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Toyin Dorcas Alabi,1 Charon de Villiers,2 Stephan S du Plessis,3 Thomas K Monsees,4 Nicole L Brooks,5 Oluwafemi Omoniyi Oguntibeju1 1Phytomedicine & Phytochemistry Group, Oxidative Stress Research Centre, Department of Biomedical Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville, South Africa; 2PUDAC-Delft Animal Facility, South African Medical Research Council, Cape Town 7505, South Africa; 3Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg 7505, South Africa; 4Department of Medical Biosciences, University of the Western Cape, Bellville 7535, South Africa; 5Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Cape Town, South AfricaCorrespondence: Oluwafemi Omoniyi Oguntibeju Tel +27 219538495Email oguntibejuo@cput.ac.zaBackground: 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.Keywords: Anchomanes difformis, diabetes mellitus, male fertility, sperm function

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.

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., (© 2020 Blackwell Verlag GmbH.)

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.

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.

Flavonoids are major compounds of Aspalathus linearis and Camellia sinensis. They are classified as endocrine disruptors and some have been shown to inhibit testosterone production. TM3 Leydig cell cultures were treated with 250-5000 μg mL(-1) A. linearis (unfermented or fermented rooibos) or Camellia sinensis (green or black tea) for 24 h in the absence or presence of 6 mIU/200 μl human chorionic gonadotropin (hCG). Under nonstimulated conditions, all teas tend to decrease testosterone production (3.9-31.8%). However, under hCG-stimulation, a significant reduction in testosterone production was observed at all concentrations by both rooibos and tea (16.3-37.9%). MTT assay and phase contrast microscopy, revealed that at 250-1000 μg ml(-1) , both plants maintained the viability, proliferation and morphology of the cells, while 5000 μg ml(-1) was cytotoxic to the cells (P < 0.05). In conclusion, the results here demonstrate the anti-androgenic property of A. linearis and C. sinensis., (© 2014 Blackwell Verlag GmbH.)

Aspalathus linearis (rooibos tea) may improve sperm function owing to its antioxidant properties. To test this hypothesis, male rats were given 2% or 5% rooibos tea for 52 days. No significant alterations were observed in body and reproductive organs weight, serum antioxidant capacity and testosterone level. Seminiferous tubules displayed complete spermatogenesis. However, a significant (P < 0.05) decrease in tubule diameter and germinal epithelial height was observed. Epithelial height of caput epididymides showed a significant increase. Unfermented rooibos significantly enhanced sperm concentration, viability and motility. Fermented rooibos also significantly improved sperm vitality (P < 0.01), but caused a significant increase in spontaneous acrosome reaction (P < 0.05), whereas unfermented did not. Creatinine was significantly enhanced in all treated rats, consistent with significant higher kidney weights. Rooibos significantly reduced alanine transaminase level, while 2% fermented rooibos significantly decreased aspartate transaminase level (P < 0.01). In conclusion, treatment with rooibos improved sperm concentration, viability and motility, which might be attributed to its high level of antioxidants. However, prolonged exposure of rooibos might result in subtle structural changes in the male reproductive system and may induce acrosome reaction, which can impair fertility. Intake of large amounts of rooibos may also harm liver and kidney function., (© 2013 Blackwell Verlag GmbH.)

We evaluated the effect of the methanol extract of Basella alba (MEBa) on testosterone level and fecundity/fertility in male rats exposed in utero to flutamide - an androgen receptor antagonist. For this purpose, 1.5- and 2.5 -month-old male rats exposed in utero to flutamide were treated with the MEBa (1 mg kg(-1) ) for 2 and 1 month respectively. Five days before the end of treatment, rats were housed with females to assess their fecundity/fertility. Thereafter, rats were sacrificed and blood collected for the quantification of testosterone. Flutamide-exposed male rats showed a decrease in their ano-genital distance (AGD, P < 0.05) and were infertile. In normal (methylcellulose-exposed) animals, MEBa provoked an increase in testosterone level in 1.5- (P < 0.008) and 2.5 -month-old rats (P < 0.01) concomitantly with the improvement in their fecundity by 25%. In flutamide-exposed male rats, MEBa increased testosterone level in 1.5 -month-old rats (P < 0.001) without any effect on their fecundity; while in 2.5- month-old rats, MEBa did not affect the testosterone level but improved fecundity (by 25%) and fertility (P < 0.001). This study demonstrated the positive effect of MEBa to enhance fecundity/fertility in normal male rats and in rats exposed to the antiandrogen flutamide during their foetal life., (© 2011 Blackwell Verlag GmbH.)

