Abstract
Atrazine is a herbicide of the chloro-s-triazine family. It inhibits photosynthesis in plants and is an endocrine disruptor, but its effects on human health are controversial. Fenoxaprop-ethyl, an aryloxy phenoxyalkanoic acid herbicide, inhibits the biosynthesis of fatty acids and provokes depolarization of membranes. The aim of this study is to evaluate the in vitro effects of both herbicides on capacitation, spontaneous acrosome reaction (SAR) and progesterone-induced acrosome reaction (PIAR) in boar sperm. Sperm capacitation is done in TALP-HEPES media for 4 hours. Capacitation and SAR are evaluated immediately; PIAR, 30 minutes later. LC50 for fenoxaprop-ethyl is 60 mM and 40 mM for atrazine. Fenoxaprop-ethyl induces capacitation at 60 mM and SAR at all concentrations, also increases significantly PIAR. Atrazine decreased capacitation whereas increase significantly SAR and PIAR at all concentrations. It seems that fenoxaprop-ethyl and atrazine accelerate the capacitation and the acrosomal reaction, possibly via plasma membrane destabilization.
Endocrine-disrupting substances are exogenous substances that mimic, antagonize, impair, enhance, or inhibit the actions of endogenous hormones. In turn, endocrine-disrupting substances cause abnormalities of growth, reproduction, development, behavior, and immune function, as well as malignancy. 1–3 Endocrine-disrupting substances act at several sites via multiple mechanisms of action; receptor-mediated mechanisms are most commonly involved, but hormone synthesis, transport, and metabolism have also been implicated. 4,5
Atrazine is a compound of the chloro-s-triazine family (eg, simazine, cyanazine). Chlorotriazines disrupt the hormonal control of ovarian cycles, primarily via the action of these compounds on the central nervous system. 6 Atrazine enhances the growth of mammary tumors in female rats in the absence of ovarian hormones, partly by increasing cell proliferation in the promotion/progression stages of tumorigenesis. 7 Treating mice with atrazine also resulted in clear dimorphic neurodegenerative patterns in some brain areas. 8 Biochemical and histopathological findings show that subacute exposure of female pigs to a low dose of atrazine prolonged their estrous cycle. 9 Atrazine is also associated with decreased semen quality and fertility in men living in agricultural areas 10 and increased breast cancer incidence in women drinking water contaminated with atrazine. 11
Fenoxaprop-ethyl (FE) is an aryloxy phenoxyalkanoic acid herbicide, a family of compounds that inhibit fatty acid biosynthesis in plant meristems, 12 affect the acetyl-CoA carboxylase enzyme, 13 and cause depolarization of membrane potentials. 14
Atrazine and FE are commonly used in the United States. 15 Few reports document the effects of FE on animal or human health. 16 Despite reports that atrazine displays endocrine-disrupting activity in wild animals and humans, 17 atrazine is the pesticide most frequently found in groundwater in the United States. 18 There are 24 facilities that manufacture or process atrazine in the United States. Louisiana houses facilities that process the greatest amounts of atrazine (22 000 000–25 000 000 kg), with activities including production, processing, manufacturing, reaction, sale and distribution, and other ancillary uses. 19,20 Although Italy and Germany banned atrazine in 1991, with no decrease in corn yields or harvested area, atrazine was prohibited only in some regions of the United States. 18
Few reports exist on the effects of atrazine or FE on sperm physiology. 21 In pigs, ejaculated sperm express aromatase as well as estrogen and androgen receptors, with different intracellular localizations of these proteins suggesting a species-specific expression pattern. Thus, pig sperm could be a potential estrogen source, whereas the different cellular sites of hormone action suggest distinct roles of androgens and estrogens in pig sperm physiology 22 that could be affected by atrazine or FE.
