Comparative Studies On The Sex Development Of Two Hermaphroditic Fish Species——Epinephelus Akaara And Kryptolebias Marmoratus

Fish is now known to be the most abundant and diverse vertebrate group in the world.In order to adapt to the complex aquatic environment and produce more offspring,the reproduction strategies of fish are also very diverse and complex,including almost all known sex determination and sex differentiation patterns in vertebrates.This feature makes fish a good model for studying sex developmental mechanisms.Different from that tetrapods are all gonochoristic,functional hermaphrodism exists in many fish species.However,most research about sex development are still focus on limited model fish species(e.g.medaka,zebrafish).What are the similarities and differences between gonochoristic and hermaphroditic fish species,or between different types of hermaphroditic species is still unclear.Moreover,research on fish determination and differentiation are mainly restricted to the gonadal sex-related genes.So far,no studies have yet examined sex-biased gene expression in hypothalamus or pituitary.Therefore,in this study,we chose two hermaphroditic fish species,Epinephelus akaara and Kryptolebias marmoratus,as representative for sequential hermaphrodism and synchronous hermaphrodism,respectively,focus on their sex development mechanisms.Detailed results and main conclusions are listed as follow:1.Comparison of gonadal structures and ultrastructures of two hermaphroditic fishspeciesLike most other teleosts,both E.akaara and K.marmoratus have bilobed gonads.In E.akaara,both the male-and female-phased gonads can be ovo-testis that containing both ovarial and testicular tissues.In male-phased gonad,excepts cells in different stages of spermatogenesis,oocytes in perinucleolus stage or earlier stages can be found mosaicked inside the testicular tissue.Similar situation can be found in female-phased gonad:small pieces of testicular tissues are mosaicked inside the ovarian tissue.Ultrastructure observation showed that sperms in male-and female-phased gonads have no morphological difference,and in the female-phased gonad,no connective tissue or basal membrane can be found between testicular and ovarian tissues.In K.marmoralus,the male testis has the restricted lobular structure,which is similar to other species in Order Cyprinodontiformes.In the hermaphroditic ovo-testis,the testicular tissue located on one side of ovo-testis,attached to the lumen,and has the similar structure with male testis.Observation of ultrastructure showed that thick basal membrane separated the ovarian and testicular tissues in ovo-testis.This physical barrier may be a key point for the ovo-testis development in synchronous hermaphroditic K.marmoratus.2.Cloning of sex-related genes and the expression patterns in two hermaphroditicfish speciesIn this study,we cloned Vasa,Dmrt1,Foxl2 and Foxl3 genes in E.akaara,and investigate their expression patterns in both E.akaara and K.marmoratus gonads.Our results indicated that Vasa have similar expression pattern in both species,signals can be detected in all germ cells in the gonad,including oogonia,pre-vitellogenic oocytes,spermatogonia and spermatocytes,which proved that it can be a good germ cells marker in both species.Dmrt1 gene expressed in the spermatogonia,primary and secondary spermatocytes and the Sertoli cells in both E.akaara and K.marmoratus gonads,which means that Dmrt1 gene may have important function in the development and maintain of testis.However.Dmrt1 signals can also be detected in the Cortical Alveoli stage oocytes and the vitellogenic oocytes in the female-phased gonad of E.akaara.and the functions of Dmrll gene in the oocytes still need further exploration.Foxl3 and Foxl2 are paralogous genes due to the early genome duplication event(1R or 2R).Foxl2 is reported to play important role in follicle development,and has conserved sequences and expression patterns between species.In both E.akaara and K.marmoratus gonads,Foxl2 signals can be detected in the follicular cells.In E.akaara,Foxl3 gene expressed in the spermatogonia,primary and secondary spermatocytes and the Sertoli cells in the male-phased gonad,but cannot be detected in the suspected testicular tissue in the female-phased gonad.However,in both male testis and the testicular part of ovo-testis of K.marmoratus,Foxl3 signals can be found,with the similar expression pattern as Dmrtl.Therefore,we suspect that Foxl3 may be involved in the activation of testicular function in hermaphroditic fish species.3.Hypothalamus and pituitary transcriptome profiling of male and female E.akaara.In this study,we used RNA-seq technology to investigate transcriptomes of both tissues in immature and mature male and female adults.Differential expression analysis revealed 275 unigenes that were differentially expressed between immature male and female adults and 561 between mature male and female adults.According to annotation and KEGG information,these differentially expressed genes were involved in development,metabolism,and regulation of transcription.Among them,AESa and YY1b gene was significantly upregulated in male and female individuals,respectively,may be involved in male/female reproductive function during development.