 1) Expression pattern and cellular localization of Sf-1 in developing gonads. Transcriptome analysis revealed that sf-1 was expressed in gonads from as early as 5 dah, with higher expression in the XY than the XX fish. Sf-1 was up-regulated from 30 dah and maintained at very high level in the adult testis. In the XX gonads it was up-regulated and reached the highest level at 30 dah, then down regulated to very low level at 90 dah and maintained this level till 180 dah. The specificity of the Sf-1 polyclonal antibody was confirmed by Western blot. Specific bands, corresponding to the calculated molecular weights of the tilapia Sf-1 C-terminal fragment(35.88 KDa) and native protein(55.01 KDa), were recognized using our Sf-1 antibody. Sf-1 specific immunostaining was observed by IHC in the cytoplasm of the germ cells and in both cytoplasm and nucleus of the somatic cells of the XX and XY gonads. At 90 and 180 dah expression was observed in the oogonia, interstitial, granulosa and theca cells of the ovary, and in the spermatogonia and Leydig cells of the testis. IHC staining of adjacent sections in series from XX and XY gonads confirmed that Sf-1 was often co-expressed with Cyp19a1 a in the interstitial, granulosa and theca cells of the ovary. Sf-1 and Cyp11b2 were co-expressed in the Leydig cells of the testis. 2) Efficient site-directed disruption of Sf-1 by CRISPR/Cas9 resulted in gonadal dysgenesis, female to male sex reversal, higher condition factor and survival rate in F0. Genomic DNA from 20 pooled injected embryos and from fin of 90 dah mutants of the two target sites was used as template for PCR amplification and mutation assays. Complete digestion produced fragments of 151 and 321bp(Bmr I) and 247 and 315bp(Bcod I) in the control group. An intact DNA fragment was observed in embryos injected with both Cas9 mRNA and target gRNA. The mutation frequencies induced by CRISPR/Cas9 in pooled embryos were approximately 90.9% and 82.1% for target site 1 and 2, respectively. Frame-shift and in-frame mutations were identified by Sanger sequencing. The ratio of frame-shift to total mutations in embryos was approximately 70.6% and 76.9% from target site 1 and 2, respectively. The hatching rate was 52.1% and 53.5% in the target site 1 and 2 group, significantly lower than that(71.0%) of the control group. The screening results indicated that 94.7%(54/57) of the micro-injected XX fish and 94.8%(37/39) of the micro-injected XY fish were mutated for target site 1, with mutation frequencies ranging from 72% to 95%. Most of Sf-1 deficient XX and XY fish displayed gonadal dysgenesis(GD) with very small and thin gonads. Mutation of Sf-1 resulted in loss of steroidogenic cells and blockage of germ cell meiosis in GD XX and XY gonads. At 90 and 150 dah the GD XX gonad was filled with mainly somatic cells and oogonia with an underdeveloped ovarian cavity. The GD XY gonad was mainly composed of somatic cells and spermatogonia with underdeveloped efferent duct. While sex reversed male(SRM) Sf-1 deficient XX fish displayed a large but short gonad, steroidogenic cells, spermatogonia, spermatocytes and spermatids were observed in the SRM gonads 90 dah. Sf-1 deficiency resulted in decreased Cyp19a1 a, Foxl2 expression and serum E2 in XX fish. In XY fish, Sf-1 deficiency increased Cyp19a1 a and Foxl2 expression but decreased Cyp11b2 expression and serum 11-KT levels. Sf-1 deficiency also increased average body weight, consequently the condition factor and survival rate of the GD XY fish. 3) E2 partially and MT completely rescued the Sf-1 deficient XX and XY gonadal phenotype. Low and high concentration of E2 treatment partially restored the ovary size, while MT treatment completely restored the testis size, some even displayed hypertrophy. E2 treatment partially rescued the development of XX GD gonad as demonstrated by the appearance of phase II oocytes and ovarian cavity; MT treatment partially rescued the development of GD gonad at 120 dah and completely rescued the development of GD gonad as demonstrated by