The pig was chosen as a model because porcine physiology is very similar to that of humans in terms of the reproductive and endocrine systems. 23 Studies on reproductive toxicology are currently done in vivo with wild or laboratory animals to evaluate toxic damage. It is necessary to carry out in vitro studies to clarify the mechanisms of action of toxic compounds; because atrazine and FE have been extensively used in Mexico and other countries, the aim of this study was to evaluate the in vitro effects of both herbicides on boar sperm capacitation and the progesterone-induced acrosome reaction (PIAR).
Methods
All chemicals were purchased from Sigma Chemical Company (St Louis, Mo) unless otherwise indicated.
Semen Samples
Semen samples were obtained from the sperm-rich ejaculated fraction of 3 healthy fertile boars using the gloved-hand method, followed by removal of the gelatinous fraction. Samples were classified as normozoospermics according to criteria described elsewhere. 24 Sperm viability was evaluated by eosin-nigrosin staining. 25–28 One hundred cells were observed to evaluate semen samples. 28 Only ejaculates showing more than 85% progressive motility, at least 90% viability, and less than 10% abnormality were used. 29
Capacitation
Semen samples were washed twice to remove the seminal plasma by adding 1 mL of phosphate-buffered saline (PBS) to an equal volume of semen followed by centrifugation at 600 g for 5 minutes. Following the 2 washes, the pellet was resuspended in 1 mL of PBS. Aliquots of 5 × 106 cells were seeded into a Nunc 4-well multidish (Nunc, Roskilde Denmark), with 1 mL capacitation medium (TALPHEPES) supplemented with bovine serum albumin fraction V (6 mg/mL) and 7 mM sodium pyruvate, pH 7.4. 30 Cells were incubated at 39°C for 4 hours in a humid atmosphere with 5% CO2.
Progesterone-Induced Acrosome Reaction
The acrosome reaction (AR) was induced by adding progesterone at a final concentration of 10 μg/mL and incubating for 30 minutes under the conditions described above. 31 The spontaneous acrosomal reaction without progesterone induction is called SAR.
Herbicide Exposure
The herbicides atrazine and FE, technical grade (Aventis Cropscience, Cajeme, Sonora, Mexico), in their commercial presentation, were used. 21 A stock solution of 10 mM was prepared in absolute ethanol. One percent ethanol in the capacitation medium during 5 hours did not show any significant change in viability in sperm treated vs the control (80 ± 4 vs 81 ± 3, P = .45). This was the biggest concentration used in these studies.
Cytotoxicity was determined using the concentrations shown in Table 1. The median lethal concentration (LC50) of each herbicide was calculated using a Probit algorithm, 32 calculated with the Probit program, version 1.5 (Probit, Evironmental Protection Agency). 33
Final concentrations of 8, 20, and 40 μM atrazine and 12, 30, and 60 μM FE were used. These concentrations correspond approximately to the LC50/5, LC50/2, and LC50, respectively, of each compound. For the capacitation and PIAR studies, the herbicides were added at the beginning of the 4-hour incubation period. The control was the same solvent without herbicide (0 μM).
Sperm Membrane Status
The status of the plasma and acrosomal membranes was assessed by chlortetracycline staining, as described by Fraser and Herod, 34 and analyzed with a 495-nm UV epifluorescence microscope (Carl Zeiss, Germany) at 1000× magnification, according to the following criteria: sperm with a uniformly distributed fluorescence over the whole head and an intact acrosome were considered noncapacitated; sperm showing bright fluorescence in the acrosomal region beside a fluorescence-free band in the postacrosomal region with an intact acrosome were considered capacitated; sperm with dull fluorescence over the whole head except for a thin, bright band in the equatorial segment were considered acrosome-reacted. 35,36 At least 200 sperm cells were analyzed per slide. 37
Statistical Analysis
LC50 values were calculated with probit analysis using software from the U.S. Environmental Protection Agency. 33 Capacitation and AR data were analyzed with the Mann-Whitney U test, and P ≤ .05 was considered statistically significant.
