Thiram inhibits human sperm motility and capacitation by increasing mitochondrial O₂⁻ and reducing PTK and PAK4 activity at environmental concentrations

Background: Environmental chemicals may suppress sperm motility (SM), hyperactivation, and capacitation, thus contributing to male infertility. This investigation was designed to examine the impacts of thiram on human SM and capacitation.

Methods: We incubated the fungicide thiram with normal human spermatozoa to investigate sperm metrics.

Results: Thiram markedly suppressed SM and hyperactivation, demonstrating half maximum effective concentration (EC₅₀) values below 5 µM. In addition, thiram reduced intracellular reactive oxygen species (ROS) and H₂O₂ with EC₅₀ values of 0.89 and 1.4 µM, respectively, while mitochondrial O₂⁻ levels were markedly elevated (EC₅₀ 0.9 µM), indicating selective disruption of mitochondrial redox balance rather than general ROS inhibition. It markedly increased mitochondrial O₂⁻ with an EC₅₀ value of 0.9 µM. Sperm DNA fragmentation remained unaffected at concentrations up to 10 M. The thiram-induced reduction in SM and forward motility (FM) was proven to be irreversible. Neither forskolin, 8Br-cAMP, pentoxifylline, progesterone, vitamin E (VE), nor A23187 was capable of preventing the thiram-induced decline in SM and FM. Thiram suppressed spontaneous capacitation processes. Additional investigation revealed that thiram reduced tyrosine protein kinase (TPK) levels with an EC₅₀ value of around 0.27 µM, decreased p21-activated kinase 4 (PAK4) levels with an EC₅₀ value of around 0.69 µM, and induced mitochondrial structural damage in normal sperm at concentrations as low as 2.5 µM.

Conclusions: Thiram causes irreversible inhibition of human SM, FM, and capacitation through elevation of mitochondrial superoxide anion levels and reduction of TPK and PAK4 activity, and may also act downstream of cAMP signaling, while compromising mitochondrial ultrastructural integrity.