Triclocarban impacts human sperm motility by inhibiting glycolysis and oxidative phosphorylation

Abstract

Triclocarban (TCC) is a broad-spectrum antimicrobial widely used in various personal care products, textiles, and children’s toys. TCC has potential reproductive and developmental toxicity in animals. However, little is known regarding the effect of TCC on human sperm function. In this study, an in vitro assay was used to investigate the effects of TCC on normal human spermatozoa and the possible underlying mechanisms involved. Semen from healthy male donors was collected and cultured in complete Biggers, Whitten and Whittingham (BWW) and low-sugar BWW media, followed by treatment with TCC at concentrations of 0, 0.1 µmol l⁻¹, 1 µmol l⁻¹, 10 µmol l⁻¹, and 100 µmol l⁻¹ for 4 h. TCC was found to reduce the sperm total motility and progressive motility. Moreover, the sperm kinematic parameters, straight-line velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL) were affected in a dose-dependent manner. After treatment with TCC at the lowest effective concentration of 10 µmol l⁻¹, TCC caused a significant decrease in mitochondrial adenosine triphosphate (ATP) production and mitochondrial membrane potential (MMP) and a significant increase in reactive oxygen species (ROS), similar to the observations with the positive control carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), suggesting that TCC may decrease sperm motility by affecting the oxidative phosphorylation (OXPHOS) pathway. In a sugar-free and low-sugar BWW culture environment, TCC enhanced the damaging effect on sperm motility and ATP, MMP, and lactate decreased significantly, suggesting that TCC may also affect the glycolytic pathway that supplies energy to spermatozoa. This study demonstrates a possible mechanism of TCC toxicity in spermatozoa involving both the OXPHOS and glycolysis pathways.

INTRODUCTION

In September 2016, the U.S. Food and Drug Administration (FDA) banned triclocarban (TCC) in over-the-counter consumer antiseptic wash products. The rationale was that there was insufficient evidence to demonstrate the safety of these ingredients for long-term daily use or that they could reduce the spread of illness and infection. Moreover, scientists from both academia and non-profit organizations co-authored The Florence Statement in 2016, which called on the international community to limit the production and use of agents such as TCC, calling for a broader consideration of the impact of antimicrobial use, especially on safety.1

TCC has broad-spectrum antimicrobial activity against most Gram-negative and Gram-positive bacteria. TCC is widely used in various personal care products (PCPs), such as antibacterial soaps, toothpastes, mouthwashes, textiles, children’s toys, and other household products.2,3 TCC persists in the environment, and transformation products and byproducts bioaccumulate in aquatic plants and animals. This compound is frequently identified in wastewater, surface water, sediment, and aquatic organisms.4 TCC has been detected in 88% of water systems in the USA, as well as in 88% of water samples and 100% of sediment samples from multiple sections of Dongjiang River Basin and its tributaries in China.5 To date, the maximum reported concentration of TCC in surface water is 6.75 μg l⁻¹ in urban streams in the USA. The content of TCC (mean ± standard deviation [s.d.]) in activated sludge in a sewage treatment plant was 51 ± 15 μg g⁻¹ (dry soil).6 Humans are exposed to TCC through direct contact with PCPs and from other sources, including food, drinking water, and dust. Excessive environmental exposure to TCC increases human exposure levels; however, research on these levels is limited.

Along with the discovery of environmental pollution, several studies have demonstrated the acute and subchronic toxicity of TCC to aquatic organisms at environmentally relevant concentrations.7,8 In addition, TCC has been shown to have endocrine-disrupting effects. TCC disrupts estrogens and androgens in the endocrine system in mammalian models9 and has estrogenic effects in vitro.10 In addition, TCC directly damages the proliferation and antioxidant status of mouse testicular Sertoli cells,11 which is hypothesized to have potentially detrimental effects on the reproductive and developmental systems of animals, especially humans. However, little is known regarding the effect of TCC on human sperm; thus, potential implications for human reproduction are of concern and merit further study.

