Role of oxidative stress in male infertility.

Abstract: Infertility affects millions of couples worldwide. Oxidative stress (OS) causes peroxidation of lipids and damage to spermatozoa, thus, reducing the quality of seminal parameters. In addition, the differences in the levels of antioxidants and reactive oxygen species (ROS) caused by intrinsic and extrinsic variables linked to lifestyle, diet, genetics, and OS also contribute to male infertility. High levels of ROS result in sperm damage of sperm parameters due to lipid peroxidation and oxidation of proteins. Other significant causes of ROS include changes in sex hormone levels, sperm DNA damage, including mutations, and immature spermatozoa. Treating the root causes of OS, by changing one's lifestyle, as well as antioxidant therapy, may be helpful strategies to fight OS-related infertility. However, the determination of male infertility induced by OS is currently a challenge in the field of reproductive health research. This review intends to describe the role of oxidative stress on male infertility and the current understanding of its management. Lay summary: The inability to conceive affects many couples globally. Oxidative stress refers to imbalances between different oxygen species which can lead to male fertility problems by damaging sperm and semen. Oxidative stress may be caused by several factors, including diets high in fats, sugars and processed foods, lifestyle (including smoking, alcohol consumption and having a sedentary lifestyle), and genetics. Treatment that focuses on the root cause may help combat male infertility. However, there is currently no consensus on the best way to treat male fertility problems, particularly those associated with oxidative stress. This paper describes the role of oxidative stress on male infertility and discusses the current techniques employed in treating male fertility issues.

The Beneficial Role of Anchomanes difformis in STZ-Induced Reproductive Dysfunction in Male Wistar Rats.

BACKGROUND: Progression of diabetes mellitus has increasingly led to several diabetic complications. Diabetes is one of the major factors implicated in male reproductive system damage. Recent approaches such as the use of medicinal plants have been explored in the management of diabetes and associated complications. Anchomanes difformis (common name: children's umbrella) has been shown to possess anti-diabetic ability in animal model. Therefore, this study seeks to investigate the potency of Achomanes difformis in ameliorating diabetes-induced reproductive dysfunction. METHODS: Type 2 diabetes was induced in male Wistar rats with 10% fructose administration for 2 weeks and an intraperitoneal injection of 40mg/kgBW of streptozotocin. Aqueous extract (200mg and 400mg/kgBW) of Anchomanes difformis leaves was administered daily for 6 weeks. The rats were randomly divided into 7 groups with a minimum of eight rats in each (8 rats in normal groups and 10 in diabetic groups). The impact of diabetes and treatment was investigated by estimating sperm concentration, motility indices, viability and morphological parameters in the normal, treatment controls and diabetic rats using CASA-SCA system. Histological examination of the testes and epididymis was performed. RESULTS: Diabetes induction resulted in significant decrease in sperm concentration, viability and some motility parameters with 40% abnormalities in sperm morphology. The administration of Anchomanes difformis significantly increased sperm concentration and sperm viability, while it significantly improved the percentage of morphologically normal sperm in diabetic rats. Anchomanes difformis ameliorated testicular damage such as vacuolization and loss of germinal epithelium in the diabetic-treated rats when compared to the diabetic controls. CONCLUSION: The potency Anchomanes difformis displayed against diabetic-induced damage in the reproductive system might be a new and promising tool in the management of male reproductive dysfunctions and associated complications in diabetes mellitus.

In vivo effects of black tea on the male rat reproductive system and functions of the kidney and liver.

This study focused on the effects of black tea on the male reproductive system as well as the kidney and liver functions. Male Wistar rats were given aqueous extract of black tea (2% and 5%) for 52 days as the only means of drinking fluid, while control rats received tap water. Black tea enhanced sperm vitality (44%-49%), total sperm motility (10%-12%) and acrosome reaction (2%-9%) (p < .05). Body weight gain, testis, epididymis, seminal vesicles, prostate, liver weight, testosterone level, sperm concentration, ferric reducing antioxidant power (FRAP) and antioxidant levels in the testes, liver and kidney remained unchanged (p > .05). Black tea (5%) increased kidney weight (p < .05). Testis and epididymis showed normal histological appearance. However, black tea significantly reduced the diameter (9%-10%) and epithelial height (9%-10%) of the seminiferous tubule, but increased the epithelial height of the cauda epididymis (8%-24%) (p < .05). A significant reduction in serum levels of alanine aminotransaminase (ALT) (38%) and aspartate aminotransaminase (AST) (23%-34%) was observed (p < .05); creatinine level, on the other hand, increased (8%-72%) (p < .05). Black tea improved several sperm parameters, but may cause subtle changes in certain reproductive organs and the kidney functions.

