【作者】 贺建宁；

【导师】 储明星；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 绵羊属于季节性发情动物，一般在短日照的秋冬季节发情配种。季节性发情不仅限制了母羊的生产力，还导致季节性产羔，致使产肉性能也呈现出一定的季节性，造成肥羔肉不能实现一年四季均衡供应。我国大部分绵羊品种表现为季节性发情，因此季节性发情成为我国养羊业高效和可持续发展以及实现国家养羊业发展模式战略转变的瓶颈。然而，要从分子水平上改变绵羊季节性发情的特性，使其能常年发情和配种，就必须首先了解常年发情的内分泌学机制和涉及的关键基因。本文旨在通过检测不同季节绵羊外周血液中褪黑激素（MLT）和雌二醇（E2）、孕酮（P4）、促卵泡激素（FSH）、促黄体激素（LH）等生殖激素的变化，同时检测绵羊卵巢组织不同繁殖状态下相关基因表达模式，期望揭示绵羊繁殖季节性的内分泌变化规律和卵巢组织中的关键基因，为进一步阐明绵羊常年发情的分子机理提供理论基础。采集小尾寒羊和滩羊春夏秋冬四个季节各一个完整发情周期的血液，检测褪黑激素（MLT）和上述4种生殖激素的浓度。春季采集小尾寒羊不同发情阶段（发情间期末、发情前期、发情期）和滩羊乏情期的卵巢组织，利用高通量测序技术RNA－Seq进行转录组水平检测。本实验获得结果如下：（1）外周血液中褪黑激素（MLT）分泌呈现一定的变化规律。MLT在夜间分泌极显著高于白昼（P<0.01），上升与下降的速度非常快，往往在日落或日出1小时左右即可完成，之后一直保持在高水平或低水平状态直至日出或日落时。小尾寒羊MLT分泌没有明显的季节性变化，而滩羊则具有显著的季节性变化特征：秋分和冬至显著高于春分和夏至（P <0.05）。进一步分析了MLT白天和夜间的季节性变化规律，白天MLT水平小尾寒羊四季变化平稳，滩羊在秋冬季节有上升趋势，但变化不显著；夜间MLT小尾寒羊四季变化平稳，滩羊秋冬季节显著上升（P<0.05）。结果提示夜间MLT水平对调控绵羊的季节性发情具有重要作用。（2）外周血液中生殖激素的分泌具有鲜明的季节性变化特征。小尾寒羊外周血液中四种生殖激素在春、夏、秋、冬四个季节变化基本稳定。滩羊生殖激素变化具有显著的季节性，E2变化基本稳定；P4分泌量秋冬季节显著高于春夏季节（P <0.05）；FSH和LH分泌量秋冬季节显著高于春季和夏季（P <0.05），夏季显著高于春季（P <0.05）。通过对两品种间激素变化研究发现，小尾寒羊与滩羊四个季节血液中E2浓度差异不显著（P>0.05）；春夏季节时小尾寒羊P4和FSH含量显著高于滩羊（P <0.05）；小尾寒羊LH含量在四个季节均高于滩羊，但仅在冬季时差异显著（P <0.05）。上述结果表明，稳定的E2水平及一定浓度的P4可能与绵羊的常年发情有关，高水平的FSH和LH对开启或维持绵羊的发情具有重要作用,或与多胎具有一定的关系。（3）本研究利用RNA-seq技术获得了绵羊不同繁殖状态下的基因表达模式。卵巢的聚类结果表明，乏情期与发情间期，发情前期与发情期分别首先聚在一起。这表明处于不同繁殖状态的绵羊，乏情期与发情间期，发情前期与发情期具有更相似的基因表达模式。（4）不同繁殖状态下（乏情期、发情间期末、发情前期、发情期）卵巢共获得差异基因767个，其中已知基因396个、未知371个，表达上调的有702个、下调的有65个。对已知的差异表达基因进行GO功能富集和KEGG Pathway分析。由GO功能分类可知，卵巢组织不同繁殖状态差异表达基因功能主要涉及结合活性、细胞器官、细胞、催化活性、生物调控、生物过程调控、代谢过程等类型。KEGG pathway富集分析表明这些基因主要参与了TGF信号通路、类固醇的生物合成、Notch信号通路、黄酮及黄酮醇的生物合成、孕酮介导的卵母细胞成熟、胰岛素信号通路等调控途径。其中TGF-β信号通路中FSTL3、SMAD1、SMAD4和TGFBRII基因及胰岛素信号通路中的BAD基因可能对常年发情有一定的调控作用，需要进一步的分析验证。更多还原

【Abstract】 Sheep are seasonal breeders. The breeding season begins at the end of summer or at the beginningof spring. This seasonal estrus limited the ewe productivity and leads to the seasonal lambing, so theprime lamb provided has the seasonal variations depend on the sheep seasonal estrus. Most of sheep areseasonal breeders in China, which limited the efficient and sustainable development of the sheepindustry. In order to change the seasonal estrus at the genetic level, the endocrine basis and the keygenes related to seasonal estrus should be understood firstly.The objective of this study is to analyze the seasonal changes of year-round and seasonal estrussheep.Melatonin, E2, P4, FSH and LH concentration in serum and the gene expression pattern of ovaryin different reproductive status were determined. Our results could provide theoretical basis forregulating the seasonal reproduction in sheep.Blood samples were collected from five Small Tail Han sheep and five Tan sheep in spring,summer, autumn and winter, respectively. The melatonin, E2, P4, FSH and LH in serum were measuredby radioimmunoassay. Ovaries were collected from Small Tail Han sheep in the different estrous periodincluding the end of dioestrus, proestrus and estrus, and Tan sheep during the anestrous in spring. TheRNA-Seq was used to screen