下丘脑DNA甲基化对母羊初情期启动的作用机制研究

Time of puberty onset is closely related to the sex maturation and later reproductive performance of ewe, and clarifying its mechanism is the key to effective control of puberty onset. Recent studies have shown that DNA methylation in hypothalamus plays an important role in the puberty onset of female mammals；however，its mechanism is still unclear. In this project, methylation sensitive amplification polymorphism (MSAP) and pharmacological methods will be used to investigate change law of the pattern and level of DNA methylation and screen the different methylation genes in hypothalamus during pubertal development of ewe. Expression and distribution of DNA methylases and methylation CpG binding protein in hypothalamus during pubertal development of ewes will also be determined to conduct qualitative and quantitative analyses its relationship with KiSS-1 and GnRH genes, using double immuhistochemical fluorescence staining and Real-time fluorescence quantitative PCR. Using bisulfite sequencing PCR(BSP) and Real-time fluorescence quantitative PCR ,the relationship between CpG island methylation status in promoter and the first exon regions of KiSS-1, NKB and GnRH genes, and their expression will be investigated. Based on the above studies, the mechanism of hypothalamic DNA methylation on puberty onset of ewes will be deciphered. The results will not only provide reference for fully understanding the regulation mechanism of puberty onset, but also lay the foundation for improving fertility of ewes.

初情期启动时间与母羊的性成熟和以后的繁殖性能密切相关，阐明启动机制是对其有效调控的关键。最近，研究表明下丘脑DNA甲基化对雌性哺乳动物初情期的启动起重要作用，但其作用机制尚不清楚。本项目拟采用MSAP和药理学等方法研究母羊初情期启动过程中下丘脑DNA甲基化模式和水平的变化规律，并获得差异甲基化基因；用荧光双标记和荧光定量PCR技术研究母羊初情期启动过程中DNA甲基化酶和甲基化CpG结合蛋白在下丘脑的表达规律及分布定位，定性和定量分析其与KiSS-1和GnRH基因的关系；用BSP技术研究母羊初情期启动过程中下丘脑KiSS-1、NKB和GnRH启动子和第1外显子区CpG岛甲基化状态及与基因表达关系，阐明下丘脑DNA甲基化对母羊初情期启动的作用机制。研究结果不仅为全面理解哺乳动物初情期启动机制提供参考，而且也为提高母羊繁殖力奠定基础。

初情期的启动是母羊从幼年发育到获得繁殖能力的标志，启动时间直接关系到母羊的性成熟早晚与繁殖性能及养羊业的经济效益,阐明启动机制是对其有效调控的关键。研究表明下丘脑DNA甲基化对雌性哺乳动物初情期的启动起重要作用，但其机制尚不清楚。本项目首先利用F-MSAP技术检测初情期启动过程中母羊下丘脑基因组DNA甲基化水平，并研究注射DNA甲基化抑制剂对母羊下丘脑DNA甲基化和初情期启动影响；随后利用荧光定量技术和免疫荧光标记技术检测DNA甲基化酶与初情期启动关键基因的表达定位，最后通过BSP技术检测初情期启动重要基因GnRH、KiSS-1和NKB的甲基化水平与表达量之间的关系。结果表明，在初情期启动的三个发育阶段中共发现了2409个条带，且主要集中于21-309bp。初情期启动时下丘脑的总体甲基化水平为61.96%，显著高于初情期前（41.08%）和临近初情期（34.20%）。注射DNA甲基化抑制剂可使母羊下丘脑DNA甲基化水平、KiSS-1基因表达量和LH分泌水平显著降低。随着初情期启动，GnRH、KiSS-1和NKB表达量显著升高，且初情期表达量水平显著高于初情前期和临近初情期,而DNA甲基转移酶1（Dnmt1）和CPG甲基化结合蛋白（MeCP2）表达量则显著降低。免疫荧光标记结果表明，GnRH和KiSS-1蛋白不仅在下丘脑的视前区和弓状核与Dnmt1、MeCP2分别呈共表达模式，而且初情期时共表达均在弓状核最高。BSP结果表明，随初情期到来，母羊下丘脑GnRH、KiSS-1和NKB的甲基化水平呈降低趋势，与其基因表达量变化呈明显负相关。本项目研究结果表明，DNA甲基化参与母羊初情期启动的调控，其作用可能通过对初情期启动关键基因（GnRH、KiSS-1和NKB）甲基化水平及Dnmt1、MeCP2表达变化的调控来实现。本项目研究结果不仅为全面理解哺乳动物初情期启动机制提供参考，而且也为提高母羊繁殖力奠定基础。