With advancing age, Leydig cells exhibit increasingdegrees of intracellular lipid peroxidation 136,137 and decreasingexpression of SOD, GPx, and glutathione . Oxidatively damaged Leydig cells are less sensitive toLH, with fewer LH receptors expressed per cell, and exhibit impairedactivation of the StAR protein, reduced activities of several enzymesof the testosterone biosynthetic pathway (CYP11A1, 3β-HSD,CYP17A1 hydroxylase, CYP17A1 lyase, 17β-HSD), and inhibitionof testosterone synthesis 61,62,127,129, . An elevated ratio of serum total 17β-estradiol concentration toserum total testosterone concentration stimulates the externalizationof phosphatidylserine on the plasma membranes of human Leydigcells and initiates premature engulfment of healthy Leydig cellsby testicular macrophages, reducing the amount of testosteronesynthesizing tissue 31-33. The rates of conversion of androstenedione and testosteroneinto 17β-estradiol are determined by both substrate availability andthe level of aromatase expression and activity 22-25. In any case, we should bear in mind that beyond the possible effect of testosterone on beak colour, this androgen has also been repeatedly reported to affect other sexual displays in male zebra finches such as song rate (e.g. Cynx et al. 2005) and courtship behaviour (e.g. Springer & Wade 1997). Whatever the physiological mechanism behind the observed results, our findings support the idea that the honesty of testosterone-based sexual signals might be reinforced by multiple, possibly additive, costs. First, testosterone can affect metabolic rate (e.g. Feuerbacher & Prinzinger 1981; Fryburg et al. 1997; Buchanan et al. 2001; but see Buttemer & Astheimer 2000). The effect of testosterone on resistance to free radicals might involve different mechanisms. In addition to smoking, excessive alcohol consumption also has a negative effect of testicular function through the induction of oxidative stress and the concomitant disruption of testicular antioxidant status.108,109 Furthermore, the ability of antioxidants such as vitamin C or lecithin to ameliorate this pathology, confirms the importance of oxidative stress in this context.110–111 In addition to inducing low sperm counts and poor sperm motility, it also appears that the oxidative stress created in the Leydig cells as a consequence of chronic alcohol exposure diminishes the steroidogenic capacity of the testes, lowering circulating testosterone levels.112 Repletion of the ascorbate levels in the diet had the reverse effect and decreased DNA damage by 36%.106 Experimental exposure of rats to cigarette smoke also induces lipid peroxidation in the testes in association with disturbances in testicular antioxidant enzyme activity.2 The testicular damage induced by cigarette smoke exposure in rats is certainly oxidative in nature because it can be reversed by concomitant exposure to an antioxidant (caffeic acid phenethyl ester).107 Physical exercise has been shown to up-regulate antioxidant activities in the testes of aging rats and may represent a practical way in which the detrimental effects of age on testicular function can be ameliorated.90 A similar case could be argued for the ability of moderate exercise to ameliorate the degree of oxidative damage inflicted on the testes by chronic ethanol ingestion.91 However, excess exercise can have the opposite effect, causing oxidative stress in the testes and generating high levels of lipid peroxidation in association with significant declines in the activities of key antioxidant enzymes including SOD, catalase, GST and GPx.92 Such stress has a significant inhibitory effect on the both steroidogenesis and germ cell differentiation within the testes. These effects could be attenuated by the administration of antioxidants such as ascorbic acid, melatonin, taurine or an herbal mixture containing extracts from Musa paradisiaca, Tamarindus indica, Eugenia jambolana and Coccinia indica.84–86 In light of recent data showing an increased level of DNA damage in the spermatozoa of diabetic patients compared with non-diabetic controls,87 causative links between diabetes, oxidative stress in the male germ line and DNA damage appears both likely and clinically, extremely important. Conversely, antioxidant defenses can be augmented by dietarysupplementation with specific antioxidant and mitochondrialprotective nutrients that reduce cell-wide oxidative damage,support redox balance within Leydig cells, release Leydig cells fromoxidative inhibition of testosterone synthesis, and increase the rateof testosterone secretion. The Leydig cells of rats fed the polychlorinated biphenyl,Arochlor-1254, exhibit decreased activities of antioxidant enzymes,increased generation of H2O2, lipid peroxidation products andother ROS, and inhibition of the StAR protein, P450scc, HSD3B2,and testosterone synthesis . This increase in circulatingantioxidant capacity is accompanied by increased circulatingglutathione concentration ; decreased circulating concentrationsof oxidized glutathione, oxidized proteins, and lipid peroxides ;and less oxidative