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Endocrinology Vol. 142, No. 12 5277-5285
Copyright © 2001 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Gene Expression in Brown Norway Rat Leydig Cells: Effects of Age and of Age-Related Germ Cell Loss

Patrick Syntin1,2, Haolin Chen2, Barry R. Zirkin and Bernard Robaire

Departments of Pharmacology and Therapeutics, and Obstetrics and Gynecology, McGill University (P.S., B.R.), Montréal, Québec, Canada H3G 1Y6; and Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University School of Hygiene and Public Health (H.C., B.R.Z.), Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Dr. B. Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, Canada H3G 1Y6. E-mail: brobaire{at}pharma.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is a marked reduction in circulating T and a commensurate decrease in Leydig cell function in males during aging. Aging is also accompanied by progressive loss of germ cells, leading to testicular atrophy. However, in aged animals, there is no difference in T production by Leydig cells from nonregressed testes and from regressed testes. We hypothesize that there are changes in Leydig cell gene expression that accompany aging, and that different changes in gene expression result from testicular regression. To test this hypothesis, the expression of stress response genes was compared in Leydig cells isolated from young rat testes, from aged testes that were not regressed, and from aged testes that were regressed, using an array approach. Similar numbers of transcripts (n = 56–63) were detected in Leydig cells isolated from all three groups of rats. Among these, 21 transcripts were increased in Leydig cells of testes from aged nonregressed animals compared with cells from young animals; 23 were increased with subsequent testicular regression. Only 3 of these transcripts were in common. Thus, age and testicular regression affected Leydig cell transcripts in dramatically different ways. Furthermore, none of the transcripts that decreased when comparing Leydig cells of young and aged nonregressed animals were the same as those that decreased when comparing aged nonregressed and aged regressed animals. In individual gene families, the steady state concentrations of transcripts in Leydig cells from aging and aging regressed testes often differed. Thus, there are major differences in the expression of a wide variety of stress response genes in Leydig cells associated with aging and testicular regression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE RAT, as in the human, aging is accompanied by a reduction in the serum T concentration (1, 2, 3). In the human, reduced T may have significant adverse consequences, including osteoporosis and reduced muscle strength and libido (4, 5, 6, 7). Recent studies using the Brown Norway rat model have demonstrated that aging is accompanied by functional deficits of individual Leydig cells (8, 9, 10, 11), the cells that are responsible for producing T in the mammalian testis. It is probable that these Leydig cell deficits are responsible for the reduced levels of T in the blood. Although the mechanism by which individual Leydig cells become hypofunctional remains uncertain, age-related changes in Leydig cell gene and protein expression have been reported, including, not surprisingly, changes in steroidogenic enzyme transcripts and in their protein products and activities (9, 11, 12).

In addition to reduced steroidogenesis, testicular aging typically is also accompanied by a decrease in the number of spermatogenic cells within the seminiferous tubules. In both humans and rodents, age-related germ cell losses begin focally, with atrophic tubules often observed adjacent to tubules exhibiting normal spermatogenesis (3, 13, 14, 15, 16). The loss of germ cells may be due to changes that are intrinsic to the germ cells, to decreases in the ability of the somatic cells of the seminiferous tubules, the Sertoli cells, to support germ cell survival and differentiation, or to as yet unknown extrinsic factors. In the Brown Norway rat, as in other rat strains, age-related germ cell loss begins focally at about 18 months of age (13). Subsequently, the loss of germ cells spreads throughout the testis, thereby causing progressive losses in testis weight. In early stages, it is not unusual to find individual rats in which one testis is entirely normal with respect to germ cell content while the other testis is partially to fully regressed, containing both normal and atrophic seminiferous tubules.

In a previous study we demonstrated that in response to LH, T production by the Leydig cells of aged rats was reduced by about 50% from the young value whether the Leydig cells were isolated from normal or regressed testes (17). This observation indicated that age-related decreased steroidogenesis was independent of germ cell loss. However, this does not mean that the Leydig cells of aged normal and aged regressed testes are necessarily comparable in all ways, i.e. that the loss of germ cells has no effect on the function of the aging Leydig cells.

We hypothesized herein that changes in Leydig cell gene expression accompany aging, and that further changes in gene expression result from testicular regression, presumably due to the loss of germ cells. To address these hypotheses, we examined the expression of stress response genes in Leydig cells isolated from young rat testes, aged testes that were not regressed, and aged testes that were regressed. Gene expression was examined using gene expression arrays. We report that of the 216 stress response genes that were studied, about 10% responded (i.e. increased or decreased) to aging, prominent among which were stress-related transcription genes, and that another 10%, primarily heat shock stress response genes, responded to testicular regression. Some genes, including those involved in metabolism, were affected both by aging and by testicular regression. These results indicate that although aging results in comparably reduced T production by Leydig cells of both nonregressed and regressed testes, there is differential expression of genes in these cells. Thus, aging and subsequent testicular regression result in distinct changes in the induction or suppression of the expression of a number of Leydig cell genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Brown Norway rats, aged 4 and 22 months, were purchased from the NIA (Bethesda, MD) and were supplied by Harlan Sprague Dawley, Inc. (Indianapolis, IN). Rats were maintained at 22 C at The Johns Hopkins School of Hygiene and Public Health facilities in a 14-h light, 10-h dark cycle and were provided with food and water ad libitum. All animal handling and care were performed in accordance with protocols approved by the institutional care and use committee of The Johns Hopkins School of Hygiene and Public Health.

