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Methyl parathion (Me-Pa) is an extremely toxic organophosphorus pesticide still used in developing countries. It has been associated with decreased sperm function and fertility and with oxidative and DNA damage. The blood-testis barrier (BTB) is a structure formed by tight junction (TJ) proteins in Sertoli cells and has a critical role in spermatogenesis. We assessed the effect of repeated doses of Me-Pa (3-12 mg/kg/day for 5 days, i.p.) on sperm quality, lipid oxidation, DNA integrity, and BTB permeability in adult male mice and explored oxidation as a mechanism of toxicity. Me-Pa caused dose-dependent effects on sperm quality, lipoperoxidation, and DNA integrity. Testis histology results showed the disruption of spermatogenesis progression and atrophy of seminiferous tubules. The pesticide opened the BTB, as evidenced by the presence of a biotin tracer in the adluminal compartment of the seminiferous tubules. This effect was not observed after 45 days of exposure when a spermatogenic cycle had completed. The coadministration of the antioxidant α-tocopherol (50 mg/kg/day for 5 days, oral) prevented the effects of Me-Pa on sperm quality, DNA and the BTB, indicating the importance of oxidative stress in the damage generated by Me-Pa. As evidenced by immunochemistry, no changes were found in the localization of the TJ proteins of the BTB, although oxidation (carbonylation) of total proteins in testis homogenates was detected. Our results show that Me-Pa disturbs the BTB and that oxidation is involved in the observed toxic effects on sperm cells., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Follicle growth culminates in ovulation, which allows for the expulsion of fertilizable oocytes and the formation of corpora lutea. Bisphenol A (BPA) is present in many consumer products, and it has been suggested that BPA impairs ovulation; however, the underlying mechanisms are unknown. Therefore, this study first evaluated whether BPA alters ovulation by affecting folliculogenesis, the number of corpora lutea or eggs shed to the oviduct, ovarian gonadotropin responsiveness, hormone levels, and estrous cyclicity. Because it has been suggested (but not directly confirmed) that BPA exerts toxic effects on the fertilization ability of oocytes, a second aim was to evaluate whether BPA impacts the oocyte fertilization rate using an in vitro fertilization assay and mating. The possible effects on early zygote development were also examined. Young adult female C57BL/6J mice (39 days old) were orally dosed with corn oil (vehicle) or 50 μg/kgbw/day BPA for a period encompassing the first three reproductive cycles (12-15 days). BPA exposure did not alter any parameters related to ovulation. Moreover, BPA exposure reduced the percentage of fertilized oocytes after either in vitro fertilization or mating, but it did not alter the zygotic stages. The data indicate that exposure to the reference dose of BPA does not impact ovulation but that it does influence the oocyte quality in terms of its fertilization ability., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate MET's effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis-vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43-57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Paternal germline exposure to organophosphorous pesticides (OP) has been associated with reproductive failures and adverse effects in the offspring. Methyl-parathion (Me-Pa), a worldwide-used OP, has reproductive adverse effects and is genotoxic to sperm, possibly via oxidative damage. This study investigated the stages of spermatogenesis susceptible to be targeted by Me-Pa exposure that impact on spermatozoa function and their ability to fertilize. Male mice were exposed to Me-Pa (20 mg/kg bw, i.p.) and spermatozoa from epididymis-vas deferens were collected at 7 or 28 days post-treatment (dpt) to assess the effects on maturing spermatozoa and spermatocytes, respectively. Spermatozoa were examined for DNA damage by nick translation (NT-positive cells) and SCSA (%DFI), lipoperoxidation (LPO) by malondialdehyde production, sperm function by spontaneous- and induced-acrosome reactions (AR), mitochondrial membrane potential (MMP) by using the JC-1 fluorochrome, and fertilization ability by an in vitro assay and in vivo mating. Alterations on DNA integrity (%DFI and NT-positive cells) in spermatozoa collected at 7 and 28 dpt, and decreases in sperm quality and induced-AR were observed; reduced MMP and LPO were observed at 7 dpt only. Negative correlations between LPO and sperm alterations were found. Altered sperm functional parameters evaluated either in vitro or in vivo were associated with reduced fertilization rates at both times. These results show that Me-Pa exposure of maturing spermatozoa and spermatocytes affects many sperm functional parameters that result in a decreased fertilizing capacity. Oxidative stress seems to be a likely mechanism of the detrimental effects of Me-Pa exposure in male germ cells.

