ABSTRACT
The epididymis is responsible for sperm maturation, but is sensitive to the action of external agents, especially during the peripubertal period. Chlorpyrifos is an organophosphate class pesticide to which humans are constantly exposed. Currently, there are few studies that evaluate the effects of this insecticide on the epididymis; therefore, the objective of the present study was to assess the effects of chlorpyrifos on the postnatal development of the epididymis during the peripubertal period. Thirty Wistar rats were divided into three groups: Control, C5, and C15. The control group received only the vehicle and the C5 and C15 groups received, respectively, 5 and 15 mg/kg of chlorpyrifos via gavage during 40 days (DPN25–DPN65). On DPN 66, the animals were euthanized. The epididymis was used for sperm count, histopathological, stereological, and oxidative stress analyses. Sperm from the vas deferens was used for motility and mitochondrial activity analyses. There was a difference in the proportion of luminal, stromal, and epithelial compartments in the groups exposed to chlorpyrifos. An increase in sperm transit time in the caput epididymis was also observed in the C5 group and a decrease in mitochondrial activity in sperm from the C15 group. Oxidative stress analyses showed a decrease in GST activity in the C5 and C15 groups. Sperm motility remained unchanged. These results suggest that the exposure to chlorpyrifos during peripuberty harms the postnatal development of the epididymis. This study is the first to evaluate the morphology and function of the epididymis in peripubertal rats.
1. Introduction
The sperm produced in the testicles are morphologically complete, but are not yet functional. It is during transit in the epididymis, through interactions with proteins secreted by the epididymal epithelium, that sperm acquire their motility and the ability to fertilize the female gamete [1].
During the juvenile and peripubertal periods in rats, the cellular population of the epididymal epithelium is established. Between postnatal days 16 and 44, the period of differentiation in the epididymis occurs, in which there is the differentiation of epididymal duct cells as well as an increase in the epithelium height in the initial and intermediate segments [2]. However, these developmental windows are more fragile to the action of toxic agents, and there may be adverse consequences that may already be visible during puberty or later, during adult life [3].
Organophosphates encompass several compounds that are used for a variety of purposes, from insecticides to flame retardants [4, 5]. The contamination of soil and aquatic ecosystems with these compounds inevitably leads to the entry of these molecules into the human body [6, 7].
Among the organophosphates with insecticidal action, there is chlorpyrifos (CPS), which acts by inhibiting irreversibly the enzyme acetylcholinesterase (AchE) in the synapses of the central nervous system [7]. Thus, there is an accumulation of acetylcholine in the synaptic clefts and the consequent interruption of the nervous impulse, leading to paralysis and death of the insects [7, 8].
Several studies demonstrate that chlorpyrifos causes damage to the reproductive system in male rats [9, 10, 11, 12, 13]. Adedara et al. [9] reported that rats exposed daily to 5 mg/kg of chlorpyrifos during 35 days caused epithelial hyperplasia in the epididymis, reduced sperm motility and decreased the activity of antioxidant enzymes in the testes and epididymis. Another study showed that treatment with chlorpyrifos at doses of 2.7, 5.4, and 12.8 mg/kg during 90 days caused a reduction in sperm motility and in the concentration of testosterone, FSH and LH in rats [10]. A decrease in fertility was observed in male rats exposed orally to 9 mg/kg/day of chlorpyrifos for 70 days, along with a reduction in testosterone levels and sperm motility [11]. The literature has shown that chlorpyrifos caused a decrease in testosterone levels and sperm count in the testes and epididymis after a 30‐day exposure at 7.5, 12.5, and 17.5 mg/kg/day [12], and 2.5, 5, and 10 mg/kg/day [13]. However, none of the studies forementioned evaluate the juvenile and pubertal periods in its entirety.
Currently, there are no studies evaluating the impact of chlorpyrifos exposure on the pubertal epididymis and its postnatal development, morphology, and function. Considering the information presented, the objective of this study was to evaluate the consequences of exposure to chlorpyrifos during the juvenile and peripubertal windows on the postnatal epididymal development of rats.
