Atrazine Induces Reproductive Toxicity in an In Vitro Spermatogenesis (IVS) Model

Background/Objectives: Atrazine (ATZ) is a widely used herbicide, and most studies of its reproductive toxicity have been conducted in vivo using animal models, where ATZ disrupts redox homeostasis, leading to male reproductive dysfunction. However, its molecular mechanisms of action in human spermatogenic cells remain poorly understood. Huntington's disease (HD), an autosomal dominant disorder caused by abnormal CAG repeat expansion in the HTT gene, exhibits heightened oxidative stress sensitivity and mitochondrial dysfunction, which may further impair reproductive function. This study investigated ATZ effects on human spermatogenesis using an in vitro spermatogenesis (IVS) model derived from human induced pluripotent stem cells (hiPSCs), focusing on Nrf2-mediated oxidative responses and apoptotic regulation during spermatogonial stem cell-like cell (SSCLC) differentiation in wild-type (WT) and HD hiPSC lines. Methods: Two WT and two HD hiPSC lines carrying 44 (HD1) and 180 (HD2) CAG repeats were treated with ATZ (0, 0.01, 1, or 10 μM) for 30 days, followed by differentiation into SSCLCs for 15 days under continuous exposure. Expression of pluripotency (OCT4, SOX2), oxidative stress (NFE2L2, SOD1, GPX1, NQO1), cell cycle (CDK1), apoptosis (BCL2, BAX, CASP3, CASP9, FAS, FASLG), and spermatogenic markers (DAZL, ZBTB16, GFRA1, PIWIL2) were assessed by immunocytochemistry and qRT-PCR. Results: Long-term ATZ exposure affected pluripotency markers in hiPSCs and SSCLC differentiation in a cell line-dependent manner. WT cells exhibited early differentiation suppression without significant apoptosis. HD1 cells were highly sensitive: low ATZ doses (0.01-1 μM) partially activated intrinsic and extrinsic apoptotic pathways, whereas high-dose ATZ (10 μM) reduced Nrf2-target and spermatogenic gene expression, strongly impairing SSCLC maturation. HD2 cells showed pronounced oxidative stress with robust Nrf2-driven antioxidant responses and BCL2 that supported differentiation at low doses. However, excessive oxidative or proliferative signaling, including CDK1 upregulation at high ATZ concentrations, disrupted redox balance and SSCLC differentiation in HD2 cells. Conclusions: ATZ exerts dose- and genotype-dependent effects on IVS through coordinated regulation of oxidative stress and apoptosis. These findings highlight the interplay between Nrf2-mediated antioxidant defenses, apoptotic signaling, and genetic background in shaping spermatogenic outcomes, providing mechanistic insight into ATZ-induced reproductive toxicity in a human-relevant in vitro spermatogenesis model.