Age-related increase in mitochondrial superoxide generation in the testosterone-producing cells of Brown Norway rat testes: relationship to reduced steroidogenic function?
Introduction
It is well established that aging in the human male typically is accompanied by reduced serum levels of testosterone, and that, as a consequence of this, and perhaps of decline in other hormones (growth hormone, for example), aging men may suffer osteoporosis, reduced muscle strength, reduced libido, and mood changes (Swerdloff and Wang, 1993, Tenover, 1999, Hermann and Berger, 1999, Matsumoto et al., 2000, Veldhuis, 2000). In aging men, reduced serum testosterone levels are considered to result in large part from primary testicular deficits, rather than secondary to reduced luteinizing hormone (LH), though changes in the hypothalamic-pituitary axis also have been reported (Kaufman and Vermeulen, 1997, Hermann and Berger, 1999). Aging in rats also is accompanied by reduced serum levels of testosterone (for review, see Chen et al., 1996b). In most rat strains, however, reduced testosterone results from testicular changes that are secondary to progressive age-related decreases in LH (Kaler and Neaves, 1981, Harman et al., 1978, Miller and Riegle, 1978), a situation that differs from that in the human male. In contrast, in male Brown Norway rats, reduced serum testosterone concentration is accompanied by increased levels of FSH (Wang and Leung, 1993, Zirkin and Chen, 2000) and unchanged LH levels (Chen et al., 1994, Chen et al., 1996a, Gruenewald et al., 1994), suggesting that, as in the human, the reduced serum testosterone levels in this rat strain result from a primary gonadal defect, and perhaps also from changes in the hypothalamic-pituitary axis.
Leydig cells, situated in the interstitial compartment of the mammalian testis, are responsible for most of the testosterone produced by males. Recent studies have shown that the production of testosterone per Leydig cell is reduced with aging of the Brown Norway rat, but that the numbers of Leydig cells per testis do not change, suggesting that changes to individual cells account for reduced testosterone levels in the serum (Chen et al., 1994). As yet, the mechanism by which aging Leydig cells lose steroidogenic function is not known.
During normal oxidative metabolism, cells continually convert molecular oxygen into superoxide anions (O2−) and hydrogen peroxide (H2O2). Mitochondrial respiration, which typically consumes about 90% of the oxygen utilized by cells, is considered to be the major source of cellular reactive oxygen (Freeman and Crapo, 1982, Shigenaga et al., 1994); superoxide is generated by mitochondria when electrons are released from the electron transport chain and transferred directly to molecular oxygen. Under circumstances in which reactive oxygen species are produced beyond the capability of a cell to detoxify them, the result may be damage to protein, lipid and/or DNA (for review, see Beckman and Ames, 1998). Mitochondrial-derived reactive oxygen species have been implicated in a number of diseases and disorders, including neurodegenerative diseases (Simonian and Coyle, 1996), cancers (St. Clair, 1996), ischemia/reperfusion injury (Ambrosio et al., 1993), and aging (Sohal and Weindruch, 1996).
Reactive oxygen species undoubtedly are produced in Leydig cells, as in other cells, via the mitochondrial electron transport chain. Additionally, however, reactive oxygen has been shown to be produced as a by-product of steroidogenesis (Hornsby, 1989, Peltola et al., 1996), in particular during steroid hydroxylations by the cytochrome P450 enzymes, which are localized in the mitochondria and in the smooth endoplasmic reticulum (SER) (Hall, 1994, Hornsby and Crivello, 1983). There is evidence that reactive oxygen species may have a detrimental effect on critical components of the steroidogenic pathway (Georgiou et al., 1987, Quinn and Payne, 1984, Quinn and Payne, 1985, Stocco et al., 1993). Consequently, it seemed plausible to us to hypothesize that free radical damage to critical components of the steroidogenic pathway might result in age-related functional deficits in Leydig cells, thereby accounting at least in part for the reduced steroidogenic ability of the old cells. As yet, this hypothesis has received little attention.
In the present study, we examined the production of mitochondrial reactive oxygen species in intact Leydig cells from young and old Brown Norway rats. To detect and quantify mitochondria-derived reactive oxygen generation, Leydig cells were incubated with lucigenin (bis-N-methylacridinium nitrate), a chemiluminigenic probe that enters cells, localizes to mitochondria, and yields a significant chemiluminescent response following its reaction with intramitochondrial superoxide (Li et al., 1998, Li et al., 1999). Leydig cells from old rats were found to elicit significantly greater lucigenin-derived chemiluminescence (LDCL) than Leydig cells from young rats; this despite the fact that the absolute volume of mitochondria in the old cells, as measured by stereological methods at the electron microscopic level, was reduced from that in the young. Taken together, these results suggest that the mitochondria of the Leydig cells of old rats produce significantly greater levels of reactive oxygen species than those of young rat Leydig cells. The results are consistent with the proposal that mitochondrial-derived reactive oxygen may play a role in the irreversible decline in the ability of the old cells to produce testosterone by damaging the steroidogenic machinery of aging Leydig cells.
Section snippets
Animals
Male Brown Norway rats of ages 4–6 months (young) and 21–24 months (old) were obtained through the National Institute on Aging, supplied by Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Rats were housed in controlled light (14 h light/10 h dark) and temperature (22°C), and had free access to rat chow and water. All procedures were in accord with the NIH Guide for the Care and Use of Laboratory Animals, with protocols approved by the Johns Hopkins Animal Care and Use Committee.
Leydig cell purification
Leydig cells were
Reactive oxygen production by Leydig cells from young vs. aged rats
Typical lucigenin-derived chemilunescence (LCDL) profiles for Leydig cells isolated from young and old rats are shown in Fig. 1. Incubation of Leydig cells with lucigenin elicited LDCL responses that were significantly above the control (‘lucigenin only’), which lacked cells. Peak LDCL was seen 10–20 min after adding lucigenin. As is evident by observing the traces, cells from aged rats produced more luminiscence than cells from young rats. Quantification of the response curves revealed that
Discussion
It is now well established that the Leydig cells of Brown Norway rats become steroidogenically hypofunctional as they age (Chen et al., 1994). As yet, the mechanism(s) by which this occurs is not known. There is considerable evidence, gathered over the course of many years, to support the contention that aging cells, in general, undergo deleterious, irreversible changes as a consequence of reactive oxygen-induced free radical reactions (Harman, 1956, Harman, 1981, Sohal and Weindruch, 1996).
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