Abstract
Testis, as a key organ for maintaining male fertility, are extremely sensitive to ionizing radiation (IR). IR-induced testicular dysfunction and infertility are common adverse effects of radiation therapy in patients with pelvic cancer. To study the phenotype and mechanism of IR-induced testicular injury, the mice were irradiated with different radiation doses (0, 2 and 5 Gy) below the semi-lethal dose for one month. Our present results showed that testicular weight and the serum testosterone levels significantly decreased with the structural injury of the testis in an IR dose-dependent manner, indicating that IR caused not only the structural damage of the testis, but also the functional damage. Further analysis by TUNEL staining and Western blotting found that IR induced the apoptosis in a dose-dependent manner as indicated by increased expressions of cleaved caspase3, p53 and Bax on Day 15 after IR treatment. Combined with significantly increased oxidative stress, these results indicated that IR-induced testicular damage may be a long-term, progressively aggravated process, accompanied by apoptosis. Given the role of autophagy in apoptosis, the present study also detected and analyzed the localization and expressions of autophagy-related proteins LC-3I/II, beclin1, p62 and Atg12 in testicular cells, and found that LC-3II, beclin1 and Atg12 expressions significantly increased in the testicular cells of mice irradiated with 2 Gy and 5 Gy, while p62 expression significantly decreased with 5 Gy, implying autophagy was involved in the apoptosis of testicular cells induced by IR. Furthermore, the expressions of HIF-1α and BNIP3 were significantly enhanced in the testis cells of mice irradiated with 2 Gy and 5 Gy, suggesting the potential role of HIF-1α/BNIP3-mediated autophagy in the apoptosis of testicular cells induced by IR. Taken together, our findings demonstrated that HIF-1α/BNIP3-mediated autophagy not only plays a protective effect on the testicular cells of mice irradiated with 2 Gy, but also induces the apoptosis of the testicular cells of mice irradiated with 5 Gy, indicating the double effects on apoptosis, which will help us further understanding the molecular mechanisms of IR-induced testicular injury, and will facilitate us further studies on testicular radioprotection.
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References
Abraham MC, Shaham S (2004) Death without caspases, caspases without death. Trends Cell Biol 14(4):184–193. https://doi.org/10.1016/j.tcb.2004.03.002
Adewoyin M, Ibrahim M, Roszaman R, Isa MLM, Alewi NAM, Rafa AAA, Anuar MNN (2017) Male infertility: the effect of natural antioxidants and phytocompounds on seminal oxidative stress. Diseases 5:1. https://doi.org/10.3390/diseases5010009
Agarwal A, Sekhon LH (2011) Oxidative stress and antioxidants for idiopathic oligoasthenoteratospermia: Is it justified? Indian J Urol 27(1):74–85. https://doi.org/10.4103/0970-1591.78437
Alizadeh E, Orlando TM, Sanche L (2015) Biomolecular damage induced by ionizing radiation: the direct and indirect effects of low-energy electrons on DNA. Annu Rev Phys Chem 66:379–398. https://doi.org/10.1146/annurev-physchem-040513-103605
Azad MB, Chen Y, Henson ES, Cizeau J, McMillan-Ward E, Israels SJ, Gibson SB (2008) Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3. Autophagy 4(2):195–204. https://doi.org/10.4161/auto.5278
Anway MD, Folmer J, Wright WW, Zirkin BR (2003) Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function. Biol Reprod 68(3):996–1002. https://doi.org/10.1095/biolreprod.102.008045
Bae MJ, Kang MK, Kye YU, Baek JH, Sim YJ, Lee HJ, Kang YR, Jo WS, Kim JS, Lee CG (2021) Differential effects of low and high radiation dose rates on mouse spermatogenesis. Int J Mol Sci 22:23. https://doi.org/10.3390/ijms222312834
Bellot G, Garcia-Medina R, Gounon P, Chiche J, Roux D, Pouyssegur J, Mazure NM (2009) Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol 29(10):2570–2581. https://doi.org/10.1128/MCB.00166-09
Bujan L, Walschaerts M, Moinard N, Hennebicq S, Saias J, Brugnon F, Auger J, Berthaut I, Szerman E, Daudin M, Rives N (2013) Impact of chemotherapy and radiotherapy for testicular germ cell tumors on spermatogenesis and sperm DNA: a multicenter prospective study from the CECOS network. Fertil Steril 100(3):673–680. https://doi.org/10.1016/j.fertnstert.2013.05.018
