| Aromatase is the key enzyme for estrogen synthesis,and functions as a key regulator in female primary sexual differentiation in non-mammalian vertebrates.Cyp19a1 is the coding gene of aromatase and participates in female sexual differentiation in turtles.Foxl2 is a member of fork head transcription factor family,and it acts as an important role in female primary gonadal differentiation and functional maintenance of ovary.Existing researches show that Foxl2 locates in the upstream of Cyp19a1,which regulates the expression of aromatase gene,therefore,this two genes function together as regulator of female gonadal differentiation.In this study,the expression pattern of Cyp19a1 was examined in adult different tissues and gonads of different developmental stages;the functional roles of Cyp19a1 and Foxl2 were emphatically investigated through aromatase inhibitor(AI)treatment and lentivirus-mediated RNA interference/over-expression systems.The Results are as follows:(1)Functional research of Cyp19a1 geneRT-PCR and Real-time PCR results showed that Cyp19a1 gene was robustly expressed in adult ovary,however,Cyp19a1 mRNA was not or weakly detected in testis,heart,liver,spleen,lung,kidney,muscle and intestine.Moreover,Cyp19a1 exhibited female specific expression pattern in embryonic gonads of stage 18,preceding the onset time of gonadal differentiation(stage 19).Loss-of-function analysis revealed that ZW embryos with AI treatment or Cyp19a1 knockdown exhibited female to male sex reversal,characterized by obviously masculinized gonads with well organized testis cord structure in the medulla and a degenerated cortex,a decline in ovarian marker Foxl2 and significant upregulation of testicular marker Amh.Gain-of-function analysis showed that ZZ gonads over expressing Cyp19a1 were feminized,determined by a thickened outer cortex and a vacuolated medulla,as well as upregulation of Foxl2 and downregulation of Amh.Immunofluorescence analysis showed that the testicular marker,Sox9 protein,highly expressed in medulla of ZZ gonads,however,its expression signal was not detected in ZW gonads.In ZW gonads with Cyp19a1 knockdown,Sox9 protein was induced to express in medulla;on the contrary,almost no Sox9 expression was detected in ZZ gonads overexpressing Cyp19a1.(2)Functional research of Foxl2 geneMoreover,the lentivirus-mediated over-expression system was utilized to investigate the functional role of Foxl2 gene.Results showed that ZZ embryos overexpressing Foxl2 appeared male to female sex reversal,characterized by highly developed cortex and degenerated medulla with cavities.In addition,real time PCR results showed that the testicular markers,Dmrt1 and Sox9,were downregulation in ZZ gonads overexpressing Foxl2,and ovarian marker,Cyp19a1,was remarkable upregulation;immunofluorescence analysis also showed that Sox9 protein expression was significantly down-regulated or even not detected in ZZ gonads overexpressing Foxl2.In this study,the expression pattern of Cyp19a1 gene was analyzed in the process of sexual differentiation of Pelodiscus sinensis;the sex reversals can be induced through Cyp19a1 knockdown/over-expression and Foxl2 over-expression;Cyp19a1 and Foxl2 were necessary to initiate ovarian development,thereby functioning as a key regulator of female sexual differentiation.We provide a new method for gene functional research of turtles,which supplies the basis for the subsequent elucidation of molecular mechanisms of sex determination and gonadal differentiation in Pelodiscus sinensis;meanwhile,we also provide a new way for all-male offspring breed of Pelodiscus sinensis: all-male offspring can be acquired through mating between ZZ individuals and ZZ pseudogyna individuals which are produced by Cyp19a1 or Foxl2 over-expression in ZZ embryos. |
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