The impact of diabetes mellitus type 2 on the steroidogenesis of male Zucker Diabetic Fatty rats.

The aim of this study was to evaluate the impact of diabetes mellitus type 2 (DM2) on the male endocrine system of Zucker Diabetic Fatty (ZDF) rats. Sexually mature ZDF rats were divided to a lean (control) and obese group, and had diabetes confirmed by blood tests. For the in vivo experiment, fasting blood was collected to obtain blood plasma. In case of the in vitro experiments, testicular fragments were cultured for 24 h, and the culture medium was collected. The concentrations of testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA-S), estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were quantified in the blood plasma and the medium by the ELISA method, while cholesterol (CHOL) was assessed spectrophotometrically. A significant decline of T (36.31 %), A4 (25.11 %) and FSH (26.99 %) as well as a significant increase of CHOL and E2 (36.17 %) was observed in the blood plasma of obese ZDF rats in comparison to the control. Under in vitro conditions, a significant decrease of FSH (23.35 %) accompanied by an increase of E2 was observed in the obese group compared to the control. In the case of CHOL, LH, T, DHEA and A4 no significant differences were observed. Our results suggest that except for FSH and E2 all steroid biomolecules were synthetized normally by the testicular tissue, however a dramatic endocrine disturbance was observed at the system level. We may conclude that DM2 has negative effects on systemic hormone secretion and these alterations are more pronounced in combination with obesity.

Antioxidative effect of dietary flavonoid isoquercitrin on human ovarian granulosa cells HGL5 in vitro.

This study aimed to examine the effect of dietary flavonoid isoquercitrin on ovarian granulosa cells using the immortalized human cell line HGL5. Cell viability, survival, apoptosis, release of steroid hormones 17beta-estradiol and progesterone, and human transforming growth factor-beta2 (TGF-beta2) and TGF-beta2 receptor as well as intracellular reactive oxygen species (ROS) generation were investigated after isoquercitrin treatment at the concentration range of 5-100 microg.ml-1. It did not cause any significant change (p>0.05) in cell viability as studied by AlamarBlue assay in comparison to control. No significant change was observed (p>0.05) in the proportion of live, dead and apoptotic cells as revealed by apoptotic assay using flow cytometry. Similarly, the release of 17beta-estradiol, progesterone, TGF-beta2 and its receptor were not affected significantly (p>0.05) by isoquercitrin as detected by ELISA, in comparison to control. Except for the highest concentration of 100 microg.ml-1, which led to oxidative stress, isoquercitrin exhibited antioxidative activity at lower concentration used in the study (5, 10, 25, and 50 microg.ml-1) by hampering the production of intracellular ROS, in comparison to control, as detected by chemiluminescence assay (p<0.05). Findings of the present study indicate an existence of the antioxidative pathway that involves inhibition of intracellular ROS generation by isoquercitrin in human ovarian granulosa cells.

Riboflavin recovery of spermatogenic dysfunction via a dual inhibition of oxidative changes and regulation of the PINK1-mediated pathway in arsenic-injured rat model.

Arsenic trioxide (As2O3) poisoning and associated potential lesions are of a global concern. Inversely, riboflavin (vitamin B2, VB2) as a component of flavoproteins could play a vital role in the spermatogenic enzymatic reactions. Thus, this research aimed to explore potential beneficial roles of VB2 during As2O3-injured-toxicity. Rats were randomly allocated into 4 groups (n=8/group) and challenged as follows (for 30 days continuously): Group 1 received normal saline; Group 2 was treated with 3 mg As2O3/L; Group 3 received 40 mg VB2/L; Group 4 received 3 mg As2O3/L + 40 mg VB2/L. Both As2O3 and VB2 were dissolved in deionized water. Malondialdehyde (MDA), Glutathione Peroxidase (GSH-Px), Superoxide dismutase (SOD), and Catalase (CAT) were assessed for the oxidative profile, while TAS (Total Antioxidative Status) levels were evaluated for the antioxidant system, in both serum and testicular tissue. P<0.05 was considered statistically significant. The results show that As2O3 significantly decreased the body weight, testicular weight and testis volume, semen quality and testicular cell count (p<0.05). Furthermore, MDA content in the testicular tissue of the As2O3 group rats was significantly higher in comparison to the vehicle group (p<0.05). Likewise, TAS and the activities of GSH-Px, CAT and SOD were reduced (p<0.05) when compared to the control. As(2)O(3) induced testicular damage and seminiferous tubular atrophy. Monodansylcadaverine assays mirrored the histopathology observations. Meanwhile, As2O3 upregulated the expression of mitophagy-related genes including PINK1, Parkin, USP8, LC3-I, Fis1 and Mfn2. The p38 gene, responsible to stress stimuli, was also upregulated by As2O3 administration. Meanwhile, exposure to VB2 led to a significant decrease of the expression levels of mitophagy related genes. Our study revealed that VB2 supplementation protected testicular structures against As2O3-induced injury via a dual inhibition of oxidative changes and a regulation of the PINK1-mediated pathway.

