Abstract：Nutrition and energy metabolism affect the reproductive ability of male animals, and AMP-activated protein kinase (AMPK) plays an important role in its reproductive function as an energy receptor molecule. This study aims to study the effect of AMPK on autophagy and apoptosis of sheep (Ovis aries) leydig cells (LCs) induced by starvation. Sheep LCs were used as experimental materials and cultivated to the fourth generation. After 0, 3, 6, 12, and 24 h in serum-free medium, used cell counting kit-8 to detect cell viability and selected appropriate treatment time. After starving for 12 h, the autophagy and apoptosis of LC were observed by transmission electron microscopy and flow cytometry, qRT-PCR and Western blot were used to detect the changes in gene and protein expression related to autophagy and apoptosis. The results showed that compared with the control group, there were more autophagosomes in starvation-induced LCs, and the apoptosis rate was significantly higher (P<0.05), programmed cell death 1 (Beclin1) and microtubule-associated protein 1 light chain 3 (LC3) and other genes expression level were significantly increased (P<0.05), Becline1 protein expression and LC3Ⅱ/LC3Ⅰ ratio were significantly increased (P<0.05), pro-apoptosis related genes Bcl-2 related X protein (BAX) and other genes expression level were significantly increased (P<0.05), anti-apoptosis related genes B-cell lymphoma 2 (Bcl-2) gene expression level was significantly reduced (P<0.01), and the ratio of BAX/Bcl-2 protein was increased; AMPK/mammalian target of rapamycin (mTOR)/UNC-51-like kinase 1 (ULK1) pathway key genes expression level were increased extremely significantly (P<0.01). In summary, starvation-induced sheep LCs could activate the AMPK/mTOR/ULK1 pathway and promote the occurrence of autophagy and apoptosis. This research provides a theoretical reference for further exploring the impact of AMPK on testicular development.
汪棋, 庞静, 蔡玉, 王锋, 张艳丽. AMPK对饥饿诱导的绵羊睾丸间质细胞自噬和凋亡的作用研究[J]. 农业生物技术学报, 2021, 29(4): 631-640.
WANG Qi, PANG Jing, CAI Yu, WANG Feng, ZHANG Yan-Li. Effect of AMPK on Starvation-induced Autophagy and Apoptosis of Sheep (Ovis aries) Leydig Cells. 农业生物技术学报, 2021, 29(4): 631-640.
[1] 陈亮, 贾金铭, 钟伟, 等. 2011. 自噬信号因子P70S6K参与Cox7a2调控睾酮生成机制研究[J]. 中华男科学杂志, 17(04): 291-295. (Chen L, Jia J M, Zhong W, et al.2011. P70S6K is involved in the inhibition of testosterone production in TM3 mouse leydig cells overexpressing Cox7a2[J]. National Journal of Andrology, 17(04): 291-295.) [2] 刘芮吟, 吴梦鸽, 曾汶, 等. 2019. EBSS饥饿人鼻咽癌CNE-2细胞株诱导自噬的机制研究[J]. 现代肿瘤医学, 27(01): 30-35. (Liu R Y, Wu M G, Zeng w, et al.2019. Research of EBSS starvation induced autophagy and its mechanism in CNE-2 cells[J]. Journal of Modern Oncology, 27(01): 30-35.) [3] 刘通, 于佳妮, 刘悦, 等. 2019. 去血清饥饿条件下肌卫星细胞增殖及自噬蛋白LC3、Beclin1的表达[J]. 中国组织工程研究, 23(11): 1657-1661. (Liu T, Yu J N, Liu Y, et al.2019. Effects of serum-free starvation on proliferative capacity of muscle satellite cells and expression of autophagy-related proteins LC3 and Beclin1[J]. Chinese Journal of Tissue Engineering Research, 23(11): 1657-1661.) [4] 马于林, 李亚, 彭媛媛, 等. 2020. 虾青素对氧化应激下平滑肌细胞自噬与凋亡的影响[J]. 武汉大学学报(医学版).41(2): 193-198. (Ma Y L, Li Y, Peng Y Y, et al.2020. Effets of astaxanthin on autophagy and apoptosis of smooth muscle cells under oxidative stress[J]. Medical Journal of Wuhan University. 41(2): 193-198.) [5] 单培仁, 余灵芳, 周昌钻, 等. 2018. 激动核受体RXR对大鼠心肌细胞饥饿损伤中自噬和凋亡的双重调控作用[J]. 浙江医学, 40(22): 2445-2449. (Shan P R, Yu L F, Zhou C Z, et al.2018. Activating RXR regulates serum and glucose deprivation induced autophagy and apoptosis in rat cardiomyocytes[J]. Zhejiang Medical Journal , 40(22): 2445-2449.) [6] 上官文姬, 李展春, 程光齐, 等. 2016. 骨碎补总黄酮对饥饿诱导血管内皮细胞凋亡的影响及机制研究[J]. 中药新药与临床药理, 27(6): 756-762. (Shangguan W J, Li Z C, Cheng G Q, et al.2016. Effect and mechanism of drynaria total flavonoids on apoptosis of vascular endothelial cells induced by serum starvation[J]. Traditional Chinese Drug Research and Clinical Pharmacology, 27(6): 756-762.) [7] 徐高青, 李志强, 王友元, 等. 2019. 褪黑素对小鼠睾丸间质细胞凋亡的影响[J]. 中国畜牧杂志, 55: 10-13. (Xu G Q, Li Z Q, Wang Y Y, et al.2019. Effect of melatonin on apoptosis of TM3[J]. Chinese Journal of Animal Science, 55: 10-13.) [8] 杨方晓, 李莲, 赵若含, 等. 2020. 褪黑素对HT-2毒素诱导的牛卵巢颗粒细胞内质网应激与自噬的影响[J]. 