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

Introduction

Sperm count among human males globally has declined by more than 50% over the past four decades (1). This decreasing trend has continued into the 2020s, highlighting alarming deterioration in sperm quality (2). Currently, approximately 5% of men experience reduced fertility and require clinical consultation. Although there are genetic factors for male infertility, exposure to environmental pollutants also markedly contributes to this condition (3).

Tetramethylthiuram disulfide (thiram) is one such environmental chemical. Thiram is called tetramethylthiuram, belonging to the dithiocarbamate fungicide, and it is widely used in agricultural seed treatment, soil treatment, and foliar sprays on grains, fruits, and vegetables. It is also used as an ingredient in certain medicated soaps and suntan and antiseptic sprays (4,5). Despite the U.S. Environmental Protection Agency’s assessment of dithiocarbamate fungicides as presenting minimal mammalian toxicity risk (6), multiple studies demonstrates that thiram and ziram induce dose-dependent toxic effects, including mutagenicity, teratogenicity, and reproductive toxicity in rodents at oral doses of 50–200 mg/kg/day, neurotoxicity, hepatotoxicity, nephrotoxicity, and lung toxicity (7-9).

Thiram has the potential to infiltrate the food chain. Human exposure to thiram occurs through ingestion or dermal contact, occasionally through respiratory pathways (7). Thiram may impact male reproductive function and contribute to male infertility (10). While prior studies have shown that dialkyl dithiocarbamates possess spermicidal properties against human sperm (11), and other environmental chemicals such as lead and bisphenol S affect reproductive function (5), certain plant extracts and antioxidants like garlic extract may mitigate sperm oxidative stress (12), the specific effects of thiram on human sperm motility (SM), hyperactivation, capacitation, and acrosome reaction (AR), along with underlying mechanisms, remain poorly understood.

Human spermatozoa require physiological alterations within the female reproductive system, particularly capacitation, to accomplish effective egg fertilization. This capacitation mechanism may be achieved through a chemically defined medium that contains energy metabolites and serum albumin serving as cholesterol receptors (13). Capacitation enables spermatozoa to exhibit vigorous motility patterns, termed hyperactivation (13), and triggers AR (14). Both hyperactivation and AR are deemed essential for successful spermatozoa fertilization of eggs. Reactive oxygen species (ROS) are generated within cells as by-products of diverse metabolic processes. At low concentrations, these species provide benefits through participation in cellular signaling pathways and various physiological functions. However, at elevated concentrations, they may cause biological system damage through oxidation of biomolecules, encompassing lipids, proteins, and DNA. ROS demonstrate significant importance in male fertility. Elevated ROS generation has been linked to various male reproductive disorders, encompassing leukocytospermia, varicocele, and idiopathic infertility (8). The resulting oxidative stress damage to spermatozoa may present as inadequate energy metabolism, lipid peroxidation, and DNA damage, consequently causing motility and viability loss (9). Nevertheless, multiple studies have shown that physiological ROS levels demonstrate important functions in spermatozoa maturation, capacitation, hyperactivation, and AR processes (13). Molecular alterations linked to capacitation encompass elevated intracellular pH, heightened intracellular Ca²⁺ levels, cyclic adenosine monophosphate (cAMP) pathway stimulation, hyperpolarization of sperm plasma membrane potential, reduction of membrane cholesterol content, and augmented protein tyrosine phosphorylation (12,15,16). The cAMP cascade demonstrates criticality for SM and forward motility (FM) (17). The elevated intracellular cAMP levels convey signals that enable protein kinase A (PKA) to stimulate tyrosine protein kinase (TPK) (17,18). P21-activated kinase 4 (PAK4) represents a pluripotent serine/threonine kinase and demonstrates criticality for early development, functioning as a signal integrator controlling numerous fundamental cellular processes, including cell morphology and motility (13). In this investigation, thiram was identified as a potent inhibitor of human SM and FM. Various aspects of human spermatozoa parameters were further examined, including motility, FM, hyperactivation, capacitation, and AR, while exploring potential mechanism(s) of thiram action. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-940/rc).

Methods

Reagents and patients

The residual standards for thiram in nuts, vegetables, and fruits are 15 ppm (19). In this investigation, 10 µM (approximately 3 ppm) of thiram was employed as the maximum concentration. Eosin-melanin staining solution was procured from Huakang (Shenzhen, China). The DNA break index measurement kit, mitochondrial membrane potential (MMP) measurement kit and AR measurement kit were procured from Cellpro Biotech (Zhejiang, China). Sperm ROS staining (DCFH-DA and MitoSOX Red) kit was procured from Puhua Technology (Chengdu, China). TPK ELISA kit and PAK4 ELISA kit were procured from Camilo Biological (Nanjing, China). Calcimycin (also called A23187), forskolin (FSK), 8Br-cAMP (8BR-cAMP), pentoxifylline (PTX), progesterone (P4), vitamin E (VE) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). The BWW (Biggers-Whitten-Whittingham) medium was formulated following previously published protocols (20). Semen samples from 8 normal men aged 25 to 45 years attending infertility clinics of Sichuan Jinxin Xi’nan Women and Children Hospital were collected. Power analysis indicated that n=8 provides 80% power to detect a 20% change in SM at α=0.05. Liquefaction assessment and semen processing conducted according to the WHO laboratory manual (5th edition) (21). The semen parameters and supplementary patient characteristics that influence motility in normal men were presented in Table S1. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Sichuan Jinxin Xi’nan Women and Children Hospital (2021: Number 4), and all patients provided informed consent. All reagents included batch numbers for reproducibility (e.g., Eosin Y, Batch No. HX202105; JC-1, Batch No. CPB-0034).

