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| The Role and Mechanism of DUOX2-mediated Oxidative Stress in Testicular Heat Stress Injury in Camelus bactrianus |
| LIU Yuan-Yuan, NAN Jing-Hong, MA Wen-Rui, WANG Hao-Tian, ZHANG Yong, ZHAO Xing-Xu*, WANG Qi* |
| College of Veterinary Medicine, Gansu Agricultural University/Gansu Provincial Key Laboratory of Animal Reproductive Physiology and Reproductive Regulation, Lanzhou 730070, China |
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Abstract The Bactrian camel (Camelus bactrianus) is a typical seasonal breeding animal, under natural conditions, its anestrus period usually coincides with the season of high ambient temperature, during which reproductive function is cyclically suppressed. The purpose of this study was to explore the mechanism of the differentially expressed dual oxidase 2 (DUOX2) gene in testicular heat stress response of the Bactrian camel. The testicular tissues of Bactrian camels in estrus and anestrus were collected. Differentially expressed genes were screened by RNA-seq, and the expression and localization of DUOX2 were verified by qRT-PCR, Western blot, immunohistochemistry (IHC) staining, and ELISA and oxidative stress-related indicators and hormone levels were detected. Testicular Sertoli cells (SCs) were isolated and cultured in vitro, and heat stress model (39 ℃ and 42 ℃) was constructed. The cell model was treated with DUOX2 inhibitor diphenyleneiodonium (DPI). Using cell counting kit-8 (CCK-8) to screen for an appropriate DPI concentration and using flow cytometry to analyze cell apoptosis and cell cycle distribution. A total of 291 differentially expressed mRNA were identified by transcriptome analysis, and the expression of DUOX2 was significantly up-regulated in the anestrus period (P<0.05). KEGG analysis showed that DUOX2 was enriched in oxidative stress and other related pathways. The mRNA transcription and protein level verification showed that the expression level of DUOX2 in testis in anestrus was significantly higher than that in estrus (P<0.05). The hematoxylin-eosin staining (HE) results showed that the seminiferous tubules appeared vacuolization and the spermatogenesis process was stagnant during anestrus. IHC results showed that DUOX2 protein was mainly distributed in the seminiferous epithelium, SCs and interstitial cells. ELISA results showed that the oxidative indexes (H2O2 and malondialdehyde (MDA)) were significantly (P<0.05) and extremely significantly (P<0.01) increased, while the levels of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) and reproductive hormones (testosterone (T) and luteinizing hormone (LH)) were extremely significantly decreased (P<0.01). Cell experiments showed that under heat stress, inhibition of DUOX2 activity could significantly reduce the apoptosis rate of SCs, and the G1 phase block changed to G2/M phase block. The up-regulated expression of DUOX2 in the testis of Bactrian camels during anestrus may aggravate heat stress injury by mediating oxidative stress, and then induce apoptosis and cycle arrest of SCs. Inhibition of DUOX2 activity could alleviate heat stress injury to a certain extent. This study revealed the key role of DUOX2 in heat stress-induced testicular SCs injury in Bactrian camels, and provides a new theoretical basis for elucidating the molecular regulatory mechanism of seasonal reproduction in Bactrian camels.
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Received: 16 October 2025
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Corresponding Authors:
*zhaoxx@gsau.edu.cn; wangqi@gsau.edu.cn
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