GnRH-1通过 MMP13调控鸡等级卵泡血管发育的作用机制

The egg production performance of domestic chicken (Gallus domesticus) depends on the developmental of large preovulatory hierarchy follicles in the functionally mature ovary. The follicular development includes follicle development, selection of dominant follicle into the ovulation hierarchy, preovulatory maturation, ovulation and postovulatory regression, all of these require the extracellular matrix (ECM) remodeling and angiogenesis and microvascular permeability, and the matrix metalloproteinases (MMPs) play an important role in these processes. Our previous study showed that MMP13 was distributed around the vascular in theca cells, and the expression of MMP13 was similar to that of vascular endothelial growth factor (VEGFA), GnRH-1 can down down-regulated MMP13 and KDR. Based on previous studies on Gonadotropin-releasing hormone (GnRH-1) and MMPs in chicken ovary, we speculated that GnRH-1 may be involved the development of follicular angiogenesis of chicken by regulating the expression of MMP13, and play an important role in the development of the preovulate follicles. In this study, we will analyze the molecular mechanism of GnRH-1 regulating MMP13 expression in follicular theca cells of chicken at cell and living levels using immunohistochemistry, overexpression and RNAi, qRT-PCR and Western blot, etc, as well as their effect on the development of follicular angiogenesis and preovulated hierarchical follicles, so as to clarify the role of GnRH-1 in the development of follicular angiogenesis and the relationship with the formation of the dominant follicle by regulating MMP13 expression in chicken ovary.

禽类卵巢中优势等级卵泡发育的数目决定了其产蛋性能的高低。卵泡血管的生成和通透性对卵泡生长和等级卵泡选择具有重要影响，而细胞外基质重塑在血管新生调节中发挥重要作用，但确切调控机制都还不清楚。我们前期研究发现，在鸡等级卵泡的膜细胞中MMP13的表达定位在血管周围且与血管内皮生长因子A（VEGFA）有相似的表达趋势，过表达GnRH-1能下调MMP13和VEGFA受体KDR的表达。我们推测：GnRH-1可能通过调控MMP13参与鸡卵泡血管发育，从而在等级卵泡的生长中发挥重要作用。本研究拟采用免疫组化、过表达及RNAi、qRT-PCR等技术，从细胞及活体水平解析鸡卵泡膜细胞中GnRH-1下调MMP13表达的分子机制，以及此调控影响鸡等级卵泡血管发育和卵泡等级化生长的作用，以期阐明GnRH-1通过调控MMP13在鸡等级卵泡血管发育中的作用机制及其与优势等级卵泡生长之间的联系。

家禽卵泡血管系统的形成与发育状态对于卵泡的生长和排卵具有重要影响。本项目组前期研究发现GnRH-1可能通过MMP13参与了鸡卵泡血管系统的调控，但具体作用和分子机制尚不清楚。本研究采用免疫组化、qRT-PCR以及Western Blot分析了GnRH-1、MMPs和VEGFs在不同发育时期鸡卵巢卵泡中的表达特征并对这些基因在高、低产蛋性能的鸡品种卵巢卵泡中的表达进行了比较；采用过表达及GnRH激动剂处理卵泡细胞及鸡胚分析了GnRH-1对MMPs和血管发育相关基因的表达调控以及对血管发育的影响；采用过表达、双荧光素酶报告基因方法研究了GnRH-1与MMP11/13之间的调控关系及相关基因启动子区多态与鸡产蛋性状的关联。结果表明GnRH-1、MMP11/13和VEGFA等血管发育相关基因在不同发育阶段卵泡的膜细胞和颗粒细胞中均有表达，但家族成员的表达模式随卵泡发育阶段而不同；在鸡胚中GnRH激动剂对血管发育存在负调控作用，在等级卵泡颗粒细胞及膜细胞中GnRH-1分别调控不同的VEGFs及其受体基因；GnRH-1调控转录因子FOXP1进而下调MMP11/13的表达。本项目还分析了miRNA对VEGFA和FOXP1的调控，筛选到3个与产蛋性状显著关联的SNP标记。综上，本项目研究结果明确了鸡卵泡中GnRH-1与MMP11/13之间的调控关系以及影响血管发育相关基因表达的潜在作用机制，为进一步阐明鸡卵泡血管发育及决定产蛋性能的遗传机理奠定分子基础。