Dose- and time-dependent effects of bisphenol A on bovine spermatozoa in vitro.

The present study was to evaluate the effect of bisphenol A (BPA) at the doses 1, 10, 100 and 200 µg mL(-1) on the bovine spermatozoa motility, viability and production of superoxide radical. The CASA system was used to determine the spermatozoa motility. The initial motility showed the significant differences (P < 0.001) between the groups higher than 100 µg BPA mL(-1) and the control group. Evaluation of the spermatozoa motility after 6 h of cultivation at the doses > 10 µg BPA mL(-1) was found to decrease motility significantly. After 24 h it was observed that the doses < 10 µg BPA mL(-1) statistically increased motility, while the doses > 100 µg BPA mL(-1) significantly decreased motility in comparison to control. The viability of spermatozoa as detected by the MTT assay decreased in all experimental groups, but significant differences were noted only at the highest doses of BPA after 24 h of in vitro cultivation. The intracellular superoxide production was observed by the NBT test after 24 h of BPA exposure. The results indicated that in all experimental groups the amount of superoxide increased as compared to the control group; significant changes were observed at the doses > 100 µg BPA mL(-1). In conclusion, the results from our experiments suggest the negative effects of BPA at the highest doses used on motility and viability of bovine spermatozoa and production of superoxide radical.

Dose- and Time-Dependent In Vitro Effects of Divalent and Trivalent Iron on the Activity of Bovine Spermatozoa.

This in vitro study was designed to assess the impact of divalent (Fe(2+)) or trivalent (Fe(3+)) iron on the activity and oxidative balance of bovine spermatozoa at specific time intervals (0, 2, 8, 16, and 24 h) during an in vitro culture. Forty-five semen samples were collected from adult breeding bulls and diluted in physiological saline solution supplemented with different concentrations (0, 1, 5, 10, 50, 100, 200, 500, 1000 µmol/L) of FeCl2 or FeCl3. Spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Both divalent and trivalent iron exhibited a dose- and time-dependent impact on the spermatozoa physiology and oxidative balance. Concentrations ≥50 µmol/L FeCl2 and ≥100 µmol/L FeCl3 led to a significant decrease of spermatozoa motility (P < 0.05) and mitochondrial activity (P < 0.001 with respect to 200-1000 µmol/L FeCl2/FeCl3; P < 0.01 in case of 100 µmol/L FeCl2/FeCl3), accompanied by a significant superoxide overproduction (P < 0.001 in terms of 200-1000 µmol/L FeCl2 and 500-1000 µmol/L FeCl3; P < 0.01 with respect to 100 µmol/L FeCl2 and 100-200 µmol/L FeCl3). On the other hand, concentrations below 10 µmol/L FeCl2 and 50 µmol/L FeCl3 proved to stimulate the spermatozoa activity, as shown by a significant preservation of the motility and viability characteristics (P < 0.001 in case of the motility parameters; P < 0.01 with respect to the spermatozoa viability), alongside a significant decline of the superoxide generation (P < 0.05). In a direct comparison, divalent iron has been shown to be more toxic than trivalent iron. Results from this in vitro study show that high concentrations of both forms of iron are toxic, while their low concentrations may have spermatozoa activity-promoting properties. In vitro concentrations of divalent or trivalent iron that could be regarded as critical are 50 µmol/L FeCl2 and 100 µmol/L FeCl3 when iron ceases to be an essential micronutrient in order to become a toxic risk factor.

Endocrine disruptive effects of cadmium on steroidogenesis: human adrenocortical carcinoma cell line NCI-H295R as a cellular model for reproductive toxicity testing.

Cadmium (Cd) is a known endocrine disruptor with the ability to affect the production of hormones involved in the regulation of reproductive processes. In this study human adrenocortical carcinoma cell line NCI-H295R was used as an in vitro biological model to study the effect of cadmium (CdCl2) on steroidogenesis. The cell cultures were exposed to different concentrations of CdCl2 (1.90, 3.90, 7.80, 15.60, 31.20 and 62.50 µM) and compared to control (medium without CdCl2). Cell viability was measured by the metabolic activity (MTT) assay for estimation of mitochondria structural integrity. Quantification of sexual steroid production directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Following 48 h culture of the cells in the presence of CdCl2 a concentration-dependent depletion in progesterone production was observed at the lower concentrations of CdCl2. The lowest amount of progesterone was significantly detected in groups with the higher doses (≥ 31.20 µM) of CdCl2, which elicited significant (P < 0.01) cytotoxic action, too. Cadmium decreased testosterone release in the whole applied range even at the lower concentration of CdCl2. The release of 17ß-estradiol decreased as well, but the decline was less pronounced compared to decrease of progesterone and testosterone. The cytotoxic effect was significantly (P < 0.01) detected at all concentrations of CdCl2 (1.90-62.50 µM) used in the study. However, the cell viability remained relatively high (>75%) up to 7.80 µM of CdCl2 and significantly (P < 0.01) decreased at 15.60 µM and higher concentrations of CdCl2. These results suggest that cadmium has endocrine disruptive effects on sexual steroid synthesis even at very low concentrations.

The impact of lead and cadmium on selected motility, prooxidant and antioxidant parameters of bovine seminal plasma and spermatozoa.

