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秦德哲, 蔡卉, 何晨, 杨栋慧, 孙静, 何文来, 李巴仑, 华进联, 彭莎. 2021: 褪黑素通过抑制ATF6和PERK信号通路缓解热应激诱导的小鼠睾丸精母细胞凋亡. 动物学研究, 42(4): 514-524. DOI: 10.24272/j.issn.2095-8137.2021.041
引用本文: 秦德哲, 蔡卉, 何晨, 杨栋慧, 孙静, 何文来, 李巴仑, 华进联, 彭莎. 2021: 褪黑素通过抑制ATF6和PERK信号通路缓解热应激诱导的小鼠睾丸精母细胞凋亡. 动物学研究, 42(4): 514-524. DOI: 10.24272/j.issn.2095-8137.2021.041
De-Zhe Qin, Hui Cai, Chen He, Dong-Hui Yang, Jing Sun, Wen-Lai He, Ba-Lun Li, Jin-Lian Hua, Sha Peng. 2021: Melatonin relieves heat-induced spermatocyte apoptosis in mouse testes by inhibition of ATF6 and PERK signaling pathways. Zoological Research, 42(4): 514-524. DOI: 10.24272/j.issn.2095-8137.2021.041
Citation: De-Zhe Qin, Hui Cai, Chen He, Dong-Hui Yang, Jing Sun, Wen-Lai He, Ba-Lun Li, Jin-Lian Hua, Sha Peng. 2021: Melatonin relieves heat-induced spermatocyte apoptosis in mouse testes by inhibition of ATF6 and PERK signaling pathways. Zoological Research, 42(4): 514-524. DOI: 10.24272/j.issn.2095-8137.2021.041

褪黑素通过抑制ATF6和PERK信号通路缓解热应激诱导的小鼠睾丸精母细胞凋亡

Melatonin relieves heat-induced spermatocyte apoptosis in mouse testes by inhibition of ATF6 and PERK signaling pathways

  • 摘要: 睾丸是精子发生的场所,哺乳动物的睾丸位于体腔外的阴囊中,成年哺乳动物包括人类,睾丸的正常温度均比身体核心温度要低。如果外界环境温度过高会影响睾丸中的精子生成,降低精子的质量和数量,甚至导致不育。当机体受到外界刺激时,内质网内会产生大量错误折叠或未折叠蛋白,使得内质网对蛋白的加工能力与载量失衡,从而导致内质网功能紊乱,即发生内质网应激(endoplasmic reticulum stress,ERS),ERS被认为是导致大量病理的关键因素。目前已有研究报道称热应激会引发ERS,但哪条ERS信号通路在热应激诱导的睾丸损伤中发挥主要作用尚不明确。褪黑激素(褪黑素)是一种具有抗氧化、抗凋亡能力的神经内分泌激素,它的抗氧化活性有助于防止应激、炎症引起的睾丸损伤,然而,褪黑素能否能缓解热应激所致的睾丸损伤尚未见报道。该研究通过建立小鼠睾丸热应激模型,并利用组织学、分子生物学检测发现,热应激会导致睾丸曲细精管内的生殖细胞数量减少、生精上皮紊乱及凋亡的产生,TUNEL染色结果显示凋亡主要发生在睾丸精母细胞中,通过实时荧光定量PCR(qRT-PCR)和免疫荧光技术发现热应激也会刺激小鼠睾丸发生ERS。在随后体外(GC2细胞系)试验中,我们通过qRT-PCR和Western blotting从RNA与蛋白水平进一步证实了上述结果,并发现ATF6和PERK信号通路在热诱导后的细胞中均被激活,而IRE1信号通路在该过程变化不明显。通过si-RNA干扰上述基因均可有效减少由热应激引起的精母细胞损伤。在此基础上继续探索褪黑素对热应激引起的精母细胞凋亡和ERS的影响,通过免疫荧光、流式细胞术、Western blotting等实验,发现褪黑素能通过褪黑素受体(MT1/MT2)抑制Atf6、Perk和凋亡相关基因Caspase12、Caspase3及Chop的表达。最后通过小鼠体内试验证实褪黑素可以缓解热应激引起的曲细精管生殖细胞数量减少和精母细胞凋亡。综上,该研究表明热应激能引起睾丸损伤并诱导精母细胞的ERS与凋亡的发生,褪黑素可以通过ATF6和PERK信号通路缓解热应激引起的精母细胞的凋亡,并对损伤的睾丸起到修复作用。该研究证明褪黑素可在一定程度上预防高温引起的精母细胞凋亡,并保护小鼠睾丸免受热应激的损害。

     

    Abstract: Normal spermatogenic processes require the scrotal temperature to be lower than that of the body as excessive heat affects spermatogenesis in the testes, reduces sperm quality and quantity, and even causes infertility. Endoplasmic reticulum stress (ERS) is a crucial factor in many pathologies. Although several studies have linked ERS to heat stress, researchers have not yet determined which ERS signaling pathways contribute to heat-induced testicular damage. Melatonin activates antioxidant enzymes, scavenges free radicals, and protects the testes from inflammation; however, few studies have reported on the influence of melatonin on heat-induced testicular damage. Using a murine model of testicular hyperthermia, we observed that heat stress causes both ERS and apoptosis in the testes, especially in the spermatocytes. These observations were confirmed using the mouse spermatocyte cell line GC2, where the Atf6 and Perk signaling pathways were activated during heat stress. Knockout of the above genes effectively reduced spermatocyte damage caused by heat stress. Pretreatment with melatonin alleviated heat-induced apoptosis by inhibiting the Atf6 and Perk signaling pathways. This mitigation was dependent on the melatonin receptors. In vivo experiments verified that melatonin treatment relieved heat-induced testicular damage. In conclusion, our results demonstrated that ATF6 and PERK are important mediators for heat-induced apoptosis, which can be prevented by melatonin treatment. Thus, our study highlights melatonin as a potential therapeutic agent in mammals for subfertility/infertility induced by testicular hyperthermia.

     

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