Exceptional Properties of Lepidium sativum L. Extract and Its Impact on Cell Viability, Ros Production, Steroidogenesis, and Intracellular Communication in Mice Leydig Cells In Vitro.

The prevalence of reproductive dysfunction in males has risen in the last few years, and alternative therapies are gradually gaining in popularity. Our in vitro study aimed to evaluate the potential impact of Lepidium sativum L. on mice TM3 Leydig cells, concerning basal parameters such as cell viability, cell membrane integrity, and lysosomal activity, after 24 h and 48 h exposure. Moreover, reactive oxygens species generation, sex-steroid hormone secretion, and intercellular communication were quantified. In the present study, the microgreen extract from Lepidium was rich in ferulic acid, 4-OH benzoic acid, and resveratrol, with a significant antioxidant activity. The results showed that lower experimental doses (62.5-250 µg/mL) could positively affect the observed parameters, with significant differences at 250 µg/mL after 24 h and 48 h, respectively. Potential risks could be associated with higher concentrations, starting at 500 µg/mL, 1000 µg/mL, and 2000 µg/mL of Lepidium. Nevertheless, biochemical quantification indicated a significant antioxidant potential and a rich content of biologically active molecules at the applied doses, and time determined the intracellular response of the cultured model.

Resveratrol attenuates hydrogen peroxide-induced oxidative stress in TM3 Leydig cells in vitro.

The objective of present study was to investigate in vitro protective potential of resveratrol in TM3 Leydig cells with induced oxidative stress using hydrogen peroxide (H2O2). Leydig cells experiencing oxidative stress exhibit reduced activities in androgens production, and become hypofunctional with age, which is also related to growing oxidative stress, while resveratrol has received growing attention as a cytoprotective agent. TM3 mouse Leydig cells were cultivated during 24 h in the presence of resveratrol (5, 10, 25, 50 and 100 µM) alone, or in combination with H2O2 (300/600 µM) to induce oxidative stress. Mitochondrial activity was evaluated using MTT test, triple assay was used in order to assess cell viability parameters, intracellular generation of superoxide was determined by the nitroblue-tetrazolium assay, and quantification of steroid hormones was performed by the enzyme- linked immunosorbent assay. Resveratrol alone treatment led to the most significantly improved values of all tested parameters in the cells of experimental group with addition of 10 µM of resveratrol in comparison to the control group. In the case of cells with induced oxidative stress (300 µM H2O2) resveratrol administration resulted in significantly increased (P < 0.05) metabolic activity, as well as cell membrane integrity at concentration 10 µM. Significantly improved (P < 0.001) lysosomal activity showed cells treated with 5 and 10 µM of resveratrol, and the level of both measured hormones was significantly higher (P < 0.05) in cells supplemented with 10 µM of resveratrol. Significant decline of superoxide radical production was observed in all experimental groups in comparison to the control exposed to H2O2 alone. With respect to cells exposed to higher concentration of H2O2 (600 µM), results showed positive effect of resveratrol only in biosynthesis of both androgens with significant increased values in experimental group treated with 5 µM (P < 0.05) and 10 µM (P < 0.01) of resveratrol, in addition, in the case of testosterone we recorded significant higher (P < 0.05) values in cells with addition of 25 and 50 µM resveratrol when compared to H2O2 control. More specific and systematic research focused especially on androgen biosynthesis is necessary related to the biological activity of resveratrol in male reproductive system due to inconsistent results of studies.

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.