The aim of this study was to investigate the effects of lead (Pb) and cadmium (Cd) content on basic motility characteristics (motility-MOT, progressive motility-PROG) as well as selected markers of the prooxidant-antioxidant balance (catalase-CAT, glutathione-GSH, malondialdehyde-MDA) in bovine seminal plasma and spermatozoa. Twenty five semen samples were collected from breeding bulls and used in the study. Motility analysis was carried out using the Computer Assisted Sperm Analysis (CASA) system. The samples were centrifuged, fractions of seminal plasma and spermatozoa were separated, lysates were prepared from the sperm cell fractions. Pb and Cd concentrations were determined by the voltametric method (ASV), antioxidants and MDA were analyzed by UV/Vis spectrophotometry. The analysis showed that the average concentration of Pb in the seminal plasma was 0.23 ± 0.02 µg/mL, while its amount in the sperm cells was significantly higher (0.41 ± 0.07 µg/mL; P < 0.05). The average Cd content in bovine seminal fractions was similar and non-significant: 0.09 ± 0.01 µg/mL in the seminal plasma and 0.11 ± 0.01 µg/mL in spermatozoa (P > 0.05). The correlation analysis revealed that both heavy metals were significantly negatively correlated with MOT and PROG (P < 0.05; P < 0.01; P < 0.001), CAT (P < 0.05; P < 0.01) as well as GSH (P < 0.05; P < 0.01) but significantly positively associated with MDA as the marker of lipid peroxidation (P < 0.05; P < 0.01). Moreover the samples were categorized in three quality groups (Excellent, Good, Moderate) according to their motility values. The lowest Pb, Cd and MDA concentrations but the best antioxidant characteristics were found in samples of the best quality, moderate quality samples exhibited the highest Pb, Cd and MDA content together with the worst antioxidant capacity. This study demonstrates that Pb and Cd are serious toxic elements, which are able to increase the risk of seminal oxidative stress development and a subsequent decrease of male fertility.

The effect of nonylphenol on the motility and viability of bovine spermatozoa in vitro.

The objective of this in vitro study was to determine the effect of nonylphenol (NP) as an environmental toxicant on the spermatozoa motility and viability. The dose- and time-dependent effect of nonylphenol (1, 10, 100 and 200 µg/mL) dissolved either in 0.1% dimethyl sulfoxide (DMSO) or 0.1% ethanol (ETOH) on the motility and viability of bovine spermatozoa, as a cell model, during several time periods (0 h, 2 h, 4 h and 6 h) were examined. The motility of spermatozoa was determined by the Sperm Vision(TM) CASA (Computer Assisted Semen Analyzer) system. The results showed decreased spermatozoa motility in all experimental groups with the addition of NP dissolved in 0.1% DMSO and 0.1% ETOH (P < 0.001 and P < 0.05). The lowest spermatozoa motility was found at doses > 100 µg/mL of NP in comparison with the control group. The viability of bovine spermatozoa detected by the MTT cytotoxicity assay was decreased significantly (P < 0.001) in all experimental groups with NP dissolved in 0.1% ETOH. The viability in groups with NP dissolved in 0.1% DMSO was significantly (P < 0.05) decreased at 1 µg/mL of NP and significantly decreased (P < 0.001) at doses > 10 µg/mL of NP. After 6 h of culture the MTT assay proved a negative effect of all NP doses the on cell viability. The obtained data clearly indicate the negative effect of NP as an endocrine disruptor on spermatozoa motility and viability, which should be seriously considered in the case of exposure to NP in animals and humans and as a reason of male reproductive dysfunction.

Effects of mercury on the steroidogenesis of human adrenocarcinoma (NCI-H295R) cell line.

In this study the NCI-H295R human adrenocortical carcinoma cell line was used as an in vitro biological model to study the effect of mercury (HgCl(2)) on the steroidogenesis. The cells were cultured for 48 h with addition of 1.0; 5.0; 25; 50 or 100 µM of HgCl(2) and compared to control. Cell viability was measured by the MTT (metabolic activity) assay estimation for the mitochondria structural integrity. Quantification of testosterone and progesterone directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Concentration-dependent depression in testosterone production was detected particularly for higher concentration of Hg(2+). Progesterone production was also decreased, but at the lower concentrations (1.0 and 5.0 µM) of Hg(2+) this decline was lower compared to depression of testosterone. The cell viability significantly decreased at 25 µM and higher concentration of Hg(2+). However, at 25 µM Hg(2+) exposure the cell viability remained relatively high (> 80%). Results of the study indicate dose-dependent decreases in both testosterone and progesterone production of H295R cell culture following a 48 h in vitro HgCl(2) exposure. The results suggest that Hg has detrimental effects on steroid hormone synthesis also at very low concentrations and consecutively on reproductive physiology.

Impact of Seminal Chemical Elements on the Oxidative Balance in Bovine Seminal Plasma and Spermatozoa.

Mutual relationships between selected chemical elements (Na, K, Fe, Cu, Mg, and Zn), basic motility characteristics (motility and progressive motility), and markers of the oxidative balance (superoxide dismutase, catalase, glutathione, albumin, and malondialdehyde) were investigated in bovine seminal plasma and spermatozoa. Computer assisted sperm analysis was used to assess the motility parameters; mineral concentrations were determined by the voltammetric method and flame absorption spectrophotometry; antioxidants and malondialdehyde were evaluated by UV/VIS spectrophotometry. Concentrations of chemical elements in both seminal fractions were in the following descending order: Na > K > Zn > Mg > Fe > Cu. Higher amounts of all minerals and nonenzymatic antioxidants were detected in the seminal plasma (P < 0.01; P < 0.001), while higher MDA concentration and activity of enzymatic antioxidants were recorded in the cell lysates (P < 0.01; P < 0.001). Na, Fe, Cu, Mg, and Zn were positively correlated with the motility and antioxidant parameters (P < 0.05; P < 0.01; P < 0.001). Inversely, K exhibited the positive associations with malondialdehyde (P < 0.05). This study demonstrates that most chemical elements are integral components of bovine semen and are needed for the protection against oxidative stress development.