the differently expressed genes in ovary. Our results were listed as follows:(1) Secretion pattern of Serum MLT. MLT level at night was significantly higher (P<0.05) than that inthe day. The increase and decrease of the speed was fastand normally completed in1hours.Then theMLT remained at either a high level or low level until sunrise or sunset. The MLT level of Small TailHan sheep showed no significant changes around the year. However, it was different in Tan sheep. MLTlevel in the autumnal equinox and winter solstice were significantly higher (P<0.05) than that in springequinox and summer solstice. The day and night MLT levels of Small Tail Han sheep showed nosignificant changes during four seasons. However, the night MLT level of Tan sheep showedsignificantly increased (P<0.05) in autum and winter. The results indicated that the night MLT levelplays an important role in seasonal reproduction regulation in sheep.(2) Secretion pattern of Serum reproductive hormone. The results showed that the concentrations ofthese four hormones in Small Tail Han sheep had no significant changes around the year. However, itwas different in Tan sheep. P4, FSH and LH concentration in estrous season were significantly higher(P<0.05) than those in anestrous season. According to the analysis between two breeds, E2level has nosignificant difference（P>0.05）around the year. The serum P4and FSH concentration in Small TailHan sheep were significantly higher (P<0.05) than in Tan sheep during spring and summer. The LHlevel in Small Tail Han sheep was higher than in Tan sheep, however, has no significantdifference(P>0.05) except in winter. These results indicated that a certain level of E2and P4wereneeded for sheep year-round estrous, and high level of FSH and LH were necessary for year-roundestrus or polyembryony in sheep.(3) RNA-Seq was used for determining the gene expression pattern in the different reproductive status.According to the hierachical clustering results of differentially expressed genes from the different reprodctive status. The results showed that the genes of anestrus and diestrus, proestrus and estrus wereclustered together firstly. These indicated that sheep in anestrus and diestrus, proestrus and estrus had asimilar gene expression pattern.(4) RNA-Seq analysis of differentially expressed genes generated a list of767genes with396as knownand371as unknown, and with702as up-regulated and65as down-regulated. The most significantlyoverrepresented GO terms are related to binding, organell, cell, cell part, cell process and metabolicprocess, etc. Moreover, KEGG pathway analysis revealed that many pathways such as TGFβ signalingpathway, Steriod biosynthesis, insulin signaling pathway, Notch signaling pathway, Flavone andflavonol biosynthesis as well as Progesterone-mediated oocyte maturation, etc. had been regulatedremarkably at the different reproductive status of sheep ovary. And FSTL3, SMAD1, SMAD4andTGFBRII and BAD gene may the important candidate gene for year-round estrus regulation.更多还原