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From the * Division of Reproductive Biology,
Department of Biochemistry and Molecular Biology and the
Division of Toxicological Sciences, Department
of Environmental Health Sciences of Johns Hopkins University Bloomberg School
of Public Health, Baltimore, Maryland.
| Correspondence to: Dr Lindi Luo, Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 (e-mail: lluo{at}jhsph.edu). |
| Received for publication June 2, 2005; accepted for publication August 30, 2005. |
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Abstract |
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Key words: Testosterone, free radical aging, pro-oxidants, antioxidants, aging
Recent studies of Leydig cells of the Brown Norway rat have demonstrated that aging is accompanied by functional deficits of individual Leydig cells (Chen et al, 1994, 1996). Exposing the aging cells to LH, whether in vivo (Grzywacz et al, 1998) or in vitro (Chen et al, 2002), did not "restore" the aged cells to the high levels of testosterone production that are characteristic of young cells. In contrast, when the aged cells were incubated with dbcAMP, testosterone production increased to levels close to those of young cells (Chen et al, 2004; Luo et al, 2005), indicating that deficits in the signal transduction mechanism that lead to cAMP production may explain age-related reductions in steroidogenesis. As yet, the mechanism by which signal transduction deficits occur is uncertain.
The free radical theory of aging posits that cells are in a chronic state of oxidative stress as a consequence of imbalance between pro-oxidants and the antioxidant defense system and that as a consequence of this imbalance, oxidative damage may occur over time to lipids, DNA, and/or proteins (Miro et al, 2000; Sastre et al, 2000; Stadtman and Levine, 2000; Stadtman, 2001). We have shown previously that Leydig cells from the testes of aged Brown Norway rats produce significantly greater levels of reactive oxygen species (ROS) than cells from young rats (Chen et al, 2001). Additionally, the possibility that there may be deficits in the antioxidant defense system of aging Brown Norway rat Leydig cells was indicated by microarray analysis, which revealed age-related down-regulation of genes of the free radical scavenger family, including the glutathione transferase subunits GST12 and GSTM2 and copper zinc (CuZn) superoxide dismutase (SOD1), the most prominent antioxidant enzyme in Leydig cells (Syntin et al, 2001; Chen et al, 2004). In addition to SOD, which catalyzes the dismutation of superoxide radicals into hydrogen peroxide and oxygen, major antioxidant defense molecules that are present in Leydig cells include catalase, which catalyzes the conversion of hydrogen peroxide to oxygen and water, and members of the glutathione peroxidase (GPx) family, which use reduced glutathione (GSH) as a nonenzymatic cofactor to convert hydrogen peroxide to water. Two forms of SOD coexist, a tetrameric enzyme containing manganese (MnSOD) and a dimeric enzyme containing copper and zinc (CuZnSOD). There are at least 5 GPx isoenzymes found in mammals (Schwaab et al, 1998), among which GPx1 is considered to be the major enzyme responsible for removing hydrogen peroxide. Alhough the relationships among ROS production, antioxidant defense, and age-related reductions in steroidogenesis are not known, it is plausible to hypothesize that the ability of Leydig cells to withstand damaging free radicals may be compromised by age-related imbalance between ROS production and the antioxidant defense system and, therefore, that oxidant-induced damage to Leydig cells may be involved in age-related reductions in steroidogenesis.
The present study was predicated on the hypothesis that the reduced ability of aging Brown Norway rat Leydig cells to respond to LH, and therefore the reduced ability of these cells to produce testosterone, is associated with the age-related impairment of the antioxidant defense system of these cells. To begin to test this hypothesis, the enzyme activities and mRNA and protein levels of the antioxidant enzymes CuZn, MnSOD, and GPx and the cellular content of the nonenzymatic antioxidant molecule glutathione were measured in freshly isolated Leydig cells from the testes of young (4-month-old) and aged (20-month-old) rats. With respect to the aged rats, Leydig cells were obtained separately from testes of normal size and cellular content and from testes that had regressed as a consequence of age-related germ cell loss. The rationale for studying aged regressed and nonregressed (normal) testes separately is that Syntin and colleagues (2001) reported that gene expression by Leydig cells isolated from the 2 types of testes differs, though testosterone production is reduced equivalently.
