Physiological and Pathological Roles of Free Radicals in Male Reproduction

Oxidative stress (OS) is a condition caused by an imbalance between reactive oxygen species (ROS) overgeneration and decreased antioxidant defense mechanisms in the cell. OS has become a prominent factor in male reproductive dysfunction as ROS cause damage to sperm DNA, lipids and proteins, alterations to critical sperm structures and signaling pathways, leading to a decreased sperm activity and fertilizing capacity. At the same time, small amounts of ROS play vital roles in events leading to sperm maturation and acquisition of functional activity, which is why a proper oxidative balance is of paramount importance for a proper male fertility. Understanding the physiological and pathological roles of ROS in male reproduction has become an essential pillar of modern andrology; however, numerous questions related to the controversial behavior of ROS in male reproductive cells and tissues still remain unanswered. This chapter aims to summarize current evidence available on the relationships between free radicals, antioxidants and male reproduction and to trigger more scientific interest, particularly with respect to the design of efficient strategies to diagnose or treat male sub- or infertility associated with OS

ZDF Rats: A Suitable Model to Study Male Reproductive Dysfunction in Diabetes Mellitus Type 2 Patients

This chapter examines the impact of diabetes mellitus type 2 (DM 2) on the vitality of male reproductive cells collected from Zucker diabetic fatty (ZDF) rats which could be a suitable experimental model for simulating this metabolic disorder. Epididymal spermatozoa were subjected to the assessment of motility, membrane integrity, mitochondrial activity, DNA fragmentation, and oxidative profile. Our results show that DM 2 in combination with obesity negatively affects the sperm vitality and increases the chances of oxidative damage to male gametes. In conclusion we may state that DM 2 has a negative impact on the spermatogenic aspect of male fertility and decreases the sperm quality

Male Reproduction: One of the Primary Targets of Bisphenol

Infertility is a major health issue affecting human life. The most notable factors causing male infertility is exposure to environmental contaminants. Bisphenol A (BPA) is a common toxic environmental contaminant. Human population is exposed to bisphenol A through air, water, food and a variety of industrial products. Growing evidence from research on laboratory animals supports the hypothesis that bisphenol A is able to adversely affect male reproductive function. The specific mechanisms of action of bisphenol A are wide but not definite. Bisphenol A interferes with the hormonal metabolism and regulation, binding affinity or enzymatic activity, resulting in a deviation from a normal reproductive behaviour. Binding ability to androgen and oestrogen receptors, as well as other properties, is currently investigated. A decreased sperm count, inhibition of sperm motility and reduction of organ weights were observed and linked with oxidative stress after bisphenol A treatment. In addition, prenatal exposure to bisphenol A may lead to adverse effects in the offspring. In this review, we address the topic of BPA effects on male reproductive function and emphasize its effects on testicular steroidogenesis and spermatogenesis. A considerably more detailed and systematic research focusing on bisphenol A toxicology is required for a better understanding of risks associated with exposure to this endocrine disruptor

Účinok 4-octylphenolu na bazálnu a stimulovanú produkciu testosterónu myších Leydigových buniek