Semen treatment

Following liquefaction, the semen underwent washing with 0.3% BWW solution, and the sperm level was standardized to 1×10⁷/mL. Afterward, various dilution media (1.5% BWW solution), vehicle solvent (DMSO), or thiram (dissolved in DMSO, achieving a final DMSO concentration of 0.1%) were introduced to the semen. The semen samples were then maintained at 37 ℃ within a 5% CO₂ incubator with controlled humidity for 3 h.

Sperm vitality assessment

Sperm vitality evaluation was conducted utilizing eosin-melanin staining methodology following established protocols (21,22). The staining solution was prepared by dissolving eosin Y and nigrosin in sodium chloride within distilled water. Following the liquefaction process, semen was combined with an equivalent volume of staining solution in a balanced proportion. The combined solution underwent a brief incubation period of 30 s at ambient temperature. Subsequently, microscope slides were prepared by spreading the mixture to create sperm smears. Once the smears had air-drying, slides underwent direct microscopic examination. A minimum of 200 sperm cells underwent comprehensive assessment through microscopic examination at 1,000× magnification utilizing an oil-immersion high-resolution 100× bright field objective. Sperm specimens presenting white pigmentation were classified as viable, whereas those demonstrating pink or red pigmentation were categorized as non-viable. Assessment reproducibility was verified through internal quality control procedures, maintaining inter-assay coefficient of variation (CV) below 10%.

Computer-assisted sperm analysis (CASA) of SM, FM, and hyperactivation

A CASA system (Hamilton IVOS II, USA) was employed to assess SM and FM following previously established protocols (23,24). Following 3 h incubation, a sperm aliquot was introduced into a microcell slide chamber measuring 20 µm in depth (Goldcyto Biotech Corp., Guangzhou, China). The slide underwent analysis under pseudo-dark-field illumination conditions. For each specimen, 10 arbitrarily selected microscopic fields encompassing >200 trajectories of motile spermatozoa were assessed at 60 Hz. The following kinematic parameters were recorded: curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), linearity % (LIN, calculated by VSL/VCL multiplied by 100), beat-cross frequency (BCF), the proportion of FM, and the motility percentage. Spermatozoa demonstrating VCL ≥150 mM/s, ALH ≥7.0 mM, and LIN ≤50% were identified as exhibiting hyperactive motility.

Assessment of capacitation and AR detection utilizing FITC-PSA fluorescent labeling

The spontaneous AR in sperm was assessed through fluorescein isothiocyanate-conjugated pea lectin (FITC-PSA) staining following established protocols (23,25). Spermatozoa samples were distributed across glass microscope slides, allowed to air-dry, and subsequently immobilized utilizing 95% ethanol for 30 min at 4 ℃. FITC-PSA working solution was applied and maintained at 4 ℃ for 15 min. After incubation, slides underwent rinsing with deionized water, air-drying, and microscopic examination using a 1,000× oil immersion lens for sperm enumeration. Spontaneous AR rate was calculated as AR count × 100% / total sperm count (AR + AI), including both intact and reacted sperm, and all images include a scale bar of 2 µm. AI denotes spermatozoa showing intact acrosome, whereas AR denotes spermatozoa demonstrating AR.

Quantification of sperm MMP

Sperm MMP was assessed per the supplier’s protocol (26). After incubating sperm with thiram for 3 h, a 20 µL aliquot of the sperm suspension was pipetted into a 1.5-mL centrifuge tube. Solution A (100 µL) was then introduced, succeeded by the addition of 485 µL of JC-1 dye solution. The components were carefully intermixed. The resulting preparation was subsequently kept shielded from light and maintained at 37 ℃ for a duration of 15 min. Subsequently, flow cytometric analysis was performed on the cells.

Quantification of sperm ROS

ROS levels were assessed through flow cytometry utilizing a ROS detection kit per the supplier’s protocols (27). Briefly, 30 µL of sperm suspension underwent washing with 1.5 mL phosphate buffer saline at 200 g for 3 min. A 300 µL staining solution comprising DCFH-DA and MitoSOX Red mitochondrial superoxide indicator was combined with 300 µL of sperm suspension to determine ROS levels and mitochondrial superoxide anion (O₂⁻) concentrations within cells. The mixture was maintained at 37 ℃ for 30 min. Subsequently, the cellular solution underwent centrifugation at 200 g for 3 min to harvest spermatozoa. The collected spermatozoa were reconstituted in 300 µL phosphate buffered saline and subjected to flow cytometric analysis.

Sperm chromatin structure assay

DNA fragmentation index (DFI) of spermatozoa was assessed through flow cytometry utilizing a DFI kit per the supplier’s protocols (28). Briefly, 10 µL sperm suspension underwent processing by sequentially introducing 100 µL of solution A and 200 µL of solution B from the designated kit. Subsequently, 600 µL of acridine orange staining solution was integrated. The cells underwent subsequent examination through flow cytometry.

Enzyme-linked immunosorbent assay of cAMP, protein tyrosine, serine and threonine kinases

The cAMP ELISA kits, employing a double antibody sandwich methodology, were utilized to measure sperm cAMP content at 450 nm wavelength per the supplier’s protocols (24). Following 3 h cultivation with thiram, 150 µL of sperm suspension was transferred into a 1.5 mL centrifuge tube and subjected to centrifugation at 600 g for 10 min, with the resulting supernatant employed for analysis. 50 µL of standard or sample was introduced to a 96-well plate, subsequently complemented by 100 µL horseradish peroxidase (HRP) conjugated antibody and incubation at 37 ℃ for 1 h. Subsequently, the plate underwent five washing cycles before adding 100 µL of substrate and incubating at 37 ℃ under dark conditions for 15 min. Afterward, 100 µL of stop solution was incorporated, and absorbance measurements were recorded utilizing a microplate spectrophotometer. By establishing a standard curve derived from reference absorbance values, the cAMP concentration in samples could be precisely quantified through comparative analysis.

