As shown in Figures 9A, B, orchidectomy significantly increased mitochondrial protein carbonylation in IFM, but not in SSM. SOD-Cu-Zn in the heart was not changed after castration; however, ACE-1 protein expression was significantly increased in the OQT group. In the IFM, the expression of mitochondrial Mn-SOD (Figure 7C) and catalase (Figure 7E) were significantly decreased after castration and this reduction was prevented by testosterone replacement. As mitochondrial biogenesis is regulated by PGC-1α and PGC-1α translocation to mitochondria is regulated by AMPK-α, we also measured the expression and phosphorylation of these two proteins. Particularly in obese populations, educational level and hypertension serve as significant mediators between obesity and testosterone deficiency (43). Furthermore, males with type 2 diabetes often exhibit testosterone deficiency, which provides new insights into the prevention of testosterone deficiency in non-diabetic individuals (42). Males with testosterone deficiency who struggle with adverse socioeconomic conditions appear less likely to seek or adhere to formal treatment regimens (41). In addition, oxidizing environmentalinsults add to local and systemic oxidative stress and contributeto alterations the redox environment of the aging Leydig cells.Oxidatively damaged Leydig cells exhibit decreased responsivenessto LH, with inhibition of testosterone synthesis. Chronic zinc deprivation generally results in a low intracellularzinc concentration 364,365, increased intracellular oxidative stress , and increased sensitivity to oxidative stress 365,366. An elevated level of systemic oxidative stress inhibits Leydig cellsynthesis of testosterone 61,62,127,129,134. In addition to itscofactor roles, α-lipoic acid penetrates both cell membranes andaqueous compartments throughout the body, allowing α-lipoic acidto act as a multi-purpose nonenzymatic antioxidant that protectsmitochondria and surrounding cellular elements from oxidationby the free radicals produced by mitochondria during oxidative metabolism 335,340,341. The supernatants were collected carefully and used for ELISA (GenAsia, Shanghai, China) and protein measurements. After a 10-min shaking, the 2–nitro–5 thiobenzoic acid formation was monitored at 405 nm. In a 96 wells plate, 40 µL sample or standard, 20 µL 5,5′ dithio–bis–2–nitrobenzoic acid and 140 µL NADPH were added. The triglyceride and total cholesterol levels were measured at 546 nm and expressed as mmol/L. The serum testosterone and blood parameters were immediately measured, while the heart left ventricle (LV) samples were excised, frozen, and kept at −80 °C until the biochemical analyses. Dependence on the mitochondrial electron transfer systemfor the energy to drive testosterone synthesis exposes Leydig cellmitochondria to oxidative stress 3,4, and the generation of ROSwithin Leydig cells increases when testosterone synthesis is stimulated6,125. Reliance on the mitochondrial electrontransfer system for the energy to drive testosterone synthesis exposes Leydigcell mitochondria to oxidative stress. In these studies, the oxidative stress induced by LPS stimulated lipid peroxidation in Leydig cell membranes as well as significant reductions in steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase isomerase (3β-HSD) activity. Thus rats fed a zinc deficient diet experience a decrease in testicular antioxidant potential and a concomitant increase of lipid peroxidation in this tissue.19 Conversely, zinc administration will counteract the oxidative stress created in the testes by exposure to lead.20–21 as well as the peroxidative damage induced by ischemia-reperfusion as a consequence testicular torsion-detorsion.22 Zinc administration has also been shown to attenuate the testicular oxidative DNA damage induced by cadmium as well as the decline in sperm production and testosterone secretion induced by this heavy metal.23 Animals fed on a selenium deficient diet exhibit a significant reduction of testicular GPx activity and an accompanying loss of germ cells from the germinal epithelium of the testes.16 Moreover, selenium administration prior to the creation of oxidative stress in the testes using the torsion/detorsion model (see later) to create ischemia-reperfusion injury, has been found to suppress lipid peroxidation and improve the histopathological profile.17 In order to determine the relative potential of different antioxidants to address oxidative stress in the testes, the testicular torsion-detorsion model has been repeatedly used. Heavy paternal smoking, for example, is known to generate oxidative DNA damage in the male germ line in association with a 32% reduction in the α-tocopherol content of the seminal plasma.105 The role of oxidative stress in the genesis of this DNA damage is supported by the observation that in individuals subjected to an ascorbate depleted diet, the seminal plasma ascorbate levels decreased by a half, while DNA damage levels in the spermatozoa increased by 91%. Exposure of rat Sertoli cells to RA led to activation of ROS generation, lipid peroxidation and, ultimately, a loss of cell viability.99 There is also some evidence to suggest that retinol might stimulate ROS generation in rat Sertoli cells99,100 and that this effect is accompanied by an upregulation of testicular antioxidant enzymes including SOD, GPx and catalase.101 There may be nothing particularly specific about this effect since retinoids have been shown to stimulate ROS generation in a variety of other cellular systems.102 Nevertheless, the free radical generation triggered by retinoids in the testes may explain the testicular degeneration induced by hypervitaminosis A in the rat103 and the association between excess beta carotene intake and infertility in human males.104 The fact that these effects can be reversed by the administration of an antioxidant, α-tocopherol succinate, confirms the importance of oxidative stress in the aetiology of such exercise-dependent testicular dysfunction.40 In animal models prolonged testicular torsion results in excessive ROS generation, depletion of antioxidant enzymes and the appearance of oxidative damage in the contralateral testes.62–64 In light of these data, surgical removal of the ipsilateral testes would seem warranted if the period of ischaemia has been extensive. A re-evaluation of the Health Professionals Follow-Up Studydata after an additional 8 years of observation initially reportedthat compared to the effects of the consumption of less than 90mg of vitamin C daily, the daily consumption of 1000 mg or moresignificantly increased the relative risk of forming a kidney stone by41% . In addition,these environmental toxins downregulate the activities of testicularHSD3B2 and HSD17B3 283, ; this effect is prevented byconcurrent consumption of vitamin C 283,285,288,289, suggestingthat vitamin C may directly upregulate testosterone synthesisin addition to protecting the synthetic pathway from oxidativeinhibition. Oral vitamin C increases LH secretion by isolated pituitary cellsin the absence of hypothalamic LH releasing hormone andstimulates testosterone synthesis 278,282 and increased serum totaltestosterone concentrations in otherwise unmanipulated healthymale rats. In Leydig cells, testosterone synthesisis stimulated by phosphatidylserine through a sequence in whichphosphatidylserine induces the translocation of cytosolic Akt (protein kinase B) to the plasma membrane and interacts directly with Akt toalter its conformation and allow it to be activated via phosphorylationby mTOR2 249,250.
