| In vertebrates, normal reproductive function of gonads requires expression of numerous genes, and those genes are in turn regulated by many transcriptional factors. GATA proteins, containing two highly conserved zinc finger domains (C-X2-C-X17-C-X2-C) which recognize the WGATAR motif in the promoter of the target gene, are members of an ancient family of transcription factors that mediate process of development, differentiation and gene expression in multiple tissues. Of six Gata members, Gata4 and Gata6 are expressed in ovaries and testes.Previous studies in vertebrates showed that Gata4 and Gata6 are expressed in follicle cells of the ovary and the Sertoli cells and Leydig cells of the testis. Gata6 can be occasionally detected also in germ cells in human and mouse. Deficiencies in either Gata4 or Gata6 in mice ovaries/testes result in a strong down-regulation of genes involved in the gonadal steroidogenesis, as well as reduced the number of germ cells and impaired gametogenesis. In teleosts, only the expression of Gata4/6, in vitro function of Gata4 in tilapia and its function in cardiac duct in zebrafish have been studied. In tilapia, Gata4 is expressed in the granulosa and theca cells of adult ovary, as well as in Sertoli and epithelial cells of the efferent duct of adult testis. Gata4 regulated steroidogenesis related gene, including amh, cyp19a1a, and star by in vitro experiment. There has been no research on Gata4/6 functions in the reproduction of teleosts.The genome sequence of the Nile tilapia (Oreochromis niloticus) was published, and eight gonadal transcriptomes have been sequenced and mapped to this reference genome by our group. To provide further insights into the function of Gata4 and Gata6 in teleost, here we examine the expression profile of Gata4/6 by real-time PCR. We document the cellular localization of Gata6 during various stages of gonad development by immunohistochemistry. In addition, we used the CRISPR/Cas9 system to knockdown Gata4 and Gata6 in both sexes of the Nile tilapia to investigate their roles in steroidogenesis and gametogenesis. The results are as follows:In the present study of tilapia, gata6 mRNA expression were found to be higher in the ovary than the testis at 5 days after hatching (dah),but lower in the ovary than the testis from 10 to 70 dah by real-time PCR and gonadal transcriptomes. We successfully produced the polyclonal antibody of tilapia Gata6. Immunohistochemistry and in situ hybridization showed that Gata6 are expressed in Leydig cells of the testis, as well as in interstitial and granulosa cells of the adult ovary. Weak Gata6 signals were also observed in the oogonia and spermatogonia. We knockout Gata4/6 successfully by using CRISPR/Cas9. In XX fish, knockout of either Gata4 or Gata6 alone resulted in impaired oogenesis, as demonstrated by impairment of granulosa cells development and vitellogenesis, and consequently reduced levels of cyp19a1a/Cypl9ala, cypl7al/Cyp17a1,foxl2, StARII and 17β-estradiol (E2). In XY fish, knockout of either gene resulted in delayed spermatogenesis, disorganized seminiferous lobules, reduced numbers of Leydig cells, and consequently decreased levels of cyp11b2/Cyp11b2, cyp17a1/Cyp17a1, cypl1a1,StARⅠ/Ⅱ and serum 11-ketotestosterone(11-KT). Furthermore, vacuolated epithelial cells of the efferent duct were observed in Gata4 deficient testis. We use Gata6 deficient XY fish cross normal XX fish, and produce Gata6 deficient Fl generation. It also means Gata6 deficient male fish are fertile. Taken together, these results demonstrated that Gata4 and Gata6 are involved in steroidogenesis and gametogenesis of both sexes. |
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