2. Methods
2.1. Experimental Design
Male Wistar rats (n = 30), from the Central Animal House of the State University of Londrina—UEL, were acclimatized in the Animal House of the Laboratory of Toxicology and Metabolic Disorders of Reproduction, CCB‐UEL from postnatal day (PND) 22–25. On DPN 25, the animals were arbitrarily distributed into three groups (n = 10 animals/group): control group, C5 group and C15 group. Animals in groups C5 and C15 received chlorpyrifos (Sumitomo Chemical—Klorpan 480 EC [48%], São Paulo, Brazil) diluted in corn oil (vehicle) at concentrations of 5 or 15 mg/kg b.w., respectively, via gavage for 40 days (DPN 25–65). Animals in the control group received only the vehicle. The choice of the doses was based on the LOAEL for the nervous system of rats, in order to carry out an asymptomatic experimental protocol [14]. Previous study of our group has shown that the animals tolerated these concentrations well, with no changes in body weight, water and feed consumption and no signs of cholinergic toxicity were observed [15]. On DPN 66, the rats were anesthetized by isoflurane inhalation and euthanized by cardiac puncture. From each animal, the left epididymis (n = 10 animals/group) were used for oxidative profile analysis. The right epididymis (n = 05 animals/group) were weighed and used to determine the sperm transit time through the epididymis. The other right epididymis (n = 05 animals/group) were used for histopathological and morphometric analyses. Spermatozoa from the left vas deferens of these animals (n = 10/group) were used to evaluate sperm motility and mitochondrial activity.
2.2. Sperm Transit Through the Epididymis
This analysis was carried according to the descriptions by Fernandes et al. [16]. The epididymis had the caput/body and cauda epididymal portions separated and was homogenized. After 20‐fold dilution, sperm were counted in the Neubauer chambers. The arithmetic mean of the counted fields results in the number of spermatozoa expressed in millions per milliliter, which, multiplied by the dilutions used, results in the number of spermatozoa/organs which, divided by the daily sperm production (DSP)—was estimated according to the method described by Robb et al. [17] and Johnson et al. [18]. Briefly, the number of homogenization‐resistant spermatids was determined in testicular homogenates, and the value obtained was divided by 6.1, which corresponds to the duration (in days) of steps 17–19 spermatid development in the seminiferous epithelium cycle of rats, expressing the sperm transit time (number of days required for them to cross the different epididymal portions) in days.
2.3. Sperm Motility
Sperm motility analysis was performed according to Siervo et al. [19], with some modifications. Spermatozoa from the vas deferens were collected in a solution of PBS and 0.75% BSA at a temperature of 37°C. An aliquot of the sperm solution was placed in a Makler chamber, observed under an optical microscope, and 100 sperm per animal were classified according to their motility as motile or immobile.
2.4. Sperm Mitochondrial Activity
Sperm were collected from the vas deferens in a PBS solution with BSA. An aliquot was separated into a microtube for addition of DAB (3,3′ Diaminobenzidine tetrahydrochloride) dye and incubation at 37°C for 1 h. Smears were made and fixed with formaldehyde (10%). Two‐hundred cells were counted and classified as: DAB I, DAB II, and DAB III [20].
2.5. Processing of Histological Samples
The protocol was carried out according to Fernandes et al. [16]. The epididymis (n = 5) was immersed in Methacarn for a 4‐h pre‐fixation. Then, the organs were subdivided into caput + body and cauda portions, which were cut longitudinally and left to immerse for another 4 h. After the fixation stage, the organs were immersed in 70% alcohol and further processed for inclusion in Paraplast. The tissues were sectioned at 5 μm with intervals of 25 μm and later stained with hematoxylin and eosin.
In the histopathological analysis, the appearance of the epithelium and interstitium was analyzed qualitatively. The stereological analysis was carried out according to the Weibel method [21], in which a test system of lines and points is used in a graticulate with 168 points and 60 lines. Thus, the proportion of the section occupied by the lumen, epithelium, and interstitium is obtained.
2.6. Analysis of Oxidative Stress in Epididymis
The samples were kept at −80°C until the time of analysis, when they were thawed and the epididymis were separated into caput + body portion and cauda portion. The samples were homogenized in PBS (pH 7.2) and the protocols for the determination of lipid peroxidation, GSH concentration and antioxidant enzyme activity were conducted according to da Costa et al. [15].
2.7. Statistical Analysis
All data were subjected to the analysis of variance test (Brown–Forsythe) and the homoscedasticity test (Shapiro–Wilk). The ANOVA test was performed with Tukey's a posteriori test for parametric data and the Kruskall–Wallis test with Dunn's a posteriori test for non‐parametric data. Differences between treatments were considered statistically significant when p < 0.05.