the appearance of spermatocytes, spermatids and efferent duct with free sperms inside at 150 dah. There were numerous vacuoles in the MT rescued gonads. By immunofluorescence staining, no Cyp19a1 a and Cyp11b2 immunostaining was observed in the E2 rescued GD XX fish(EGD) and MT rescued GD XY fish(MGD) gonad. To examine the effects of Sf-1 deficiency on male pathway gene expression, gonads of control, GD and MGD XY fish were examined by IHC. In the XY control, Dmrt1 was observed in the Sertoli cells, and Amh was observed in the myoid cells and Sertoli cells. In the GD XY gonad, strong Dmrt1 immunostaining was observed in the Sertoli cells, and sparse Amh immunostaining was observed in the myoid cells and Sertoli cells. In the MGD gonads, only sparse Dmrt1 immunostaining was observed in the Sertoli cells, and no Amh was observed indicating MT treatment could not rescue their expression. Consistent with the IHC results, the expression of dmrt1 in the GD XY gonads showed no significant difference from the XY control. However, the expression of amh and sf-1 was significantly down-regulated in the GD XY gonads compared with the XY control. As a whole, the expressions of the male pathway genes remained low and unchanged in the GD XX gonads, while they were down-regulated in the GD XY gonads 4) Haploinsufficiency of Sf-1 causes female to male sex reversal. F1 fish were generated by MGD XY male mated with a wild type female(XX). In contrast to various somatic mutations, only two types of mutation were detected in the sperm and F1 fish. Genotyping of F1 fish was performed by polyacrylamide gel electrophoresis(PAGE). Due to high mutation rate of the MGD XY parent, all of F1 generation were sf-1+/- mutants, with frame-shift mutation of 11 and 7 bp deletions in one allele causing premature truncation at the hinge region of Sf-1. Of the 80 sf-1+/- F1 fish examined, 42 were XY and 38 were XX. All XY fish developed as male. Thirty-five of the 38 sf-1+/-XX mutants were found to be sex reversed, and the remaining 3 were female with ovarian insufficiency. Both XX and XY sf-1+/- testes displayed delayed spermatogenesis and disordered efferent duct formation at 90 dah but become normal and fertile at 150 dah. Strong Cyp11b2 while no Cyp19a1 a was observed in the Leydig cells of the sf-1+/-XX and XY testis at 90 dah. 5) Sf-1+/- XX and XY fish displayed similar gene expression pattern to that of the sf-1+/+ XY fish. By qRT-PCR and Western blot, the gonadal expression of cyp19a1a/Cyp19a1 a and the serum E2 level in the sf-1+/+ XX was significant higher than those of the sf-1+/+ XY, sf-1+/- XX and XY fish, and those of the latter three showed no significant difference. In contrast, the gonadal expression of cyp11b2/Cyp11b2 and the serum 11-KT level in the sf-1+/+ XX was significant lower than those of the sf-1+/+ XY, sf-1+/- XX and XY fish, and those of the latter three showed no significant difference. Additionally, Sf-1 was found to be expressed highly in the sf-1+/+ XY testis, moderately in the sf-1+/+ XX ovary, and low in the SRM, sf-1+/- XX and XY testis.In summary, we produced tilapia Sf-1 antibody and investigated its expression in gonads. We also generated Sf-1 deficient F0 and haploinsufficient F1 tilapia using CRISPR/Cas9 to study the function of the Sf-1 in teleost. Our results are consistent with the findings in mammalian models such as disrupted steroidogenesis, gonadal dysgenesis and enhanced body growth. In addition, we demonstrated that Sf-1 deficient XX and XY fish with gonadal dysgenesis could be partially rescued by E2 and completely rescued by MT treatment, respectively, and haploinsufficiency of Sf-1 causes female to male sex reversal in XX tilapia. The results of the present study support that Sf-1 is also a major regulator of steroidogenesis, gonadal development and sex differentiation in fish. Our results not only enriched our knowledge about molecular mechanism of sex determination and differentiation in teleost, but also provided theoretical basis for tilapia sex control in aquaculture.