Octylphenol is biodegradation product of alkylphenolethoxylates frequently used in detergents, paints and other industrial applications. This compound is classified as an endocrine disruptor. Recent studies have hypothesized that occupational exposure to octylphenol poses adverse effects on reproductive system of humans and wildlife species. Enzymes involved in the steroid biosynthesis pathway are really sensitive targets for the action of various endocrine-disrupting chemicals. Aim of in vitro study was determined the effect of 4-octylphenol on basal and human chorionic gonadotropin stimulated testosterone formation of ICR mice Leydig cells. On the other hand, was classified potential impact of mentioned endocrine disruptor on Leydig cell viability after 44 h of cultivation. Cell suspension was cultured with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg*mL-1 of 4-octylphenol and compared to the control. Hormone quantification from the medium was performed by enzyme-linked immunosorbent assay. Viability of cell suspension was determined by the metabolic activity assay. Unstimulated testosterone production significantly (P˂0.001) increased with 2.5 and 5.0 μg*mL-1 4-octylphenol. Cell viability was also significantly (P˂0.001; P˂0.05) stimulated by 4-octylphenol. Although human chorionic gonadotropin stimulated testosterone secretion was significantly (P˂0.05) affected by the lowest concentration (0.04 μg*mL-1) in the cell viability was recorded significantly (P˂0.001; P˂0.05) higher mitochondrial activity (1.0; 2.5 and 5.0 μg*mL-1). Considerably more detailed and systematic research in this area is required for a better understanding of potential risk to humans or animals.Octylphenol je biodegradačný produkt alkylphenol-ethoxylátov, ktorý je široko využívaný v detergentoch, farbivách a ďalších industriálnych produktoch. Zároveň je klasifikovaný ako endokrinný disruptor. Nedávne štúdie poukazujú na fakt, že pôsobenie octylphenolu vyvoláva negatívne účinky v reprodukčnom systéme ľudí a zvierat. Enzýmy, ktoré sú zapojené do procesu biosyntézy steroidov sú veľmi citlivé na pôsobenie endokrinných disruptorov. Táto in vitro štúdia bola zameraná na sledovanie účinku 4-octylphenolu na bazálnu a hCG stimulovanú produkciu testosterónu u ICR myších Leydigových buniek. Rovnako bol klasifikovaný účinok spomínaného endokrinného disruptora na viabilitu týchto buniek po 44 hodinovej kultivácií. Bunková suspenzia bola kultivovaná s prídavkom 0,04; 0,2; 1,0; 2,5 a 5.0 μg*mL-1 4-octylphenolu a porovnávaná s kontrolou. Kvantifikácia množstva testosterónu bola vykonávaná priamo z média pomocou imunoanalýzy. Pre stanovenie viability Leydigových buniek bol využitý MTT test. Bazálna produkcia testosterónu bola signifitantne (P˂0.001) zvýšená v koncentráciách 2,5 a 5,0 μg*mL-1 4-octylphenolu. Pri sledovaní životaschopnosti bol zaznamenaný signifikantný (P˂0.001; P˂0.05) nárast v koncentráciách 1,0 ; 2,5 a 5,0 μg*mL-1 4-octylphenolu v porovnaní s kontrolou. Stimulovaná produkcia testosterónu pomocou hCG spolu s pôsobením 4-octylphenolu spôsobila siginifikantý nárast produkcie v najnižšej koncentrácií testovanej látky. V prípade bunkovej viability bola zaznamenaná signifikantne(P˂0.001; P˂0.05) vyššia mitochondriálna aktivita exponovaných buniek (1,0; 2.5 a 5.0 μg*mL-1). Ďalší systematický výskum v oblasti pôsobenia endokrinných disruptorov považujeme za veľmi potrebný pre lepšie pochopenie potenciálnych rizík pre ľudí a zvieratá

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 ( < 0.01; < 0.001), while higher MDA concentration and activity of enzymatic antioxidants were recorded in the cell lysates ( < 0.01; < 0.001). Na, Fe, Cu, Mg, and Zn were positively correlated with the motility and antioxidant parameters ( < 0.05; < 0.01; < 0.001). Inversely, K exhibited the positive associations with malondialdehyde ( < 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

In vitro impact of macrolide antibiotics on the viability of selected mammalian cell lines

The aim of this study was to evaluate the in vitro cytotoxicity of different concentrations of macrolide  antibiotics (tilmicosin-TILM, tylosin-TYL and spiramycin-SPI) on selected animal cell cultures. VERO cells (kidney cells from Macacus rhesus), FE cells (feline embryonal cells) and BHK 21 cells (cell line from young hamster kidneys) were used in the study and subjected to concentrations of macrolides ranging 50-1000 µg/mL, depending on the specific antibiotic and cell line. The cell viability expressed as the mitochondrial activity of living cells was assessed using the metabolic mitochondrial MTT test. The effect of tilmicosin: FE cells were the most sensitive with a significant decrease of mitochondrial activity at 100-150 µg/mL (P<0.001) TILM. VERO cells were the most resistant, as no significant decrease of viability was observed at any TILM dose. The effect of tylosin: FE cells showed the highest sensitivity to TYL, as 1000 µg/mL reduced the cell viability to a half (P<0.001) when compared to the untreated control. Similarly, a decreased viability of BHK 21 cells was observed following the supplementation of 1500 (P<0.001) and 900 (P<0.05) µg/mL TYL. VERO cells exhibited the highest resilience to the TYL treatment, with no significant differences of viability in comparison to the control. The effect of spiramycin: BHK 21 cells exhibited the highest sensitivity to the antibiotic, as all concentrations (150, 200, 300 µg/mL SPI) led to a significant decrease (P<0.001) of the mitochondrial activity.  Similarly, the viability of FE cells significantly (P<0.05) decreased after the administration of 350 and 540 µg/mL SPI. On the other hand, VERO cells revealed the highest resistance to the antibiotic, with no significant effects in comparison to the control. Our data reveal that macrolides have a significant adverse negative effect on the cell viability, and may provide more information to our knowledge on the specific effects medication has on the organism

Changes in the Antioxidant Capacity and Iron-Binding Properties of Bovine Spermatozoa Following In Vitro Incubation with Ferrous or Ferric Iron

