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

De-Zhe Qin Hui Cai Chen He Dong-Hui Yang Jing Sun Wen-Lai He Ba-Lun Li Jin-Lian Hua Sha Peng

De-Zhe Qin, Hui Cai, Chen He, Dong-Hui Yang, Jing Sun, Wen-Lai He, Ba-Lun Li, Jin-Lian Hua, Sha Peng. Melatonin relieves heat-induced spermatocyte apoptosis in mouse testes by inhibition of ATF6 and PERK signaling pathways. Zoological Research, 2021, 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. Melatonin relieves heat-induced spermatocyte apoptosis in mouse testes by inhibition of ATF6 and PERK signaling pathways. Zoological Research, 2021, 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

doi: 10.24272/j.issn.2095-8137.2021.041

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

Funds: This work was supported by the National Natural Science Foundation of China (32072815), General Project of the Key R & D Plan of Shaanxi Province (2019NY-091), Program of Shaanxi Province Science and Technology Innovation Team (2019TD-036), and Fundamental Research Funds for the Central Universities (2452020157)
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  • 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。
  • Figure  1.  Heat stress causes testicular damage in male mice

    A: Hematoxylin and eosin staining showing testicular damage at 6, 12, and 24 h after heat treatment. B: Testicular weight/body weight ratios of all mouse groups presented as a bar graph at 6, 12, and 24 h after heat treatment. C: Morphology of testes after 12 h of recovery. D: TUNEL staining. Red fluorescence indicates TUNEL positive. Hoechst 33342 (blue)-stained cell nuclei. E: qRT-PCR of Hsp70, Caspase3, Caspase9, and Caspase12 expression. F: Western blotting of HSP70 and c-CASPASE3 expression at 6, 12, and 24 h after heat treatment. Con: Control. Data are mean±SD (n=3); statistical significance is expressed as: *: P<0.05; **: P<0.01.

    Figure  2.  Atf6 and Perk signaling pathways are enhanced by heat stress in testes

    A, B: mRNA expression of ERS-related genes (Grp78, Ire1, Perk and Atf6) in testes at 6, 12, and 24 h after heat treatment. C: Immunofluorescence and TUNEL double staining showing spermatocyte apoptosis after 12 h of recovery from heat stress. D, E, F: Representative immunofluorescent images of pIRE1 (D), pPERK (E), and ATF6 (F) All images were merged from pIRE1, pPERK, or ATF6 (green) and Hoechst 33342-stained nuclei (blue). Scale bar: 200 μm. Con: Control. Data are mean±SD (n=3); statistical significance is expressed as: **: P<0.01. NS: Not significant.

    Figure  3.  Heat stress induces spermatocyte apoptosis and endoplasmic reticulum stress

    A: mRNA expression of heat shock genes (Hsp27 and Hsp70) in spermatocytes (GC2 cell line) at 0, 2, 4, and 6 h after heat treatment for 2 h. B: mRNA expression of apoptosis genes (Caspase3 and Chop) and Grp78 in spermatocytes at 0, 2, 4, and 6 h after heat treatment for 2 h. C: mRNA expression of ERS-related genes (Ire1, Perk and Atf6) in spermatocytes at 0, 2, 4, and 6 h after heat treatment for 2 h. D: Western blotting of ERS-related proteins (p-IRE1, p-PERK, and ATF6), apoptosis protein (CASPASE12), and heat shock protein (HSP70) in spermatocytes at 0, 2, 4, and 6 h after heat treatment for 2 h. E: Image J analysis of (D). F: TUNEL staining for apoptosis at 4 h after heat treatment for 2 h. Cells showing red fluorescence are positive. Hoechst 33342 (blue)-stained cell nuclei. G: Immunofluorescent images showing GRP78 and CHOP in spermatocytes at 4 h after heat treatment for 2 h. H: Immunofluorescent images showing heat shock proteins (HSP27 and HSP70) in spermatocytes at 4 h after heat treatment for 2 h. I: Immunofluorescent images showing ERS-related proteins (p-IRE1, p-PERK, and ATF6) in spermatocytes at 4 h after heat treatment for 2 h. All images were merged from pIRE1, pPERK, or ATF6 (green) and Hoechst 33342-stained nuclei (blue). Scale bar: 200 μm. Con: Control, hs: Heat stress. Data are mean±SD (n=3); statistical significance is expressed as: **: P<0.01, NS: Not significant.

