Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry.

An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17ß-estradiol (E(2)), and 5α-androstan-3α,17ß-diol (3α-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-µL samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E(2). Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E(2) and 3α-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.

Cyclooxygenases in rat Leydig cells: effects of luteinizing hormone and aging.

Previous studies suggested that increased Leydig cell cyclooxygenase (COX)2 expression may be involved in the reduced testosterone production that characterizes aged Leydig cells. Our objective herein was to further elucidate the relationships among LH stimulation, Leydig cell COX2 and COX1 expression, aging, and testosterone production. Incubation of Leydig cells from young or aged rats with LH or dibutyryl cAMP resulted in increases in both intracellular COX2 protein expression and testosterone production. COX1 expression did not respond to LH or dibutyryl cAMP. Incubation of adult cells with a protein kinase A inhibitor suppressed the stimulatory effects of LH on COX2 and testosterone production. Short-term incubation of Leydig cells with TGF-alpha or IL-1beta also increased COX2 protein levels; IGF-I had no effect. In vivo, LH also was found to stimulate both COX2 and testosterone, but not COX1. As reported previously, COX2 expression was greater in old than in young cells, and old Leydig cells responded to inhibition of COX2 in vitro with increased testosterone production. However, the effects of the COX2 inhibitors were not restricted to old cells; young Leydig cells also responded to COX2 inhibition with increased testosterone production. This and the observation that the incubation of young or old cells with LH resulted in increased COX2 and testosterone production in both cases suggests that the relationship between COX2 and testosterone production is not unique to aged Leydig cells. Moreover, the close correlation between increases in COX2 and testosterone in LH-stimulated young and aged Leydig cells is difficult to reconcile with the contention that the increased expression of COX2 in aged cells is responsible for age-related suppression of Leydig cell testosterone production.

Aging and the brown Norway rat leydig cell antioxidant defense system.

Previous studies have shown that testosterone production by the Leydig cells of aged Brown Norway rats is reduced from the relatively high levels produced by Leydig cells of young rats and that this reduction is not secondary to decreased serum luteinizing hormone concentration. The free radical theory of aging proposes that imbalance between pro-oxidants and the antioxidant defense system ultimately results in oxidative damage to cellular processes. With this in mind, we hypothesized herein that age-related reductions in steroidogenesis by Brown Norway rat Leydig cells may be associated with the impairment of the antioxidant defense system of these cells. To begin to test this hypothesis, we compared the activities and steady-state mRNA and protein levels of the antioxidant enzymes copper zinc (CuZn) superoxide dismutase (CuZnSOD, SOD1), manganese (Mn) superoxide dismutase (MnSOD, SOD2), and glutathione peroxidase (GPx) and the levels of reduced and oxidized glutathione in Leydig cells isolated from the testes of young (4-month-old) and aged (20-month-old) Brown Norway rats. For some studies, Leydig cells were isolated separately from aged testes that either had regressed because of age-related losses of germ cells or that were nonregressed. SOD (total) and GPx activities were found to decrease significantly with age whether or not the testes were regressed. CuZnSOD and MnSOD mRNA levels decreased with aging, though the magnitude of the decreases were considerably lower than the respective decreases in enzyme activities. GPx mRNA levels also decreased, which is consistent with the decreases seen in enzyme activity. MnSOD protein expression declined with age, and to a lesser extent, CuZnSOD did as well. Reduced and oxidized glutathione also exhibited age-related reductions in cells from both normal and regressed aged testes. The age-related decreases in Leydig cell antioxidant enzyme activities, gene expression, and protein levels and in glutathione were consistent with the hypothesis that the loss of steroidogenic function that accompanies Leydig cell aging may result in part from a decrease in the fidelity of the cellular antioxidant defense system.

Vitamin E, aging and Leydig cell steroidogenesis.

