繁殖力差异绵羊卵巢微血管构筑特征与血管发生有关因子表达的研究

Differences of reproductivity in sheep are attributed to the different hereditary characters as well as ecological environments, so physiological features in sheep will be able to be changed to some extent due to environmental impacts and it will affect the genetic potential of reproductivity. Sheep breeds with different reproductivity show different ovulation rates. Developmental performances and nutrition supply ability of ovarian vascular system are associated with the amount of matured follicles in ovaries, and that has been regulated by some angiogenesis genes. So the difference of reproductivity in sheep is caused by the interfacing impacts from individual hereditary characters, ovarian vascular physiology and environmental influences. Four sheep breeds including Tibetan sheep, Aohan fine-wool sheep, Hu sheep and Small tailed han sheep who come from different ecological conditions will be selected as experimental animals. Their ovarian samples will be collected from two populations including three-month-old lambs and adult sheep each breed. Ovarian vascular corrosion casts and capillary perfusion models will be made respectively. The gross anatomic features of ovarian vascular system will be described, and ovarian capillary distribution pattern and microvascular ultrastructure will be scanned and observed by SEM. Additionally, the expression levels of some genes that influence the development of ovarian vascular system including VEGF, CTGF and Ang will be detected quantitatively and compared by RealTime-PCR and FISH & IHC methods among different sheep populations. According to the expectant results, we will analyze the correlation of the microvascular anatomical structures, angiogenesis genes’ expression levels respectively with the ovine ovulation rates and follicles’ development, and explore the impacts of different living environments on ovarian microvascular development and follicles’ developmental characteristics.

繁殖力有差异的绵羊品种除了遗传特性的差别，所处生态环境也各不相同，环境因素会引起生理特性改变并影响遗传效应。不同繁殖力绵羊有不同的排卵数，成熟卵泡数也与卵巢微血管的发育及营养供给有关，并受血管发生有关因子的调节，故绵羊排卵数受品种遗传特性、卵巢血管生理和生态环境因素的交互影响。根据繁殖力的差异，选择不同产地的4个绵羊品种西藏羊、敖汉细毛羊、湖羊和小尾寒羊，采集3月龄羔羊和成年羊卵巢；制作卵巢血管立体构筑和微血管灌注标本，观察其动脉解剖形态特征，比较大卵泡微血管密度及分布规律，研究微血管超微结构特征；采用RealTime-PCR、原位杂交和免疫组化双标记方法，定量检测血管内皮生长因子（VEGF）、结缔组织生长因子（CTGF）和血管生成素（Ang）在卵巢中的表达差异；研究卵巢微血管解剖生理、血管发生因子表达与绵羊卵泡发育及排卵数的相关性，揭示生态环境对卵巢微血管发育及卵泡发育特性的影响。

环境因素会引起哺乳动物生理特性改变并影响遗传效应，特别是高海拔、低氧等不利因子能够诱导血管发生相关因子的表达差异，可能会造成绵羊卵巢微血管发育程度的不同及养分输送效率的改变，最终导致成熟卵泡数等繁殖性状的改变，故母羊的繁殖力受遗传特性、卵巢血管生理和环境因素的交互影响。. 本研究采集了3月龄和成年藏羊的卵巢样本，采用血管灌注方法，获得卵巢动脉构筑标本，研究了羔羊及成年羊卵巢动脉的发育特征；采集成年的小尾寒羊、湖羊、敖汉细毛羊及藏羊卵巢样本，获得卵巢动脉血管构筑标本，比较了构筑特征的差异，对卵巢螺旋微动脉上皮的超微特征通过SEM扫描观察；利用实时荧光定量PCR技术，对4个绵羊品种与卵巢血管发生有关的基因及其通路包括VEGF、Ang-1、Ang-2及Tie-2等进行表达差异检测，同时检测原产地及引入地小尾寒羊卵巢中VEGF, CTGF, Ang/Tie-2, Ang-1, Ang-2, Tie-2 基因的相对表达量；利用免疫组化和Western blot技术，分析了VEGF蛋白在小尾寒羊、湖羊及藏羊卵巢组织中的表达与分布特征。. 研究发现，小尾寒羊、湖羊繁殖力高的原因部分是由于发达的卵巢微血管有利于血氧运输使其成熟卵泡增加的结果；生活在高海拔环境的藏羊，其卵巢动脉及其分支不够发达，会引起卵巢及其外周供血减少，导致卵泡的发育及优势化能力降低；但藏羊卵巢微动脉内皮的解剖学特征，则有利于增加卵巢内部的氧供，通过调节血供而适应高原环境。. 研究认为，促进血管发生的Ang/Tie-2通路对绵羊排卵率有影响，同时受其他血管生成因子特别是VEGF的调节；绵羊的繁殖力特别是排卵数差异与卵巢动脉血管发育状况有关，而卵巢动脉构筑特点又与环境因子如高海拔、低氧有关；藏羊繁殖力低不仅是遗传因素，也是高海拔低氧引起卵巢动脉不发达、养分供应受限的结果；饲养环境不同会引起小尾寒羊卵巢血管发生因子的表达差异，在一定程度上影响繁殖潜能的发挥。. 该研究结果为深入了解绵羊卵巢动脉的血供特点及功能研究提供解剖学基础，同时为进一步研究与绵羊繁殖力有关的遗传、生理及环境交互作用提供理论参考。