Cigarette smoking in men has been associated with increased chromosomal abnormalities in sperm and with increased risks for spontaneous abortions, birth defects and neonatal death. Little is known, however, about the reproductive consequences of paternal exposure to second-hand smoke. We used a mouse model to investigate the effects of paternal exposure to sidestream (SS) smoke, the main constituent of second-hand smoke, on the genetic integrity and function of sperm, and to determine whether male germ cells were equally sensitive to mainstream (MS) and SS smoke. A series of sperm DNA quality and reproductive endpoints were investigated after exposing male mice for two weeks to MS or SS smoke. Our results indicated that: (i) only SS smoke significantly affected sperm motility; (ii) only MS smoke induced DNA strand breaks in sperm; (iii) both MS and SS smoke increased sperm chromatin structure abnormalities; and (iv) MS smoke affected both fertilization and the rate of early embryonic development, while SS smoke affected fertilization only. These results show that MS and SS smoke have differential effects on the genetic integrity and function of sperm and provide further evidence that male exposure to second-hand smoke, as well as direct cigarette smoke, may diminish a couple's chance for a successful pregnancy and the birth of a healthy baby.

Organophosphorous (OP) pesticides are considered genotoxic mainly to somatic cells, but results are not conclusive. Few studies have reported OP alterations on sperm chromatin and DNA, and oxidative stress has been related to their toxicity. Sperm cells are very sensitive to oxidative damage which has been associated with reproductive dysfunctions. We evaluated the effects of methyl-parathion (Me-Pa; a widely used OP) on sperm DNA, exploring the sensitive stage(s) of spermatogenesis and the relationship with oxidative stress. Male mice (10-12-weeks old) were administered Me-Pa (3-20 mg/kg bw/i.p.) and euthanized at 7- or 28-days post-treatment. Mature spermatozoa were obtained and evaluated for chromatin structure through SCSA (Sperm Chromatin Structure Assay; DNA Fragmentation Index parameters: Mean DFI and DFI%) and chromomycin-A(3) (CMA(3))-staining, for DNA damage through in situ-nick translation (NT-positive) and for oxidative stress through lipid peroxidation (LPO; malondialdehyde production). At 7-days post-treatment (mature spermatozoa when Me-Pa exposure), dose-dependent alterations in chromatin structure (Mean DFI and CMA(3)-staining) were observed, as well as increased DNA damage, from 2-5-fold in DFI% and NT-positive cells. Chromatin alterations and DNA damage were also observed at 28-days post-treatment (cells at meiosis at the time of exposure); suggesting that the damage induced in spermatocytes was not repaired. Positive correlations were observed between LPO and sperm DNA-related parameters. These data suggest that oxidative stress is related to Me-Pa alterations on sperm DNA integrity and cells at meiosis (28-days post-treatment) and epididymal maturation (7-days post-treatment) are Me-Pa targets. These findings suggest a potential risk of Me-Pa to the offspring after transmission.

Human paraoxonase (PON1) plays a role in detoxification of organophosphorus (OP) compounds by hydrolyzing the bioactive oxons, and in reducing oxidative low-density lipoproteins, which may protect against atherosclerosis. Some PON1 polymorphisms have been found to be responsible for variations in catalytic activity and expression and have been associated with susceptibility to OP poisoning and vascular diseases. Both situations are of public health relevance in Mexico. Therefore, the aim of this study was to evaluate PON1 phenotype and the frequencies of polymorphisms PON1 -162, -108, 55, and 192 in a Mexican population. The studied population consisted of unrelated individuals (n = 214) of either gender, 18-52 years old. Serum PON1 activity was assayed using phenylacetate and paraoxon as substrates. PON1 variants, -162, 55, and 192, were determined by real-time PCR using the TaqMan System, and PON1 -108 genotype by PCR-RFLP. We found a wide interindividual variability of PON1 activity with a unimodal distribution; the range of enzymatic activity toward phenylacetate was 84.72 to 422.0 U/mL, and 88.37 to 1645.6 U/L toward paraoxon. All four PON1 polymorphisms showed strong linkage disequilibrium (D% >90). PON1 polymorphisms -108, 55, and 192 were independently associated with arylesterase activity; whereas the activity toward paraoxon was related only with PON1 192 polymorphism, suggesting that this polymorphism is determinant to infer PON1 activity. A better understanding of the phenotype and genotypes of PON1 in Mexican populations will facilitate further epidemiological studies involving PON1 variability in OP poisoning and in the development of atherosclerosis.

Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A(3) (CMA(3)). Increases in DFI (15%), DFI% (4.5-fold), and CMA(3) (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA(3) provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.