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.

To improve the biocompatibility of polyurethane (PUR), we modified the surface by irradiation with different ions (Carbon; C, Oxygen; O, Nitrogen; N, or Argon; Ar) at 0.3-50 keV energy and doses of 1,00E+13 - 1,00E+15 ions/cm(2). The effects of ion implantation using different ion energies and densities were observed on adhesion, proliferation, and viability of human umbilical vein endothelial cells (HUVECs). The long-term in vitro stability of ion-implanted PUR was also investigated. Ion irradiation moderately affected the surface roughness (R(a)), but strongly enhanced the work of adhesion (W(a)). Cell adhesion was markedly improved on O-, N-, and Ar-, but not on C-implanted PUR surfaces. Medium ion energies and lower ion doses produced the best HUVEC attachment and proliferation, indicating the importance of choosing the proper range of energy applied during ion irradiation. In addition, apoptosis rates were significantly reduced when compared with unmodified PUR (uPUR). N implantation significantly protected the surface, although C implantation led to stronger surface erosions than on uPUR. In total, ion implantation on flexible PUR surfaces strongly improved the material surface characteristics and biocompatibility. Electron beam ion implantation within an appropriate energy window is thus a key to improving flexible PUR surfaces for clinical use to support endothelial cell performance. Thus, it can contribute to designing small-diameter grafts, which are in great demand, towards vascular tissue engineering applications.

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.

The aim of this study was to develop and characterize novel metal-polymer constructs to improve the biocompatibility of flexible but hydrophobic polyurethane (PUR) implants. Using a physical vapor deposition (PVD) technique, thin films (< or =100 nm) of zirconium (Zr) or titanium (Ti) were deposited on the polyurethane surface. Both coatings displayed good stability when subjected to cross-cutting test and especially Zr showed only minor and superficial cracks in the scanning electron microscopy analysis. PVD coating resulted in significantly lowered contact angles and the standard surface free energy of wetting (Delta(wet)G degrees ) turned to more favorable negative values (Ti: -40; Zr: -30; untreated PUR (uPUR): +10.1 mN/m). This may lead to the highly enhanced adhesion and proliferation properties observed with human umbilical vein endothelial cells (HUVECs). In addition, the novel coatings had no toxic effect and even drastically reduced apoptosis rates of HUVECs. Cell morphology, nitric oxide production, and mitochondrial membrane potential--both at static and flow conditions--were superior compared with uPUR, thus demonstrating intact physiological functions. Therefore, we suggest that combining PUR as a flexible material with a thin coating of Zr or Ti as the improved biocompatible surface may have advantages for use, for example, vascular graft material., (Copyright 2008 Wiley Periodicals, Inc.)

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.

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., (Copyright 2006 S. Karger AG, Basel.)

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.

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., (Copyright (c) 2005 S. Karger AG, Basel.)

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.

The setting of a local tissue kallikrein kinin system (tKKS) within the reproductive organs of the male rat was investigated by analysing bradykinin subtype 2 receptor (B2R) gene expression and cellular distribution of B2R protein and the kinin-liberating protease tissue kallikrein (tK). Reverse transcription-polymerase chain reaction showed B2R expression in testis, epididymis and prostate from prepubertal and sexually mature rats. In mature testis, in situ hybridization and immunohistochemistry localized B2R mRNA and protein besides endothelial cells of blood vessels exclusively on pachytene spermatocytes and round and elongated spermatids. B2R expression within the seminiferous tubules was found to be dependent on the stage of the spermatogenic cycle. In pre-pubertal rat testis, B2R mRNA and protein were additionally located in peritubular cells. In the testis, specific staining for tK occurred in addition to endothelial cells of blood vessels on the acrosomal cap of round and elongated spermatids. This immunostaining was also stage-dependent. In the epididymis, tK was detected on epithelial cells near the apical surface. The stage-dependent specific expression of tK and bradykinin B2Rs in developing germ cells and peritubular cells suggests a potential role of the tKKS in the local regulation of spermatogenesis and seminiferous tubule function.