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Materials and Methods |
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CuZn and MnSOD Enzyme Activities
Total SOD activity was measured as described previously
(Spitz and Oberley, 1989). In
brief, Leydig cells (1 x 106) were suspended in 50 mM
potassium phosphate buffer/0.1 mM EDTA buffer, pH 7.8, and were then
sonicated. The supernatant was collected after centrifugation and assayed as
follows: Supernatants (2–20 µL) were added to the reaction buffer (50
mM potassium phosphate buffer, pH 7.8; 1 mM diethylene triamine pentaacetic
acid; 1 U of catalase; 5.6 x 10-5 M nitroblue tetrazolium;
0.1 mM xanthine; 0.13 mg bovine serum albumin, and 50 µM bathocuproine
disulfonic acid). The reaction was initiated by adding 100 µL of xanthine
oxidase solution, which was diluted until the absorbance rate in tubes without
SOD (blanks) was between 0.016 to 0.025/min at 560 nm. Absorbance changes were
monitored at 30-second intervals for 4 minutes at 560 nm in a Beckman DU 800
spectrophotometer. One unit of activity was defined as the amount of protein
resulting in half-maximal inhibition. To measure MnSOD in this assay, samples
were preincubated (1 hour) with potassium cyanide (5 mM). The MnSOD activity
was subtracted from the total SOD activity to calculate the CuZnSOD
activity.
GPx Activity
To measure GPx activity, Leydig cell pellets were suspended in buffer (50
mM Tris-HCl, pH 7.6; 5 mM EDTA; 1 mM dithiothreitol) and disrupted by
sonication. The supernatant was collected after centrifugation and assayed as
follows: Sample (20 µL) was added to the assay (50 mM Tris-HCl, pH 7.6; 5
mM EDTA; 1 mM GSH; 0.2 mM NADPH; and 0.4 U of glutathione reductase) and the
reaction was initiated by adding tert-butyl hydroperoxide to a final
concentration of 0.22 mM. NADPH consumption was monitored for 4 minutes at
30-second intervals at 340 nm in a Beckman DU 800 spectrophotometer
(Fullerton, Calif). One unit of GPx activity was defined as the oxidation of 1
µmol NADHP/min.
Northern Blot Analysis of CuZnSOD, MnSOD, and GPx
RNA was purified from adult rat Leydig cells by the Trizol method
(Invitrogen Corporation, Carlsbad, Calif). A Leydig cell cDNA library was
generated by performing first-strand synthesis from total RNA. Briefly, RNA (3
µg) was reverse-transcribed in a 20 µL reaction at 46°C for 60
minutes, using 0.2 units of Superscript II (Invitrogen) and 50 ng of oligo-dT
primer in single-strength buffer, according to manufacturer specifications.
Polymerase chain reaction (PCR) was performed as previously described
(Anway et al, 2003). Primers
were designed (Table) to
amplify rat cDNA fragments of CuZnSOD
(Chen et al, 1999), MnSOD
(Csonka et al, 2000), and GPx
(Suwa et al, 2000). PCR
products were cloned into a p-GemT Easy Vector (Promega, Madison, Wisc)
according to manufacturer specifications and sequenced to verify insert
product. Northern blots were run as described previously
(Luo et al, 1996), with total
RNA from 1 x 106 cells loaded per lane and electrophoresed
through a denaturing 1.2% agarose gel.
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Immunoblotting for Detection of CuZnSOD and MnSOD Protein
Western blot analysis was performed as previously described
(Luo et al, 1996), with each
lane containing protein from equal numbers of cells (1 x
105). The primary antibodies, anti-human CuZnSOD and anti-human
MnSOD (UpState, St Louis, Mo), were used to detect the respective proteins
according to manufacturer instructions. The blots were stripped and stained
for the monoclonal anti-ß-actin (Sigma Chemical Co, Charlottesville, Va)
to correct for possible differences in protein loading of the gels.
GSH and Oxidized Glutathione
GSH and oxidized (GSSG) glutathione were measured as previously described
(Hissin and Hilf, 1976). In
brief, Leydig cells were suspended in sodium phosphate buffer (0.1 M, pH 8.0,
with 5 mM EDTA) and sonicated. Protein was precipitated in metaphosphoric acid
(HPO3) and then centrifuged at 13 000 x g for 30
minutes to obtain the supernatant. The supernatant was diluted 10 times with
sodium phosphate buffer. Diluted sample (0.05 mL) was incubated with 0.05 mL
of o-phthalaldehyde (in methanol) and 0.9 mL of phosphate buffer for
15 minutes at room temperature. Fluorescence was read with a BioRad
luminescence spectrometer at an excitation wavelength of 350 nm and an
emission wavelength of 420 nm. Leydig cell GSH content was calculated using a
concurrently run reduced GSH standard curve. To assay GSSG, the supernatant
was incubated at room temperature with 0.04 M N-ethylmaleimide to
interact with GSH present in the sample. To this mixture, 0.1 N NaOH was
added. A 100-µL portion of this mixture was taken for measurement of GSSG,
using the procedure outlined above for the GSH assay.
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Results |
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Steady-State Levels of mRNA Transcripts of Scavenging Enzymes
Northern blots of CuZnSOD, MnSOD, and GPx mRNA are shown in
Figure 3A. For these studies,
Leydig cells were isolated from the testes of 4 groups of young rats and 4
groups of aged rats with nonregressed testes (A-N). Quantification of the mRNA
levels is shown in Figure 3B.
