Zuzana Knazicka

Dose- and time-dependent effect of copper ions on the viability of bull spermatozoa in different media

The purpose of this in vitro study was to determine the effect of copper (Cu) on the motility and viability of spermatozoa in the presence of different culture media. Specifically, we examined the dose- and time-dependent effect of copper ions (Cu2+) on the motility and viability of spermatozoa during different time periods (Time 0 h, 1 h, 24 h). The percentage of motile spermatozoa and progressive motile spermatozoa was determined after exposure to concentrations of 3.9; 7.8; 15.6; 31.2; 62.5; 125; 250; 500; 1000 μM/L of Cu2+ using the Sperm VisionTM CASA (Computer Assisted Semen Analyzer) system. The cell viability was measured by the MTT (metabolic activity) assay. The initial spermatozoa motility in the presence of Cu2+ in physiological saline solution (PS) showed slight increased values at doses <31.20 μM/L of Cu2+ compared to the control group. The long-term cultivation (Time 24 h) reduced the average motility values in all experimental groups (P < 0.001) in comparison to the control group. Identical spermatozoa motility was detected for the percentage of progressive motile spermatozoa during all time periods. The culture medium containing 20 % bovine serum albumin (BSA), triladyl and 5 % glucose increased the overall percentage of spermatozoa motility after 1 h of cultivation. A concurrently maintained motility of spermatozoa at doses <62.50 μM/L of Cu2+ during the long-term in vitro cultivation confirms the protective effect of albumin. The cell viability was decreased significantly (P < 0.001) in all experimental groups with copper administration. The obtained data point out that Cu2+ at high doses is a toxic element on the spermatozoa motility, which subsequently disrupts the viability of cells. However, using a suitable culture medium containing an energy component- and protein-rich substrate, the spermatozoa motility could increase.

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 VisionTM 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 (HgCl2) on the steroidogenesis. The cells were cultured for 48 h with addition of 1.0; 5.0; 25; 50 or 100 μM of HgCl2 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 Hg2+. Progesterone production was also decreased, but at the lower concentrations (1.0 and 5.0 μM) of Hg2+ this decline was lower compared to depression of testosterone. The cell viability significantly decreased at 25 μM and higher concentration of Hg2+. However, at 25 μM Hg2+ 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 HgCl2 exposure. The results suggest that Hg has detrimental effects on steroid hormone synthesis also at very low concentrations and consecutively on reproductive physiology.

Selected heavy metals versus antioxidant parameters in bull seminal plasma – A comparative study

To investigate the effects of lead (Pb) and cadmium (Cd) content on basic motility characteristics (motility, progressive motility) and selected antioxidant parameters (total antioxidant status - TAS, superoxide dismutase - SOD, albumin - ALB) in the bovine seminal plasma 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. Subsequently, the samples were centrifuged and fractions of seminal plasma were collected. Pb and Cd concentrations were determined by the voltametric method (ASV), antioxidant parameters were analyzed by UV/VIS spectrophotometry using commercial kits. The analysis showed that the average concentrations of the trace elements were 0.57 ± 0.01μg/mL for Pb and 0.11±0.01 μg/mL for Cd. The correlation analysis revealed that both heavy metals were negatively correlated with motility (r = −0.777; P < 0.001 for Pb and r = −0.786; P < 0.001 for Cd), progressive motility (r = −0.763; P < 0.001 for Pb and r = −0.792; P < 0.001 for Cd), TAS (r = −0.375; p > 0.05 and r = −0.334; P > 0.05, respectively), SOD (r = −0.746; P < 0.001 and r = −0.537; P < 0.05, respectively) as well as with ALB (r = −0.609; P < 0.01 and r = −0.699; P < 0.001, respectively). Moreover the samples were categorized in three quality groups (Excellent, Good, Medium) according to their motility values. The lowest Pb and Cd concentrations but the best antioxidant characteristics were found in samples of excellent quality, medium quality samples were described by the highest Pb and Cd concentration and the worst antioxidant power. This study demonstrates that Pb and Cd are serious toxic elements, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.

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.

In vitro effects of radiofrequency electromagnetic waves on bovine spermatozoa motility

In this study the effects of 1800 MHz GSM–like radiofrequency electromagnetic waves (RF–EMW) exposure on bovine semen was monitored. The experimental samples were analyzed in vitro in four time periods (0, 30, 120 and 420 min) and compared with unexposed samples (control). Spermatozoa motility was determined by computer assisted semen analyzer (CASA). Evaluation of the percentage of motile spermatozoa showed significant (P < 0.001) decrease in experimental groups after 120 and 420 min of culture when exposed to microwaves, in comparison to control. Similar spermatozoa motility inhibition was detected for the percentage of progressively motile spermatozoa, too. Average path distance decreased significantly (p < 0.001) in experimental groups after 30 and 420 min of culture. Path velocity increased in the experimental groups exposed to RF–EMW after 30 minutes of culture, but subsequently decreased after 420 min of culture, in comparison to control. This indicates a possible initial stimulation and subsequent velocity inhibition of bovine spermatozoa under RF–EMW exposure. Changes in spermatozoa motility were also detected for some fine parameters, too. A significant decrease (P < 0.001) was noted for amplitude of lateral head displacement in the experimental group after 420 minutes of culture. Detailed in vitro motility analysis of bovine spermatozoa exposed to microwave radiation suggested that the parameters of path and velocity at the beginning of the culture significantly increase, but after longer culture (420 minutes) a significant decrease occur in the experimental group as compared to control. In general, results of this experiment indicate a negative time–dependent effect of 1800 MHz RF–EMW radiation on bovine spermatozoa motility.

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 17β-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.

Effects of 4-nonylphenol on the steroidogenesis of human adrenocarcinoma cell line (NCI-H295R)

ABSTRACT In this study, the human H295R adrenocarcinoma cell line was exposed to different concentrations (0.04, 0.2, 1.0, 2.5 or 5 µg/mL) of nonylphenol (NP) to investigate its impact on the inhibition or induction of the steroid hormones production during 48 h of in vitro culture. The hormone production was measured using ELISA kits. Results of this in vitro study suggest various effect of nonylphenol in relatively low concentrations on the selected steroid hormones production by the human H295R adrenocarcinoma cell line. The inhibiting impact on progesterone and androstenedione production was observed. The amount of progesterone was significantly decreased at 1.0, 2.5 and 5 µg/mL NP. Equally, the androstenedione production significantly decreased at 5 µg/mL NP. On the other hand, the amount of testosterone and 17β-estradiol was induced after nonylphenol exposition. The significant increase of testosterone level was found out at treatment with 5 µg/mL NP. 17β-estradiol production significantly increased at the doses of 2.5 and 5 µg/mL NP.