Results
Determination of Herbicide Cytotoxicity in Boar Sperm During Capacitation
Herbicide cytotoxicity was determined by incubating sperm with variable concentrations of each herbicide for 5 hours. Preliminary tests showed that a range from 0 to 100 μM was suitable for evaluating both compounds.
The effects of different herbicide concentrations on sperm viability are presented in Table 1. Treatment with FE caused sperm viability to decrease in a concentration-dependent manner with an LC50 of 55.6 μM with 95% confidence limits of 47.7 to 63.1 μM. Treatment with atrazine resulted in decreased sperm viability in a manner similar to FE treatment. LC50 was 37.5 μMas calculated by probit, with 95% confidence limits of 29.6 to 44.5 μM. The concentrations used in later experiments as the LC50 were 60 μM for FE and 40 μM for atrazine, because these values are within the confidence interval.
Effect of FE on Sperm Capacitation
The viability of capacitated sperm in the presence of FE diminished significantly in a linear manner and corroborated the data used to obtain the LC50.
The control group attained greater than 50% capacitation. In sperm exposed to 12 μM FE, capacitation was significantly reduced (P ≤ .05), resulting in failed exposure in 85% of treated sperm. Noncapacitated sperm did not change with respect to controls. SAR increased 1.6-fold over controls (P ≤ .05) (Figure 1A).
At 30 μM FE, the proportion of capacitated sperm remained equal to that during 12 μM FE treatment; sperm undergoing SAR increased to 2.3 times the proportion found in controls (P ≤ .05) (Figure 1A), which was also significant compared with 12 μM FE (P ≤ .05).
Treatment with 60 μM FE caused an increase (1.1-fold) in capacitated sperm with respect to controls (P ≤ .05) and lower concentrations of FE, whereas levels of noncapacitated sperm were reduced to 27% of controls. Nevertheless, the proportion of sperm that developed SAR diminished significantly compared with 30 μM FE (P ≤ .05) but was increased 1.3-fold over controls (Figure 1A).
Effect of FE on the Progesterone-Induced Acrosomal Reaction
The AR observed in control sperm immediately after capacitation (SAR) was 17% (Figure 1A); 30 minutes of progesterone induction of the AR significantly increased capacitation to 30% (Figure 1B) (P ≤ .05).
Treatment with 12 μM FE had no significant effect on capacitation or PIAR. With 30 μM FE treatment, noncapacitated sperm decreased with respect to both 12 μM samples and controls (P ≤ .05); capacitated sperm decreased with respect to 12 μM samples but not controls. PIAR increased significantly with respect to the 12 μM samples and was increased 1.7-fold over controls (P ≤ .05). The 60 μM concentration showed PIAR levels similar to controls and significantly different from 30 μM treatment (P ≤ .05). Furthermore, noncapacitated sperm decreased significantly with respect to 30 μM and control samples (P ≤ .05), whereas the proportion of capacitated sperm did not change (Figure 1B).
Effect of Atrazine on Sperm Capacitation
In the presence of atrazine, the viability of capacitated sperm decreased in a linear fashion, in agreement with data used to obtain the LC50 results. In sperm populations exposed to atrazine, capacitation decreased significantly only at 40 μM, to 79% of controls (P ≤ .05) (Figure 2A).
In the sperm population exposed to 8 and 20 ≤ M atrazine, SAR increased 1.9-fold with respect to controls, which was significant (P ≤ .05) (Figure 2A). The noncapacitated population was reduced at both concentrations, compared with controls (P ≤ .05). There were no significant differences between results at these 2 atrazine concentrations. Treatment with 40 μM atrazine produced a 2.2-fold increase in SAR compared with controls (P ≤ .05) (Figure 2A).
Effect of Atrazine on the PIAR
All atrazine concentrations produced a significant decrease (P ≤ .05) in noncapacitated and capacitated sperm compared with controls, but there were no differences in these parameters between the atrazine concentrations (Figure 2B). Treatment with 8 μM atrazine caused a 1.6-fold increase in PIAR (P ≤ .05) compared with controls; this increase was the result of a decrease in noncapacitated and capacitated sperm. Treatment with 20 μM atrazine increased PIAR 1.8-fold over controls and was significantly different with respect to 8 μM samples (P ≤ .05). Atrazine at 40 μM produced the same effect, increasing PIAR 1.8-fold over the control value (Figure 2B).
