Introduction

Over the past century, pesticides and agrochemicals have played an integral role in global agricultural systems, significantly enhancing crop yields and food production (Aktar et al., 2009). Pesticides, designed to kill pests and weeds, contain chemicals that can be toxic to other organisms and the environment. Chemical residues from pesticides can affect human health through food contamination and contribute to environmental pollution (Mingo et al., 2017). Mancozeb is a fungicide from the ethylene bis-dithiocarbamate (EBDC) group that contains manganese and zinc and is widely used in tropical countries, mostly to protect crops, vegetables, fruits, and nuts (Runkle et al., 2017). Mancozeb has a half-life of one to three days on fruits and vegetables, and its residue becomes undetectable in the environment within a week of application (Paramasivam and Chandrasekaran, 2013; Devi et al., 2015). Mancozeb exposure can occur through direct skin contact or consumption of contaminated food or drink (Mandić-Rajćević et al., 2020). The toxicity level of mancozeb is low and rapidly decomposes into ethylene thiourea (ETU; C₃H₆N₂S) in the presence of moisture and oxygen (Suárez et al., 2013). In contrast to the less toxic mancozeb, ETU is a primary metabolite of mancozeb. The relative stability of ETU and its designation as a hazardous compound make it a pollutant of concern (Jacobsen and Bossi, 1997; Suárez et al., 2013). ETU is persistent, polar, and weakly absorbed by soil particles. Therefore, its high soil mobility makes it a potential contaminant for groundwater. Moreover, ETU lasts longer in the soil for five to 10 weeks and has a higher water solubility than mancozeb (Dearfield, 1994). Low soil temperatures and anaerobic conditions can significantly reduce the ETU breakdown rates. Also, the absence of microorganisms reduces the ETU breakdown rates since microorganisms can break ETU down into ethylene urea (EU), which is then degraded further to CO₂ (Jacobsen and Bossi, 1997).

Pesticides can directly affect the structure of cells in the male reproductive system, and they can interfere with normal cellular functions, alter hormone signaling, and potentially lead to decreased sperm production, poor sperm quality, and overall reproductive organ dysfunction (Rey et al., 2009; Zhou et al., 2019). Similarly, mancozeb and ETU also can disrupt normal physiological processes in humans and animals (Pirozzi et al., 2016). Meanwhile, the World Health Organization (WHO) has reported that the general population’s exposure to mancozeb and ETU was 0.01 - 1 µg·kg^-1 body weight per day (WHO, 1988). Ksheerasagar and Kaliwal (2010) reported that mancozeb was absorbed rapidly into the body through the gastrointestinal system, transported to multiple target organs, and excreted completely after 96 hours. ETU accounts for approximately 24% of the bio-available dosage in urine and bile. Axelstad et al. (2011) reported that ETU inhibited thyroid peroxidase, induced malformations of the neural tube, and caused thyroid cancer in mammals. Toxicity to the thyroid gland could be one of the consequences that disrupt testicular homeostasis, and a thyroid hormone deficiency could be related to morphological and functional abnormalities in the pituitary-testicular hormonal axis that alters the structure and function of the testis and accessory sex organs (Chandrasekhar et al., 1985; Ksheerasagar and Kaliwal, 2010). Few studies have examined how pesticide metabolites affect humans and animals. However, there are no reports on the toxicity of ETU on mammalian spermatozoa. Therefore, this study assessed the effect of direct exposure to different concentrations of ETU on reproductive functions in boar spermatozoa at two different incubation time periods.

Materials and Methods

Sperm preparation

Liquid boar semen samples with more than 80% sperm motility were purchased from a local artificial insemination (AI) center. These samples were subjected to washing and resuspension in Beltsville thawing solution (BTS; Pursel and Johnson, 1976) followed by incubation without ETU (W/O), with 0.1% dimethyl sulfoxide (DMSO) as the vehicle control or with 10, 50, 100 and 200 µM ETU, at 37℃ for 30 min and 2 hrs. The stock solution was prepared by dissolving ETU (PESTIANAL, 96457, Sigma-Aldrich, Inc., USA) in DMSO. Unless stated otherwise, all other reagents used in this study were purchased from Sigma-Aldrich Chemical Co. LLC (USA).