Carvalho HA, Villar RC (2018) Radiotherapy and immune response: the systemic effects of a local treatment. Clinics (sao Paulo) 73(suppl 1):e557s. https://doi.org/10.6061/clinics/2018/e557s
Chen C, Chen Z, Xu F, Zhu C, Fang F, Shu S, Li M, Ling C (2013) Radio-protective effect of catalpol in cultured cells and mice. J Radiat Res 54(1):76–82. https://doi.org/10.1093/jrr/rrs080
Choy H, Gerber DE, Bradley JD, Iyengar P, Monberg M, Treat J, Govindan R, Koustensis A, Barker S, Obasaju C (2015) Concurrent pemetrexed and radiation therapy in the treatment of patients with inoperable stage III non-small cell lung cancer: a systematic review of completed and ongoing studies. Lung Cancer 87(3):232–240. https://doi.org/10.1016/j.lungcan.2014.12.003
Clifton DK, Bremner WJ (1983) The effect of testicular x-irradiation on spermatogenesis in man. A comparison with the mouse. J Androl 4(6):387–392. https://doi.org/10.1002/j.1939-4640.1983.tb00765.x
Corrado C, Fontana S (2020) Hypoxia and HIF signaling: one axis with divergent effects. Int J Mol Sci 21:16. https://doi.org/10.3390/ijms21165611
Chen P, Zirkin BR, Chen H (2020) Stem Leydig cells in the adult testis: characterization regulation and potential applications. Endocr Rev 41(1):22–32. https://doi.org/10.1210/endrev/bnz013
Davidoff MS, Middendorff R, Enikolopov G, Riethmacher D, Holstein AF, Müller D (2004) Progenitor cells of the testosterone-producing Leydig cells revealed. J Cell Biol 167(5):935–944. https://doi.org/10.1083/jcb.200409107
Doll R (1995) Hazards of ionising radiation: 100 years of observations on man. Br J Cancer 72(6):1339–1349. https://doi.org/10.1038/bjc.1995.513
Fukunaga H, Butterworth KT, Yokoya A, Ogawa T, Prise KM (2017) Low-dose radiation-induced risk in spermatogenesis. Int J Radiat Biol 93(12):1291–1298. https://doi.org/10.1080/09553002.2017.1355579
Gao F, Li G, Liu C, Gao H, Wang H, Liu W, Chen M, Shang Y, Wang L, Shi J, Xia W, Jiao J, Gao F, Li J, Chen L, Li W (2018) Autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells. J Cell Biol 217(6):2103–2119. https://doi.org/10.1083/jcb.201710078
Hasegawa M, Zhang Y, Niibe H, Terry NH, Meistrich ML (1998) Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in p53-deficient mice. Radiat Res 149(3):263–270
Havrankova R (2020) Biological effects of ionizing radiation. Cas Lek Cesk 159(7–8):258–260
He G, Tang A, Xie M, Xia W, Zhao P, Wei J, Lai Y, Tang X, Zou YM, Liu H (2020) Blood gene expression profile study revealed the activation of apoptosis and p53 signaling pathway may be the potential molecular mechanisms of ionizing radiation damage and radiation-induced bystander effects. Dose Response 18(1):1559325820914184. https://doi.org/10.1177/1559325820914184
Jankovic Velickovic L, Stefanovic V (2014) Hypoxia and spermatogenesis. Int Urol Nephrol 46(5):887–894. https://doi.org/10.1007/s11255-013-0601-1
Kovacs GT, Stern K (1999) Reproductive aspects of cancer treatment: an update. Med J Aust 170(10):495–497. https://doi.org/10.5694/j.1326-5377.1999.tb127853.x
Kiffin R, Bandyopadhyay U, Cuervo AM (2006) Oxidative stress and autophagy. Antioxid Redox Signal 8(1–2):152–162. https://doi.org/10.1089/ars.2006.8.152
Li J, You Y, Zhang P, Huang X, Dong L, Yang F, Yu X, Chang D (2022) Qiangjing tablets repair of blood-testis barrier dysfunction in rats via regulating oxidative stress and p38 MAPK pathway. BMC Complement Med Ther 22(1):133. https://doi.org/10.1186/s12906-022-03615-z
Liu C, Wang H, Shang Y, Liu W, Song Z, Zhao H, Wang L, Jia P, Gao F, Xu Z, Yang L, Gao F, Li W (2016) Autophagy is required for ectoplasmic specialization assembly in sertoli cells. Autophagy 12(5):814–832. https://doi.org/10.1080/15548627.2016.1159377
Liu Z, Cao K, Liao Z, Chen Y, Lei X, Wei Q, Liu C, Sun X, Yang Y, Cai J, Gao F (2020) Monophosphoryl lipid A alleviated radiation-induced testicular injury through TLR4-dependent exosomes. J Cell Mol Med 24(7):3917–3930. https://doi.org/10.1111/jcmm.14978
Makela JA, Koskenniemi JJ, Virtanen HE, Toppari J (2019) Testis development. Endocr Rev 40(4):857–905. https://doi.org/10.1210/er.2018-00140
Mazure NM, Pouyssegur J (2010) Hypoxia-induced autophagy: cell death or cell survival? Curr Opin Cell Biol 22(2):177–180. https://doi.org/10.1016/j.ceb.2009.11.015
