| The full-length c DNAs of wt1, foxl2, dax1 and amh in the hermaphrodite rice-field eel(Monopterus albus) were cloned and characterized in this study. Multiple sequence alignment revealed that Wt1, Foxl2 and Dax1 were well conserved among M. albus and other vertebrates, whereas Amh had a low degree of similarity among different vertebrates. Stability analysis of reference genes indicated that 18 s, ef1α and rpl17 are the suitable genes in the gonads of different developmental stages as well as in other tissues of M. albus. The expression of wt1 and dax1 was widespread among the different tissues, implying the complex functions of these two genes. The expression of foxl2 was detected in the brain, eyes, and gonads, suggesting the role of foxl2 in the development of ocular structures. amh has important role in gonadal differentiation and development because of its special expression in gonads. Real-time RT-PCR and immunohistochemistry analyze showed wt1 was expressed in ovaries, ovotestis and testis, and Wt1 protein mainly located in Sertoli cells of testis and granulosa cells of ovaries. It is revealed the important role of Wt1 in growth and maturity of oocytes and spermatogonium. The expression profile of foxl2 during sex reversal was female > intersexual > male, and the Foxl2 protein mainly located in granulosa cells, immature oocytes and Leydig cells. It is also showed the important role of Foxl2 during sex reversal of M. albus. In contrast, amh was scarcely detectable in ovaries whereas it was abundant in both intersexual and male stages, indicated the important role of amh in the induction of the testis development. dax1 was also involved in the development of testis and ovaries. In addtion, these sex-ralated genes may directly or indirectly regulated the expression of cyp19a1 a during sexual development of M. albus.The hormones concentration and genes expression in HPG axis, HPT axis and HPI axis have been measured in this study. There was no significant difference in E2 and cortisol during different development stages of M. albus. It is indicated that sexual development might be controlled by er and gr other than the serum E2 and cortisol. However, T3 and FT3 have high levels in females and males with a signicanlty decline in intersexual, implying the important function of THs in sex reversal. Gene expressions analyze of fshr and lhr showed that LH has the important role in maturity of oocytes and ovulation. FSH and LH has also involved in testis development and spermatogenesis. star shows the same role of fshr in sexual development. Moreover, the parallel expression levels of fshr and star, suggesting that star might be regulated by FSH.The expression profiles of vtg shows that it is mainly expressed in liver, especially in female liver. It is revealed that the vitellogenin during oocytes development was mainly from liver synthesis. The expression of vtg increased significantly when gonadal development from female III to female IV and V, implying that the increasing of vtg in liver was crucial for the accumulation of yolk and oocyte mature. The vtg significantly decreased and hardly detected when gonadal development enter into intersexual and male stages. The result indicated that the decrease of vitellogenin was crucial to the degradation of oocytes. Moreover, the expression levels of erα, erβ, ar and gr have been detected in this study. The high levels of erα have been found in liver than erβ shows the important role of erα in vitellogenin synthesis. The expression of erα was gradually lowered in female livers, implying the yolk accumulation induced by E2 decreased during the development of oocytes. However, vtg expression was significantly increased during oocytes development, revealing yolk accumulation have been effected by other hormone other than E2. erα m RNA was gradually raised in female gonads, showing the maturity of oocytes and ovulation needed to be stimulated by E2. When gonadal development entered into intesexual stages, erα increased again in liver, indicating the asynchronization of oocytes development needed vitellogenin continuous synthesis, but oocytes degeneration needed the low expression of erα in gonads. E2 is also important for testis development in M. albus. The expression of ar was increased gradually either in ovary development or in testis development, shows the important role of ar in gonadal development. The high level of gr has been found in testis implied that cortisol was involved in testis development in M. albus. |
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