3. Results
3.1. Sperm Count and Spermatic Transit
There was an increase in sperm transit time in the caput and body portion of the epididymis in the C5 group (Table 1). However, in the cauda portion of the epididymis, sperm transit remained unchanged between the experimental groups (Table 1).
TABLE 1.
Weight of the epididymis and sperm transit time in the caput and corpus portion and in the cauda portion of the epididymis of rats exposed to 5 and 15 mg/kg of chlorpyrifos.
| Parameters | Experimental groups | ||
|---|---|---|---|
| Control | C5 | C15 | |
| Epididymis weight (g) a | 0.372 [0.319–0.387] | 0.385 [0.380–0.399] | 0.355 [0.330–0.382] |
| Spermatic transit (days) | |||
| Caput + corpus portion b | 1.388 ± 0.1291 | 1.857 ± 0.1796** | 1.537 ± 0.1581 |
| Cauda portion b | 1.634 ± 0.2958 | 1.893 ± 0.1193 | 1.688 ± 0.3592 |
Data presented as median [Q1–Q3]. Mann–Whitney test.
Data presented as mean ± standard error of the mean. ANOVA with post hoc Tukey test.
p < 0.01.
3.2. Sperm Motility and Mitochondrial Activity
There was no change in the number of motile and immobile sperm between the experimental groups (Table 2). However, there was a decrease in the number of sperm with greater mitochondrial activity (DAB I) (p < 0.01) and an increase in sperm with lower mitochondrial activity (DAB II) (p < 0.05) only in the C15 group (Table 2).
TABLE 2.
Sperm mitochondrial activity of rats exposed to 5 and 15 mg/kg of chlorpyrifos.
| Parameters | Experimental groups | ||
|---|---|---|---|
| Control | C5 | C15 | |
| Sperm motility a | |||
| Motile sperm (%) | 38 [0–52.5] | 35.5 [0.75–52.5] | 33 [0–43] |
| Immotile sperm (%) | 62 [47.5–100] | 64.5 [47.5–99.25] | 67 [57–100] |
| Mitochondrial activity b | |||
| DAB I | 144 ± 10.140 | 127.8 ± 12.530 | 99 ± 28.490** |
| DAB II | 48.5 ± 7.423 | 65.67 ± 11.270 | 77.5 ± 27.700* |
| DAB III | 8.6 ± 1.342 | 7.6 ± 2.510 | 16.4 ± 10.450 |
Abbreviations: DAB I, number of sperm with the mitochondrial sheath fully stained; DAB II, number of sperm with partially stained mitochondrial sheath; DAB III, number of sperm with unstained mitochondrial sheath.
Data presented as median [Q1–Q3]. Mann–Whitney test.
Data presented as mean ± standard error of the mean. ANOVA with post hoc Tukey test.
p < 0.05.
p < 0.01.
3.3. Stereological Analysis
Stereological analysis revealed that chlorpyrifos influenced postnatal development of the epididymis. In the caput and body portion of the epididymis, an increase in the percentage of the luminal compartment (p < 0.01) and a decrease in the stromal compartment (p = 0.001) were observed in the C5 group in relation to the control group; in the C15 group there was an increase in the luminal compartment (p < 0.01) and a decrease in the epithelial compartment (p < 0.01) in relation to the control group (Table 3).
TABLE 3.
Proportion of lumen, stroma and epithelium compartments in the caput and corpus region and in the cauda region of the epididymis of rats exposed to 5 and 15 mg/kg of chlorpyrifos.
| Parameters | Experimental groups | ||
|---|---|---|---|
| Control | C5 | C15 | |
| Caput and corpus (2A region) (%) | |||
| Lumen | 29.17 [25.6–34.67] | 36.01 [29.17–43.6]** | 37.2 [24.55–47.77]** |
| Stroma | 33.93 [27.98–38.1] | 27.08 [20.09–31.55]*** | 31.25 [22.02–39.58] |
| Epithelium | 35.12 [30.95–44.2] | 37.2 [32.14–40.63] | 31.85 [26.04–35.42]** |
| Caput (5A/B region) (%) | |||
| Lumen | 57.44 [49.85–65.18] | 56.55 [52.23–62.65] | 52.08 [43.45–59.38] |
| Stroma | 23.21 [16.67–30.65] | 20.24 [13.54–27.08] | 32.14 [21.13–41.67]** |
| Epithelium | 18.45 [13.54–25.15] | 22.92 [19.49–25.74]* | 15.48 [9.374–20.24]* |
Note: Data presented as median [Q1–Q3]. Kruskall–Wallis test with post hoc Dunn test.
p < 0.05.
p < 0.01.
p = 0.001.