Human spermatozoa are highly specialized cells that are excellent in vitro targets for the study of male reproductive health and toxicity. High-quality sperm function is essential for male fertility. Adenosine triphosphate (ATP) is the main energy source for maintaining the vitality and function of sperm. Glucose metabolism is the main method of energy metabolism. Most studies have suggested that ATP is produced in sperm mainly through two pathways of glucose metabolism, glycolysis and oxidative phosphorylation (OXPHOS).12 The former occurs in the head and principal piece of the flagellum, whereas the latter occurs in the mitochondria in the midpiece of the tail.13 However, when and how sperm select metabolic pathways remains uncertain.13 Some researchers have proposed that even under aerobic conditions, intracytoplasmic glycolysis is the main source of ATP production in sperm, which is similar to the Warburg effect in tumor cells. Both the glycolysis and OXPHOS pathways are necessary for human sperm function and successful fertilization and are often used as targets to study the effects of exogenous substance exposure on sperm function.12 Under normal physiological conditions, metabolic processes are strictly and finely regulated to maintain the internal environment’s stability and adapt to physiological activities. If exogenous chemicals stimulate the sperm, changes in the plasma membrane and dynamics may occur, leading to changes in energy metabolism processes, affecting the sperm’s ability to combine with and fertilize oocytes.14

In vitro incubation is a commonly used research method to assess an organism’s response to external stimuli. Using this method, researchers can control experimental conditions, such as the dose and duration of exposure, to study the effects of xenobiotics on the biological properties of spermatozoa.15,16,17 For example, in vitro oregano essential oil supplementation significantly improved sperm motility in bulls, dogs, and rabbits, which can have important implications for the success of artificial insemination.15 By contrast, some exogenous chemicals that are considered endocrine disruptors (EDs), such as cadmium, have been reported to inhibit mouse sperm motility and ATP levels.16 The herbicide roundup was found to exert a deleterious effect on the progressive motility and mitochondrial dysfunction of human sperm.17

In 2018, TCC was identified as a mitochondrial toxicant that can significantly decrease the mitochondrial membrane potential (MMP), respiratory control ratio (RCR), and ATP of HepG2 cells and rat hepatocytes.18 The mitochondrion, as the powerhouse of the cell, is also a biomarker of sperm health and fertility.19 TCC can potentially reduce mitochondrial activity in human spermatozoa and adversely affect sperm motility and function. Glycolysis is another important way for sperm to obtain energy.13 By contrast, low-glycemic media can mimic the physiological environment and help study sperm metabolism and function under energy-limited conditions, providing an important tool for in-depth studies of sperm motility.20 This study aimed to characterize the effects of TCC on human sperm motility, kinematic parameters, and mitochondrial function. The results obtained help improve our understanding of the toxicity pathways of TCC in human spermatozoa and provide a critical reference for reproductive problems caused by exogenous substances.

PARTICIPANTS AND METHODS

Medium

Biggers, Whitten and Whittingham (BWW) medium was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). The complete glucose-containing BWW medium (BWW+) consisted of 5.6 mmol l⁻¹ D-glucose, 44 mmol l⁻¹ sodium lactate, 0.27 mmol l⁻¹ sodium pyruvate, 95 mmol l⁻¹ NaCl, 25 mmol l⁻¹ NaHCO₃, 4.6 mmol l⁻¹ KCl, 1.7 mmol l⁻¹ CaCl₂, 1.2 mmol l⁻¹ KH₂PO₄, 1.2 mmol l⁻¹ MgSO₄, 3.5 mg ml⁻¹ bovine serum albumin (BSA), 10 U ml⁻¹ penicillin, and 10 mg ml⁻¹ streptomycin, pH 7.4. BWW stock solution (BWW-) is devoid of any kind of glucose, lactate, or pyruvate. We also prepared low-glucose BWW medium, containing 2.75 mmol l⁻¹ D-glucose (BWW-G2.75+), and modified BWW medium, containing 5.5 mmol l⁻¹ D-glucose (BWW-G5.5+), based on BWW stock medium.