The effect of Apium graveolens L., Levisticum officinale and Calendula officinalis L. on cell viability, membrane integrity, steroidogenesis, and intercellular communication in mice Leydig cells in vitro.

Several plants have the potential to protect essential reproductive processes such as spermatogenesis or steroidogenesis, however, effective concentrations and main mechanisms of action are still unknown. This in vitro study was aimed to assess the effects of Apium graveolens L., Levisticum officinale, and Calendula officinalis L. extracts on the structural integrity, functional activity and gap junctional intercellular communication (GJIC) in mice Leydig cells. TM3 cells were grown in the presence of experimental extracts (37.5; 75; 150 and 300 µg/ml) for 24 h. For the present study, high-performance liquid chromatography analysis was used to quantify flavonoids or phenolic acids. Subsequently, Leydig cell viability was assessed by alamarBlue assay, while the cell membrane integrity was detected by 5-carboxyfluorescein diacetate-acetoxymethyl ester. The level of steroid hormones production was determined by enzyme-linked immunosorbent assay. Additionally, GJIC was assessed by scalpel loading/dye transfer assay. According to our results, Apium graveolens L. significantly increased the viability and cell membrane integrity at 75 µg/ml (109.0±4.3%) followed by a decline at 300 µg/ml (89.4±2.3%). In case of Levisticum officinale and Calendula officinalis L. was observed significant decrease at 150 µg/ml (88.8±11.66%; 87.4±6.0%) and 300 µg/ml (86.2±9.3%; 84.1±4.6%). Furthermore, Apium graveolens L. significantly increased the progesterone and testosterone production (75 and 150 µg/ml) however, Levisticum officinale and Calendula officinalis L. significantly reduced steroid hormones synthesis at 150 and 300 µg/ml. Finally, the disturbance of GJIC was significantly affected at 300 µg/ml of Levisticum officinale (82.5±7.7%) and Calendula officinalis L. (79.8±7.0%). The balanced concentration ratio may support the Leydig cell function, steroidogenesis as well as all essential parameters that may significantly improve reproductive functions.

Investigation of the Properties and Effects of Salvia Officinalis L. on the Viability, Steroidogenesis and Reactive Oxygen Species (ROS) Production in TM3 Leydig Cells in Vitro.

The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.

Sperm DNA fragmentation in donors and normozoospermic patients attending for a first spermiogram: Static and dynamic assessment.

Static assessment of sperm DNA Fragmentation (SDF at the time of ejaculation or sperm thawing when cryopreserved) and the dynamic assessment of SDF (SDF assessed after T2 hr, T6 hr and T24 hr of sperm thawing) were used to establish cut-off values associated with sperm donors when compared with closely related normozoospermic patients. Cryopreserved samples from donors revealed SDF levels two times lower in comparison with the patients. Donor sperm DNA exhibited a 2.5 times higher longevity when compared with the patients. Static values of SDF after thawing of approximately 11% identify the donors with a 71% of sensitivity and 84% specificity. With respect to the dynamic assessment, SDF increases of 2.3 per hr during the first 2 hr of incubation identify the donors with 70% of sensitivity and 66% of specificity. Creating the Rate of Combined Damage (RCD) defined as the product of SDF-T0 by the increase in the damage registered during the first 2 hr of incubation (r-SDF-T0-2 ), an index of RCD = 22.2 units has an identification capacity of donors with a 78% sensitivity and 77% specificity. Such cut-off values could be used to characterise donors with high chromatin resistance to damage when meeting the above-established criteria.

Taurine does not improve the quality of short-term stored rabbit spermatozoa in vitro.

