【作者】 黄英；

【导师】 龚炎长；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2021， 博士

【摘要】 动物有两种主要繁殖方式,即胎生与卵生;胎生的和卵生方式在分子层面差异明显,另外卵生动物和胎生动物的性激素在合成场所及富集趋势也存在差异。为探讨雄性激素受体(AR)对鸡卵泡发育的具体作用,本研究以人、牛、鼠为胎生动物的代表,以鸡为卵生动物的代表,构建了物种间卵泡发育各阶段的对应关系;通过比较物种间卵泡发育相关基因、相关生物学过程的差异,鉴定鸡特有的卵泡发育相关基因;基于超级增强子判断雄激素受体是否为鸡特有的核心转录因子;在细胞水平和个体水平验证雄激素/雄激素受体对鸡卵泡选择和排卵的作用;基于转录组数据分析AR、鸡特有的卵泡发育相关基因、卵泡选择三者之间的关系;基于UCSC中各物种多序列比对数据比较鸡中鉴定的卵泡超级增强子在物种间的保守性。主要研究结果如下:(1)基于人、牛、鸡转录组数据,构建了胎生动物和卵生动物间卵泡发育各阶段对应关系,结果显示卵生动物排卵前卵泡阶段对应到胎生动物有腔卵泡阶段,二者排卵前卵泡阶段对应关系不一致。值得注意的是,鸡F5卵泡阶段对应到胎生动物有腔卵泡阶段,提示鸡排卵前卵泡阶段可能需要进一步细分;(2)基于人、牛、鸡转录组数据,比较物种间卵泡发育相关基因和相关生物学过程的异同。结果发现,物种间卵泡发育相关基因不保守,较多卵泡发育相关生物学过程是物种特异的,其中,鸡特有的卵泡发育相关生物学过程在选择阶段的膜细胞中、分化阶段的颗粒细胞和膜细胞中占比较高。在此基础上,本研究鉴定了鸡特有的卵泡发育相关基因;(3)基于人卵巢、鼠颗粒细胞、鸡颗粒细胞H3K27ac组蛋白修饰Ch IP-Seq结果,比较胎生动物和卵生动物核心转录调控因子的异同,结果显示胎生动物和卵生动物间卵泡核心转录调控因子大部分是保守的,二者保守性好的核心转录调控因子倾向于占据调控网络关键中介节点的位置,保守性差的核心转录调控因子倾向于占据调控网络中对外调控能力强的节点;这提示物种间的共性可通过占据网络关键中介节点的核心调控因子来维持,物种间的个性可通过占据网络对外调控能力强的节点来体现。研究还发现胎生动物的雌激素受体和卵生动物的雄激素受体则分别占据二者调控网络对外调控能力强的节点,胎生动物和卵生动物间卵泡发育的差异可能与此有关;(4)通过细胞水平实验和个体水平实验,本研究检测了雄激素受体对鸡颗粒细胞增殖、产蛋、卵泡选择和卵泡形态的影响,结果显示抑制雄激素受体导致鸡小黄卵泡减少、产蛋停止,过量刺激雄激素受体导致鸡排卵前卵泡增多、产蛋停止,说明雄激素受体是调控卵泡选择和排卵的关键调节因子;(5)通过比较卵泡选择正常组(未处理或添加雄激素类似物)和异常组(添加雄激素抑制剂)中鸡特有的卵泡发育相关基因的表达情况发现,卵泡选择异常组中鸡特有的卵泡发育相关基因在F5阶段的卵泡颗粒细胞中显著低表达。在体外培养的颗粒细胞中敲低雄激素受体的表达也显著降低了鸡特有的卵泡发育相关基因的表达。这些结果说明,雄激素/雄激素受体可通过维持鸡特有的卵泡发育相关基因的表达来维持卵泡选择的正常进行;(6)基于UCSC各物种多序列比对数据,比较了鸡雄激素受体超级增强子在各物种同源区域的保守性,结果显示其同源区域仅在鸟类中保守,在爬行动物次之,在其他物种中保守性极差,大多为基因组缺失区域,这提示通过雄激素受体超级增强子来调控卵泡发育的模式可能是鸟类特有的。总体而言,本研究发现,与哺乳动物不同,雄激素受体可通过鸡特有的卵泡发育相关基因来调节卵泡的选择,其中鸡特有的卵泡发育相关基因的表达变化可能会影响跨膜转运蛋白的表达水平,从而影响鸡卵泡选择过程中卵黄物质的积累,进而影响卵泡选择。更多还原