The TPK or PAK4 ELISA kits, employing a double antibody sandwich methodology, were employed to assess sperm TPK or PAK4 content for evaluating sperm protein tyrosine, serine and threonine kinase levels at 450/630 nm wavelength per the supplier’s protocols (29). Following 3-hour thiram cultivation, 150 µL of sperm suspension was placed into a 1.5 mL centrifuge tube and subjected to centrifugation at 600 g for 10 min, with the resulting supernatant utilized for analysis. Standard or sample aliquots of 100 µL were dispensed into 96-well plates and maintained at 37 ℃ for 90 min, followed by two washing cycles and addition of 100 µL biotinylated human TPK or PKA4 antibody working solution (antibody diluent: biotinylated antibody =100:1) with 37 ℃ incubation for 60 min. Post plate cleansing, 100 µL of enzyme conjugate solution was dispensed and subsequently maintained at 37 ℃ under light-shielded conditions for a 30-minute duration. Following additional washing procedures, 100 µL of TMB chromogenic solution was introduced and maintained at 37 ℃ in a dark environment for 30 minutes. Subsequently, 100 µL of stop solution was incorporated, and absorbance readings were determined utilizing a microplate spectrophotometer. A standard curve was constructed using standard absorbance values, enabling calculation of sample TPK or PAK4 content through standard curve analysis.

Testing the protection of FSK, 8BR, PTX, P4, and VE of thiram-mediated inhibition of SM and FM

The cAMP cascade has been demonstrated to serve a crucial function in SM, FM, capacitation, and the AR (15,16). FSK acts as an activator of transmembrane adenylate cyclase, capable of elevating intracellular cAMP levels, resulting in enhanced SM and FM (30). 8BR possesses the ability to penetrate cellular membranes and substitute for intracellular cAMP to enhance SM and FM (30). PTX functions as a phosphodiesterase inhibitor, capable of preventing cAMP degradation, thus increasing cAMP production and leading to improved SM and capacitation (31). P4 stimulates SM and FM through activation of the calcium ion CatSper channel in human sperm (32). VE possesses the capacity to prevent ROS-mediated impairment of SM (33). To investigate whether these agents could shield human spermatozoa against thiram-induced suppression of SM and FM, isolated sperm cells were exposed to 20 µM FSK, 1 mM 8BR, 10 µM PTX, 20 µM P4, and 400 µg/mL VE individually, in the absence and/or presence of 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 µM thiram, followed by incubation at 37 ℃ for 3 h. Subsequently, a 5 µL portion of sperm suspension from each specimen was introduced into a 20 µm-depth microporous slide cavity, then examined under pseudo-dark field illumination. Motility percentage and FM were documented. Stability controls were included for FSK, 8Br-cAMP, PTX, P4, VE, and A23187 over the 3 h incubation to confirm compound stability.

Assessment of A23187 protection against thiram-mediated SM and FM inhibition

An aliquot (10 µL) of A23187 was administered to spermatozoa at a final level of 10 µM based on established protocols (21) and subjected to concurrent exposure with different thiram concentrations over 3 h. Thereafter, a 5 µL portion of the sperm suspension from each specimen was placed into a 20 µm-depth microporous slide cavity, followed by examination utilizing pseudo-dark field illumination. Both motility and FM parameters were documented.

Impact of thiram on sperm mitochondrial architecture

To examine thiram’s impact on sperm mitochondrial structure, sperm samples exposed to thiram treatment for 3 h underwent pre-fixation using 2.5% glutaraldehyde and were subsequently submitted to the company (Chengdu Lilai Biotechnology, China) for electron microscopy analysis (34). Electron microscopy images were quantified by measuring the percentage of mitochondria showing cristae disruption and vacuolation per 100 sperm mitochondria.

Statistical analysis

GraphPad software (version 6, GraphPad Software Inc., San Diego, CA) enabled calculation of the half maximum effective concentration (EC₅₀) and the half maximum inhibitory concentration (IC₅₀) via nonlinear regression modeling of dose-response relationships on a logarithmic scale. The lowest-observed-adverse-effect level (LOAEL) was ascertained (35). Quantitative analysis was executed employing GraphPad software (35). Results are denoted as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with subsequent Dunnett’s multiple comparisons test evaluated differences versus the control group. Significance threshold was developed at P<0.05. Statistical significance was reported with exact P values (e.g., P=0.032, P<0.001) for each key comparison.

Results

Thiram reduces SM and FM of normal human spermatozoa

Human sperm underwent thiram treatment for 3 h (Figure 1A). Following 3 h of exposure, thiram showed no impact on sperm vitality at concentrations of 5 µM or lower, though it produced a slight yet significant reduction in sperm vitality at 10 µM (Figure 1B). Thiram substantially suppressed human SM with a LOAEL of 2.5 µM (Figure 1C) and an IC₅₀ value of 4.95 µM (Table 1). The compound suppressed sperm FM with a LOAEL of 2.5 µM (Figure 1D) and an IC₅₀ value of 4.23 µM (Table 1). These findings demonstrate that thiram suppresses SM and FM at low concentrations. The reversibility of thiram-mediated suppression on motility and FM was further investigated. Human spermatozoa underwent treatment with diverse levels of thiram for 3 h, succeeded by washing with BWW and subsequent incubation in BWW for 1 h (Figure 2A). The SM (Figure 2B) and FM (Figure 2C) showed no recovery with a LOAEL of 2.5 µM and IC₅₀ values of 4.70 and 4.16 µM for motility and FM, respectively. This suggests apparent irreversibility, although thiram’s lipophilic nature may limit complete removal during washing, which should be considered when interpreting these results.