南京农业大学学报, 43(1): 143-150. (Yang F X, Li L, Zhao R H, et al.2020. Effects of melatonin on endoplasmic reticulum stress and autophagy in bovine ovarian granulosa cells induced by HT-2 toxin[J]. Journal of Nanjing Agricultural University , 43(1): 143-150.) [9] 张康. 2019. 饥饿对小鼠子宫颈上皮细胞中脂滴脂解和噬脂的影响[D]. 硕士学位论文, 山东农业大学, 导师: 葛立江, pp. 21-27. (Zhang K.2019. The effect of starvation on lipolysis and lipophagy of lipid droplets in mice cervical epithelial cells[D]. Thesis for M. S., Shandong Agricultural University, Supervisor: Ge L J, pp. 21-27.) [10] 朱文胤, 陈倩倩, 刘祁, 等. 2015. 饥饿诱导下小鼠成骨细胞自噬与凋亡相互作用研究[J]. 川北医学院学报, 23(11): 206-211. (Zhu W Y, Chen Q Q, Deng Q, et al.2015. Study on the interaction between autophagy and apoptosis of MC3T3-E1 at starvation state[J]. Journal of North Sichuan Medical College, 23(11): 206-211.) [11] Chen X W, Dong Y Y, Tian E, et al.2018. 4-Bromodiphenyl ether delays pubertal leydig cell development in rats[J]. Chemosphere[J], 211: 986-997. [12] Chen Y, He J, Tian M, et al.2014. UNC51-like kinase 1, autophagic regulator and cancer therapeutic target[J]. Cell Proliferation 47(6): 494-505. [13] Cheng Y, Chen G J, Wang L, et al.2018. Triptolide-induced mitochondrial damage dysregulates fatty acid metabolism in mouse sertoli cells[J]. Toxicology Letters, 292: 136-150. [14] Duan P, Hu C H, Quan C, et al.2016. 4-Nonylphenol induces apoptosis, autophagy and necrosis in sertoli cells: Involvement of ROS-mediated AMPK/AKT-mTOR and JNK pathways[J]. Toxicology, 341: 28-40. [15] Egan D F, Shackelford D B, Mihaylova M M, et al.2011. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy[J]. Science, 331(6016): 456-461. [16] Furuya D, Tsuji N, Yagihashi A, et al.2005. Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity[J]. Experimental Cell Research, 307(1): 26-40. [17] Gao X X, Yao X L, Yang H, et al.2018. Role of FGF9 in sheep testis steroidogenesis during sexual maturation[J]. Animal Reproduction Science, 197: 177-184. [18] Hallows K R, Alzamora R, Li H, et al.2009. AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells[J]. American Journal of Physiology. Cell Physiology, 296(4): C672-C681. [19] Ham S, Brown K A, Simpson E R, et al.2016. Immunolocalisation of aromatase regulators liver kinase B1, phosphorylated AMP-activated protein kinase and cAMP response element-binding protein-regulated transcription co-activators in the human testis[J]. Reproduction Fertility and Development, 229(1): 68-72. [20] Jung C H, Jun C B, Ro S H, et al.2009. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery[J]. Cellular and Molecular Biology, 20(7): 1992-2003. [21] Kim S Y, Song X X, Zhang L, et al.2014. Role of Bcl-xL/Beclin-1 in interplay between apoptosis and autophagy in oxaliplatin and bortezomib-induced cell death[J]. Biochemical Pharmacology, 88(2): 178-188. [22] Komatsu M, Kageyama S, Ichimura Y.2012. p62/SQSTM1/A170: Physiology and pathology[J]. Pharmacologica Research, 66(6): 457-462. [23] Nakashima A, Aoki A, Kusabiraki T, et al.2017. Role of autophagy in oocytogenesis, embryogenesis, implantation, and pathophysiology of pre-eclampsia[J]. The Journal of Obstetrics and Gynaecology Research, 43(4): 633-643. [24] Nakatogawa H, Suzuki K, Kamada Y, et al.2009. Dynamics and diversity in autophagy mechanisms: Lessons from yeast[J]. Nature Reviews. Molecular Cell Biology, 10(1): 458-467. [25] Pattingre S, Tassa A, Qu X, et al.2005. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy[J]. Cell, 122(6): 927-939. [26] Shang Y, Wang H, Jia P, et al.2016. Autophagy regulates spermatid differentiation via degradation of PDLIM1[J]. Autophagy, 12: 1575-1592. [27] Shi L, Song R, Yao X, et al.2017. Effects of selenium on the proliferation, apoptosis and testosterone production of sheep leydig cells in vitro[J]. Theriogenology, 93: 24-32. [28] Su H, Wang X.2011. p62 stages an interplay between the ubiquitin-proteasome system and autophagy in the heart of defense against proteotoxic stress[J]. Trends in Cardiovascular Medicine, 21(8): 224-228. [29] Xia H G, Zhang L, Chen G, et al.2010. Control of basal autophagy by calpain1 mediated cleavage of ATG5[J]. Autophagy, 6: 61-66. [30] Xie Z, Nair U, Klionsky D J.2008. Atg8 controls phagophore expansion during autophagosome formation[J]. Cellular and Molecular Biology, 19(8): 3290-3298. [31] Zachari M, Ganley I G.2017. The mammalian ULK1 complex and autophagy initiation[J]. Essays in Biochemistry, 61(6): 585-596.