Regarding the cauda portion, in the C5 group there was an increase in the percentage of the epithelial compartment (p < 0.05) and in the C15 group there was an increase in the stromal compartment (p < 0.01) and a decrease in the proportion of epithelium (p < 0.05) (Table 3).
3.4. Histopathological Analysis
The present analysis showed that exposure to the insecticide chlorpyrifos did not because histopathological changes in the epididymal compartments analyzed since the results are qualitatively similar to the organs of animals in the control group (Figure 1).
FIGURE 1.
Histology of caput and cauda region of the epididymis. Magnification: ×40. Hematoxylin and eosin staining. E, epithelium; L, lumen; S, stroma.
3.5. Oxidative Profile
3.5.1. Lipid Peroxidation
There was no change in the concentration of malondialdehyde between the experimental groups, both in the caput and body portion and in the cauda portion of the epididymis (Figures 2A and 3A).
FIGURE 2.
Oxidative stress parameters in the region of the caput and body of the epididymis. Data presented as mean ± standard error of the mean. ANOVA with post hoc Tukey test. p value considered significant when < 0.05. (A) Tiobarbituric acid reactive substances (nmol/protein mg). (B) Glutathione‐S‐transferase activity (nmol/min/mL/mg of protein). (C) Total glutathione (μg/mg protein). (D) Oxidized glutathione (μg/mg protein). (E) Reduced glutathione concentration (μg/mg protein). (F) Superoxide dismutase activity (nmol/min/mL/mg of protein). (G) Catalase activity (nmol/min/mL/mg of protein).
FIGURE 3.
Oxidative stress parameters in the cauda epididymis region. Data presented as mean ± standard error of the mean. ANOVA with post hoc Tukey test. p value considered significant when < 0.05. (A) Tiobarbituric acid reactive substances (nmol/protein mg). (B) Glutathione‐S‐transferase activity (nmol/min/mL/mg of protein). (C) Total glutathione (μg/mg protein). (D) Oxidized glutathione (μg/mg protein). (E) Reduced glutathione concentration (μg/mg protein). (F) Superoxide dismutase activity (nmol/min/mL/mg of protein). (G) Catalase activity (nmol/min/mL/mg of protein).
3.5.2. Antioxidant Capacity
The glutathione‐S‐transferase activity was reduced in the two groups exposed to chlorpyrifos in the caput and body portion of the epididymis (Figure 2B) (p < 0.05) and in the C15 group in the cauda portion of the epididymis (Figure 3B) (p < 0.05).
The concentrations of total glutathione (Figures 2C and 3C), oxidized glutathione (Figures 2D and 3D), and reduced glutathione (Figures 2E and 3E) and the activities of superoxide dismutase (Figures 2F and 3F) and catalase (Figures 2G and 3G) remained unchanged between the experimental groups in the caput and body portions and in the cauda portion of the epididymis.
4. Discussion
This study shows that exposure to chlorpyrifos caused changes in the postnatal development of the epididymal duct, but these changes are not related to oxidative stress, since we did not observe important changes in the antioxidant profile, suggesting that other mechanisms are involved in the changes observed.
During sperm capacitation throughout the epididymis, an elevated generation of reactive oxygen species is common [22], however excessive generation of ROS can cause damage to sperm [23, 24]. Chlorpyrifos did not induce generalized oxidative stress, although specific defenses such as glutathione‐S‐transferase (GST) were compromised. In relation to GST activity, which was reduced in the groups exposed to chlorpyrifos, this molecule is an enzyme that acts in the detoxification of several exogenous and endogenous organic compounds, catalyzing the conjugation of the reduced form of glutathione with the metabolites produced, which makes them more water‐soluble and easier to excrete [25]. Similar to our results, Adedara et al. [9] also observed a decrease in GST activity in the epididymis of pubescent rats after daily exposure to 5 mg/kg of chlorpyrifos for 35 days.