Assessment of sperm motility

The sperm motility was measured using a computer-assisted sperm analysis (CASA) system (Sperm Class Analyzer^Ⓡ, Microptic S.L., Spain). Boar spermatozoa were incubated at 37.5℃ for 30 min and 2 hrs, either with or without ETU. A 2 µL aliquot of semen was deposited on a pre-warmed (38℃) Leja-counting slide (Leja Products BV, Netherlands). Ten fields were examined at 37.5℃, assessing a minimum of 500 spermatozoa per sample. The percentages of total motile spermatozoa (%), progressive motile spermatozoa (%), and hyperactive spermatozoa (%) were determined. The kinetic parameters were measured for each spermatozoon. They included the curvilinear velocity (VCL, µm·s^-1), straight-line velocity (VSL, µm·s^-1), average path velocity (VAP, µm·s^-1), percentage linearity (LIN, %), percentage straightness (STR, %), and wobble percentage (WOB, %).

Evaluation of sperm viability

The LIVE/DEAD^Ⓡ Sperm Viability Kit (Molecular Probes, Eugene, USA) was used to assay sperm viability. The kit contains the DNA dyes SYBR14 (100 nM) and propidium iodide (PI; 10 µM). First, the incubated spermatozoa (1 × 10⁸ cells·mL^-1) were rinsed twice with a phosphate-buffered saline solution containing 0.1% (w·v^-1) polyvinyl alcohol (PBS-PVA). The spermatozoa were stained, and images were then taken using a fluorescence microscope (Nikon Eclipse Ci microscope, Nikon Instruments Inc., Japan), a camera (DS-Fi2, Nikon, Japan), and imaging software (version 4.30, Nikon, Japan). The spermatozoa were categorized as viable (SYBR14) or dead (PI).

Measurement of acrosome integrity

The boar spermatozoa were fixed in a 95% ethanol solution and then kept at 4℃ for 30 min. The fixed sperm cells were then dried onto slides and stained with fluorescein isothiocyanate-labeled Pisum sativum agglutinin (FITC-PSA) at 5 µg·mL^-1 for 10 minutes (Berger, 1990). The acrosome integrity was determined using a fluorescence microscope camera and imaging software (Nikon Eclipse Ci microscope, Nikon Instruments Inc., Japan). The images were classified into two categories. The sperm heads displaying green fluorescence were considered to have an intact acrosome, while partial green fluorescence or its absence in the head indicated damaged or acrosome-reacted spermatozoa based on the fluorescent signals observed in the sperm heads.

Assessment of chromatin stability

The sperm chromatin stability was evaluated by acridine orange staining, as described elsewhere (Martins et al., 2007), with some modifications. Slides with sperm samples were prepared and air-dried following overnight fixation in Carnoy’s solution (methanol : glacial acetic acid = 3 : 1) under chilled conditions. The fixed slides were incubated with a tampon solution (composed of 80 mM citric acid and 15 mM Na₂HPO₄, pH 2.5) at 75℃ for five minutes. Subsequently, the slides were stained with acridine orange (0.2 mg·mL^-1. The excess background stain was removed with water, and coverslips were placed. The prepared slides were observed under a fluorescence microscope equipped with a camera and imaging software (Nikon Eclipse Ci microscope, Nikon Instruments Inc., Japan). The green fluorescence indicated that the spermatozoa contained normal DNA, whereas abnormal DNA exhibited yellow-green to red fluorescence.

Measurement of intracellular reactive oxygen species (ROS)

Spermatozoa were washed twice with 0.1% PBS-PVA, incubated with 1 µM 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA, Invitrogen, Eugene, OR, USA) at 37℃ for 10 min, and rinsed twice with 0.1% PBS. The stained spermatozoa were mounted in a Vectashield solution (Vector Laboratories, Burlingame, USA) and examined using a fluorescence microscope equipped with imaging software (Nikon Eclipse Ci microscope, Nikon Instruments Inc., Japan), which assessed the fluorescence intensity of ROS generation in the spermatozoa (Yi et al., 2021).