Figure 1 Treatment protocol and impacts of thiram on viability, motility, and FM of normal human spermatozoa along with reversibility assessment. Spermatozoa underwent incubation with different thiram concentrations for 3 h [(A) this figure and subsequent figures]; (B) sperm viability; (C) SM; (D) sperm FM. Mean ± SEM, n=8 (independent human semen samples). *P<0.05, ***P<0.001 denote significant differences versus the control (0 µM thiram). All concentration values are expressed in µM. CASA, computer-assisted sperm analysis; FM, forward motility; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; SEM, standard error of the mean; SM, sperm motility.

Figure 2 Reversibility assessment of thiram-induced suppression on human SM and FM. (A) Treatment regimen: spermatozoa were exposed to varying levels of thiram for 3 h, subsequently washed and incubated with BWW for an extra 1 h. (B) Motility. (C) FM. Mean ± SEM, n=8. ***P<0.001 denotes a significant difference versus the control (BWW). All concentration values are expressed in µM. BWW, Biggers-Whitten-Whittingham; FM, forward motility; SEM, standard error of the mean; SM, sperm motility.

Thiram diminishes sperm hyperactivation in normal human spermatozoa

CASA evaluation demonstrated that thiram substantially diminished sperm VCL, VSL, LIN, ALH, VAP, and BCF with LOAEL of 2.5 µM for VCL, VSL, LIN, and VAP (Figure 3) and LOAEL of 5 µM for ALH and BCF. The lower sensitivity of ALH and BCF at low thiram concentrations indicates that lateral head displacement and beat frequency are less affected than velocity parameters, suggesting differential impact on hyperactivation kinematics. IC₅₀ values of thiram for VCL, VSL, LIN, ALH, VAP, and BCF were 9.92, 5.89, 7.20, 7.44, 5.50, and 11.07 µM, respectively (Table 1). These observations suggest that thiram suppresses the hyperactivation of human spermatozoa.

Figure 3 CASA analysis of thiram-treated human spermatozoa as described in Figure 1A. (A) VCL. (B) VSL. (C) LIN % (VSL/VCL). (D) ALH. (E) VAP. (F) BCF were examined. Mean ± SEM, n=8. *P<0.05, **P<0.01, ***P<0.001 denote significant differences versus the control (BWW). All concentration values are expressed in µM. ALH, amplitude of lateral head displacement; BCF, beat-cross frequency; BWW, Biggers-Whitten-Whittingham; CASA, computer-assisted sperm analysis; LIN, linearity; SEM, standard error of the mean; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity.

Thiram inhibits capacitation and the spontaneous AR of normal human spermatozoa

Sperm AR and AI (Figure 4A) following 3 h of incubation were assessed. Thiram markedly suppressed sperm capacitation with a LOAEL of 2.5 µM (Figure 4B) and an IC₅₀ value of 4.59 µM (Table 1), as evidenced by suppression of the transition from AI mode to AR mode (Figure 4A).

Figure 4 Effects of thiram on spontaneous AR and ultrastructure of mitochondria of normal human spermatozoa. Spermatozoa were exposed to varying thiram concentrations for 3 h, washed, and subsequently analyzed. (A) Image of AI and AR. (B) Spontaneous AR. Mean ± SEM, n=8. *P<0.05, ***P<0.001 denote significant differences versus the control (BWW). Mitochondrial images: (C) 0 µM thiram: the mitochondrial sheaths of sperm tails displayed orderly arrangement; (D) 2.5 µM: sperm mitochondria exhibited sparse arrangement with swelling and vacuolation; (E) 5 µM: mitochondria showed disorganized arrangement with swelling and vacuolation; (F) mitochondria underwent disintegration. Magnification ×25,000. Arrows indicate the alterations. All concentration values are expressed in µM. AI, acrosome intact; AR, acrosome reaction; BWW, Biggers-Whitten-Whittingham; SEM, standard error of the mean.

Thiram causes damage of mitochondrial structure of normal human spermatozoa

Under transmission electron microscopy, the mitochondrial sheaths surrounding sperm tails in the control group displayed well-organized architecture featuring clearly defined mitochondrial cristae structure (Figure 4C). In the 2.5 µM (Figure 4D), 5 µM (Figure 4E) and 10 µM (Figure 4F) thiram groups, sperm mitochondria exhibited swelling and vacuolation, with disrupted mitochondrial cristae and blurred mitochondrial architecture.

Thiram fails to elicit DNA damage in normal human spermatozoa

The DFI was measured to reflect sperm DNA integrity. Following 3 h of treatment, thiram showed no effect on DFI at any tested concentration (Figure 5A). Despite increased mitochondrial O₂⁻, the lack of DFI changes suggests that short-term oxidative stress induced by thiram is insufficient to cause detectable DNA fragmentation in human spermatozoa.

Figure 5 Impact of thiram on sperm DFI, MMP, ROS, H₂O₂ levels, mitochondrial O₂⁻, cAMP, TPK and p21-activated kinase 4 activity. Spermatozoa received thiram treatment as described in Figure 1A. (A) DFI. (B) MMP. (C) ROS. (D) H₂O₂ levels. (E) Mitochondrial O₂⁻. (F) cAMP. (G) TPK. (H) PAK4. Mean ± SEM, n=8 for (A)-(E); n=4 for (F)-(H). *P<0.05, **P<0.01, ***P<0.001 denote significant differences versus the control (BWW). All concentration values are expressed in µM. BWW, Biggers-Whitten-Whittingham; cAMP, cyclic adenosine monophosphate; DFI, DNA fragmentation index; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; SEM, standard error of the mean; TPK, tyrosine protein kinase.

Thiram diminishes sperm MMP of normal human spermatozoa

SM and FM correlate with energy production by sperm mitochondria. The MMP of sperm mitochondria was assessed. After 3 h of exposure, thiram substantially diminished MMP at 10 µM (Figure 5B), consistent with an IC50 value >100 µM as shown in Table 1, confirming the high resistance of MMP to thiram at environmentally relevant doses.

