Autophagy is a conserved intracellular degradation process that has been shown to have important biological implications in a variety of organisms. However, the study of autophagy in the plateau characteristic species of Bosgrunniens has hasn't been reported. Therefore, in order to explore the expression of autophagy-related genes in the reproductive process of yak under normal physiological conditions, the main reproductive organs (fallopian tubes, ovaries and uterus) of different breeding cycles (follicular phase, luteal phase and pregnancy) of yak were collected. Autophagy related 5 (Atg5) gene was cloned ，and analyzed by bioinformatics; qRT- PCR was used to detect the relative expression of Atg5 gene in tissues; Western blotting (West-blot, WB) was used to detect the expression of Atg5 and Atg5-Atg12 complex in different tissues; immunohistochemical method was used to analyze the distribution characteristics of Atg5 in various reproductive organs. We successfully cloned the yak Atg5 gene (GenBank: MK531791), which was highly conserved during the evolution process, and the encoded protein was a soluble non-transmembrane protein. The results of qRT-PCR and WB showed Atg5 and Atg5-Atg12 complex is expressed in the main reproductive organs of calves. The expression of Atg5-Atg12 in the ovarian follicles during follicular phase was significantly higher than that in the luteal phase and gestational phase, while the expression of Atg5 in the follicular phase was significantly lower than that in the luteal phase and gestational phase. The

expression of Atg5-Atg12 in the oviduct during luteal phase was significantly higher than that in the follicular phase and pregnancy. The expression of Atg5 in the fallopian tube during gestational phase was significantly higher than that in the follicular phase and the luteal phase. The expression of Atg5-Atg12 in the uterus during follicular phase and pregnancy was significantly higher than that in the luteal phase, while the expression of Atg5 in the uterus of the luteal phase is significantly higher than that during pregnancy and follicular phase. There was a negative correlation between the expression of Atg5 and Atg5-Atg12 at the protein level. Immunohistochemistry results showed that Atg5 was expressed in the oviductal mucosa epithelium, ovarian follicle membrane, granular layer, reproductive epithelium, luteal cells, endometrium and uterine gland. The results showed that autophagy was similar to other species in yak by detecting the expression of Atg 5 and Atg 12 in yak reproductive organs, and it was speculated that autophagy was involved in the regulation of yak reproductive physiology. The results of this study have implications for the study of autophagy in other large animals, specifically mammals in the alpine and hypoxic environment, and contribute to the study of autophagy involved in the reproductive physiology of other animals.

自噬是一种保守的细胞内降解过程，在多种生物体内证实自噬有重要的生物学意义。但是自噬在牦牛这一高原特色物种体内的研究未见报道。因此为探索正常生理条件下自噬相关基因在牦牛生殖过程中的表达，本研究分别采集牦牛不同繁殖周期（卵泡期、黄体期及妊娠期）的主要生殖器官（输卵管、卵巢、子宫），克隆牦牛自噬相关基因5（Autophagy related 5，Atg5）基因并进行生物信息学分析；利用qRT-PCR检测Atg5 基因在组织中的相对表达量；并采用蛋白质免疫印迹（Western-blot, WB）检测Atg5和Atg5-Atg12（Autophagy related 12，自噬相关基因12）复合体在不同组织中的表达水平；免疫组织化学方法分析Atg5在各生殖器官中的分布特征。结果显示，成功克隆牦牛Atg5基因在进化过程中高度保守，编码的蛋白质为可溶性的非跨膜蛋白；qRT-PCR和WB检测结果显示Atg5和Atg5-Atg12复合体在牦牛输卵管、卵巢和子宫中均有表达。其中卵泡期卵巢Atg5-Atg12表达显著高于黄体期和妊娠期，卵泡期Atg5的表达却显著低于黄体期和妊娠期；黄体期输卵管Atg5-Atg12表达量显著高于卵泡期和妊娠期，妊娠期输卵管中Atg5的表达量显著高于卵泡期和黄体期；卵泡期和妊娠期子宫中Atg5-Atg12的表达量显著高于黄体期，而黄体期子宫中的Atg5的表达量又显著高于妊娠期和卵泡期，在蛋白水平上Atg5和Atg5-Atg12的表达呈负相关。免疫组织化学结果显示Atg5在输卵管黏膜上皮，卵巢卵泡膜、颗粒层、生殖上皮、黄体细胞，子宫内膜和子宫腺体均有表达。研究结果表明自噬在牦牛体内与其他物种相似具有保守性，通过检测Atg5-Atg12复合体在牦牛生殖器官中的表达，推测自噬可能参与牦牛生殖生理过程的调控。该研究结果对自噬在其他大型哺乳动物以及高寒低氧环境中动物的研究具有借鉴意义，有助于自噬参与其他动物生殖生理作用机制的研究。