Previous studies have suggested that oxidant-induced damage may play a role in the reduced ability of aged Brown Norway rat Leydig cells to produce testosterone. We reasoned that if this was the case, antioxidants such as vitamin E (VE) would be expected to have protective effects on steroidogenesis. To test this hypothesis, the effects of VE on Leydig cell steroidogenesis were examined both in vitro and in vivo. In vitro studies were conducted using Leydig cells isolated from the testes of young adult Brown Norway rats. In one experiment, isolated cells were incubated with luteinizing hormone (LH) alone or with LH plus VE (1.3-40 microg/ml). At each of 3, 5 and 7 days thereafter, the ability of the cells to produce testosterone was greater in the presence of VE than in its absence, and depended upon VE dose. Culturing the Leydig cells with the antioxidants melatonin or N-tert-butyl-alpha-phenylnitrone also protected Leydig cell steroidogenic function. Additionally, VE was found to suppress Fe2+/sodium ascorbate-induced lipid peroxidation in Leydig cells. These studies strongly supported the contention that VE has a protective effect on Leydig cell steroidogenesis. These in vitro results prompted us to ask whether, in vivo, VE also would affect steroidogenesis as Leydig cells age. To this end, rats were provided one of three diets, begun when the rats were 6 months of age and carried out through age 25 months: VE-deficient, VE-control, or VE-supplemented. The VE-deficient diet had no effect on the age-related reductions in Leydig cell testosterone production observed in VE-control rats. The VE-supplemented diet did not prevent age-related reductions in steroidogenesis, but the reductions at ages 23 and 25 months were significantly less than those seen in Leydig cells from VE-control or VE-deficient rats. Taken together, the results of the in vitro and in vivo studies reported herein are consistent with the conclusion that vitamin E exerts a protective effect on Leydig cell steroidogenesis.

Aging and caloric restriction: effects on Leydig cell steroidogenesis.

We have shown previously that testosterone concentration in the blood serum of Brown Norway rat becomes reduced with aging, and that this results from reduced testosterone production by individual Leydig cells. Herein we examine the effects of caloric restriction (CR), an intervention shown to delay or inhibit age-associated pathologic and biologic changes in a number of systems and organisms, on Leydig cell steroidogenic function. CR (40%) was initiated in 4 month-old Brown Norway rats, and continued through age 34 months. Serum testosterone concentration in the ad libitum (AL)-fed controls was reduced by 30% from 5 to 13 months, by another 67% through 25 months, and then was sustained through 34 months. For the CR rats, the serum testosterone level was reduced to 45% of AL controls by 5 months, only 6 weeks after the initiation of the CR regimen. There was no further reduction through 25 months, at which time serum testosterone concentration in CR animals was significantly higher than in AL controls. By age 28-34 months, there was no significant difference between the two diets. The weights of prostate and seminal vesicle, two biomarkers of serum androgen levels, were consistent with the changes in serum testosterone concentration in both AL and CR animals. The ability of isolated Leydig cells to produce testosterone in vitro also paralleled the age- and CR-related changes in serum testosterone concentration. CR resulted in a rapid, 36% reduction in testosterone production from control by age 5 months. In contrast to cells from the AL rats, there were no further decreases in testosterone production through age 25 months. Indeed, Leydig cells from the 25 month-old CR rats produced significantly greater amounts of testosterone than cells from the 25 month-old AL rats. These results indicate that short-term CR results in the suppression of Leydig cell function and in reduction in serum testosterone levels. The significantly higher concentrations of serum testosterone concentration, and increased Leydig cell testosterone production, elicited by CR in 25 month-old rats compared to AL controls suggest that long-term CR can transiently suppress the reductions in steroidogenesis that are characteristic of aging.

Temporal relationships among testosterone production, steroidogenic acute regulatory protein (StAR), and P450 side-chain cleavage enzyme (P450scc) during Leydig cell aging.

Previous studies have shown that the capacity of Leydig cells from aged (21-24-month-old) Brown Norway rats to produce testosterone is reduced from young (4-month-old) levels, and that this is correlated with reductions in steroidogenic acute regulatory protein (StAR), peripheral benzodiazapine receptor (PBR), and the levels and activities of the steroidogenic enzymes. The age(s) at which particular changes in the steroidogenic pathway occur, and the relationship of particular changes to reduced testosterone production, are not known. We examined 3 critical components of the steroidogenic pathway, cyclic adenosine monophosphate (cAMP) production, StAR, and P450 side-chain cleavage enzyme (P450scc) in relationship to age-related decreases in testosterone production. Leydig cells isolated from Brown Norway rats of increasing ages (4, 9, 15, and 20 months) were evaluated. The ability of Leydig cells to produce testosterone was reduced at 9 months, although not significantly. Significant reductions in testosterone production were first seen in cells isolated from rats of 15 months of age, and further reductions occurred thereafter. Reduced testosterone was correlated with reductions in StAR, P450scc mRNA, and protein. Significant decline in luteinizing hormone-stimulated intracellular cAMP levels was seen by 9 months, before significant reductions in testosterone, StAR, and P450scc. Further declines in cAMP levels were seen at 15 and 20 months. These studies suggest that age-related reductions in intracellular cAMP may lead to the reduced testosterone production that characterizes aged Leydig cells. This suggestion is supported by recent studies from our lab demonstrating that long-term (3 days) culture of old Leydig cells with dbcAMP restored testosterone production to levels approximating those of young cells.