Sputtered Si and Mg doped hydroxyapatite for biomedical applications.

Hydroxyapatite (HAP) coatings are applied on metallic implant materials to combine mechanical properties of metallic material with bioactivity abilities of HAP ceramic. In this study, HAP coatings with additions of Si and Mg are proposed to be deposited on Ti6Al4V substrates by RF magnetron sputtering. Chemical bonding, morphology, topography and corrosion resistance in simulated body fluids (SBF) of the coatings were investigated. Additionally, mechanical and biological properties of the coatings were evaluated. It was found that the addition of Si and Mg does not influence the formation of a HAP phase. All the coatings exhibited smooth surface and uniform growth, without defects or cracks. Both hardness and elastic modulus of the coated samples decrease with Mg addition in the HAP-Si structure. Both Mg and Si addition into HAP coatings were found to enhance the corrosion resistance of the Ti6Al4V alloy in the SBF solution. Coatings with low Mg content exhibited better corrosion performance. All the coatings investigated were biocompatible, as demonstrated by SaOS-2 bone cell attachment and growth. However, cell proliferation and morphology were inferior on samples with the highest Mg content.

The para isomer of dinitrobenzene disrupts redox homeostasis in liver and kidney of male wistar rats.

BACKGROUND: Para-Dinitrobenzene (p-DNB) is one of the isomers of dinitrobenzene which have been detected as environmental toxicants. Skin irritation and organ toxicities are likely for industrial workers exposed to p-DNB. This study evaluated the effect of sub-chronic exposure of rats to p-DNB on cellular redox balance, hepatic and renal integrity. METHODS: Forty eight male Wistar rats weighing 160-180 g were administered 50, 75, 1000 and 2000 mg/kg b.wt (body weight) of p-DNB or an equivalent volume of vehicle (control) orally and topically for 14 days. After the period of treatment, the activities of kidney and liver catalase (CAT), alkaline phosphatase (ALP) and superoxide dismutase (SOD) as well as extent of renal and hepatic lipid peroxidation (LPO) were determined. Serum ALP activity and plasma urea concentration were also evaluated. RESULTS: Compared with control animals, p-DNB -administered rats showed decrease in the body and relative kidney and liver weights as well as increased renal and hepatic hydrogen peroxide and lipid peroxidation levels accompanied by decreased superoxide dismutase and catalase activities. However, p-DNB caused a significant increase in plasma urea concentration and serum, liver and kidney ALP activities relative to control. In addition, p-DNB caused periportal infiltration, severe macro vesicular steatosis and hepatic necrosis in the liver. CONCLUSIONS: Our findings show that sub-chronic oral and sub-dermal administration of p-DNB may produce hepato-nephrotoxicity through oxidative stress.

Effects of the methanol extract of Basella alba L (Basellaceae) on steroid production in Leydig cells.

In this study, Leydig cells were purified from 70 day-old Sprague Dawley male rats and incubated with 10 and 100 µg/mL of methanol extract of Basella alba (MEBa) for 4 hours followed by the evaluation of cell viability, steroid (testosterone and estradiol) production, and the level of aromatase mRNA. Results showed that MEBa did not affect Leydig cell viability. At the concentration of 10 µg/mL, MEBa significantly stimulated testosterone and estradiol production (p < 0.01 and p < 0.03, respectively), and enhanced aromatase mRNA level (p < 0.04). These observations suggest that MEBa directly stimulated testosterone, estradiol and aromatase mRNA levels in isolated Leydig cells.

Local calcium elevation and cell elongation initiate guided motility in electrically stimulated osteoblast-like cells.

BACKGROUND: Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10-15 V/cm) and weak (< or = 5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation. CONCLUSION/SIGNIFICANCE: Taken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.

Effects of electromagnetic fields on cells: physiological and therapeutical approaches and molecular mechanisms of interaction. A review.

This review concentrates on findings described in the recent literature on the response of cells and tissues to electromagnetic fields (EMF). Models of the causal interaction between different forms of EMF and ions or biomolecules of the cell will be presented together with our own results in cell surface recognition. Naturally occurring electric fields are not only important for cell-surface interactions but are also pivotal for the normal development of the organism and its physiological functions. A further goal of this review is to bridge the gap between recent cell biological studies (which, indeed, show new data of EMF actions) and aspects of EMF-based therapy, e.g., in wounds and bone fractures.