Human semen samples and sperm processing

This study was conducted with the approval of the Human Subjects Ethics Committee of the National Research Institute for Family Planning (Beijing, China; Approval No. NRIFP2023024) and the Medical Ethical Review Committee of the National Institute for Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention (Beijing, China; Approval No. NIOHP202206). All donors provided written consent before participating in the study and were recruited from the Reproductive Health Research Centre/Human Sperm Bank at the National Research Institute for Family Planning and the Center for Assisted Reproductive Medicine at the Sixth Medical Center, Chinese PLA General Hospital (Beijing, China). The donors were aged 22–48 years and were in good health. Those at high risk for sexually transmitted infections, genetic diseases, and occupational exposure to hazardous factors were excluded. One hundred and ten semen samples from healthy donors were collected by masturbation according to the 5^th edition World Health Organization (WHO) manual.21

After allowing at least 30 min for liquefaction to occur, a preliminary assessment of sperm viability and motility was conducted via computer-aided semen analysis (CASA; Hamilton Thorne-IVOS CASA, Beverly, MA, USA). Normal sperm criteria were defined as a concentration ≥15 × 10⁶ ml⁻¹, progressive motility (PM) ≥32%, total motility (TM) ≥40%, normal sperm morphology forms ≥4%, and leukocytes <1 × 10⁶ ml⁻¹ according to the WHO manual.21 The normal semen was separated on a discontinuous two-step Percoll gradient (80%:40%) as described in the WHO recommended procedure.21 The spermatozoa in the bottom region of the gradient were washed twice with 1 ml of BWW media, centrifuged at 300g for 10 min (3K15; Sigma, Yangzhou, China), and finally resuspended in BWW+, BWW-, or low-glucose BWW media at room temperature. All experiments were performed with high-density sperm to ensure sufficient sperm and good motility. Unless otherwise mentioned, 5 × 10⁶ ml⁻¹ cells were aliquoted into each tube for each experiment.

Chemicals and reagents

The chemicals and reagents used were of research grade. TCC, rhodamine 123 (Rh123), fluorescein isothiocyanate-labeled pisum sativum agglutinin (FITC-PSA), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Percoll, D-glucose, and 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Beijing Solarbio Science & Technology Co., Ltd.. The TCC, Rh123, and FCCP stock solutions were prepared in DMSO. FITC-PSA was dissolved in pure water.

TCC treatment and selection of the optimal TCC concentration

Stock solutions of TCC prepared with DMSO were stored at 4°C and serially diluted to 0.1 μmol l⁻¹, 1 μmol l⁻¹, 10 μmol l⁻¹, or 100 μmol l⁻¹ with BWW+ media. The maximum DMSO concentration in the treatments was maintained below 0.1% (v/v), which did not affect sperm motility. The corresponding vehicle control was used in each experiment, with an equivalent DMSO concentration as the negative control. The sperm suspensions were incubated with TCC for 30 min, 1 h, 2 h, 3 h, 4 h, or 12 h at the room temperature. After the pre-experiment, 4 h was chosen as the most suitable incubation time. FCCP, an uncoupler of OXPHOS, was used to treat the sperm suspension at a final concentration of 5 μmol l⁻¹ for 4 h as a positive control.

The TM, PM, curvilinear velocity (VCL), average path velocity (VAP), and straight-line velocity (VSL) of sperm treated with TCC were evaluated via CASA using Hamilton Thorne-IVOS CASA. On the basis of these results, the optimal effective concentration of TCC exposure was selected for further experiments.

Evaluation of sperm motility

As mentioned above, sperm motility and kinematics were evaluated via CASA. Ten microliters of each sperm sample at a concentration of 20 × 10⁶ ml⁻¹ was placed into a prewarmed (37°C) Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). At least 500 spermatozoa per sample were analyzed in five different randomly selected fields. The sperm TM, PM, and kinematic parameters VCL (μm s⁻¹), VAP (μm s⁻¹), and VSL (μm s⁻¹) were recorded. Spermatozoa exhibiting VAP >5 μm s⁻¹ were categorized as progressively motile spermatozoa, whereas spermatozoa with VAP <5 μm s⁻¹ or VSL <11 μm s⁻¹ were considered nonprogressive spermatozoa. The sum of progressive and nonprogressive spermatozoa constituted the TM.22

Assessment of sperm treated with the optimal TCC concentration in low-glucose BWW media

On the basis of the changes in sperm motility and kinematics after exposure to TCC (0.1 μmol l⁻¹, 1 μmol l⁻¹, 10 μmol l⁻¹, or 100 μmol l⁻¹), 10 μmol l⁻¹ TCC was selected as the optimal effective concentration for further experiments. To assess the effects of low-glucose media on sperm motility, the sperm suspensions were exposed to 10 μmol l⁻¹ TCC and incubated for 4 h in BWW-, BWW-G2.75+, BWW-G5.5+, or BWW+ media.

