Long-term Atrazine exposure promotes ovarian dysfunction by enhancing the inflammatory microenvironment and inducing fibrosis

This study investigated the effects of long-term exposure to the herbicide Atrazine (ATZ) on ovarian function in rats and its underlying mechanisms. Following 180-day exposure to 2 µM and 10 µM ATZ via drinking water, ATZ reduced ovarian weight and index, disrupted follicular development, increased atretic follicles, and altered serum hormone levels, including decreased estradiol (E2) and increased follicle-stimulating hormone (FSH), indicating diminished ovarian reserve (DOR). Histological analysis revealed that ATZ exposure induced ovarian fibrosis, characterized by increased collagen deposition and upregulation of fibrosis-related proteins (collagen I/III, fibronectin, a-SMA). Mechanistically, ATZ activated the Wnt/ß-catenin signaling pathway, upregulating the expression of Wnt1, MMP2, MMP9, and Cyclin D1 in ovarian granulosa cells. Additionally, ATZ promoted M2 macrophage polarization in the ovarian microenvironment, accompanied by increased anti-inflammatory cytokine IL-4 and decreased pro-inflammatory cytokine IL-6, suggesting that ATZ induces fibrosis through inflammatory regulation of the Wnt pathway. These findings reveal a novel mechanism by which long-term low-dose ATZ exposure impairs ovarian function via the inflammation-fibrosis axis, providing experimental evidence for the reproductive toxicity of environmental pollutants. Overall design: The SD rats (21 days old) were intraperitoneally injected with 60 IU of pregnant mare serum gonadotropin (PMSG), after 48 h the rats were euthanized by cervical dislocation under anesthesia. Bilateral ovaries were removed from abdominal cavity of rats and washed with precooled PBS for 3 times. GCs were isolated using sterile scissors to cut the ovaries into 2 mm pieces, placed in the precooled medium [DMEM/F12 (HyClone)+10% fetal bovine serum (FBS, Gibico)] and centrifuged at 1200 rpm for 5 min. The precipitation was resuspended with 0.25% pancreatic enzyme containing 0.02% EDTA and placed in an incubator at 37 ? for 1 hour. After 40 µm sterile cell sieve filtration, the suspension was added into complete medium [DMEM/F12+10% FBS+1% Insulin-Transferrin-Sodium Selenite (100×, Procell)+1% MEM Non-Essential Amino Acids, NEAA (100×, Procell)+1% Penicillins-streptomyci (100×)] for culture. In order to identify the GCs, its positive marker FSHR was detected by immunofluorescence.