Autophagy involved in the activation of the Nrf2-antioxidant system in testes of heat-exposed mice

Hyperthermia causes oxidative stress in testes, which triggers antioxidant signals including autophagy and nuclear factor erythroid 2-related factor 2 (Nrf2). However, their relationship in testes under oxidative stress is unclear. In this study, we conducted testes injection for autophagy alteration and heat exposure to reveal the interaction between autophagy and the Nrf2-antioxidant system. Male mice were injected once with normal saline as control (Cont group), autophagy inhibitor 3-methyladenine (3-MA group) or autophagy inducer rapamycin (Rapa group). Then, each group was divided into two parts: one received a 2-h 42 °C heat treatment for eight days (HT groups), and the other was kept thermal neutral (NT groups). Heat-exposed mice showed significantly increased rectal, scrotal surface and body surface temperatures. Histology of the testes revealed many vacuoles inserted in the seminiferous tubules in the HT Cont group and two 3-MA groups. Ultrastructural changes in germ cells revealed autophagosomes in two 3-MA groups. Immunohistochemical detection of Nrf2 and p62/SQSTM1 proteins showed prominent expression in Leydig cells. Heat exposure increased Nrf2 protein and mRNA levels. 3-MA and Rapa testes injection also resulted in Nrf2 cytoplasm accumulation. Massive conversion of LC3 (microtubule-associated protein light chain 3)Ⅰ to LC3Ⅱ was detected in two 3-MA groups, accompanied by decreased ATG5 (autophagy related gene 5) mRNA levels in the HT 3-MA group. These results indicated autophagy alteration triggered the Nrf2 signaling pathway with consequences such that the autophagy inducer protected the testes and the autophagy inhibitor enhanced the detrimental effects caused by heat exposure.