damage to DNA in circulating white blood cells. Patients with private insurance can utilize telemedicine for the diagnosis and treatment of testosterone deficiency, indicating that individuals require a certain level of financial resources and capability (40). In males, there exists an inverse correlation between obesity and testosterone levels (37). Moderate physical activities can increase testosterone levels, thereby maintaining male physiological functions and combating aging (36). Once cotinine levels in the body reach a certain threshold, they are negatively correlated with testosterone (34). Cotinine, a metabolite of cigarette smoke, exhibits a non-linear relationship with testosterone levels. Long-term alcohol consumption can impair the male gonadal axis and lead to a reduction in testosterone levels (33). Fold change for mRNA was calculated using the ΔΔ cycle threshold (ΔΔCT) method (27) and expressed relative to balance values within a treatment. Percutaneous muscle biopsies of the vastus lateralis were collected under rested/fasted conditions on study days 14 (BAL) and 42 (DEF). Hematocrit was calculated by multiplying red blood cell count by mean cell volume. Activity factor was determined as 24-h energy expenditure divided by sleep energy expenditure (25). Total testosterone was measured using a Siemens Immulite 2000 (Llanbeis, United Kingdom). Samples were collected between 0600 and 0900 h to limit circadian rhythm confounding total testosterone concentrations. Body composition was determined by dual-energy x-ray absorptiometry (DXA; Lunar iDXA; GE Healthcare, Madison, WI). The plethora of physical, chemical, and pathological factors that can apparently contribute to the induction of oxidative stress in the testes is impressive and suggests that the clinical picture will be extremely complex, with each individual being subject to a unique range of causative factors as a result of differences in occupational and environmental exposures, the presence of other pathological factors such as infection or diabetes, and genetic factors that could influence everything from the way in which specific xenobiotics are metabolised to the endocrine environment in which the testes have to function. Although oxidative stress is clearly a dominant feature in the aetiology of male infertility, the underlying causative mechanisms remain unresolved. One of the most effective antioxidants for the protection of testicular function is melatonin. In addition to the major ROS processing enzymes, the testes rely heavily on small molecular weight antioxidant factors for protection against oxidative damage. Given the importance of SOD in this defence strategy, it is not surprising that the testes contain not only the conventional cytosolic (Cu/Zn) and mitochondrial (Fe/Mn) forms of SOD but also feature an unusual form of extracellular SOD, (SOD-Ex) which is produced by both Sertoli and germ cells, particularly the former. Superoxide can be generated by specialized enzymes, such as the xanthine or NADPH oxidases, or as a by-product of cellular metabolism, particularly the mitochondrial electron transport chain. This activity is critical in the detoxification of peroxidised lipids as well as the metabolism of xenobiotics. The elimination of H2O2 is either effected by catalase or glutathione peroxidase, with the latter predominating in the case of the testes.9,10 GST on the other hand involves a large and complex family of proteins that catalyse the conjugation of reduced glutathione via the sulfhydryl group to electrophilic centres on a wide variety of substrates in preparation for excretion from the cell. The high rates of cell division inherent in this process imply correspondingly high rates of mitochondrial oxygen consumption by the germinal epithelium. On the other hand, testosterone-dependent immunossuppression could also be the consequence of an ‘indirect pathway’ (Owen-Ashley et al. 2004) mediated by the pro-oxidant properties of this androgen, as firstly proposed by von Schantz et al. (1999). This is also in agreement with recent studies (Buchanan et al. 2001; Duckworth et al. 2001; see also Mougeot et al. 2004), which have shown that the cost of high testosterone concentration is not exclusively based on immunosuppression, as proposed by the immunocompetence handicap hypothesis (Folstad & Karter 1992). To assess the sensitivity of the results to these extreme values, we also ran the analyses excluding birds that had testosterone values higher than 20 ng ml−1. In this study, the highest value for control birds (with empty implants) was 7.66 ng ml−1, whereas the highest value for T-individuals was 35 ng ml−1. To manipulate testosterone, we used implants filled with this steroid and flutamide, a testosterone antagonist that binds to testosterone receptors. Male zebra finches whose testosterone receptors were blocked by the anti-androgen (flutamide) showed the strongest resistance to free radicals and the strongest immune response, whereas T-implanted males had the weakest resistance to free radicals and the weakest immune response.