Mehdipour A, Yousefi-Ahmadipour A, Kennedy D, Kazemi Arababadi M (2021) Ionizing radiation and toll like receptors: a systematic review article. Hum Immunol 82(6):446–454. https://doi.org/10.1016/j.humimm.2021.03.008
Nakayama K, Milbourne A, Schover LR, Champlin RE, Ueno NT (2008) Gonadal failure after treatment of hematologic malignancies: from recognition to management for health-care providers. Nat Clin Pract Oncol 5(2):78–89. https://doi.org/10.1038/ncponc1016
Qi L, Li J, Le W, Zhang J (2019) Low-dose ionizing irradiation triggers apoptosis of undifferentiated spermatogonia in vivo and in vitro. Transl Androl Urol 8(6):591–600. https://doi.org/10.21037/tau.2019.10.16
Rikka S, Quinsay MN, Thomas RL, Kubli DA, Zhang X, Murphy AN, Gustafsson AB (2011) Bnip3 impairs mitochondrial bioenergetics and stimulates mitochondrial turnover. Cell Death Differ 18(4):721–731. https://doi.org/10.1038/cdd.2010.146
Roca-Agujetas V, de Dios C, Leston L, Mari M, Morales A, Colell A (2019) Recent insights into the mitochondrial role in autophagy and its regulation by oxidative stress. Oxid Med Cell Longev 2019:3809308. https://doi.org/10.1155/2019/3809308
Rowley MJ, Leach DR, Warner GA, Heller CG (1974) Effect of graded doses of ionizing radiation on the human testis. Radiat Res 59(3):665–678
Tang Z, Chen J, Zhang Z, Bi J, Xu R, Lin Q, Wang Z (2021a) HIF-1alpha activation promotes luteolysis by enhancing ROS levels in the corpus luteum of pseudopregnant rats. Oxid Med Cell Longev 2021:1764929. https://doi.org/10.1155/2021/1764929
Tang Z, Xu R, Zhang Z, Shi C, Zhang Y, Yang H, Lin Q, Liu Y, Lin F, Geng B, Wang Z (2021b) HIF-1alpha protects granulosa cells from hypoxia-induced apoptosis during follicular development by inducing autophagy. Front Cell Dev Biol 9:631016. https://doi.org/10.3389/fcell.2021.631016
Tang Z, Zhang Z, Lin Q, Xu R, Chen J, Wang Y, Zhang Y, Tang Y, Shi C, Liu Y, Yang H, Wang Z (2020) HIF-1alpha/BNIP3-mediated autophagy contributes to the luteinization of granulosa cells during the formation of corpus luteum. Front Cell Dev Biol 8:619924. https://doi.org/10.3389/fcell.2020.619924
Vignard J, Mirey G, Salles B (2013) Ionizing-radiation induced DNA double-strand breaks: a direct and indirect lighting up. Radiother Oncol 108(3):362–369. https://doi.org/10.1016/j.radonc.2013.06.013
Wang M, Zeng L, Su P, Ma L, Zhang M, Zhang YZ (2022) Autophagy: a multifaceted player in the fate of sperm. Hum Reprod Update 28(2):200–231. https://doi.org/10.1093/humupd/dmab043
Wu K, Chen Z, Peng Q, Chen G, Yan W, Chen X (2019) Ku86 alleviates human umbilical vein endothelial cellular apoptosis and senescence induced by a low dose of ionizing radiation. J Int Med Res 47(2):893–904. https://doi.org/10.1177/0300060518805302
Yin J, Ni B, Tian ZQ, Yang F, Liao WG, Gao YQ (2017) Regulatory effects of autophagy on spermatogenesis. Biol Reprod 96(3):525–530. https://doi.org/10.1095/biolreprod.116.144063
Zhang ZY, Fu SL, Xu SQ, Zhou X, Liu XS, Xu YJ, Zhao JP, Wei S (2015) By downregulating Ku60, hsa-miR-526b suppresses non-small cell lung cancer. Oncotarget 6(3):1462–1477. https://doi.org/10.18632/oncotarget.2808
Acknowledgements
This research was funded by the Fujian Natural Science Foundation (Nos. 2020J01176, 2021J02028 and 2022J01172), Key Projects of Scientific and Technological Innovation in Fujian Province (Nos. 2022G023 and 2022G028), Special Funds of the Central Government Guilding Local Science and Technology Development (No. 2020L3008) and the Innovation and Entrepreneurship Project of Fujian Normal University (Nos. I202003009 and I202102008).
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RX, ZY and ZW designed the experiment. RX, SS, DW, JY and SS performed the experiment and analyzed the data. RX wrote the draft manuscript. ZY and ZW revised the final manuscript. All authors have read and agreed to the published version of the manuscript.
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Xu, R., Shen, S., Wang, D. et al. The role of HIF-1α-mediated autophagy in ionizing radiation-induced testicular injury. J Mol Histol 54, 439–451 (2023). https://doi.org/10.1007/s10735-023-10153-6
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DOI: https://doi.org/10.1007/s10735-023-10153-6