Abstract：Bisphenol A (BPA), an environmental estrogen analogue, is capable of inducing perturbations within the antioxidant system of sertoli cells (SCs), thereby giving rise to oxidative stress. Concurrently, SCs which serve as the pivotal cells responsible for nourishing and supporting spermatogenic cells, exhibit a high susceptibility to alterations in temperature. To explore whether heat stress exacerbates the occurrence of BPA-induced ferroptosis in SCs in Holstein cattle (Bos taurus), bovine testis SCs were divided into blank control group (Con), BPA treatment group (BPA), heat stress group (HS), BPA and heat stress treatment group (HS+BPA) in this study. Western blot and RT-qPCR methods were used to detect the proliferation and secretion factors of bovine testis SCs and the genes and proteins related to ferroptosis, the content of reactive oxygen species (ROS) and free iron and mitochondrial membrane potential was detected by fluorescent probe method, and the change of malondialdehyde (MDA) content was detected by colorimetric method. The results showed compared with the control group, HS+BPA group reduced the expression of glial cell-derived neurotrophic factor (GDNF), bone morphogenetic protein 4 (BMP4), C-X-C motif chemokine ligand 12 (CXCL12) and proliferating cell nuclear antigen (PCNA), androgen-binding protein (ABP) related to proliferation and secretion of SCs. The expression of ferroptosis-related genes and proteins ferritin heavy chain 1 (FTH1), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4) were extremely significantly decreased (P<0.01). Meanwhile, the expression levels of prostaglandin-endoperoxide synthase 2 (PTGS2), acyl-coA synthetase long-chain family member 4 (ACSL4) and transferrin receptor (TFRC) in SCs of testis were extremely significantly increased (P<0.01) and the difference was significant compared with BPA group and HS group (P<0.05). Compared with the control group, the content of ROS, MDA and free iron in testicular SCs in the co-treatment group increased, while the content of GSH and the relative value of JC-1 staining decreased, and the difference was significant compared with the BPA group and HS group (P<0.05). These results indicated that 43 ℃ for 1 h increased the incidence of ferroptosis and decreased the expression of cell proliferation and secretory factors in Holstein bovine testis SCs after heat stress with 160 μmol/L BPA pretreated. This study provides research directions and fundamental materials for addressing the issue of reduced fertility in dairy cows during summer production.
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