For example, the50- to 91-year old male participants in the Baltimore LongitudinalStudy of Aging exhibited significant correlations between their initialserum total testosterone concentrations and measures of visualmemory, verbal memory, and visuospatial functioning measured 10years later . Although definitions of "testosterone deficiency" vary, mid-morning serumtotal testosterone concentrations less than 7 nmol/L to 10.5 nmol/Lare considered to reflect "testosterone deficiency" 68,76-78. Male sexual performanceand function are dependent upon testosterone adequacy 63-73,while relative testosterone inadequacy reduces male sexual desire,function, performance, and potency 63-67. Significantly reduced reactive oxygen species (ROS) generation, lipid peroxide contents and hypoxia induction factor (HIF)-1α stabilization and activation were found with 100-nmol l−1 testosterone treatment. Cytoprotective effects were identified with 100-nmol l−1 testosterone treatment, but cytotoxic effects were found with ≥500-nmol l−1 testosterone supplementation. However, the effects of exogenous testosterone on Leydig cells are still unclear and need to be clarified. The lowest concentration detectable was 0.1 ng ml−1 and the intra-assay coefficient of variation was 5%. A 20 μl aliquot was immediately transferred to another tube containing 730 μl of KRL buffer (150 mM Na+, 120 mM Cl−, 6 mM K+, 24 mM HCO3−, 2 mM Ca2+, 340 mosm, pH 7.4). The androgen-receptor antagonist activity of flutamide in this species is supported by the results from a recent study (Tomaszycki et al. 2006). Same-sized implants, filled with T and F, have been successfully used in studies on zebra finches and other passerines (Schwabl & Kriner 1991; Adkins-Regan et al. 1994; Williams et al. 2003; Van Roo 2004; McGraw et al. 2006). Fourteen birds per group received a 10 mm subcutaneous implant (1.47 mm i.d., 1.96 mm o.d.; Silastic laboratory tubing, Dow Corning) filled with the crystalline testosterone (SIGMA, ref. T1500), flutamide (SIGMA ref. F9397) or left empty. In line with our predictions, we found that experimental manipulation of testosterone levels, affected both red blood cell resistance to free radicals and T-cell mediated immune response. Final values of resistance to oxidative stress for male zebra finches treated with anti-androgen (F; flutamide), control (C) or testosterone (T) implants during a 28-day period. This experimental design allowed us to assess the effect of testosterone on sexual signalling (beak colour), body mass, T-cell mediated immune response and red blood cell resistance to a free radical attack. Von Schantz et al. (1999) were the first to suggest a role for oxidative stress in the trade-off between the benefits of sexual signalling and the costs derived from sustaining high testosterone levels. Clinical studies on healthy men have shown an average increase in total testosterone levels of approximately 20% after 90 days of consistent supplementation. By neutralizing free radicals, shilajit protects the Leydig cells, allowing them to function at optimal capacity. The testes are highly susceptible to oxidative damage, which can impair testosterone production. Pomegranates and their extracts contain a large number ofnutritional antioxidants, including punicalagins, hydrolyzableellagitannins (ellagic esters of glucose), anthocyanins, ascorbic acid,and α-tocopherol . With advancing age, Leydig cells exhibit increasingdegrees of intracellular lipid peroxidation 136,137 and decreasingexpression of SOD, GPx, and glutathione . ROS and other oxidizing molecules generated byenvironmental insults (e.g., ultraviolet irradiation, cigarette smoke,and air pollutants ), drugs , and ethanol add tolocal and systemic oxidative stress. Following RT, complementary (c)DNA was applied in a fluorescence-based real-time polymerase chain reaction (PCR) to determine alterations in StAR and AR mRNA expressions in testosterone-treated cells. In brief, 200-µl aliquots of suspensions of 2×104 testosterone-treated cells were placed into wells of microtiter plates. Different concentrations of testosterone were added to cells, while only alcohol (solvent) was added to the control group. In addition to the central effect (along the hypothalamus–pituitary–testis axis), the peripheral effect of exogenous testosterone on Leydig cells has not been clarified. There was a 1.72-fold increase in ROS generation in the 500-nmol l−1 compared to the 100-nmol l−1 testosterone treatment.