Detection of the mitochondrial membrane potential (MMP)

The MMP was measured with a Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit (AAT Bioquest, Sunnyvale, CA, USA), as per the manufacturer’s instructions. JC-10, a fluorescent cationic lipophilic dye, has been developed to be a superior alternative to JC-1 and has much better water solubility. JC-10 can selectively enter the mitochondria and reversibly change color from green to orange as the membrane potential increases. In normal cells with a high MMP, JC-10 is concentrated in the mitochondrial matrix, forming red or orange fluorescent aggregates. However, in apoptotic and necrotic cells with a low MMP, JC-10 exists in monomeric form and stains cells green. After TCC treatment, the sperm suspensions (500 μl) containing 1 × 10⁶ spermatozoa in the different groups were stained with JC-10 (1×) at 37°C in the dark for 30 min and then analyzed via a flow cytometer (Accui™ C6; BD Biosciences, San Diego, CA, USA). Green fluorescence (480–530 nm) was detected in the FL-1 channel, and orange-red fluorescence (580–630 nm) was detected in the FL-2 channel. The FL2/FL1 ratio was recorded. The staining effect and cell localization were observed via a fluorescence microscope (Nikon, Tokyo, Japan).

Measurement of ATP

The intracellular ATP content was determined via an EZScreen™ ATP Colorimetric Assay Kit (BioVision, Milpitas, CA, USA), per the manufacturer’s directions. The sperm samples were centrifuged at 18 000g for 10 min at 4°C (3K15; Sigma), and the resulting supernatant was used for the assay. The kit utilizes a series of enzymatic reactions to form a product that is easily quantified at an optical density (OD) of 570 nm. The relative light unit was measured by a Tecan Infinite® 200 Pro Microplate Reader (TECAN, Grodig, Austria). The standard curve was plotted each time, and the ATP content was calculated according to the curve.

Measurement of intracellular reactive oxygen species (ROS) production

DCFH-DA, an oxidation-sensitive fluorescent probe, was used to analyze ROS generation. DCFH-DA is hydrolyzed into DCFH by esterase after it enters cells. Intracellular ROS can oxidize nonfluorescent DCFH to generate fluorescent DCF. The treated sperm samples were incubated with 10 μmol l⁻¹ DCFH-DA for 30 min at 37°C. The mean fluorescence intensity (MFI) was recorded via flow cytometry. The smear was prepared by washing and resuspension, and the staining effect and cell localization were observed via a laser scanning confocal microscope (LSM 800; Zeiss, Oberkochen, Germany).

Lactate determination

Lactate levels in the culture media from spermatozoa were determined via a commercial kit (Lactate Assay Kit K607; BioVision). Lactate specifically reacts with an enzyme mixture to generate a product, which interacts with the lactate probe to produce color (OD 570 nm) and fluorescence (Ex/Em = 535/587 nm). After preparing the reaction mixture according to the supplier’s instructions, the OD was measured at 570 nm via a microplate reader (TECAN, Grodig, Austria). Lactate standard curves were constructed using solutions containing known concentrations of lactate. Then, the corrected OD was applied to the standard curve to obtain the lactate levels in the sample well.

Evaluation of acrosome integrity

Acrosome integrity was evaluated according to the WHO-recommended procedure.21 The sperm suspension concentration was adjusted to 2 × 107 ml⁻¹. Then, 5 μl sperm suspension from each sample was made into a smear, and after being air-dried and fixed with 95% ethanol, 10 μl of FITC-PSA (1 μg μl⁻¹) was added to each smear for >1 h at 4°C. An anti-fluorescence attenuation agent was used to seal the smear. The acrosome integrity rate (AI%) was recorded via a fluorescence microscope. The sperm were classified as follows: (1) acrosomal integrity (AI): more than half of the head was stained with fluorescence brightly and evenly; (2) acrosomal reaction (AR): fluorescence only appeared in the equatorial zone, or there was no fluorescence staining in the acrosomal region; and (3) acrosomal abnormality (AA): all sperm except the above-mentioned two types.

Statistical analyses

Statistical analysis was performed via SPSS version 25.0 statistical software (SPSS Inc., Chicago, IL, USA). Variables were assessed for a normal distribution via the Kolmogorov‒Meier test. Given the normality of the distribution, statistical analysis was performed via one-way analysis of variance (ANOVA), followed by Dunnett’s t-test for multiple comparisons when appropriate. The result is presented as the mean ± s.d. P < 0.05 was considered to indicate significance. GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used to construct the diagrams.