Real-time PCR

The sperm were washed three times with PBS-PVA after 30 min of incubation with or without ETU. The total RNA was produced using the guidelines of the PureLink™ RNA Mini Kit (Thermo Fisher Scientific, USA) with minor modifications. The RNA concentrations were determined using a nanodrop spectrophotometer (DeNovix DS-11FX, DeNovix Inc., USA). The cDNA was synthesized from purified RNA using the TOYOBO ReverTra Ace qPCR RT kit (TOYOBO Inc., Japan) according to the manufacturer’s instructions. The SYBR™ Premix Ex Taq™ II (Bioneer Corporation, Korea) and MyGo Pro PCR cycler (Diagnostic Technology Pty Ltd., Australia) were used in triplicate for the quantitative real-time polymerase chain reaction (qRT-PCR). The mRNA expression levels of the target genes were compared with those of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences (Table 1) used to measure the relative expression of A-kinase anchor proteins 3 and 4 (AKAP3/AKAP4), outer dense fiber of sperm tails 2 protein (ODF2), and zona pellucida binding protein 2 (ZPBP2) were produced using Primer Blast software (National Centre for Biotechnology Information, http://www.ncbi.nlm.nih.gov/).

Statistical analysis

All experimental data are expressed as the mean ± standard error of means (SEM) and analyzed using one-way ANOVA in GraphPad PRISM^® (GraphPad Software, USA). A completely randomized design was used, and a Tukey’s multiple comparison test was used to compare the values between individual treatments. The statistical significance is indicated by p-values of *p < 0.05, **p < 0.01, and ***p < 0.001.

Results and Discussion

The effects of ETU on boar spermatozoa functions were assessed by measuring the sperm motility and motion kinetic parameters following incubation with ETU (10 - 200 µM) for 30 min and 2 hrs at 37℃ (Table 2; Fig. 1). At 30 min of incubation, a gradual decrease in sperm motility was observed in all ETU-treated groups (85.7 - 86.1% controls vs. 41.0 - 73.4% ETU [10 - 200 µM], p < 0.05 and p < 0.001; Fig. 1A). Spermatozoa incubated with ETU for 2 hrs showed a similar reduction in motility (72.2 - 73.2% controls vs. 36.2 - 63.1% [10 - 200 µM] ETU, p < 0.001; Fig. 1B). At both incubation times, the percentages of sperm progressive motility (PR), straightness index (STR), curve speed (VCL), linear speed (VSL), and average path velocity (VAP) decreased in the spermatozoa incubated with ETU compared to the controls (p < 0.05, p < 0.01, and p < 0.001; Table 2).

Fig. 1.

Sperm motility was assessed in boar sperm exposed to different doses of ethylene thiourea (ETU) or the controls (without ETU [W/O] or with dimethyl sulfoxide [DMSO]) after 30 min (A) and 2 hrs (B). The values are presented as the mean ± SEM (standard error of the mean). ** p < 0.01, *** p < 0.001.

The sperm viability was evaluated after staining with SYBR14 and propidium iodide (PI; Fig. 2). Sperm exposed to 10 - 200 µM ETU for 30 min and 2 hrs observed a statistically significant dose-dependent decrease in viability compared to the control and DMSO at all doses and both incubation times. At 30 min incubation, the lowest viability was detected in the samples treated with 200 µM ETU (41.0%) than the control (85.7 - 86.1%, p < 0.001; Fig. 2A). Similarly, after 2 hrs incubation, the sperm exposed to ETU-treated groups showed a dose-dependent reduction in viable sperm compared to the controls (78.6 - 80.9% in controls vs. 27.0 - 52.4% in ETU [10 - 200 µM], p < 0.01 and p < 0.001; Fig. 2B).

Fig. 2.

Sperm viability was assessed after treatment with varying concentrations of ethylene thiourea (ETU) or controls (without ETU [W/O] or with dimethyl sulfoxide [DMSO]) and incubation times of 30 min (A) and 2 hrs (B). The values are presented as the mean ± SEM (standard error of the mean). ** p < 0.01, *** p < 0.001.

The acrosome integrity was assessed and is shown in Fig. 3. Lower rates of in-contact acrosome were observed after exposure to 10 - 200 µM ETU (94.7 - 98.2% in controls vs. 64.8% at 200 µM ETU, p < 0.001; Fig. 3A) for 30 min. The decreasing rates of intact acrosome were higher in the sperm treated with 10 - 200 µM ETU for 2 hrs (95.3 - 98.1% in controls vs. 56.4 - 89.5% in ETU in 10 - 200 µM ETU, p < 0.05, p < 0.01, and p < 0.001; Fig. 3B).

