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Lead (Pb) exposure at high concentrations is associated with poor sperm quality, acrosome alterations, and low fertilization rate. Sperm capacitation and the acrosome reaction (AR) are required for successful fertilization. Actin polymerization is crucial for correct capacitation, and small GTPases, such as RhoA, Rac1, and Cdc42, are involved. This study aimed to evaluate the effects of Pb on sperm fertilization ability, capacitation, AR, and the mechanisms involved in mice exposed to low Pb concentrations. CD1 mice were exposed to 0.01% Pb 2+ for 45 days through their drinking water and their spermatozoa were collected from the cauda epididymis-vas deferens to evaluate the following: AR (oAR: initial, sAR: spontaneous, and iAR: induced) using the PNA-FITC assay, sperm capacitation (P-Tyr levels), actin polymerization (phalloidin-TRITC), MDA production (stress oxidative marker), the RhoA, Rac1, and Cdc42 protein levels, and the in vitro fertilization (IVF). After the treatment, the blood Pb (PbB) concentration was 9.4 ± 1.6 μg/dL. Abnormal sperm morphology and the oAR increased (8 and 19%, respectively), whereas the iAR decreased (15%) after a calcium ionophore challenge, and the actin polymerization decreased in the sperm heads (59%) and tails (42%). Rac1 was the only Rho protein to significantly decrease (33%). Spermatozoa from the Pb-treated mice showed a significant reduction in the fertilization rate (19%). Our data suggest that Pb exposure at environmental concentrations (PbB < 10 μg/dL) decreases the acrosome function and affects the sperm fertilization ability; this is probably a consequence of the low Rac1 levels, which did not allow adequate actin polymerization to occur., (Copyright © 2019 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.

Extensive use of organophosphorous pesticides (OP) by young men represents a public health problem. Toxicity of OP mainly results in neurotoxicity due to their oxygen analogues (oxons), formed during the OP oxidative activation. OP alter semen quality and sperm chromatin and DNA at different stages of spermatogenesis. Oxons are more toxic than the parent compounds; however, their toxicity to spermatogenic cells has not been reported. We evaluated sperm DNA damage by several OP compounds and their oxons in human spermatozoa from healthy volunteers incubated with 50-750 microM of methyl-parathion (MePA), methyl-paraoxon (MePO), chlorpyrifos (CPF), chlorpyrifos-oxon (CPO), diazinon (DZN) or diazoxon (DZO). All concentrations were not cytotoxic (evaluated by eosin-Y exclusion), except 750 microM MePO. Oxons were 15% to 10 times more toxic to sperm DNA (evaluated by the SCSA parameter, %DFI) than their corresponding parent compounds, at the following order: MePO>CPO=MePA>CPF>DZO>DZN, suggesting that oxon metabolites participate in OP sperm genotoxicity.

Lead (Pb) alters sperm chromatin condensation (CC) and the mechanisms are investigated. During spermatogenesis, protamines replace histones and disulfide bonds formation during epididymal maturation condense the chromatin. We evaluated sperm Pb uptake in testis and epididymis and the effects on CC in mice (0.06% Pb(2+)/16 weeks/drinking water). Spermatozoa from caput epididymis (CP) and cauda epididymis-vas deferens (CE-VD) were obtained and CC was measured by SCSA. Lead levels in spermatozoa from CP were lower than those from CE-VD, and correlated with a decreased CC, while Pb in CE-VD spermatozoa correlated with an increased CC. Lead accumulation into the nucleus was observed and Pb binding to nuclear sulfhydryl groups decreased chromatin decondensation in vitro. Our results suggest that spermatozoa take up Pb during testicular development and epididymal transport and alter CC, depending of the timing of Pb incorporation into the sperm nucleus, which finally may interfere with the chromatin decondensation process after fertilization.

We evaluated environmental-lead (Pb) effects on semen quality and sperm chromatin, considering Pb in seminal fluid (PbSF), spermatozoa (PbSpz), and blood (PbB) as exposure biomarkers in urban men (9.3 microg/dL PbB). Several individuals (44%) showed decreases in sperm quality; sperm concentration, motility, morphology and viability associated negatively with PbSpz, whereas semen volume associated negatively with PbSF. Multiple linear regression estimated PbSF and PbSpz thresholds for alterations in semen quality. Forty-eight percent of samples showed high values of nuclear chromatin condensation (NCD) positively associated with PbSF and zinc in spermatozoa (ZnSpz). ZnSpz values were higher than in fertile men. These results suggest that Pb may affect sperm chromatin by altering sperm Zn availability. PbB was not associated with semen quality or NCD, suggesting that Pb in semen compartments assesses better the amount of Pb in the reproductive tract; therefore, these are better biomarkers to evaluate toxicity at low Pb-exposure levels.