RESULTS

Effects of TCC on the motility of human spermatozoa in complete BWW medium

Aliquots of the sperm suspensions were exposed to increasing concentrations of TCC (0.1 μmol l⁻¹, 1 μmol l⁻¹, 10 μmol l⁻¹, or 100 μmol l⁻¹) in BWW+ media for 4 h. The sperm exposed to FCCP (5 μmol l⁻¹) were used as a positive control. As shown in Figure 1a, exposure to 10 μmol l⁻¹ and 100 μmol l⁻¹ TCC significantly decreased the TM% compared with that of the negative control (P < 0.0001). Compared with the negative control, all the concentrations except 0.1 μmol l⁻¹ TCC reduced the PM% (P < 0.001). Compared with the negative control, incubation of sperm in the presence of FCCP resulted in significantly decreased TM% and PM% (both P < 0.0001). The effects of TCC on sperm kinematic parameters were also determined. After 4 h of incubation, over 10 μmol l⁻¹ TCC significantly decreased VAP, VSL, and VCL (all P < 0.05). Similarly, compared with the negative control, FCCP significantly reduced VAP, VSL, and VCL (all P < 0.05; Figure 1b–1d).

Figure 1.

Effects of TCC on human sperm motility and kinematic parameters. Human spermatozoa were incubated for 4 h in complete BWW media supplemented with TCC (0, 0.1 µmol l⁻¹, 1 µmol l⁻¹, 10 µmol l⁻¹, or 100 µmol l⁻¹) or FCCP (5 µmol l⁻¹). (a) TM and PM, (b) VAP, (c) VCL, and (d) VSL were measured. ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001 (n = 8 for each group, with different donors), the indicated value compared with that in the control group. The results are presented as the mean ± standard deviation. TM: total motile spermatozoa; PM: progressive motile spermatozoa; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight line velocity; TCC: triclocarban; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone.

The motility and kinematic parameters of normal spermatozoa in low-glucose BWW medium

To evaluate sperm motility in media-containing low glucose and the absence of substrate media, normal human spermatozoa were incubated in different media for 4 h: (1) BWW-, (2) BWW-G2.75+, (3) BWW-G5.5+, and (4) BWW+. As shown in Figure 2, the TM%, PM%, VAP, and VSL of sperm were significantly lower in media containing low glucose (BWW-G2.75+) or in the absence of substrate media, such as BWW-G5.5+ and BWW-, than in the BWW+ group (all P < 0.05). The VCLs of the BWW- and BWW-G2.75+ groups were significantly lower (both P < 0.0001).

Figure 2.

Human sperm motility and kinematic parameters in different media. Normal human spermatozoa were incubated for 4 h in BWW-, BWW-G2.75+, BWW-G5.5+, and BWW+ groups. (a) TM and PM, (b) VAP, (c) VSL, and (d) VCL were measured. ^*P < 0.05, and ^****P < 0.0001 (n = 8 for each group), the indicated value compared with that in the BWW+ group. BWW-: BWW stock solution, free of glucose, lactate and pyruvate; BWW-G2.75+: BWW stock medium containing 2.75 mmol l^-1 D-glucose; BWW-G5.5+: BWW stock medium containing 5.5 mmol l^-1 D-glucose; BWW+: complete glucose BWW. TM: total motile spermatozoa; PM: progressive motile spermatozoa; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight line velocity; BWW: Biggers, Whitten and Whittingham.

Effects of TCC on the motility of human spermatozoa in low-glucose BWW medium

According to the results of the motility of human spermatozoa exposed to TCC in BWW+ media, a dose of TCC greater than 10 μmol l⁻¹ significantly impaired sperm motility and kinematic parameters. Thus, 10 μmol l⁻¹ could be regarded as the effective concentration. Aliquots of the sperm suspensions were exposed to 10 μmol l⁻¹ TCC for 4 h in different media: BWW-, BWW-G2.75+, BWW-G5.5+, and BWW+ (Figure 3). In addition, sperm motility decreased in a dose-dependent manner, that is, the lower the sugar content in the media, the lower the sperm motility.

Figure 3.