Exposure to chlorpyrifos caused structural changes during the postnatal development of the epididymis, both in the caput and body and cauda portions, evidenced by the difference in the proportions of the stromal, epithelial, and luminal compartments in stereological analysis. As our analyses did not show an increase in lipid peroxidation or changes in antioxidant concentrations, the structural changes observed are not related to an increase in oxidative stress. The region of the caput and body of the epididymis is responsible for carrying out the initial maturation of sperm, while the cauda has the primary function of storing these sperm [1]. The main cells responsible for sperm maturation are the main cells, which secrete proteins that are capable to bind to the sperm's membrane and/or interact with already existing proteins so that their functional competence is acquired [26, 27]. Considering the information presented, the decrease in the epithelial compartment in the epididymal duct of the C15 group may be related to the decrease in the mitochondrial activity of sperm from the same group.
In sperm, mitochondria are present only in the mitochondrial midpiece sheath. As in other cell types, mitochondria have the function of producing ATP, supplied to the sperm axoneme for tail motility [28]. However, the decrease in mitochondrial activity was not sufficient to reduce sperm motility in the C15 group. However, intact motility does not necessarily indicate that the sperm exhibit an adequate motility pattern (trajectory and velocity), as these parameters also depend on mitochondrial activity. This can be explained by the fact that mitochondrial ATP is not the sperm's only energy source. Studies in mice have demonstrated that, even when mitochondrial activity is impaired, fertilization can still occur, as sperm continue to produce ATP, albeit at lower levels, and maintain motility, although reduced [29]. Furthermore, studies have demonstrated that glycolysis is a complementary pathway sustaining motility, assisting mitochondrial oxidative phosphorylation [30, 31]. Knockout models lacking sperm‐specific glycolytic enzymes exhibit complete loss of motility despite preserved mitochondrial function [32]. Therefore, even with decreased mitochondrial activity, glycolytic ATP production may have compensated, maintaining motility in the C15 group.
The contractile capacity of the epididymal smooth muscle cells contributes to the sperm's transit time through the epididymis. This capacity is controlled by autonomous neurotransmitters, including acetylcholine and androgens [33, 34]. Chlorpyrifos irreversibly inhibits AchE and causes an accumulation of acetylcholine in synaptic clefts and, thus, causes changes in the contractility pattern in skeletal striated muscle [35] and smooth muscle [36]. Considering the mechanism of action of chlorpyrifos, we can infer that the increase in transit time observed in the caput and body region in the C5 group is due to cholinergic signaling in the epididymis and consequent change in the contractile activity of epididymal smooth muscle cells. However, the same was not observed in the C15 group, possibly due to some adaptive mechanism, such as desensitization or internalization of acetylcholinergic receptors. This interpretation is speculative and requires further confirmation. However, the region of the cauda of the epididymis seems to be more resistant to the effects of chlorpyrifos and there was no change in transit time.
It is interesting to highlight that the administration of chlorpyrifos showed a non‐linear dose–response relationship. This is worrying, since when it comes to pesticides, the population is normally exposed for a prolonged period to very low doses, which do not cause the appearance of acute signs and symptoms [37, 38].
5. Conclusion
From the results of this experimental model, it is evident that the daily exposure to both doses of chlorpyrifos altered the peripubertal development of the epididymis and increased transit time through the epididymis, although oxidative stress markers were not consistently altered, suggesting involvement of additional or alternative mechanisms.
Author Contributions
Ivana Regina da Costa: writing – original draft, visualization, investigation, formal analysis. Débora Hipólito Quadreli: investigation. Lucas Marcelo Meira da Silva: investigation. Fábio Goulart de Andrade: writing – review and editing, methodology, conceptualization. Glaura Scantamburlo Alves Fernandes: writing – review and editing, supervision, project administration, methodology, conceptualization.
Funding
The authors have nothing to report.
Ethics Statement
The experimental protocol received approval from the Ethics Committee on the Use of Animals at the State University of Londrina (OF. CIRC. CEUA No. 034/2021, protocol No. 013.2021), adhering to the Ethical Principles in Animal Experimentation established by the National Council for the Control of Animal Experimentation (CONCEA).
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments
The authors are grateful to CAPES (Coordinating Body for the Improvement of Postgraduate Studies in Higher Education) for providing a master scholarship to IR Costa and partial financial support (Finance Code 001). To the Government of the State of Paraná, Paraná Council of Science and Technology, and State Secretariat for Science, Technology, and Higher Education (budget allocation # 4560.19.571.06.6153; eprotocol 21.234.745‐0). We also thank the CMLP‐UEL (Multiuser Research Laboratory Center of the State University of Londrina) for technical support.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.