Microplastics (MPs), as emerging persistent environmental pollutants, have widely infiltrated terrestrial ecosystems. MPs can be absorbed by crops or mixed into feed, subsequently bioaccumulating in ruminants such as sheep via the food chain, posing potential threats to male reproductive function. Recent studies indicate that ingested MPs can not only accumulate in organs but may also cross the intestinal barrier into the circulatory system. These micron-sized particles can disrupt the blood-testis barrier, induce oxidative stress, and potentially impair testicular development and spermatogenesis. Therefore, elucidating their toxicological mechanisms and health impacts is urgently needed.

Three-month-old male Hu sheep were used as experimental animals. The low-dose exposure group received 75 mg/kg polystyrene microplastics (PS-MPs) daily in their diet, the high-dose group received 150 mg/kg, and the control group received 0 mg/kg (n = 18). A 14-day pre-trial period was followed by a 120-day exposure period. Blood and testicular tissues were collected post-experiment. Mechanistic effects of MPs on the testis were investigated using ELISA, metabolomics, single-cell sequencing, and immunofluorescence.

MP exposure led to significant reductions in body weight and testicular index. Serum hormones related to testosterone synthesis and spermatogenesis were decreased. Concurrently, serum antioxidant enzyme activity and immune factor levels were significantly reduced, whereas inflammatory cytokine levels were elevated. Histological analysis revealed increased abnormal seminiferous tubules, partial loss of seminiferous epithelium, vacuolization of germ cells, membrane rupture in some Sertoli cells, mitochondrial swelling, and loss of mitochondrial cristae. Fluorescence quantification, Western blotting, and immunofluorescence demonstrated decreased expression of testosterone synthesis-related factors and increased expression of inflammatory factors in exposed groups.

Metabolomic analysis revealed significant downregulation of key metabolites such as glutathione and L-methionine, essential for normal testicular function, in both testis and serum. Differential metabolites were significantly enriched in cysteine and methionine metabolic pathways. Single-cell RNA sequencing indicated a reduction in spermatogenic cell numbers after exposure. Subclustering and functional analysis of spermatogonia revealed that MPs hindered their differentiation, with differentially expressed genes significantly enriched in the Nrf2 signaling pathway, which is closely related to oxidative stress. Experimental validation confirmed that MPs inhibited the Nrf2 pathway.

This study demonstrates that long-term oral exposure to polystyrene MPs significantly damages testicular structure and reproductive function in Hu sheep, with a dose-dependent effect. Exposure reduced body weight and testicular index, decreased testosterone and spermatogenesis-related hormone levels, suggesting suppressed steroidogenesis and impaired spermatogenesis. Additionally, reduced antioxidant enzyme activity, elevated inflammatory cytokines, and mitochondrial ultrastructural damage indicate that oxidative stress, inflammation, and mitochondrial dysfunction jointly mediate MP toxicity. Metabolomics showed downregulation of glutathione and L-methionine, with enrichment in cysteine and methionine metabolism, suggesting redox imbalance in the testis. Single-cell sequencing further revealed that
MPs reduce spermatogenic cell numbers and inhibit spermatogonial differentiation, potentially via suppression of the Nrf2 pathway.

Long-term exposure to polystyrene MPs significantly damages Hu sheep testicular structure and function, manifested as inhibited testosterone synthesis, impaired spermatogenesis, and structural disruption. The underlying mechanisms involve enhanced oxidative stress, activation of inflammatory responses, and suppression of the Nrf2 signaling pathway, leading to disrupted spermatogonial differentiation and energy metabolism homeostasis, ultimately resulting in male reproductive toxicity.