Effects of TCC (10 µmol l^-1) on human sperm motility and sperm kinematics in different media. (a) TM and PM and (b) VAP, VSL, and VCL were measured. ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001 (n = 8 for each group), the indicated value compared with that in the BWW+ group. The group description is shown in Figure 2. TM: total motile spermatozoa; PM: progressive motile spermatozoa; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight line velocity; TCC: triclocarban; BWW: Biggers, Whitten and Whittingham.

Influence of TCC on ROS production

The effect of TCC on reactive oxygen species (ROS) production was evaluated. The sperm suspensions were exposed to TCC (10 μmol l⁻¹) for 4 h in different media: BWW-, BWW-G2.75+, BWW-G5.5+, and BWW+. Normal human spermatozoa not treated with TCC were also incubated in BWW+, which was used as a negative control. Normal human spermatozoa were incubated in BWW+ supplemented with FCCP (5 μmol l⁻¹), which was used as a positive control. Compared with the negative control, the MFI of ROS in the BWW-G2.75+, BWW-G5.5+, BWW+, and FCCP groups was significantly greater (Figure 4); that is, TCC induced an increase in ROS in sperm. Compared with those in the FCCP group, the MFIs of ROS in the BWW-G2.75+, BWW-G5.5+, and BWW+ groups were significantly greater (all P < 0.001). The MFI of ROS in the BWW- and negative control groups decreased. However, the MFI of ROS did not differ between these two groups (both P > 0.05).

Figure 4.

(a) Effects of TCC (10 µmol l⁻¹) on the MFI of human spermatozoa showing ROS production. Normal spermatozoa not treated with TCC were incubated in BWW+ as a negative control (n = 6 for each group). ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001, the indicated value compared with that in the negative control group. ^#P < 0.05, the indicated value compared with that in the positive control group (FCCP). (b) Representative confocal microscopy image of ROS production. The samples were stained with DCFH-DA reagent. Intracellular ROS oxidize nonfluorescent DCFH to generate fluorescent DCF, which is located in the midpiece of spermatozoa. The group description is shown in Figure 2. TM: total motile spermatozoa; PM: progressive motile spermatozoa; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight line velocity; TCC: triclocarban; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; MFI: mean fluorescence intensity; ROS: reactive oxygen species; DCFH-DA: 2’,7’-dichlorodihydrofluorescein diacetate fluorescent probe; DCF: 2’,7’-dichlorofluorescein; BWW: Biggers, Whitten and Whittingham.

Effects of TCC on ATP production

The effects of TCC on ATP production are shown in Figure 5a. Compared with those of the negative control group, the ATP levels of all the treated groups were significantly lower (all P < 0.05). Compared with those of the FCCP group, the ATP levels of the BWW- and BWW-G2.75+ groups were significantly lower ( all P < 0.05).

Figure 5.

Effects of TCC (10 µmol l⁻¹) on (a) ATP production and (b) the MMP of sperm. ^*P < 0.05 and ^****P < 0.0001, the indicated value compared with that in the negative control group. ^#P < 0.05 and ^####P < 0.0001, the indicated value compared with that in the positive control (FCCP) group. (c) Representative fluorescence microscopy image of the MMP and samples were stained with JC-10 reagent. When the MMP is high, JC-10 is polymerized in the mitochondrial matrix and produces orange or red fluorescence; when the MMP is low, JC-10 is monomeric, and the midpiece of spermatozoa is green. The group description is shown in Figure 2. MMP: mitochondrial membrane potential; ATP: adenosine triphosphate; TCC: triclocarban; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; BWW: Biggers, Whitten and Whittingham.

Effects of TCC on the MMP of sperm

The effects of TCC on the MMP of sperm are shown in Figure 5b. Compared with those of the negative control group, the MMPs of all the TCC-treated groups were significantly lower (all P < 0.05). Representative fluorescence microscopy image of the MMP in Figure 5c.

Lactate levels of sperm exposed to TCC in different media

To evaluate the effect of TCC on lactate production, which is a biomarker of the end product of glycolysis, aliquots of the sperm suspensions were exposed to TCC (10 μmol l⁻¹) for 4 h in different media: BWW-, BWW-G2.75+, BWW-G5.5+, and BWW+. The results revealed significant differences in the lactate levels between the BWW- and BWW-G2.75+ groups and the BWW+ group (P < 0.0001; Figure 6a).