Collageneous matrix coatings on titanium implants modified with decorin and chondroitin sulfate: characterization and influence on osteoblastic cells.

Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM), utilizing the natural self-assembly potential of collagen in combination with further ECM components in close analogy to the situation in vivo. Aiming at load-bearing applications in bone contact, collagen type I in combination with the proteoglycan decorin and the glycosaminoglycan chondroitin sulfate (CS) was used; fibrillogenesis, fibril morphology, and adsorption of differently composed fibrils onto titanium were assessed. Both decorin and CS could be integrated into the fibrils during fibrillogenesis, the amount bound respectively desorbed depending on the ionic strength of fibrillogenesis buffer. Including decorin always resulted in a significant decrease of fibril diameter, CS in only a slight decrease or even increase, depending on the collagen preparation used. No significant changes in adsorption to titanium could be detected. Osteoblastic cells showed different reactions for cytoskeletal arrangement and osteopontin expression depending on the composition of the ECM, with CS enhancing the osteoblast phenotype.

Effects of Basella alba and Hibiscus macranthus extracts on testosterone production of adult rat and bull Leydig cells.

AIM: To determine the androgenic effects of Basella alba and Hibiscus macranthus extracts in the rat and the bull, and to develop a novel in vitro test system using Leydig cells from bull testes. METHODS: The effect of methanol extracts from both plants on testosterone production in isolated Leydig cells from the rat and the bull was analyzed using 125I-radioimmunoassay (125I-RIA). Rat Leydig cells were obtained by common methods, whereas a novel technique was used to purify Leydig cells from bull testes. RESULTS: Bull testes from the slaughter house were a cheap source of pure Leydig cells. In culture, these cells produced testosterone for 5-6 days, which can be stimulated by human chorionic gonadotrophin (hCG). Basella alba extracts significantly enhanced testosterone production in bull and rat Leydig cells in a concentration-dependent manner. Hibiscus macranthus showed no androgenic effect but was shown to inhibit testosterone production at higher concentrations. CONCLUSION: Leydig cells purified from bull testes can be used as an alternative tool in experimental animal research. Certain fractions of Basella alba extract demonstrated androgenic potential whereas Hibiscus macranthus extracts did not.

Effects of different titanium alloys and nanosize surface patterning on adhesion, differentiation, and orientation of osteoblast-like cells.

To test nanosize surface patterning for application as implant material, a suitable titanium composition has to be found first. Therefore we investigated the effect of surface chemistry on attachment and differentiation of osteoblast-like cells on pure titanium prepared by pulsed laser deposition (TiPLD) and different Ti alloys (Ti6Al4V, TiNb30 and TiNb13Zr13). Early attachment (30 min) and alkaline phosphatase (ALP) activity (day 5) was found to be fastest and highest, respectively, in cells grown on TiPLD and Ti6Al4V. Osteoblasts seeded on TiPLD produced most osteopontin (day 10), whereas expression of this extracellular matrix protein was an order of magnitude lower on the TiNb30 surface. In contrast, expression of the corresponding receptor, CD44, was not influenced by surface chemistry. Thus, TiPLD was used for further experiments to explore the influence of surface nanostructures on osteoblast adhesion, differentiation and orientation. By laser-induced oxidation, we produced patterns of parallel Ti oxide lines with different widths (0.2-10 microm) and distances (2-20 and 1,000 microm), but a common height of only 12 nm. These structures did not influence ALP activity (days 5-9), but had a positive effect on cell alignment. Two days after plating, the majority of the focal contacts were placed on the oxide lines. The portion of larger focal adhesions bridging two lines was inversely related to the line distance (2-20 microm). In contrast, the portion of aligned cells did not depend on the line distance. On average, 43% of the cells orientated parallel towards the lines, whereas 34% orientated vertically. In the control pattern (1,000 microm line distance), cell distribution was completely at random. Because a significant surplus of the cells preferred a parallel alignment, the nanosize difference in height between Ti surface and oxide lines may be sufficient to orientate the cells by contact guiding. However, gradients in electrostatic potential and surface charge density at the Ti/Ti oxide interface may additionally influence focal contact formation and cell guidance.

Effects of single non-ortho, mono-ortho, and di-ortho chlorinated biphenyls on cell functions and proliferation of the human prostatic carcinoma cell line, LNCaP.