Fig. 3.

Acrosome integrity of spermatozoa incubated with different concentrations of ethylene thiourea (ETU) or controls (without ETU [W/O] or with dimethyl sulfoxide [DMSO]) after incubation of 30 min (A) and 2 hrs (B). The values are presented as the mean ± SEM (standard error of the mean). ** p < 0.01, *** p < 0.001.

Fig. 4 shows the chromatin stability in both incubation periods. After 30 minutes incubation, the significantly lowest rate of normal chromatin stability was observed after exposure to 200 µM ETU (96.2 - 96.5% in controls vs. 57.9% at 200 µM ETU, p < 0.001; Fig. 4A) and the rate of normal chromatin decreased significantly after exposure to ETU compared to the controls after 2 hours of incubation (92.8 - 94.2% in the controls vs. 28.5 - 83.4% in 10 - 200 µM ETU, p < 0.01 and p < 0.001; Fig. 4B).

Fig. 4.

Chromatin stability of sperm incubated with varying concentrations of ethylene thiourea (ETU) or controls (without ETU [W/O] or with dimethyl sulfoxide [DMSO]) after 30 min (A) and 2 hrs (B) of incubation. The values are presented as the mean ± SEM (standard error of the mean). ** p < 0.01, *** p < 0.001.

The intracellular ROS production was assessed by staining the spermatozoa with a carboxy-2′,7′-dichlorofluorescein diacetate solution and measuring the fluorescence intensity under a fluorescence microscope (Fig. 5). Compared to the control group, the sperm treated with 10 - 200 µM ETU showed higher fluorescence intensity. Intracellular ROS production was increased significantly at concentrations from 10 - 200 µM ETU after both incubation times (p < 0.01 and p < 0.001; Fig. 5).

Fig. 5.

Production of intracellular reactive oxygen species (ROS) was evaluated in boar spermatozoa exposed to different concentrations of ethylene thiourea (ETU) or controls (without ETU [W/O] or with dimethyl sulfoxide [DMSO]) after 2 hrs of incubation. The values are presented as the mean ± SEM (standard error of the mean). *p < 0.05, *** p < 0.001.

The mRNA expressions of AKAP3, AKAP4, ODF2, and ZPBP2, which were associated with regulating the fertilization capacity of spermatozoa, showed significantly lower expression in sperm exposed to ETU (p < 0.05 and p < 0.01; Fig. 6A-D), as shown in Fig. 6.

Fig. 6.

Sperm mRNA expression is associated with the fertility capacity. Spermatozoa were incubated with different concentrations of ethylene thiourea (ETU) or controls (without ETU [W/O] or with dimethyl sulfoxide [DMSO]). (A) A-kinase anchor protein 3 (AKAP3), (B) A-kinase anchor protein 4 (AKAP4), (C) the outer dense fiber sperm tails protein 2 (ODF2), and (D) zona pellucida binding protein 2 (ZPBP2). The relative mRNA expression levels from the target genes were compared with the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The values are presented as the mean ± SEM (standard error of the mean). * p < 0.05, ** p < 0.01, *** p < 0.001.