Figure 6.

Effects of TCC (10 µmol l⁻¹) on (a) the lactate content and (b) AI of human sperm in different media. ^****P < 0.0001 (n = 6 for each group), the indicated value compared with that in BWW+ group. (c) Representative image under a fluorescence microscope for the assessment of AI. The samples were stained with the FITC-PSA reagent. The group description is shown in Figure 2. AI: acrosome integrity (more than half of the head was stained with fluorescence brightly and evenly); AR: acrosomal reaction (fluorescence only appeared in the equatorial zone, or there was no fluorescence staining in the acrosomal region); AA: acrosomal abnormality (all sperm except the above-mentioned two types); TCC: triclocarban; FITC-PSA: fluorescein isothiocyanate-labeled pisum sativum agglutinin.

Acrosome integrity of sperm exposed to TCC in different media

To evaluate the effect of TCC on the AI of human sperm, 10 μmol l⁻¹ TCC was added to aliquots of the sperm suspensions for 4 h in different media: BWW-, BWW-G2.75+, BWW-G5.5+, and BWW+. The results revealed no differences in the AI% among the groups (all P >0.05; Figure 6b). Representative image under a fluorescence microscope for the assessment of AI (Figure 6c).

DISCUSSION

TCC is an antibacterial and antiseptic agent commonly used in PCPs. It is among the top 10 contaminants of emerging concern (CEC) due to its difficult biodegradation and extensive environmental accumulation.23 As an ED, it can interfere with the body’s hormonal system and affect metabolic enzyme-related functions.24 This disruption can lead to various female reproductive health problems. It affects female hormones, interferes with the menstrual cycle, and affects female development, reproduction, and offspring health. As research has progressed, researchers have focused on the following effects on male reproductive health: lowered testosterone levels, impaired testicular spermatogenesis, and reduced sperm count and quality.25 Previous studies on the estrogenic or androgenic effects of TCC in rodents and aquatic animals, in addition to in vitro studies involving cell lines such as MCF-7, MDA-MB-231, and H295R, have provided more evidence on endocrine endpoints.26 However, research on the reproductive endpoints of TCC, especially its effects on sperm and related mechanisms, is lacking. This study investigates TCC’s effects on sperm motility and energy metabolism in vitro.

In previous in vitro studies on the effects of TCC on reproductive, endocrine, and developmental endpoints, the dose range was 1 × 10⁻⁹ mol l⁻¹–2 × 10^–3 mol l⁻¹.26 In accordance with the dose range used in our preliminary experiments, the doses of TCC used in this study were 0.1 μmol l⁻¹, 1 μmol l⁻¹, 10 μmol l⁻¹, and 100 μmol l⁻¹. TCC decreased the motility (TM and PM) and kinetic parameters (VAP, VSL, and VCL) of normal human spermatozoa. After treatment with TCC at the lowest effective concentration of 10 μmol l⁻¹, TCC caused a significant decrease in mitochondrial ATP production and the MMP, and a significant increase in ROS, which were similar to the toxic effects of FCCP,27 suggesting that TCC may cause a decrease in sperm motility by affecting the mitochondrial OXPHOS pathway. Parabens, which belongs to the category of EDs, inhibited sperm motility and viability in a dose-dependent manner at the concentrations used in commercially available formulations.28 These compounds increase the generation of mitochondrial ROS and stimulate the formation of oxidative DNA adducts. Human sperm exposed to Aroclor 1254 (a commercial polychlorinated biphenyl [PCB] mixture) exhibited reduced sperm motility and kinematic parameters, increased levels of ROS, and general mitochondrial dysfunction.29 High bisphenol-A (BPA) concentrations inhibited sperm motility and motion kinematics by significantly decreasing ATP levels in spermatozoa.30 These EDs impair sperm by affecting mitochondrial function, possibly through the OXPHOS pathway.