Thiram diminishes intracellular ROS level but increases mitochondrial O₂⁻ in normal human spermatozoa

ROS represents an oxygen metabolite. Physiological ROS concentrations serve a crucial function in sustaining male reproductive physiological functions. ROS concentrations in spermatozoa were assessed following thiram exposure. Following 3 h of exposure, thiram markedly decreased ROS concentrations with a LOAEL of 1.25 µM (Figure 5C) and an IC₅₀ value of 0.89 µM (Table 1). Thiram additionally reduced intracellular H₂O₂ concentrations with a LOAEL of 1.25 µM (Figure 5D) and an IC₅₀ value of 0.90 µM (Table 1). Nevertheless, thiram markedly elevated mitochondrial O₂⁻ concentrations with a LOAEL of 2.5 µM (Figure 5E) and an EC₅₀ value of 1.4 µM (Table 1).

Thiram lowers cAMP level of normal human spermatozoa

After 3 h of exposure, thiram markedly decreased cAMP at 10 µM (Figure 5F). This suggests that at the highest concentration thiram also affects SM and FM by reducing intracellular cAMP level.

Thiram decreases TPK and PAK4 activity in normal human spermatozoa

During sperm capacitation, TPK and PAK4 may participate in this process. To investigate whether thiram influences sperm capacitation through the inhibition of TPK and PAK4 activity, the enzymatic activities of both proteins were assessed. Following 3 h of exposure, thiram substantially decreased TPK activity with a LOAEL of 0.625 µM (Figure 5G) and an IC₅₀ value of 0.27 µM, while it also notably suppressed PAK4 activity with a LOAEL of 2.5 µM (Figure 5H) and an IC₅₀ value of 0.69 µM (Table 1). These results suggest that TPK and PAK4 are sensitive molecular targets in human sperm; however, direct binding of thiram to these kinases has not been experimentally confirmed, and further biochemical studies are required. These findings suggest that TPK and PAK4 represent highly sensitive molecular targets for thiram.

Impact of thiram on cAMP signal in normal human spermatozoa

A series of chemicals capable of affecting intracellular cAMP levels in human spermatozoa was employed. FSK (an agent elevating intracellular cAMP levels), 8Br-cAMP (a cAMP analogue capable of direct cellular penetration to mimic cAMP activity), and PTX (a phosphodiesterase inhibitor that augments intracellular cAMP levels through inhibition of cAMP degradation) were applied to treat normal human spermatozoa. During co-incubation of these chemicals with thiram for 3 h, they failed to prevent thiram-induced suppression of SM and FM (Figure 6 and Table 1), with the LOAEL of thiram for both motility and FM remaining at 2.5 µM and IC₅₀ values of thiram continuing to be approximately 4–6 µM (Table 1).

Figure 6 Impact of thiram on SM and FM in normal human spermatozoa following incubation with cAMP-associated compounds. Spermatozoa underwent incubation with varying thiram concentrations alongside 8Br-cAMP, FSK, and PTX for 3 h. (A,C,E) SM. (B,D,F) FM; Mean ± SEM, n=6. ***P<0.001 denotes a significant difference versus the control (BWW). All concentration values are expressed in µM. 8Br-cAMP, 8-Bromo-cAMP; BWW, Biggers-Whitten-Whittingham; cAMP, cyclic adenosine monophosphate; FM, forward motility; FSK, forskolin; PTX, pentoxifylline; SEM, standard error of the mean; SM, sperm motility.

Effect of thiram on Ca²⁺ signal of normal human spermatozoa

P4 (a chemical compound activating the Ca²⁺ channel CatSper for elevating intracellular Ca²⁺ levels) was employed to treat normal human spermatozoa and co-incubated with thiram for 3 h. P4 failed to prevent thiram-induced suppression of SM and FM (Figure 7 and Table 1). The LOAEL remained at 2.5 µM and the IC₅₀ values of thiram were still approximately 4–5 µM (Table 1). Ca²⁺ ionophore A23187 has the capacity to promote extracellular Ca²⁺ entry into sperm, thus enhancing SM and the AR (15,16). Washed spermatozoa underwent simultaneous incubation with A23187 and thiram for 3 h. A23187 failed to prevent thiram-induced suppression of SM and FM (Figure 7 and Table 1). The LOAEL remained at 2.5 µM and the IC₅₀ values of thiram were still approximately 3 µM (Table 1).

Figure 7 Impact of thiram on SM and FM in normal human spermatozoa following co-incubation with calcium and ROS signal modulators. Spermatozoa underwent incubation with varying thiram concentrations alongside progesterone (P4), A23187, and VE for 3 h. (A,C,E) SM. (B,D,F) FM; mean ± SEM, n=6. ***P<0.001 denotes a significant difference versus the control (BWW). All concentration values are expressed in µM. BWW, Biggers-Whitten-Whittingham; FM, forward motility; ROS, reactive oxygen species; SEM, standard error of the mean; SM, sperm motility; VE, vitamin E.

The effect of VE on thiram-induced suppression of human SM and FM

Since thiram potentially influences mitochondrial ROS levels, the antioxidant VE was employed to treat normal human spermatozoa and co-incubated with thiram for 3 h. VE failed to prevent the thiram-induced suppression of SM and FM (Figure 7 and Table 1). The LOAEL was 2.5 µM and the IC₅₀ values of thiram were still around 4 µM (Table 1).

Discussion

In this study, we demonstrated that the fungicide thiram markedly suppressed human SM and FM. Further examination of thiram’s dose-dependent effects revealed that thiram acts as a potent inhibitor of SM and FM in normal human spermatozoa. Thiram also inhibited sperm capacitation and the AR. The inhibition of thiram is irreversible and may be associated with suppression of TPK and PAK4 activity.

