【作者】 贾永芳；

【导师】 常重杰；

【作者基本信息】 河南师范大学 ， 生物学、动物学， 2017， 博士

【摘要】 生殖是生命有机体非常重要的生物特征,而卵巢的发育分化是有性生殖的基本前提。鱼类是脊椎动物中种类最多的类群,具有重要的经济价值。鱼类卵巢分化发育的类型众多,影响其分化发育的遗传因素和环境因素繁杂。研究鱼类卵巢分化发育的分子机制不仅有利于渔业生产的发展,对于人们了解生物性别决定机制也具有重要意义。黄河鲤(Cyprinus carpio)是河南省乃至全国重要的淡水养殖鱼类,其生长具有明显的性别二态性,雌性生长较雄性快。目前鲤鱼卵巢分化发育的机制研究较少,影响其分化发育的遗传因素和环境因素也不清楚。为了全面研究黄河鲤卵巢分化发育的调控机制,寻找调控卵巢分化发育的分子机制和关键基因,本研究首先从组织学水平上确定了黄河鲤原始性腺向卵巢分化的关键时间,通过分析卵巢不同发育阶段的转录物组信息,找到了与卵巢分化发育过程密切相关的候选基因和信号通路,通过研究这些基因结构与时空表达模式,确定了其在卵巢分化发育中的作用;同时研究了信号通路对卵巢分化的影响及其作用机制。本文还对环境内分泌干扰物TPT对黄河鲤性腺分化的影响进行了分析,通过分析TPT处理后性腺的转录物组信息,发现了其影响卵巢分化的分子机制。获得以下创新结果:1.确定了黄河鲤性腺分化的关键时间。通过组织学切片,原始性腺在幼鱼40 dpf时可以从显微镜下分离,其在55 dpf之前保持未分化状态,在70 dpf出现了卵巢的分化。我们对性腺发育相关基因的表达水平从原始性腺到幼稚卵巢期间进行检测,发现在多数基因在50 dpf发生显著变化。根据原始性腺分化为精卵巢的关键时间早于性腺发生的组织结构变化,我们认为黄河鲤性腺分化的关键时期在45-50 dpf。2.分析比较了原始性腺、幼稚卵巢和成熟卵巢的转录物组,发现与卵巢分化高度相关的差异表达基因和信号通路。将45 dpf原始性腺,80 dpf幼稚卵巢和排卵前的成熟卵巢进行转录物组测序。在原始性腺、幼稚卵巢和成熟卵巢中分别发现了1788,1681和1256个特异表达的基因。研究发现原始性腺和幼稚卵巢的差异基因有29679个,原始性腺和成熟卵巢的基因间表达差异的基因有31150个,幼稚卵巢和成熟卵巢间有21201个差异基因。通过对差异表达基因(degs)进行go和kegg分析,得到卵巢分化发育密切相关的基因,如foxl2、dax1、lhx8、dnmt1等基因。对4个与卵巢早期发育相关的候选基因foxl2、dax1、lhx8、dnmt1进行了详细的基因结构和表达模式分析。4个基因均为首次在黄河鲤报道其结构和表达模式。这些基因与卵巢分化有着密切的关系,推测其可能参与黄河鲤性腺分化和精卵巢早期的分化调控。通过degs的kegg显著富集分析,发现notch、nf-κb、范可尼贫血、类固醇生物合成,孕酮介导的卵母细胞成熟、ppar等信号通路参与了卵巢的分化。3.研究notch和nf-κb信号通路在黄河鲤卵巢发育中的作用,发现抑制notch信号可以促进卵巢分化,抑制nf-κb信号则抑制卵巢分化。利用notch信号通路抑制剂dapt对原始性腺进行短期体外处理和对幼鱼为期30天的水体暴露试验。短期处理结果显示发现dapt可以抑制原始性腺中的notch信号和amh,dmrt1,sox9a,sf1,dax1基因的表达,增强foxl2,gdf9,nobox基因表达。长期暴露实验显示,dapt可以抑制notch信号,并促进原始性腺向卵巢的分化,这也是首次证明该信号通路参与调控鱼类的性腺分化,抑制该通路可以显著促进卵巢分化。利用nf-κb信号通路激活剂doc和抑制剂nai,在黄河鲤卵巢分化的关键时期,对原始性腺分别短期处理和长期水体暴露试验。结果发现激活nf-κb信号通路,诱导了il-8,tnf-α、抗凋亡基因xiap和雌性发育相关基因zp2,foxl2表达上调,凋亡相关基因casp3a,smac基因表达下调,雌性比例增加。抑制nf-κb信号通路,则nf-κb,tnf-α,il8,xiap基因表达下调,并提高了促进了amh,sox9a,dax1基因,凋亡基因smac和casp3a的表达,降低了雌性的性别比例。说明该通路参与黄河鲤卵巢的早期分化发育。另外zp2,foxl2、amh,sox9a,sf1,dax1基因在处理后表达显著变化,说明这些基因受到nf-κb信号通路的调控,从而在性腺分化中发挥作用。这一结果不仅验证了黄河鲤性腺分化的关键时间,同时还发现了新的信号通路可能通过调控炎症分子和细胞调亡参与黄河鲤卵巢分化。4.分析了tpt对黄河鲤幼体的长期体内处理和不同时期性腺的短期体外处理结果,发现tpt可以显著改变幼体性腺分化的比例,其可能的分子机制是通过改变原始性腺中调控卵巢分化的关键基因和信号通路尤其是类固醇激素生物合成通路而实现的。综上所述,本研究确定了黄河鲤性腺发育的关键时间,发现了与卵巢分化早期的调控基因和信号通路,详尽研究了这些基因和信号通路在卵巢发育过程中的作用;同时发现了与TPT影响黄河鲤性腺分化有关的基因和信号通路,揭示了TPT作为性激素类似物的分子机理。这些结果为揭示鱼类的卵巢分化的分子机理奠定了坚实基础,对于水产养殖有着重要的理论意义和实践意义。更多还原

【Abstract】 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.更多还原