Dibutyryl cyclic adenosine monophosphate restores the ability of aged Leydig cells to produce testosterone at the high levels characteristic of young cells.

The wealth of knowledge about the function and regulation of adult Leydig cells, the cells within the mammalian testis that produce testosterone, make these cells ideal for studying principles and mechanisms of aging. A hallmark of mammalian aging is decreased serum testosterone concentration. In the Brown Norway rat, this has been shown to be associated with the reduced ability of aged Leydig cells to produce testosterone in response to LH. Herein, we demonstrate that culturing the aged cells with dibutyryl cAMP, a membrane-permeable cAMP agonist that bypasses the LH receptor-adenlyly cyclase cascade, restores testosterone production to levels comparable to those of young cells and also restores steroidogenic acute regulatory protein and P450scc, the proteins involved in the rate-limiting steps of steroidogenesis. These results strongly suggest that signal transduction deficits are responsible for reduced steroidogenesis by aged Leydig cells and that bypassing signal transduction reverses the steroidogenic decline by the aged cells.

Leydig cell gene expression: effects of age and caloric restriction.

In mammals, the concentration of testosterone in blood serum typically becomes reduced with aging. We have shown that, in rats, this results from reduced testosterone production by individual Leydig cells. To gain an understanding of the mechanisms by which Leydig cell steroidogenic function changes with aging, we compared the gene expression profiles in Leydig cells isolated from young and old Brown Norway rats, focusing on possible changes in genes involved in, or associated with, steroidogenesis. We also explored the effects of caloric restriction, an intervention shown to delay or inhibit age-associated pathologic and biologic changes in a number of systems and organisms, on the expression of these genes. Of the total of 1176 genes arrayed on each of the five membranes per group that we used, about 500 (45%) were detectable in Leydig cells isolated from young and old rat testes, with the same genes detectable in cells from both ages. An ANOVA model was applied to statistically analyze the 500 detectable genes in the four treatment groups. The primary criterion by which interesting genes were selected was a P value of 1.5. However, when P1.5. Among the 500 genes, 17 were found to decrease in expression with aging, the most prominent among which were genes involved in steroidogenesis and stress response/free radical scavenging. Long-term caloric restriction had little effect on the 45 genes altered by age. In contrast to the effects of caloric restriction as seen in many other cell types and systems, the vast majority (44/45) of the Leydig cell genes that changed with age were not 'rescued' by this intervention.

Cholesterol transport, peripheral benzodiazepine receptor, and steroidogenesis in aging Leydig cells.

The cellular mechanisms responsible for age-related decline in the ability of Leydig cells to produce testosterone are not yet fully understood. The decline in testosterone production could result from a reduction in the Leydig cell enzymatic activities mediating testosterone synthesis, the amount of substrate available for these enzymes, or both. In the present study, we examined the effect of age on a critical early step in the steroidogenic pathway, the transport of cholesterol into mitochondria. Leydig cells were isolated from the testes of young and old Brown Norway rats and incubated with human chorionic gonadotropin (hCG) and the side-chain cleavage cytochrome P450scc inhibitor aminoglutethimide (AMG). Mitochondria were isolated from these cells in the presence of AMG. Upon removal of AMG, the mitochondria from old cells produced 80% less steroid than those from young cells, only a fraction of which could be accounted for by a decrease in P450scc activity. These results suggest that the accumulation of hormonally recruited cholesterol into mitochondria is defective in old Leydig cells. With this in mind, we turned our attention to peripheral benzodiazepine receptor (PBR), a mitochondrial cholesterol-binding protein known to be involved in mediating cholesterol transport. PBR messenger RNA (mRNA) and protein expression were decreased in old cells. Moreover, both the dissociation constant (Kd) and the number of binding sites (Bmax) of the PBR were decreased in the old cells by 50% and 30%, respectively. Taken together, these results suggest that alterations in cholesterol transport and in PBR may play critical roles in age-related decreases in testosterone production in Brown Norway rat Leydig cells.