    Figure  4.  Knockdown of ATF6 and PERK relieves heat-induced damage

    A: qRT-PCR of Perk and Atf6 mRNA expression in si-Perk and si-Ptf6 cell lines. B: Western blotting of PERK and ATF6 expression in si-Perk and si-Atf6 cell lines. C: Image J analysis of (B). D: qRT-PCR of apoptosis-related genes (Caspase12, Caspase3, and Chop) in si-Perk and si-ATF6 cell lines at 4 h after heat treatment for 2 h. E: Western blotting of apoptosis-related protein (CASPASE12, CASPASE3, CHOP) expression in si-Perk and si-ATF6 cell lines at 4 h after heat treatment for 2 h. F: Image J analysis of (E). Con: Control, hs: Heat stress. Data are mean±SD (n=3); statistical significance is expressed as: **: P<0.01.

    Figure  5.  Melatonin relieves spermatocyte damage caused by heat stress through ATF6 and PERK

    A: Flow cytometry was used to determine cell apoptosis in different melatonin treatment groups (i.e., con, 0, 10-4, 10-5, 10-6, and 10-7 mmol/L) at 4 h after heat treatment. B: PI staining was used to detect apoptosis in 10-6 mmol/L melatonin-treated spermatocytes at 4 h after heat treatment. C: Statistical analysis of (B). D: qRT-PCR of ERS-related genes (Perk and Atf6) in 10-6 mmol/L melatonin-treated spermatocytes at 4 h after heat treatment. E: qRT-PCR of apoptosis-related genes (Caspase3, Caspase12, and Chop) in different groups, i.e., con, con+mel (10-6 mmol/L melatonin-treated spermatocytes), hs (4 h after heat treatment for 2 h), and hs+mel (10-6 mmol/L melatonin-treated spermatocytes at 4 h after heat treatment). F: Western blotting of apoptosis-related proteins (CASPASE12 and c-CASPASE3) and ERS-related proteins (p-PERK and ATF6) in con, con+mel 10-6 μmol/L melatonin-treated spermatocytes), hs (4 h after heat treatment for 2 h), and hs+mel (10-6 mmol/L melatonin-treated spermatocytes at 4 h after heat treatment) groups. G, H: Image J analysis of (F). Con: Control, hs: Heat stress, mel: melatonin. Data are mean±SD (n=3); statistical significance is expressed as: *: P<0.05; **: P<0.01.

    Figure  6.  MT1/MT2 receptors are essential for melatonin to relieve heat stress in spermatocytes

    A: Western blotting of melatonin receptor protein expression (MT1 and MT2) in con, hs (4 h after heat treatment for 2 h), con+mel (10-6 mmol/L melatonin-treated spermatocytes), and hs+mel (10-6 mmol/L melatonin-treated spermatocytes at 4 h after heat treatment) groups. B: Image J analysis of (A). C: Immunofluorescent images showing melatonin receptor protein (MT1 and MT2) in con, hs, con+mel, and hs+mel groups. D: Western blotting of apoptosis-related proteins (CASPASE12, c-CASPASE3, and CHOP) in hs (4 h after heat treatment for 2 h), hs+i (luzindole), hs+mel (10-6 mmol/L melatonin-treated spermatocytes at 4 h after heat treatment), and hs+mel+i (luzindole). E: ImageJ analysis of (D). Con: Control, hs: Heat stress, mel: melatonin. Data are mean±SD (n=3); statistical significance is expressed as: *: P<0.05; **: P<0.01.

    Figure  7.  Melatonin alleviates apoptosis of testicular spermatocytes caused by heat stress in vivo

    A: Hematoxylin and eosin staining of testicular damage in con, mel, hs, and hs+mel groups. B: TUNEL staining (red fluorescence represents TUNEL positive staining), Hoechst 33342 (blue)-stained cell nuclei. C: Western blotting of ERS-related proteins (ATF6 and p-PERK) in con+mel, con, hs, and hs+mel groups. D: Immunofluorescent images showing ATF6 in con+mel, con, hs, and hs+mel groups. Con: Control, hs: Heat stress, mel: melatonin. Data are mean±SD (n=3); statistical significance is expressed as: *: P<0.05; **: P<0.01.

    Figure  8.  Illustration of melatonin relieving heat-induced spermatocyte apoptosis in mouse testes

    In this study, we concluded that heat stress causes both ERS and apoptosis in testes, especially in spermatocytes, and p-PERK and ATF6 play dominant roles in spermatocyte damage induced by high temperature. Furthermore, pretreatment with melatonin alleviates heat-induced apoptosis by inhibiting ATF6 and PERK signaling pathways. This mitigation depends on melatonin receptors (MT1/MT2).

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  • 收稿日期:  2021-02-24
  • 录用日期:  2021-07-08
  • 网络出版日期:  2021-07-13
  • 刊出日期:  2021-07-18

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