Lindi Luo

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.

Leydig cell steroidogenesis in aging rats.

Recent studies from our and other laboratories have shown that Leydig cells, the testicular cells responsible for testosterone production; become steroidogenically hypofunctional with age. Herein we review some of what we now know about the mechanisms by which this occurs, and some among the many remaining uncertainties in our understanding of Leydig cell aging. To help shed light on how Leydig cells age, we also briefly discuss the regulation of Leydig cell differentiation during puberty and of Leydig cell function in adult animals.

Aging and the Decline of Androgen Production

Human male aging is associated with progressive decreases in serum concentrations of testosterone, which are not in response to decreased circulating basal luteinizing hormone (LH) concentrations, suggesting that reduced testosterone results from a primary deficit at the gonadal rather than the hypothalamic-pituitary level. This also is true of Brown Norway rats, a strain that has become widely used for studies of Leydig cell aging. Age-related reduced testosterone was found to result from reduced Leydig cell steroidogenesiss, but not by their loss. This chapter deals with the cellular changes, which are associated with reduced testosterone, and their causes. Age-related reductions have been reported in Leydig cell LH receptor numbers, intracellular cyclic adenosine monophosphate (cAMP) formation, steroidogenic acute regulatory protein, peripheral benzodiazapine receptor, the conversion of cholesterol to pregnenolone within the mitochondria and the subsequent conversion of pregnenolone to progesterone, 17a-hydroxyprogesterone, androstenedione, and ultimately testosterone in the smooth endoplasmic reticulum. Culturing isolated Leydig cells with LH maintained high levels of testosterone production by young cells but did not restore old cells to “young” levels. In contrast, culturing old cells with dibutyryl cAMP restored testosterone production to high levels, suggesting a deficit in the signal transduction mechanism between the LH receptor and cAMP production. Long-term suppression of steroidogenesis, accomplished by administering exogenous testosterone to middle-aged rats, prevented the steroidogenic aging of the cells, suggesting that ultimately steroidogenesis itself might result in age-related reductions in steroidogenesis. As measured with lucigenin, reactive oxygen production by old Leydig cells was found to be significantly more than by young Leydig cells. Microarray and Northern blot analysis revealed that the expression of genes for Cu-Zn Superoxide dismutase 1, Cu-Zn Superoxide dismutase 2, catalase, and glutathione peroxidase, the products of which protect Leydig cells from oxidative stress, are reduced as the Leydig cells age, as does their activities and protein levels. Depletion of glutathione with buthionine sulfoximine resulted in reduced testosterone production by young adult Leydig cells. Incubation of the cells with vitamin E delayed reduced testosterone production. Taken together, the available data suggests that changes in reactive oxygen and thus, an altered redox environment in aging Leydig cells might cause the changes in Leydig cells that result in age-related reduced testosterone production, and that the reactive oxygen might derive, at least in part, from steroidogenesis itself.

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), 17β-estradiol (E(2)), and 5α-androstan-3α,17β-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.

Testicular Aging: Leydig Cells and Spermatogenesis

Significant age-related decreases in serum testosterone concentration occur in both humans and rats (1). Such changes could result from reduced numbers of Leydig cells, decreased testosterone production by Leydig cells, increased testosterone metabolism, or all of these. Herein we discuss evidence showing that the ability of the Leydig cells of Brown Norway rats to produce testosterone declines with age, and we speculate as to the mechanisms by which this might occur. As in the human (2–8), degenerative changes in the seminiferous tubules accompany aging of the Brown Norway rat (9–11). Age-related atrophy of the seminiferous epithelium (i.e., loss of germ cells) begins focally and subsequently involves most of the testis. We provide evidence that changes in follicle-stimulating hormone (FSH) and intratesticular testosterone concentration are unlikely to cause age-related germ cell loss, and we speculate as to alternative mechanisms.