Gavrilescu et al. (2015) reported that pesticide metabolites affect animals’ metabolic, physiological, and reproductive health and pose potential risks to future generations. Mancozeb can disrupt mononuclear cells and thymocytes and interfere with the neurological and endocrine systems (Srivastava et al., 2012; Pandey and Mohanty, 2015; Wang et al., 2021). The reproductive effects of mancozeb include changes in meiotic spindle shape, blastomere apoptosis, and reduced fertilization of oocytes exposed to the drug (Greenlee et al., 2004; Rossi et al., 2006). Furthermore, the weights of male reproductive organs (e.g., the testis and accessory tracts) were altered when the mice were treated with mancozeb. In addition, the histological changes, lower testosterone levels, and increased oxidative stress all led to apoptosis in mice testis (Ksheerasagar and Kaliwal, 2003; Mohammadi-Sardoo et al., 2018). Thyroid hormones (THs) play a role in testes differentiation, prostate function, and maturation in the male reproductive system (Maran et al., 2000; Wagner et al., 2009; Aruldhas et al., 2010). Moreover, they are important for identifying developing and adult testicular somatic and germ cells. Triiodothyronine (T3) plays an important role in regulating the proliferation and differentiation of Sertoli cells (SCs) during testis development while also contributing to the assembly of the blood-testis barrier (BTB) in rodents, supported by in vitro and in vivo evidence. (Gao et al., 2014; Hernandez, 2018). ETU can be classified as an endocrine disruptor because it inhibits the thyroid peroxidase activity, which interferes with thyroid hormone production (Marinovich et al., 1997; Maran et al., 2000; Wagner et al., 2009; Aruldhas et al., 2010). Male rodents treated with 0.3 mg·kg^-1 (body weight per day) ETU, the dose at which the hypothyroidism condition was found, showed significantly lower serum dihydrotestosterone (DHT) levels (Rijntjes et al., 2009). Severe hypothyroidism is related to the inhibition of testosterone conversion to DHT by 5a-reductases, with a corresponding increase in testosterone (T) levels in serum (Rijntjes et al., 2009).

Reduced antioxidant activity from decreasing enzymes, including superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), leads to an increase in ROS production and oxidative stress in cells (Ayala et al., 2014). ROS production increases the fluidity of the sperm plasma membrane and decreases the intercellular adenosine triphosphate (ATP) levels, resulting in decreased sperm motility (Asadi et al., 2017; Calogero et al., 2017). Mohammadi-Sardoo et al. (2018) reported that mancozeb causes apoptosis in the testis of male albino mice due to increased oxidative stress. These results show that the exposure of sperm samples to ETU at different doses decreased the motility gradually, and ROS production increased as the ETU doses (10 - 200 µM) were increased in both incubation times. Similarly, when mancozeb doses (0.01 - 0.5 µM) increased, motility decreased, and ROS production increased in 30 minutes and 2 hours of incubation (Adikari et al., 2022).

The sperm acrosome plays a major role in the penetration and fusion of gametes, which leads to successful fertilization. Similarly, Veeramachaneni (2000) reported that pesticide exposure during or after pregnancy can result in acrosomal abnormalities in mammals. Another study showed that greater doses of glyphosate-based compounds dramatically reduced the acrosome integrity in the sperm of stallions, suggesting that pesticides cause cellular damage in the cell membrane (Spinaci et al., 2022). The present study showed that the sperm viability and acrosomal integrity were reduced in all ETU-treated groups after 30 minutes and 2 hours incubation (Fig. 5). Similar results were obtained from the different concentrations of mancozeb (0.01 - 0.5 µM)-treated groups incubated for 30 minutes and 2 hours in boar spermatozoa (Adikari et al., 2022).

An absence of AKAP3 could disrupt the integrity of the subcellular structure and proteome of mouse spermatozoa, resulting in male infertility (Xu et al., 2020). AKAP4 is an essential component of the sperm biology, particularly in the flagellar structure and motility, and may affect other sperm functions, such as chemotaxis and capacitation (Blommaert et al., 2019). The lack or absence of AKAP4 expression in male mice is related to impaired sperm motility and fibrous sheath dysplasia (Miki et al., 2002). Zhao et al. (2018) reported that ODFs are part of the complex structure that surrounds the central axoneme in the flagellum. Reduced ODF2 protein expression in ETU-exposed sperm (Fig. 6) was positively correlated with decreased motility, indicating structural alterations in the sperm flagella. ZPBP2, which is expressed abundantly in sperm cells, is necessary for sperm maturation and fertilization because it binds to the zona pellucida, allowing the sperm to penetrate the oocyte (Lin et al., 2007; Kozlovsky and Gefen, 2013). In the present study, an analysis of several sperm protein expressions suggests that direct exposure to ETU may reduce sperm motility and fertilization capacity, possibly due to structural changes in sperm proteins.

Conclusion

Direct exposure of boar spermatozoa to ETU decreased motility, motion kinematics, viability, acrosome integrity, and chromatin stability while increasing intercellular ROS production. The levels of AKAP3, AKAP3, ODF2, and ZPBP2 mRNA expression decreased gradually in sperm exposed to ETU. Therefore, direct exposure of spermatozoa to different concentrations of ETU adversely affects the fertilization capacity.