| Reproduction is the most important biological characteristics of organism and the ovary development is the base of sexual reproduction.Fish was the most number and species in vertebrates,and of great significance in economics.Ovary differentiation mechanism of fish exhibits extraordinary plasticity and diversity during evolution,it should be affected by genetic and environmental factors.Study ovary differentiation of fish should promote the fishery production and help us to understand the sex determination mechanism of organisms.Yellow River carp is an importantly fresh water fish and has a significant economic importance in China,especially in Henan province.Sex dimorphic growth pattern has been found in Yellow River carp,females exhibited much faster growth rate than male under the same culturing condition.Up to date,there are few of reports about the mechanisms of ovary development,and the genetic and environmental factors for ovary differentiation and development have remained unclear.In order to study the mechanism of ovary development and search for the molecular mechanism and crucial genes of ovary development in carp.We identified the critical time of ovary differentiation by studying the morphology and the expression of genes which related to sex determination in Yellow River carp at first.Then the transcriptomes of primordial gonad,juvenile ovary and adult ovary were analyzed,and the candidate genes and signal pathways related ovary differentiation were identified.The effects of the candidate genes were analyzed by studying the structures and expression pattern of them.Meanwhile,the effects and the underlying mechanism of the signal pathway were studied.Then,the effect of endocrine disruptors TPT on gonad differentiation were studied,and the molecular mechanism of it were identified by analyzing the transcriptomes of gonad which were treated with TPT.The main innovative conclusions are shown as follows.1.Identified the critical time of ovary differentiationPrimordial gonad were identified at 40 dpf larva by histologic slice studies,it should maintain the undifferentiated state untill 55 dpf.Then the primary oocyte started appearing at 70 dpf.The expression of genes involved in gonadal differentiation in vertebrates were changed greatly from primordial gonad to juvenile ovary.According to the specific expression of gonad related genes before early primordial gonadal differentiation,we suggested 40-45 dpf is critical time for undifferentiated gonads to differentiate into either the ovary or testis in the Yellow River carp.2.Analyzed the transcriptome of primordial gonad,juvenile ovary and adult ovary,and identified the candidate genes and signal pathway related ovary differentiation.Here we explored the transcriptome of primordial gonad(PGC),juvenile ovary(OJ)and adult ovary(OA)of Yellow River carp.We identified 1788,1681 and 1256 uniquely expressed genes from primordial gonad,juvenile ovary and adult ovary,respectively.The number of differentially expressed genes(DEGs)of PGC vs OJ were 29679,the DEGs of PGC vs OA were 31150,OJ vs OA were 21201.We identified candidate genes from DEGs by GO and KEGG ananlysis,such as foxl2,dax1,lhx8,dnmt1 genes.The structures and expression pattern of 4 candidate genes were analyzed.the results of them are first reported in carp.They should be involved in primordial gonad differentiation and the development of ovary and testis,and closely associated with ovary development in carp.Furthermore,our investigation has also revealed DEGs enriched in several important functional pathways,such as notch,NF-κB,fanconi anemia,steroid biosynthesis,progesterone-mediated oocyte maturation and PPAR signaling pathway,and so on.3.Study the effect of notch andd NF-κB signal pathway on ovary development in Yellow River carp,and found inhibited notch pathway should promote ovary development and inhibited the NF-κB should inhibit the development of ovary.The primordial gonad were treated with DAPT(notch signaling pathway inhibitor)before critical time of gonad differentiation in vivo and in vito.The expression of notch related genes,amh,dmrt1,sox9 a,sf1 and dax1 genes were decreased,and foxl2,gdf9 and nobox genes were increased in short-term exposure in vitro.DAPT should inhibited the notch signal and promote the differentiation of ovary in long-term exposure.Notch signal should regulated the gonad differentiation of fish is first reported in this paper,inhibited notch pathway should promote ovary development.The primordial gonad were treated with DOC(NF-κB activator)or NAI(NF-κB activation inhibitor)before critical time of gonad differentiation in vivo and in vito.The results showed DOC activated NF-κB,promote the differentiation of ovary by increasing the expression of tnf-α,il8,foxl2,zp2,antiapoptotic gene xiap and decreasing the expression of casp3 a and smac.NAI should downregulated the expression of NF-κB,tnf-α,il8,xiap genes and upregulated the amh,sox9 a,dax1,smac,casp3 a genes,and induce the decrease of female ratio.The results indicated that NF-κB signal pathway was involved in early ovary development of Yellow River carp,and the expression of zp2,foxl2,amh,sox9 a,sf1,dax1 genes should be regulated by NF-κB signal.The results in this paper not only validate the critical time for primordial gonads to differentiate into ovary,but also find a new signal pathway which were involved in ovary differentiation by regulating the expression of inflammatory molecule and apoptotic related genes.4.Analysis of the effect of TPT on the primordial gonad in short term and long term treated in vivo and in vito.TPT should significantly changed the sex ratio of larva.The results presented indicated the ovary differentiation related genes and signal pathway,especially the steroid biosynthesis pathway were responded to TPT treatment,which was probably the molecular mechanism of TPT treatement.In summary,the critical time of ovary differentiation of Yellow River carp were identified,many ovary differentiation related genes and pathways were identified by analysis the transcriptomes of 3 critical stages of ovary,and the effects of the candidate genes and signal pathway were analyzed.We also fund genes and signal pathway were responded to TPT treatment and the molecular mechanism of TPT as a sex hormone analogue was revealed.These results provided a foundation for revealing the molecular mechanism of ovarian differentiation in fish,and have important theoretical and practical significance for aquaculture. |
PDF Full Text Request