This study investigated the effects of the non-ortho 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77), the mono-ortho 2,3',4,4',5-pentachlorobiphenyl (PCB 118), and the di-ortho substituted 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) on the human prostatic carcinoma cell line LNCaP. Activities of 5alpha-reductase and ethoxyresorufin-O-deethylase (EROD) secretion of testosterone-regulated prostatic specific antigen (PSA) and cell proliferation served as specific and general cell markers. The two non-ortho (dioxin-like) PCBs, PCB 126 and PCB 77, showed anti-androgenic properties. Both PCBs reduced androgen-depending PSA secretion and cell proliferation, and inhibited the DHT-producing enzyme 5alpha-reductase in a concentration-dependent manner. In contrast, the ortho-substituted PCBs, PCB 118 and PCB 153, had no effect on 5alpha-reductase. However, they had a biphasic effect on LNCaP cell proliferation. PCB 153 and, to some extent, PCB 118 induced cell proliferation and PSA secretion at low concentrations, whereas, these parameters were reduced at high concentrations. Since EROD induction and inhibition of 5alpha-reductase activity was not observed, these findings suggest an AhR and AR independent mechanism and post-transcriptional target sites should be considered. The effects of PCB 126 on cell proliferation, PSA secretion, and 5alpha-reductase activity may be partly a result of AhR-dependent inhibition. In conclusion, the endocrine activity of PCBs via direct steroid hormone receptor-mediated and Ah receptor-mediated pathways are well known. However, the modulation of post-transcriptional targets may account for some of the endocrine disrupting properties of single PCB congeners.

Bradykinin increases intracellular calcium levels in rat testis peritubular cells via the B2 receptor subtype.

1. RT-PCR and Western blots were used to detect bradykinin B(2) receptors in testis and isolated peritubular cells of pre-pubertal rats. RT-PCR demonstrated expression of a single transcript, whereas Western blots showed up to three specific bands that were in accordance with the described native, glycosylated and dimeric form of B(2) receptor proteins, respectively. 2. Fura-2-loaded peritubular cells responded with an instantaneous, linear and transient rise in [Ca(2+)](i) after adding bradykinin. Stimulation of cells with bradykinin concentrations between 1 micro M and 1 pM showed a dose dependent increase of [Ca(2+)](i). The calcium response to bradykinin was diminished after stimulation of peritubular cells in calcium-free buffer. After blocking the SERCA-pumps by thapsigargin and subsequent stimulation with bradykinin, no rise of [Ca(2+)](i) was appreciated. 3. Multiple stimulation of a single peritubular cell by local perfusion with a brief addition of BK (10 nM) resulted in a fast and immediate response. However, the second and third stimuli had slower rise rates and diminished [Ca(2+)](i) peaks, showing desensitization of the kinin receptor. 4. Addition of the bradykinin B(1) receptor agonist [des-Arg(9)]-bradykinin (100 nM) to Fura-2-loaded peritubular cells did not change the [Ca(2+)](i). However, the B(2) receptor antagonist HOE 140 (100 nM) strongly inhibited the bradykinin-induced calcium response. 5. We conclude that the bradykinin-induced increase in [Ca(2+)](i), in testicular peritubular cells is mediated by the stimulation of kinin receptors of the B(2) subtype.

Expression and location of the bradykinin B2 receptor in rat testis.

To investigate the possible role of the local tissue kallikrein-kinin system in spermatogenesis, we analyzed gene expression and cellular distribution of the bradykinin subtype-2 receptor (B(2) receptor) in the rat testis. Reverse transcription-polymerase chain reaction revealed B(2) receptor expression in testis and primary cultures of Sertoli cells and peritubular cells isolated from immature and mature rats. In situ hybridization of the B(2)-receptor mRNA showed intense labeling of cells on the base of the seminiferous tubule, whereas the autoradiographic signals gradually decreased toward the lumen. Immune histochemistry using testicular sections of pubertal and adult rats showed specific staining for the B(2)-receptor protein in cells of the adluminal compartment of the seminiferous tubules, especially on pachytene spermatocytes and spermatids. This immunostaining varied with the stages of the seminiferous cycle. The receptor protein was also observed on peritubular cells of pubertal rats. In conclusion, we demonstrated a stage-specific expression of the bradykinin B(2) receptor in different cells of the seminiferous tubules of the rat testis. The results point to a possible function of the tissue kallikrein-kinin system in the local regulation of spermatogenesis.