MMP2基因启动子低甲基化延缓母猪初情启动的机制研究

In gilts, the initiation of puberty indicates the sexual maturation and reproductive competence. However, previous studies suggest that about 14% of gilts are culled before being served due to the delayed puberty, which is consequent in a heavily economic burden on the modern commercial farms. Our previous results revealed that the DNA methylation of Matrix metalloproteinases 2 (MMP2) promoter could block the transcriptional regulation of E2/ERα on MMP2, induce the excessive apoptosis of granulosa cells and follicular atresia, then delay the onset of puberty in gilts. Therefore, in this project, we first try to identify the relationship among MMP2, granulosa cell apoptosis, follicular atresia, and delayed puberty by using the ovaries from the normal and delayed gilts. Then we explore and determine the mechanism that how the DNA methylation of MMP2 promoter affect the transcriptional regulation of E2/ERα on MMP2, using the porcine ovarian granulosa cells and cultured follicles. We further investigate the influences of this mechanism on the proliferation and apoptosis of granulosa cells and follicular growth. These works could not only improve the theoretical resource available for better understanding the mechanism for the emergence of DP, but also provide valuable insights for the selection of early sexual maturation in gilts.

初情启动是后备母猪性成熟和获得繁殖能力的标志，但研究表明，约14%的后备母猪因初情启动延缓而被淘汰，影响猪场的经济效益。项目申请人前期发现，MMP2启动子区低DNA甲基化状态可能影响E2/ERα对MMP2基因的转录调控，进而诱导颗粒细胞过度凋亡和有腔卵泡过量闭锁，最终导致母猪初情启动延缓的发生。本项目拟在前期基础上，以初情启动正常和延缓母猪的卵巢为实验材料，利用组织切片和bisulfite sequencing PCR等方法，明确MMP2基因与颗粒细胞凋亡、卵泡闭锁及初情启动延缓之间的关系；然后，在细胞和组织水平，采用过表达、siRNA、ChIP和启动子定点突变等方法，研究MMP2启动子区低DNA甲基化影响E2/ERα调控MMP2的转录表达，及其影响颗粒细胞功能和卵泡生长的作用机制。研究结果将从理论上揭示MMP2基因延缓母猪初情启动的作用机制，并为选育性成熟早的母猪提供理论依据。

因卵巢颗粒细胞（Granulosa cells，GCs）过度凋亡和卵泡过量闭锁引起的初情延缓，导致约14%的后备母猪未经生产便被淘汰，降低后备母猪的利用效率，但它发生的详细机制尚不清楚。前期，申请人发现DNA甲基化可能调控基质金属蛋白酶MMP2的转录，促进GCs凋亡和卵泡闭锁，延缓初情启动。利用BSP、qPCR、siRNA、Western Blot等手段，本研究发现：（1）敲低DNMT3A 或DNMT3B降低MMP2启动子区域的甲基化水平，促进MMP2转录和翻译;（2）低DNA甲基化抑制转录因子ESR1与MMP2启动子区的结合，ESR1抑制MMP2基因的转录和蛋白的合成；（3）MMP2抑制GCs的增殖、ECM合成和E2分泌，促进GCs的凋亡；（4）转录因子ESR1促进GCs的增殖、周期进程、ECM合成和E2分泌，抑制GCs的凋亡；（5）MMP2基因促进卵泡内GCs的凋亡和卵泡血管的消失，诱导卵泡闭锁；MMP2促进小鼠卵巢GCs的凋亡，抑制E2分泌以及卵泡发育，进而延缓小鼠的初情启动和降低小鼠的窝仔数和窝重。综上，敲低DNMT3A或DNMT3B的表达降低MMP2启动子甲基化水平，抑制ESR1在MMP2启动子区的结合，促进MMP2的转录，诱导GCs凋亡、卵泡重塑和卵泡闭锁，延缓初情。这些结果不仅为解析母猪初情启动机制提供新的科学依据，还为母猪初情期的遗传改良提供新的理论基础，对培育母猪性早熟的品系具有重要意义，在现代化的养猪生产中有极强的应用前景。