CuZnSOD mRNA levels in Leydig cells from the A-N testes were reduced
significantly, although modestly (by 14%), from the young rat value. MnSOD
mRNA levels also were reduced, but even more modestly (by 10%) and not
significantly. These decreases in mRNA for both CuZnSOD and MnSOD were in
contrast to the much more robust age-related decreases in enzyme activities
(Figure 1). A significant
decrease in GPx mRNA levels also was seen
(Figure 3B). In this case, the
reduction from young to A-N was more robust (34%) than the reductions in CuZn
and MnSOD mRNAs, and this reduction was consistent with the 24% decrease seen
in GPx enzyme activity. The 18S RNA did not change in aged vs young Leydig
cells.
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Discussion |
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We demonstrated recently that aging Brown Norway rat Leydig cells produce significantly greater levels of ROS than do young Leydig cells (Chen et al, 2001), and we suggested that ROS may be involved in the reduced steroidogenesis that characterizes aging Leydig cells. The extent to which cells are damaged by the free radicals that they produce is considered to be a function of the balance between free radical production and the efficacy of the cell's free radical defense system. We show in the present study that the activities of major antioxidant enzymes in Leydig cells, CuZnSOD, MnSOD, and GPx, all were reduced significantly in Leydig cells from aged rats. These reductions were seen whether the Leydig cells were from nonregressed or regressed testes, although the decreases in each case were somewhat greater in comparable numbers of cells from the aged regressed testes than from the aged nonregressed testes. The observation that age-related reductions in antioxidants occur in testes with normal germ cell content indicates that such reductions are largely, though probably not entirely, a consequence of changes within the Leydig cells and do not take place in response to extrinsic changes in the germ cell content of the seminiferous tubules.
Interestingly, mRNA and protein levels did not necessarily predict the changes seen in the activities of the antioxidant enzymes. In the case of CuZnSOD and MnSOD, for example, the mRNA levels per cell changed relatively little with aging (10%–14%), compared to the changes seen in their activities (50%–75%), whereas the age-related decline in GPx mRNA (35%) was as great in magnitude as the decline in its enzyme activity. Similarly, whereas the protein levels of CuZnSOD in the aged normal testes changed little, which is reminiscent of mRNA levels but not activity, there was a significant change in MnSOD protein level, which was reminiscent of the mRNA level but not nearly as great in magnitude as the decrease in activity level.
In addition to changes in the antioxidant enzymes, decreases both in reduced and oxidized glutathione were seen. In Leydig cells from both aged nonregressed and regressed testes, the cellular contents of GSH and of GSSG were reduced from the young cell levels, whereas the ratios of GSH to GSSG changed relatively little. Decreased GSH could be particularly important. GSH plays a particularly important role in scavenging free radicals (Toussaint et al, 1993) and protects cells against several toxic oxygen-derived chemical species. It has been reported that the intracellular concentration of GSH affects DNA by modulating DNA synthesis (Suthanthiran et al, 1990) or by protecting DNA from oxidative damage (Bellomo et al, 1992). A constant supply of GSH is necessary to repair the effects of spontaneous oxidation of sulfhydryl groups, which results in cell membrane damage. GSH also is an important co-factor for GPx activity (Spooren and Evelo, 1998). Therefore, it is believed that GSH is particularly important in protecting cells against oxidative damage (Di Mascio et al, 1991). The fact that its level is decreased in aged Leydig cells could have a significant effect on the ability of these cells to defend against oxidative stress.
The results presented herein complement and extend a recent study using microarray technology, in which we reported that the expression of a number of genes involved in protecting cells against oxidative stress was reduced in response to aging, including SOD1 and glutathione S-transferases (GST12 and GSTM2) (Syntin et al, 2001; Chen et al, 2004). Taken together, these results are consistent with published reports of age-related decreases in antioxidant enzymes in Leydig and adrenocortical cells (Azhar et al, 1995; Cao et al, 2004), with evidence for a central role for oxidative stress in steroidogenic function in the ovary (Stocco et al, 1993; Musicki et al, 1994; Diemer et al, 2003). Recently, Cao and co-workers (2004) reported reduced levels of antioxidants in Leydig cells from 24-month-old Sprague-Dawley rats compared to 5-month-old rats. Although serum testosterone levels decrease with aging in the Sprague-Dawley strain, decreased serum LH levels also occur, indicating that in contrast to both the human and Brown Norway rat, reduced testosterone in aged Sprague-Dawley rats is secondary to inadequate LH secretion. Additionally, substantial weight gain and the common occurrence of pituitary adenomas and other endocrine tumors in aging Sprague-Dawley rats (Hollander, 1976; Cohen et al, 1978; Johnson and Neaves, 1983) make results obtained with aging rats of this strain difficult to interpret. Clearly, further work needs to be done to determine the relationship among age-related increases in ROS production, decreases in the antioxidant defense system and thus in the capacity to maintain a normal oxidant/antioxidant balance, and decreases in steroidogenesis.
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Footnotes |
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