【Abstract】 The differences of reproductive processes at molecular levels between the viviparous and oviparous animals are evident,and in the oviparous animal,the locations to synthetize sex hormones and also the trends to enrich sex hormones during follicle development are different from that of viviparous animals.To explore the specific role of the androgen receptor(AR)on chicken follicle development,we constructed a correspondence between the stages of follicular development among species,using human,bovine and rat as representatives of viviparous animals and chicken as a representative of oviparous animals.By comparing the differences in follicle development-related genes and related biological processes among species,we identified follicle development-related genes specific to chicken.We investigated whether the androgen receptor is a core transcription factor specific to chickens based on super enhancers,and verified the role of androgens/androgen receptors on follicle selection and ovulation in chickens at the cellular and individual levels.The relationship among AR,chicken-specific follicle development-related genes and follicle selection was determined based on transcriptomic data.The cross-species of follicular super-enhancers in chickens was identified using multiple alignments data from UCSC.The main findings are as follows.(1)Based on human,cow and chicken transcriptome data,we constructed a correspondence between the follicular development stages of viviparous and oviparous animals,showing that the oviparous preovulatory stage corresponds to the viviparous antral stage.The inconsistency in the correspondence between the preovulatory stages of viviparous and oviparous animals suggests the immaturity of the preovulatory follicles in oviparous animals.Notably,the F5 stage in chickens corresponds to the antral stage in viviparous animal,suggesting that the preovulatory stage in chickens may need further subdivision;(2)Based on human,bovine and chicken transcriptomic data,we compared the similarities and differences of follicle development-related genes and related biological processes among species.The results showed that follicle development-related genes were unconserved among species,and more follicle development-related biological processes were species-specific,in which chicken-specific follicle development-related biological processes were more prevalent in theca cells at the selection stage,and in granulosa cells and theca cells at the differentiation stage.In turn,we identified chickenspecific follicle development-associated genes.(3)The similarities and differences of core transcriptional regulators between viviparous and oviparous animals were compared based on Ch IP-Seq results of H3K27 ac histone modification in human ovary,mouse granulosa cells and chicken granulosa cells.The results showed that most of the follicular core transcriptional regulators were conserved between viviparous and oviparous animals,and the core transcriptional regulators that were higly conserved among species tended to occupy the positions of key intermediary nodes in the regulatory network,while the core transcriptional regulators that were poorly conserved tended to occupy the nodes with high external regulatory capacity,suggesting that the commonality among species can be maintained by the core regulators that occupy key intermediary nodes in the network,and the individuality between species can be reflected by the nodes that occupy the regulators with strong external regulatory capacity in the network.The estrogen receptor in viviparous animals and the androgen receptor in oviparous animals occupy the nodes with strong external regulation in their regulatory networks,suggesting that the differences between viviparous and oviparous animals may be related to these two core transcriptional regulators.(4)We examined the effects of androgen receptors on granulosa cell proliferation capacity,egg production,follicle selection and follicle morphology in chickens through cellular and field experiments.The results showed that inhibition of androgen receptors decreased the number of small yellow follicles and halted egg production,while overstimulation of androgen receptors increased the number of preovulatory follicles but halted egg production,indicating that androgen receptors are key regulators in regulating follicle selection and ovulation.(5)A comparison of the expression of chicken-specific follicle development-related genes in the normal follicle selection group(untreated or with androgen analogues)and the abnormal group(with androgen inhibitors)revealed that chicken-specific follicle development-related genes in the abnormal follicle selection group were significantly down-expressed in follicular granulosa cells at the F5 stage.Knockdown of androgen receptor expression in granulosa cells cultured in vitro also significantly reduced the expression of chicken-specific follicle development-related genes.These results suggest that androgen/androgen receptors can maintain normal follicle selection by maintaining the expression of chicken-specific follicle development-related genes.(6)Based on the UCSC multiple sequence alignment data,we compared the conservation of homologous regions of the chicken androgen receptor super-enhancer across species and found that its homologous regions are only conserved in birds,followed by reptiles,and are extremely poorly conserved in other species,with mostly genomic deletion regions,suggesting that the mode of follicle development regulation by the androgen receptor super-enhancer may be unique to birds.Overall,this study found that,unlike mammals,the androgen receptor can regulate follicle selection through chicken-specific follicle development-related genes,where changes in the expression of chicken-specific follicle development-related genes may affect the expression levels of transmembrane transporter proteins,thereby influencing the accumulation of yolk material during follicle selection and hence regulate follicle selection process in chickens.更多还原