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.

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.

Male fertility can be impaired by various toxicants. Some of them are known to target mainly Sertoli cells, which play an essential role in spermatogenesis. In this study, the in vitro response of immature rat Sertoli cells to various environmental pollutants, including pesticides, oestrogenic compounds and heavy metals, has been investigated. Mitochondrial dehydrogenase activity has been used to measure Sertoli cell viability, while production of lactate and secretion of inhibin B have been used as general and specific cell markers. Sertoli cell viability was not affected after 24-h exposure to lindane, DDT, ethinyloestradiol or bisphenol A in the concentration range analysed (up to 100, 25 or 50 microM, respectively). In contrast, mercury(II) (EC50 = 31 microM) and cisplatin (15% decrease in viability at 100 microM) induced some cytotoxic effect. With the exception of the pesticide DDT, all chemicals investigated induced a significant dose-dependent increase in lactate production after 24-h exposure to Sertoli cells. Owing to the cytotoxic effect of mercury(II), lactate levels dropped again at concentrations above 20 microM. The pesticide lindane (but not DDT) and both oestrogens significantly increased the production of the Sertoli cell specific hormone inhibin B without affecting cell viability. In contrast, the heavy metals mercury(II) and platinum(II) markedly decreased inhibin B levels. This sharp decrease was already significant at metal concentrations that reduced Sertoli cell viability only moderately (10-15%). In conclusion, the secretion of lactate and inhibin B by immature rat Sertoli cells seems to be a useful and sensitive marker with which to explore potential Sertoli cell toxicants.

Peptide hormones are involved in the paracrine regulation of several physiological processes. A possible function of the kallikrein-kinin system (KKS) in mammalian reproduction has been discussed. To evaluate its putative role in spermatogenesis, we searched for components of the KKS (kallikrein, kininases, kinin receptor) in the rat testis. Specific immunostaining demonstrated that the kininogenase tissue kallikrein was present in round and elongated spermatids. Leydig cells, Sertoli cells, peritubular cells, spermatogonia and spermatocytes were not stained. Bradykinin in the supernatant of Sertoli cell cultures was effectively degraded. The resulting metabolites were analysed by high-performance liquid chromatography (HPLC). Specific protease inhibition in the degrading experiments confirmed the occurrence of several metalloproteases on Sertoli cell membranes, including neutral metalloendopeptidases (NEP 24.11 and NEP 24.15), kininase type II (angiotensin converting enzyme, ACE), and kininase type I (metallocarboxypeptidase). Northern blots hybridized with a bradykinin B2 receptor probe showed the presence of B2 receptor mRNA in testis homogenate and Sertoli cell extract. All components of the kallikrein-kinin system are present within the seminiferous epithelium of the rat. Therefore, this paracrine peptide system may play a role in the regulation of Sertoli cell function or in the Sertoli cell-germ cell crosstalk.

Proteolytic enzymes, which are synthesized and secreted by cells of the seminiferous tubule of the testis, have important functions in spermatogenesis. We performed metabolic studies using small peptide hormones as a substrate to investigate the activity of proteases in cultured Sertoli cells of the rat. High-performance liquid chromatographic analysis of the cell culture supernatants showed cleavage of met- and leu-enkephalin, substance P, and bradykinin. No peptidolysis was observed for the cyclic peptide oxytocin. The hormone cleavage pattern and the use of specific protease inhibitors in peptide degradation experiments demonstrated activities of several proteases in Sertoli cells. These are mainly metalloproteinases including neutral metalloendopeptidases, angiotensin-converting enzyme and aminopeptidases. In addition, activities of serine and aspartic proteases were detected. Only marginal proteolytic activities were observed in Sertoli cell conditioned supernatants, indicating that the investigated proteases are mainly located on Sertoli cell membranes. The peptide hormones used in this study have been found to play a potential role in the endocrine, paracrine or autocrine regulation of testicular cells. The membrane-associated proteases reported here may therefore be involved in the metabolism and inactivation of these peptides.