Mitochondria are the energy factories of the cell, providing energy by generating ATP. In spermatozoa, mitochondria are concentrated mainly in the mid-tail portion and provide the necessary energy for flagellar motility, which drives the spermatozoa to swim.31 Spermatozoa motility depends on the normal function of mitochondria and an adequate supply of ATP. As shown by our results, the mechanism of mitochondrial damage caused by TCC may be as follows: TCC exposure leads to excessive production of ROS in sperm cells, and these free radicals can damage the mitochondrial membrane as well as mitochondrial proteins and DNA. In addition, oxidative stress can weaken mitochondrial function and reduce the production of ATP, which in turn affects the motility of sperm.32 TCC exposure may damage the mitochondrial membrane and dysfunction of the electron transport chain, potentially affecting the OXPHOS process, leading to an MMP and ATP generation efficiency decrease.33

In this study, we treated healthy human spermatozoa with TCC in BWW media containing different concentrations of glucose to observe the effects of glucose on sperm motility. In glucose-free medium (BWW-), sperm motility was very low. This occurred because there were no major functional substrates in the culture environment. Sugar-free/low-sugar media can reflect the changes in sperm motility dependent on glycolysis/OXPHOS; however, sperm need a certain substrate, including an appropriate concentration of glucose, to maintain sperm motility and normal function.13 In a low-glucose BWW culture environment, TCC enhanced the damaging effect on sperm motility. Moreover, the ATP, MMP, and lactate decreased significantly. The PM, VSL, and ROS seemed more sensitive to the low-glucose culture environment. The high motility of mammalian sperm is often closely associated with high glycolysis rates;34 therefore, it suggests that TCC may also affect the glycolytic pathway.

ATP production in spermatozoa can occur via two pathways: glycolysis and OXPHOS; however, the stage at which the cell selects the metabolic pathway is unclear.13 Although aerobic glycolysis is an inefficient pathway for ATP production, the main segment of the sperm tail may select aerobic glycolysis in preference to mitochondrial OXPHOS and compensate for ATP production through an increase in glycolytic flux, thus providing sperm whipping motility, a rapid function.

The results of the present study suggest that TCC may affect sperm motility by interfering with energy metabolism pathways. A low-glucose environment may exacerbate this effect because spermatozoa are more susceptible to interference from external factors in the presence of a low energy supply. TCC may affect sperm motility by inhibiting mitochondrial function or interfering with ATP synthesis. In addition, the low-glucose environment may limit the ability of spermatozoa to produce ATP via the glycolytic pathway, thus increasing their sensitivity to the toxic effects of TCC. Exploring these mechanisms will contribute to a more comprehensive understanding of the effects of TCC on sperm motility.

Although the blood-testis barrier provides a highly protective environment for spermatozoa, preventing the entry of many exogenous substances, TCC, as a small-molecule lipid-soluble compound, may cross the blood-testis barrier via passive diffusion or carrier-mediated transport mechanisms. In addition, metabolites of TCC may accumulate in testicular tissues, further increasing their potential exposure to spermatozoa.35 Certain environmental pollutants can cross the blood‒testis barrier and negatively affect spermatozoa,36 which provides a reference for us to explore the mechanism of action of TCC. Future studies should investigate the distribution of TCC and its metabolites in the testis and their effects on the integrity of the blood‒testis barrier further.

This study has several limitations. First, the activities of mitochondrial respiratory chain complexes I–V were not detected, and the damaging effect of TCC on the mitochondrial respiratory chain was inferred only from the ATP, MMP, and ROS levels. Second, the possible mechanism by which TCC impairs sperm motility through two energy metabolism pathways, glycolysis and OXPHOS, was not verified through antagonists or agonists, which must be remedied in future experiments. In addition, to explore the harmful effects of TCC on sperm, the exposure doses used far exceeded environmental exposure levels. Whether TCC can affect sperm quality in vivo requires further investigation, for example, through conducting population studies.

In conclusion, this study investigated the effects of TCC on human sperm. TCC may disrupt the balance of sperm energy metabolism by damaging the structure and function of sperm mitochondria, reducing the motility of normal human spermatozoa. More in-depth studies on the influence of TCC on mitochondria and energy metabolism in human sperm should be conducted in future.

AUTHOR CONTRIBUTIONS

LYZ and LLF had full access to all study data and took responsibility for data integrity and analytical accuracy. WZW and FC were responsible for the screening and enrollment of participants and performed experimental investigations and data collection. BL and LH carried out data validation and formal statistical analyses. LLF and YF secured research funding and contributed to study design. LYZ and LC coordinated research resources and supervised project implementation. LLF developed methodology and drafted the manuscript. All authors participated in manuscript review, and read and approved the final manuscript.

COMPETING INTERESTS

All authors declared no competing interests.