Discussion
This study analyzed the in vitro effects of 2 herbicides on boar sperm capacitation and PIAR. All the concentrations used in this study were sublethal for sperm in vitro, using the LC50 as a maximum. Natural contact with herbicides is usually a chronic exposure attributable to work conditions. In this study, concentrations producing sperm damage were used. Such concentrations could not be considered environmentally relevant but could help to reveal othermechanisms that contribute to the sperm damages.
Atrazine was slightly more toxic than FE, because the LC50 of the former compound was higher. FE and atrazine are spermatotoxic compounds, because both herbicides affected gamete viability in a concentration-dependent manner. This result agrees with a study by Betancourt et al, 21 who found that these 2 herbicides affect sperm mobility and that after 1 hour of incubation with 50 μM FE or atrazine, sperm viability decreases to 13% and 24%, respectively. All these results are in close agreement with those reported previously by Campagna et al, 38 who found that sperm motility is immediately reduced in a dose-dependent manner by organochlorine treatment. The reduction in sperm quality could account for the decrease in fertilization.
The SAR observed in control sperm immediately after capacitation is 17%; after 30 minutes of progesterone induction of AR, capacitation increased to 30%. This significant difference shows that progesterone acts as an AR inducer. Our system is adequate to study the possible stimulatory or inhibitory actions of compounds in the process of in vitro capacitation–AR. Capacitation and AR data shown in this study agree with results previously obtained in our lab and others. 39,40 PIAR values are similar to those reported by other authors. 28,31,37,41–43
With 12 μM FE, the proportion of capacitated sperm diminished significantly compared with controls, whereas the noncapacitated proportion did not change. This decrease corresponds to an increase in the sperm that developed SAR.
At 30 μM FE, sperm capacitation did not diminish significantly because simultaneously, noncapacitated sperm underwent capacitation, generating a proportion of capacitated sperm equal to that found for 12 μM FE. Sperm suffering SAR increased dramatically with respect to controls, attributable to capacitated cells undergoing AR. This indicates that FE at these concentrations induces AR but not capacitation.
Treatment with 60 μM FE increased the proportion of capacitated sperm with respect to controls and both lower concentrations, whereas the noncapacitated proportion decreased. Nevertheless, the proportion of sperm that developed SAR diminished significantly compared with 30 μM, although it increased compared with controls. These results indicate that this FE concentration strongly induced capacitation and SAR but that many cells are arrested in the capacitation process.
FE induced capacitation at the higher concentration and induced SAR at all concentrations studied. Progesterone and FE (30 μM and 60 μM) increased PIAR significantly with respect to controls. The higher FE concentration duplicated control PIAR and was significantly different with respect to 30 μM FE. We found that there is a synergistic effect between FE and progesterone in the induction of AR. Our data analysis shows that FE induces capacitation, which prepares the sperm to trigger AR. We found a higher proportion of reacted sperm after 4 and 4.5 hours of incubation; these findings suggest that FE reduces capacitation time at higher concentrations.
Capacitation diminished significantly in sperm populations exposed only to 40 μM atrazine, but in the presence of progesterone, capacitation decreased significantly at all the concentrations tested.
In the sperm population exposed to atrazine, SAR and PIAR increased with respect to controls. This increase was the result of both a reduction in the noncapacitated population and an increase in AR from capacitated sperm, compared with controls.