Cottonseed gossypol is a potent male contraceptive in several mammalian species including man. Sertoli cells play a crucial role in spermatogenesis. Therefore, the antifertility competence of gossypol may reflect a change in Sertoli cell function. Rat primary cultures were used to examine the effect of gossypol on cell viability, mitochondrial dehydrogenase function, lactate production and secretion of the Sertoli cell-specific protein inhibin. Exposure for 24 h to gossypol (3-6 microM) significantly enhance secretion of lactate but reduce secretion of inhibin without affecting cell viability. At 9-15 microM, the observed decrease of both lactate and inhibin accumulation apparently resulted from Sertoli cell degeneration and death, because viability and mitochondrial function were also reduced. The results suggest that mitochondria of Sertoli cells are a possible target for gossypol-induced infertility.

Peritubular cells, Sertoli cells, and germ cells of the seminiferous tubule synthesize and secrete several proteases and protease inhibitors. Experimental evidence suggests that the complex network of proteolytic enzyme activity and their regulation by protease inhibitors play an important role in male reproduction. Interaction between protease and protease inhibitors seems to play an important role in remodeling and restructuring of the seminiferous tubule during spermatogenesis. Controlled proteolytic activity is also involved in the migration of germ cells from the basal compartment to the lumen of the seminiferous epithelium, and in the release of spermatids during spermiation. The recently reported occurrence of Sertoli cell membrane-associated proteases indicate the possible involvement of regulatory peptide systems within the testis. This view is supported by the detection of all components of one of these paracrine systems, the kallikrein-kinin system, in cells of the seminiferous tubule.

Sertoli cells play a key role in spermatogenesis. To study the involvement of the kallikrein-kinin system in the testis, the pattern of bradykinin-inactivating kininases in rat Sertoli cells was investigated. Exogenous bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is cleaved at Pro7-Phe8, Phe5-Ser6, and Gly4-Phe5, as demonstrated by high performance liquid chromatography analysis. Degradation of bradykinin was strongly inhibited by phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase-24.11. The kininase type II-specific inhibitors, captopril and enalapril, were only partially effective in preventing peptidolysis. This indicates that the main kininases responsible for rapid bradykinin inactivation are neutral metalloendopeptidase and, to a lesser extent, kininase type II. Neutral metalloendopeptidase and kininase type II were shown to be located on Sertoli cell membranes. A low degree of bradykinin degradation was detected by simultaneous inhibition of neutral metalloendopeptidase-24.11 and kininase II, pointing out the involvement of further peptidases with minor activities. This remaining activity is probably not due to the action of kininase type I or cysteine proteases, as shown by specific inhibitors. The data presented indicate the occurrence of membrane-bound kininases, which are an important part of the kallikrein-kinin system, in rat Sertoli cell cultures.

Kininases are an important part of the kallikrein-kinin system. We investigated the pattern of kininases in rat Sertoli cells. Sertoli cells are found in the seminifereous tubule of the testis and play a key role in spermatogenesis. Bradykinin was actively cleaved by cultivated Sertoli cells at the Pro7-Phe8, Phe5-Ser6 and Gly4-Phe5 bonds as demonstrated by high performance liquid chromatography analysis. Addition of phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase 3.4.24.11 (NEP), strongly inhibited the degradation of bradykinin. In contrast, the kininase type II-specific inhibitors captopril and enalapril were only partially effective in preventing peptidolysis. NEP and kininase type II were shown to be located in Sertoli cell membranes. The action of kininase type I leads to the formation of the metabolite bradykinin (1-8) which could be detected in small amounts by HPLC analysis. Cleavage of the Ph5-Ser6 bond might be caused by the action of the endopeptidases 24.15 and 24.16, which are phosphoramidon-insensitive. Our results indicate that neutral metalloendopeptidase 24.11 is the main kininase responsible for rapid bradykinin inactivation in Sertoli cells. Further kininases with minor activities are the kininases type I and II and probably the metalloendopeptidases 24.15 and 24.16.