The aim of this study was to assess the impact of ferrous (Fe2+) or ferric (Fe3+) iron on the antioxidant capacity and the ability to bind iron of bovine spermatozoa at specific time intervals (0h, 2h, 8h, 16h and 24h) during an in vitro culture. 35 semen samples were collected from 7 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 motility was assessed using the SpermVisionTM CASA (Computer aided sperm analysis) system. The ferric reducing ability of plasma (FRAP) assay was applied to study the antioxidant capacity of the samples, while the ability of the sample to bind excess iron was determined using the Total iron-binding capacity (TIBC) test. Both ferrous and ferric iron exhibited a dose- and time-dependent impact on the spermatozoa motility. Concentrations ≥50 µmol/L FeCl2 and ≥100 µmol/L FeCl3 led to a significant decrease of spermatozoa motion (P<0.001), while concentrations below 10 µmol/L FeCl2 and 50 µmol/L FeCl3 proved to preserve the parameter (P<0.001). The FRAP assay revealed that both ferrous as well as ferric iron had a similar effect on the FRAP marker of the samples: high concentrations led to a dramatic and significant (P<0.001) increase of the parameter, followed by a notable decrease of the reducing ability in the subsequent time periods, whose intensity was dependent upon the time, oxidation state of iron, as well as the time of analysis. Furthermore, supplementation of FeCl2 and FeCl3 had an impact on the capacity of the sperm culture to bind free iron, reflected in a significant decrease of the parameter (P<0.001) early on (Time 2h) in case of high doses of both oxidative states of this biometal. In a direct comparison, ferrous iron has been shown to be more toxic than ferric 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. 50 µmol/L FeCl2 and 100 µmol/L FeCl3 could be regarded as critical in vitro concentrations of ferrous or ferric iron when it critically accumulates with toxic outcomes

The Drosera Extract as an Alternative In Vitro Supplement to Animal Semen: Effects on Bovine Spermatozoa Activity and Oxidative Balance

In vitro storage and processing of animal semen is considered to be a risk factor to spermatozoa activity, possibly leading to reduced fertility and litter sizes following artificial insemination (AI). A variety of substances isolated from natural resources have the potential to exhibit protective or antioxidant properties on the spermatozoon, thus they may extend the lifespan of stored semen. Drosera (Drosera rotundifolia L.) has been shown to possess antimicrobial, anti-inflammatory and antioxidant properties, making the plant extract a potential candidate for preserving liquid animal semen during in vitro storage. This study compared the ability of different concentrations of Drosera extract on the motility, viability and superoxide production of bovine spermatozoa during different time periods (0, 2, 6, 12 and 24h) of in vitro culture. Spermatozoa motility was assessed using the SpermVisionTM CASA (Computer aided sperm analysis) system. Cell viability was examined using the metabolic activity MTT assay and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. The CASA analysis revealed that Drosera extract supplementation was able to prevent a rapid decline of spermatozoa motility, especially in the case of concentrations ranging between 1 and 5 mg/mL (P<0.001 with respect to Times 6h, 12h and 24h). At the same time, concentrations ranging between 1 and 10 mg/mL of the extract led to a significant preservation of the cell viability throughout short-term (P<0.05 in case of Time 6h) as well as long-term periods of the experiment (P<0.01 with respect to Time 12h, and P<0.001 in case of Time 24h). 10 and 5 mg/mL of the extract exhibited antioxidant characteristics, translated into a significant reduction of the intracellular superoxide production, particularly notable at Times 12h (P<0.01) and 24h (P<0.001). The results indicate that the Drosera extract is capable of delaying the damage inflicted to the spermatozoon by the in vitro environment

Associations between inflammatory factors, lipid peroxidation and antioxidant capacity in bovine seminal plasma

Oxidative stress and inflammation are cooperative events involved in male reproductive dysfunction.   In   the   present   study, we assessed the associations between the spermatozoa motility, inflammatory factors (C-reactive protein and Interleukin-6), total antioxidant status (TAS) and lipid peroxidation expressed as malondialdehyde (MDA) concentration in the seminal plasma of breeding bulls. 17 semen samples were included in the study. Computer-aided sperm analysis (CASA) system was used to assess the spermatozoa motion characteristics, and seminal plasma was collected for further analyses. Interleukin-6 (IL-6) was quantified using ELISA, while C-reactive protein (CRP) and markers of the oxidative balance were evaluated by UV/VIS spectrophotometry. The correlation analysis revealed significantly positive associations between the sperm motility and TAS (P<0.05), while both parameters were in significantly negative correlations with CRP (P<0.05), IL-6 (P<0.05) and MDA (P<0.01). At the same time, the samples were divided according to the motility characteristics into groups of Excellent (Ex) and Moderate (Mo) quality. CRP, IL-6 as well as MDA concentrations were significantly (P<0.05) higher in the Mo group, while the Ex group exhibited a significantly higher antioxidant capacity (P<0.05).  The relationships between the oxidative balance and inflammatory markers detected in our study suggest their intricate involvement in the resulting semen quality