Thiram markedly elevated the occurrence rate of abnormal sperm. Evidently, thiram suppressed SM, FM, hyperactivation, capacitation, and spontaneous AR at concentrations substantially lower than those required to suppress sperm viability and decrease MMP. The IC₅₀ values for thiram’s inhibitory effects on SM and FM in normal human spermatozoa were approximately 4 µM (Table 1). Thiram suppressed sperm hyperactivation as demonstrated by the reduction of VCL, VSL, LIN, VAP, and ALH (Table 1). Thiram suppressed sperm capacitation and spontaneous AR with IC₅₀ values below 5 µM (Table 1). Nevertheless, thiram only marginally suppressed sperm viability and MMP at 10 µM. Thiram also failed to induce sperm DNA damage at 10 µM.

The cAMP cascade demonstrates a pivotal function in regulating human SM and capacitation (13). Nevertheless, our findings revealed that thiram reduced intracellular cAMP levels at concentrations of at least 10 µM, whereas SM and FM inhibition occurred at a LOAEL of 2.5 µM. Co-culture experiments utilizing FSK, 8Br-AMP, and PTX alongside thiram showed that these compounds failed to counteract thiram-mediated suppression of SM and FM. These observations suggest that thiram’s mechanism of action operates downstream of cAMP signaling.

The Ca²⁺ influx signaling serves a crucial function in SM, FM, capacitation, and the AR, with Ca²⁺ capable of cross-talking to the cAMP cascade through activation of soluble adenylyl cyclase (13). Nevertheless, P4, a steroid that specifically triggers the sperm-specific calcium channel CatSper, together with A23187, a calcium ionophore, does not reverse the thiram-induced inhibition of SM and FM (Table 1), indicating that thiram implements its suppressive effects outside the realm of CatSper stimulation by progesterone and Ca²⁺ signaling.

ROS represents an oxygen metabolite. At physiological concentrations, ROS serves a crucial function in maintaining male reproductive physiological functions, encompassing cell signal transduction, hormone production, sperm capacitation, AR, and SM (17,36). Although thiram can reduce ROS levels to as low as 1.25 M, it demonstrates potency in inhibiting SM and FM. Previous and recent studies have shown that the thiram analog ziram can elevate ROS levels in somatic cells (37). In this investigation, thiram reduced ROS levels in normal human spermatozoa while increasing mitochondrial O₂⁻ levels at concentrations as low as 2.5 M, suggesting that thiram may impact mitochondrial ROS (38). Despite potential for ROS to exceed antioxidant capacity, triggering oxidative stress and DNA damage, thiram showed no impact on sperm DNA integrity (unaltered DFI). The antioxidant VE was proven ineffective in counteracting thiram’s inhibitory effects on SM and FM (39). This inhibitory effect may be mediated through increased mitochondrial O₂⁻ and suppression of TPK and PAK4 (40). Previous studies indicate that antioxidants can mitigate ROS-induced sperm dysfunction and that ROS exerts both beneficial and harmful effects depending on concentration, highlighting mitochondrial regulation as a key factor in motility and capacitation (39,41-43).

Sperm mitochondria serve a crucial function in SM and FM through ATP generation. During energy production, mitochondria accumulate electrochemical potential energy within the inner mitochondrial membrane. The uneven dispersion of proton and additional ion concentrations across both sides of the inner membrane establishes MMP. SM serves as the primary determinant of male fertility, with MMP being associated with SM (40,44). It has been established that thiram analog ziram can diminish MMP in somatic cells, encompassing Leydig cells (37). Furthermore, thiram markedly decreased sperm MMP at 10 M. Morphological examination via electron microscopy revealed that thiram induces mitochondrial damage at concentrations of 2.5, 5 and 10 M. These microscopic alterations may indicate the irreversible suppression of SM and FM (42).

Prior research has demonstrated that protein tyrosine phosphorylation serves a crucial function in sperm capacitation and the AR (45). Tyrosine phosphorylation may be mediated by TPK and Ser/Thr kinases (45). Notably, thiram exhibited significant inhibition of TPK at concentrations as low as 0.625 M. This suggests that TPK shows a strong correlation with thiram-induced suppression of SM, FM, and capacitation (43). Additionally, thiram influenced PAK4 levels at concentrations as low as 2.5 M, with PAK4 having been recently characterized in human sperm (46).

Thiram is a dimethyl dithiocarbamate fungicide, extensively utilized in agricultural seed treatment, soil enhancement, and foliar applications on grains, fruits, and vegetables and is also employed as a vulcanization accelerator in the rubber industry for producing plastics, paints, dressings and sterile soaps (4). Humans expose to it through food consumption, dermal contact and respiratory inhalation (47). In this investigation, the potential detrimental effects of thiram on human SM, FM, capacitation, and spontaneous AR were examined. The findings demonstrated that thiram can suppress SM, FM, capacitation, and AR at environmentally relevant concentrations. Additionally, Thiram can impair SM and FM at low concentrations. Its in vitro effects raise potential concerns for male reproductive health, although human exposure levels and epidemiological data remain limited. The study is limited by its exclusive reliance on in vitro experiments. Future studies using in vivo rodent exposure model are warranted to evaluate dose-response relationships, reproductive endpoints, and systemic toxicity of thiram at environmentally relevant concentrations (12,40). The epidemiological investigation between male sperm quality and thiram exposure levels merits further research.

Conclusions

In conclusion, thiram strongly inhibits human SM, FM, capacitation, and spontaneous AR in vitro. These effects are mediated through increased mitochondrial O₂⁻ and suppression of TPK and PAK4, providing a clear mechanistic overview. Given the widespread agricultural and industrial use of thiram, these findings underscore the importance of regulatory monitoring and risk assessment for male reproductive health.