This study examines the impact of taurine on the viability, morphology and acrosome integrity of rabbit spermatozoa in vitro. Semen samples, obtained from four to five sexually mature and healthy New Zealand White rabbits, were pooled in heterospermic semen sample. This was divided and treated with taurine in a concentration of 0 (control), 1.5, 7, 12.5, 50 mM to a final concentration of 108  spermatozoa/ml. The samples were then incubated at 37°C for 4 hr. A combination of fluorescent probes SYBR-14/propidium iodide/PNA-Alexa Fluor 647 was used to assess spermatozoa viability and acrosome integrity on a flow cytometer. The sperm morphology was evaluated under a light microscope following fixation in 1.5% paraformaldehyde. The experiment was repeated three times. According to the obtained results, the spermatozoa neither could have benefit from immediate taurine treatment, nor had they after 4-hr incubation with respect to viability and acrosome integrity. Taurine did not initially alter the total and acrosome morphology of treated spermatozoa nor has it by 4 hr upon treatment. In conclusion, taurine may have no protective effect on the viability, morphology and acrosome integrity of short-term stored rabbit spermatozoa.

Protective Effects of Quercetin on Selected Oxidative Biomarkers in Bovine Spermatozoa Subjected to Ferrous Ascorbate.

Quercetin (QUE) is a natural flavonol-type flavonoid with antibacterial, anti-inflammatory and anti-aggregatory properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. The aim of this study was to assess the effectiveness of QUE to reverse ROS-mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to QUE treatment (7.5, 25, 50 and 100 µmol/l) in the presence or absence of a pro-oxidant, that is ferrous ascorbate (FeAA; 150 µmol/l FeSO4 and 750 µmol/l ascorbic acid) during a 6-h in vitro culture. Spermatozoa motion characteristics were assessed using the SpermVision computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry, and the nitroblue tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (p < 0.001), viability (p < 0.001) and decreased the antioxidant parameters of the sperm samples (p < 0.001) but increased the ROS generation (p < 0.001), superoxide production (p < 0.001) and lipid peroxidation (p < 0.001). QUE administration resulted in a preservation of the spermatozoa vitality and antioxidant characteristics (p < 0.01 with respect to the enzymatic antioxidants, p < 0.001 in relation to GSH) with a concentration range of 50-100 µmol/l QUE revealing to be the most effective. Our results suggest that QUE exhibits significant ROS-scavenging and metal-chelating properties which may prevent spermatozoa alterations caused by ROS, and preserve the functionality of male reproductive cells.

The Impact of 4-Nonylphenol on the Viability and Hormone Production of Mouse Leydig Cells.

Exogenous substances altering the function of the endocrine system and exhibiting adverse health effects on the organism are defined as endocrine disruptors. Nonylphenol is one of the most abundant alkylphenol ethoxylate derivatives, being detected in food products. Diverse studies have classified nonylphenol as hazardous to the health, especially to male reproduction. This in vitro study aimed to examine the effects of 4-nonylphenol on androstenedione and testosterone production as well as on the viability of Leydig cells of NMRI mice. The cells were cultured for 44 h with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 µg/ml of 4-nonylphenol and compared to the control. Quantification of testosterone and androstenedione directly from aliquots of the medium was performed by enzyme-linked immunosorbent assay. Cell viability was measured by the metabolic activity assay for mitochondrial functional activity. Androstenedione production significantly (P < 0.001) increased with 1.0; 2.5 and 5.0 µg/ml 4-nonylphenol. Although cAMP-stimulated testosterone production was not significantly affected by 4-nonylphenol, a tendency to attenuate the level of testosterone in the Leydig cells treated with 2.5 and 5.0 µg/ml 4-nonylphenol was observed. The viability of mouse Leydig cells was slightly increased at the lowest doses of 4-nonylphenol (0.04 and 0.2 µg/ml). We also observed an increase at higher concentrations of the substance (1.0; 2.5 and 5.0 µg/ml), but this increase was not significant. Further investigations are required to establish the biological significance and possible reproductive implications.

The effect of transgenesis on rabbit thyroid tissue structure.

This study was aimed to compare structures of the thyroid tissue of transgenic rabbits expressing the human clotting factor VIII under the murine whey acidic protein promoter (mWAP-hFVIII rabbits) with the non-transgenic controls. Thyroid tissue samples were taken from transgenic and non-transgenic New Zealand White rabbits, examined by optical microscopy and analysed morphometrically. The analysis revealed no significant differences (P > 0.05) in the relative volume of basic thyroid structures. Furthermore, no significant differences (P > 0.05) were observed when measuring the epithelial height and nuclear diameter of the follicular cells. Altogether, this study demonstrates no negative effect of the mWAP-hFVIII transgenesis on the rabbit thyroid gland structure.