These results suggest that atrazine strengthens the progesterone effect but not in a concentrationdependent manner, because the data reveal asymptotic behavior. This effect was also reported by Flores-Maya et al 44 during the induction of sister chromatid exchange in human lymphocytes treated in vitro with the herbicide ametryn. Roberge et al 45 showed that atrazine has affinity for neither androgen nor estrogen receptor but is a competitive inhibitor of phosphodiesterase increasing cyclic adenosine monophosphate, which results in elevated transcription of the gene for human aromatase, increasing aromatase activity and subsequent estrogen production. Although this mechanism is consistent with the demasculinization and feminization effects of atrazine, some studies suggest that atrazine does not induce aromatase in some species and/or in certain cell lines. 46 Recently, Fan et al 47 showed that atrazine induces human aromatase gene expression via promoter II (ArPII) in a steroidogenic factor 1 (SF-1)-dependent manner, binding directly to the SF-1, and concomitantly enhances interactions of SF-1 with co-activator TIF2 and renders more SF-1 binding to ArPII chromatin. It is clear that effects in sperm are not genomics, but recently a report showed that atrazine treatment significantly decreased total lipid, cholesterol, and phospholipid content and caused a significant inhibition of acetylcholinesterase activity and induction of oxidative stress of erythrocyte membranes. 48 This indicates that 1 or more of these processes could happen in the sperm membrane.
The AR was affected by both herbicides, with a significant increase in the number of sperm that underwent the AR. These results suggest that capacitation may be completed in less time than normal and that premature AR may be induced. Alternatively, the acrosomal membrane could be breaking without capacitation.
Sperm acquire their fertilizing capacity through the processes of capacitation and AR; the herbicides tested in this study interfere with both of these processes. These herbicide concentrations are not in a relevant range to produce in vivo effects, but they allow us to approach investigations of the mechanisms by which herbicides impair these processes in sperm. It will be necessary to perform more investigations in vitro to elucidate the mechanism by which these herbicides damage sperm.
During sperm capacitation, several biochemical and membrane surface modifications occur, such as peripheral glycoprotein removal, integral glycoprotein redistribution, protein phosphorylation, cholesterol and potassium loss, and bicarbonate and calcium uptake. These modifications induce the activation of receptors and bicarbonate-dependent adenyl cyclase as well as membrane hyperpolarization. These events occur after the exposure of sperm to the female genital tract, making the sperm competent to undergo the acrosome reaction. Patrat et al 49 found that progesterone promotes the AR in a sperm subpopulation by increasing the number of hyperpolarized cells after a transient depolarization phase in human sperm. In addition, the family of aryloxy phenoxyalkanoic acid compounds (including FE) produce depolarization of membrane potentials. 14 Progesterone exerts its effects via receptors in sperm, 42 so we think that capacitation is affected by FE and atrazine via destabilization of the sperm membrane potential and that a synergistic effect of either herbicide with progesterone promotes AR.
Ejaculated pig sperm expresses aromatase as well as estrogen and androgen receptors with a differential intracellular localization. 22 The mechanism of action of FE is poorly characterized, but both herbicides may affect sperm at the same functional level.
Similarly, some organochlorine pesticides, such as DDT (dichloro-diphenyl-trichloroethane), induce sublethal changes in transmembrane potential and reactive oxygen species production in bovine oviduct cells. 50 The plasma membrane of the male gamete is rich in polysaturated lipids, which are sensitive to the oxidative stress that reactive oxygen species generate. 51 Also, reactive oxygen radicals produced by nicotinamide adenine dinucleotide phosphate (reduced) oxidase may be converted into hydroperoxide radicals (−OOH) that initiate a peroxidation cascade, affecting membrane lipids and causing destabilization of the plasma membrane. 52,53 Slight peroxidation is required to induce capacitation in vitro 54,55 ; however, excessive peroxidation damages the cell. 51,56–58
Footnotes
Figures and Table
Acknowledgements
The authors thank Eduardo Casas for helping with the preparation of the herbicides. This work was partially financed by CONACYT (Mexico), grant 5-37923-B to MB, grant 52689/66955-M to RF, and scholarship number 176119 to RM. All authors disclose they have no financial or personal arrangements or relationships with other persons or organizations that would inappropriately influence the work submitted.