Acknowledgments

We would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-940/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-940/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-940/prf

Funding: This study was supported by Chengdu Medical Research Projects of Chengdu Municipal Health Commission (Nos. 2021241, to Z.W. and 2021387, to R.S.G.), Sichuan Medical Research Project Program of Sichuan Medical Association (No. S21080, to Z.W.), and Chengdu Technology Innovation Research and Development Project of Chengdu Bureau of Science and Technology (No. 2022-YF05-01601-SN, to R.S.G.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-940/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Sichuan Jinxin Xi’nan Women and Children Hospital (2021: Number 4), and all patients provided informed consent.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Levine H, Jørgensen N, Martino-Andrade A, et al. Temporal trends in sperm count: a systematic review and meta-regression analysis. Hum Reprod Update 2017;23:646-59. [Crossref] [PubMed]
2. Levine H, Jørgensen N, Martino-Andrade A, et al. Temporal trends in sperm count: a systematic review and meta-regression analysis of samples collected globally in the 20th and 21st centuries. Hum Reprod Update 2023;29:157-76. [Crossref] [PubMed]
3. Soleimanzadeh A, Kian M, Moradi S, et al. Carob (Ceratonia siliqua L.) fruit hydro-alcoholic extract alleviates reproductive toxicity of lead in male mice: Evidence on sperm parameters, sex hormones, oxidative stress biomarkers and expression of Nrf2 and iNOS. Avicenna J Phytomed 2020;10:35-49.
4. Caldas ED, Conceição MH, Miranda MC, et al. Determination of dithiocarbamate fungicide residues in food by a spectrophotometric method using a vertical disulfide reaction system. J Agric Food Chem 2001;49:4521-5. [Crossref] [PubMed]
5. Soleimanzadeh A, Kian M, Moradi S, et al. Protective effects of hydro-alcoholic extract of Quercus brantii against lead-induced oxidative stress in the reproductive system of male mice. Avicenna J Phytomed 2018;8:448-56.
6. Mohammadnehjad K, Mohammadi R, Soleimanzadeh A, et al. Provitamin A carotenoid (β-cryptoxanthin) ameliorated testicular ischemia-reperfusion injury in mature rats. Vet Res Forum 2025;16:277-84. [Crossref] [PubMed]
7. Nourian A, Soleimanzadeh A, Shalizar Jalali A, et al. Bisphenol-A analogue (bisphenol-S) exposure alters female reproductive tract and apoptosis/oxidative gene expression in blastocyst-derived cells. Iran J Basic Med Sci 2020;23:576-85. [Crossref] [PubMed]
8. Soleimanzadeh A, Mohammadnejad L, Ahmadi A. Ameliorative effect of Allium sativum extract on busulfan-induced oxidative stress in mice sperm. Vet Res Forum 2018;9:265-71. [Crossref] [PubMed]
9. Kabirian A, Batavani RA, Asri-Rezaei S, et al. Comparative study of the protective effects of chicken embryo amniotic fluid, vitamin C and coenzyme Q10 on cyclophosphamide-induced oxidative stress in mice ovaries. Vet Res Forum 2018;9:217-24. [Crossref] [PubMed]
10. Nourian A, Soleimanzadeh A, Shalizar Jalali A, et al. Effects of bisphenol-S low concentrations on oxidative stress status and in vitro fertilization potential in mature female mice. Vet Res Forum 2017;8:341-5.
11. Rice EW. Morphological changes in human spermatozoa following treatment of semen with certain dialkyldithiocarbamates. Exp Cell Res 1964;34:186-8. [Crossref] [PubMed]
12. Zolfaghari S, Soleimanzadeh A, Baqerkhani M. The synergistic activity of fisetin on quercetin improves testicular recover in ischemia-reperfusion injury in rats. Sci Rep 2025;15:12053. [Crossref] [PubMed]
13. Puga Molina LC, Luque GM, Balestrini PA, et al. Molecular Basis of Human Sperm Capacitation. Front Cell Dev Biol 2018;6:72. [Crossref] [PubMed]
14. Bucak MN, Taravat M, Korkmaz O, et al. Effects of co-supplementation of trehalose with fetuin and taxifolin in freezing extender on quality parameters of bull spermatozoa following freeze-thaw process. Cryobiology 2025;121:105330. [Crossref] [PubMed]
15. Okabe M. Sperm-egg interaction and fertilization: past, present, and future. Biol Reprod 2018;99:134-46. [Crossref] [PubMed]
16. Stival C, Puga Molina Ldel C, Paudel B, et al. Sperm Capacitation and Acrosome Reaction in Mammalian Sperm. Adv Anat Embryol Cell Biol 2016;220:93-106. [Crossref] [PubMed]
17. Pereira R, Sá R, Barros A, et al. Major regulatory mechanisms involved in sperm motility. Asian J Androl 2017;19:5-14. [Crossref] [PubMed]
18. Navarro G, Gómez-Autet M, Morales P, et al. Homodimerization of CB(2) cannabinoid receptor triggered by a bivalent ligand enhances cellular signaling. Pharmacol Res 2024;208:107363. [Crossref] [PubMed]
19. EPA. Registration eligibility decision for ziram (PC Code: 034805 Case: 2180). Washington (DC): US Environmental Protection Agency; 2003:1-118.
20. Biggers JD, Whitten WK, Whittinghan DC. The culture of mouse embryos in vitro. In: Daniel JC. editor. Methods in mammalian embryology. San Francisco: Freeman Press; 1971:86-116.
21. WHO. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge: Cambridge University Press; 2010.
22. Soleimanzadeh A, Talavi N, Shafiepour Yourdshahi V, et al. Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process. Cryobiology 2020;95:29-35. [Crossref] [PubMed]
23. Mortimer ST, Swan MA, Mortimer D. Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa. Hum Reprod 1998;13:2139-46. [Crossref] [PubMed]
24. Ramazani N, Mahd Gharebagh F, Soleimanzadeh A, et al. The influence of L-proline and fulvic acid on oxidative stress and semen quality of buffalo bull semen following cryopreservation. Vet Med Sci 2023;9:1791-802. [Crossref] [PubMed]
25. Izanloo H, Soleimanzadeh A, Bucak MN, et al. The effects of varying concentrations of glutathione and trehalose in improving microscopic and oxidative stress parameters in Turkey semen during liquid storage at 5 °C. Cryobiology 2021;101:12-9. [Crossref] [PubMed]
26. Izanloo H, Soleimanzadeh A, Bucak MN, et al. The effects of glutathione supplementation on post-thawed Turkey semen quality and oxidative stress parameters and fertilization, and hatching potential. Theriogenology 2022;179:32-8. [Crossref] [PubMed]
27. Sheikholeslami SA, Soleimanzadeh A, Rakhshanpour A, et al. The evaluation of lycopene and cysteamine supplementation effects on sperm and oxidative stress parameters during chilled storage of canine semen. Reprod Domest Anim 2020;55:1229-39. [Crossref] [PubMed]
28. Shakouri N, Soleimanzadeh A, Rakhshanpour A, et al. Antioxidant effects of supplementation of 3,4-dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen. Reprod Domest Anim 2021;56:1004-14. [Crossref] [PubMed]
29. Ramazani N, Mahd Gharebagh F, Soleimanzadeh A, et al. Reducing oxidative stress by κ-carrageenan and C60HyFn: The post-thaw quality and antioxidant status of Azari water buffalo bull semen. Cryobiology 2023;111:104-12. [Crossref] [PubMed]
30. Liu JH, Li Y, Cao ZG, et al. Influences of dibutyryl cyclic adenosine monophosphate and forskolin on human sperm motility in vitro. Asian J Androl 2003;5:113-5.
31. Tarlatzis BC, Kolibianakis EM, Bontis J, et al. Effect of pentoxifylline on human sperm motility and fertilizing capacity. Arch Androl 1995;34:33-42. [Crossref] [PubMed]
32. Achikanu C, Pendekanti V, Teague R, et al. Effects of pH manipulation, CatSper stimulation and Ca2+-store mobilization on Ca2+i and behaviour of human sperm. Hum Reprod 2018;33:1802-11. [Crossref] [PubMed]
33. Ourique GM, Saccol EM, Pês TS, et al. Protective effect of vitamin E on sperm motility and oxidative stress in valproic acid treated rats. Food Chem Toxicol 2016;95:159-67. [Crossref] [PubMed]
34. Rezazadeh A, Najafpour A, Soleimanzadeh A, et al. Intra-testicular injection of titanium dioxide nanoparticles induces chemical castration in male mice. Biochem Biophys Res Commun 2025;787:152734. [Crossref] [PubMed]
35. Ashoory A, Saberivand A, Soleimanzadeh A, et al. In vitro effects of alpha-ketoglutarate and folic acid supplementation on bull sperm exposed to ammonia stress. Vet Res Forum 2025;16:629-38. [Crossref] [PubMed]
36. Soleimanzadeh A, Karvani N, Davoodi F, et al. Efficacy of silver-doped Carbon dots in Chemical Castration: a rat model study. Sci Rep 2024;14:24132. [Crossref] [PubMed]
37. Xie L, Li X, Mo J, et al. Delayed Puberty by Ziram Is Associated with Down Regulation of Testicular Phosphorylated AKT1 and SIRT1/PGC-1α Signaling. Chem Res Toxicol 2018;31:1315-22. [Crossref] [PubMed]
38. Baqerkhani M, Soleimanzadeh A, Mohammadi R. Effects of intratesticular injection of hypertonic mannitol and saline on the quality of donkey sperm, indicators of oxidative stress and testicular tissue pathology. BMC Vet Res 2024;20:99. [Crossref] [PubMed]
39. Mohammadnejad K, Mohammadi R, Soleimanzadeh A, et al. The effect of β-cryptoxanthin on testicular ischemia-reperfusion injury in a rat model: evidence from testicular histology. Iran J Vet Surg 2025;20:110-5.
40. Behnejad G, Mohammadi T, Soleimanzadeh A. Allicin and hesperidin protect sperm production from environmental toxins in mice. Sci Rep 2025;15:14224. [Crossref] [PubMed]
41. Mostahsan Z, Azizi S, Soleimanzadeh A, et al. The protective roles of Mito-TEMPO on testicular ischemia-reperfusion injury based on biochemical and histopathological evidences in mice. Iran J Vet Surg 2024;19:97-105.
42. Mostahsan Z, Azizi S, Soleimanzadeh A, et al. Protective effects of Mito-TEMPO on ischemia-reperfusion injury in a mouse testicular torsion and detorsion model. Vet Res Forum 2024;15:665-72. [Crossref] [PubMed]
43. Zeynali E, Soleimanzadeh A, Azizi S. Synergistic protection of alpha-glucosyl hesperidin and procyanidin against testicular ischemia-reperfusion injury. J Food Biochem 2025;49:5634210.
44. Agnihotri SK, Agrawal AK, Hakim BA, et al. Mitochondrial membrane potential (MMP) regulates sperm motility. In Vitro Cell Dev Biol Anim 2016;52:953-60. [Crossref] [PubMed]
45. Naz RK, Rajesh PB. Role of tyrosine phosphorylation in sperm capacitation / acrosome reaction. Reprod Biol Endocrinol 2004;2:75. [Crossref] [PubMed]
46. Castillo J, Knol JC, Korver CM, et al. Human Testis Phosphoproteome Reveals Kinases as Potential Targets in Spermatogenesis and Testicular Cancer. Mol Cell Proteomics 2019;18:S132-44. [Crossref] [PubMed]
47. Asma ST, Acaroz U, Imre K, et al. Natural Products/Bioactive Compounds as a Source of Anticancer Drugs. Cancers (Basel) 2022;14:6203. [Crossref] [PubMed]