Experimental design
Leydig cells were obtained from young rats, aged rats with nonregressed testes, and aged rats with regressed testes. Five nylon membranes (Rat Stress and Toxicology Atlas Arrays, CLONTECH Laboratories, Inc., Palo Alto, CA) were probed using RNA extracted from Leydig cells from each of young, aged nonregressed, and aged regressed testes; the testes were obtained from five different groups of young and aged rats. For each membrane, Leydig cells were pooled from six to eight testes.

Isolation and purification of Leydig cells
Rats were killed by decapitation, and testes were removed. The procedure for the isolation of Leydig cells was previously described (18). In brief, each testis was removed, weighed, and placed in dissociation buffer (medium 199 with 2.2 g/liter HEPES, 0.1% BSA, 25 mg/liter trypsin inhibitor, and 0.7 g/liter sodium bicarbonate, pH 7.4) at 4 C. The testicular artery was cannulated and perfused with collagenase (1 mg/ml) in dissociation buffer. Testes were decapsulated, and dissociation was continued at 34 C in a lower concentration (0.25 mg/ml) of collagenase, with low speed shaking (90 cycles/min). Seminiferous tubules were removed by filtration through a 100-µm pore size nylon mesh. The dissociated cells in the filtrate were subjected to centrifugal elutriation (16 ml/min, 2,000 rpm). The fraction enriched with Leydig cells was collected and centrifuged (27,000 x g, 1 h) on a 50% Percoll gradient formed in situ. Leydig cells with a density of 1.07 and heavier were harvested for subsequent RNA extraction. The percentage of Leydig cells, examined by staining for 3ß-hydroxysteroid dehydrogenase activity (19), was about 95% for both young and aged rats. Cells were stored at -80 C until RNA extraction.

Preparation of total RNA
Total RNA was extracted from 2.3–14.6 x 106 cells (the number depended upon the age of the rats) according to the technique described by Chomczynski and Sacchi (20). To reduce contamination by genomic DNA, total RNA was treated with ribonuclease-free deoxyribonuclease I for 1 h at 37 C as recommended by the manufacturer (CLONTECH Laboratories, Inc.), followed by phenol/chloroform purification (20).

Probe preparation and hybridization to cDNA Atlas arrays
To generate radiolabeled cDNA probes, total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase and radiolabeled with [{alpha}-32P]dATP (Amersham Pharmacia Biotech, Baie d’Urfe, Canada; 10 µCi/µl). The radiolabeled cDNA probes were purified from unincorporated nucleotides by gel filtration in Chroma Spin-200 columns (CLONTECH Laboratories, Inc.) and hybridized overnight at 68 C to a rat stress/toxicology microarray consisting of 216 known rat genes, as described by the manufacturer (CLONTECH Laboratories, Inc.). After a series of stringent washes (three 20-min washes in 2x SSC/1% SDS, followed by two 20-min washes in 0.1x SSC/0.5% SDS) at 68 C, the membranes were sealed in plastic (Kapak Corp., Minneapolis, MN) and exposed to phosphorimager plates for 24 h.

After scanning of the plates (Storm, Molecular Dynamics, Inc., Sunnyvale, CA), the intensity of each cDNA was quantified using AtlasImage software (version 1.5, CLONTECH Laboratories, Inc.). A representative blot is shown in Fig. 1Go. The intensity of each spot, reflecting the relative abundance of mRNA in the sample, was analyzed for a total of five different membranes (see above). The data files were then imported into GeneSpring software (Silicon Genetics, Redwood, CA) for analysis (21, 22). The level of background, representing nonspecific binding, was subtracted from the intensity of each spot on the array to generate the raw data for each gene. To minimize experimental variation, data were normalized by defining the mean level of all mRNAs on each array as 1 and normalizing the expression of each relative to 1; this is defined as the relative intensity for any given gene. A gene was considered as expressed if its intensity was at least 2-fold the average background of all replicates in that experiment. To conclude that the expression of a given gene changed with aging, differences in expression level were at least 1.5-fold, and consistent results were obtained in at least three of five replicate experiments.



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  		Figure 1. Phosphorimage of a representative Atlas rat stress array hybridized with a 32P-labeled cDNA probe made with Leydig cells from a regressed testis of an old Brown Norway rat (22 months old). This array includes 216 different rat cDNAs immobilized in duplicate dots on a nylon membrane. The first lane contains 9 housekeeping control cDNAs, 3 negative controls, and 4 calibration markers. The 6 spots at the corners and in the middle top and bottom represent orientation marks to align the membrane on a grid before quantification.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in overall Leydig cell gene expression with aging and germ cell depletion
A total of 216 genes were present on the arrays, representing gene families involved in apoptosis, cell cycle, intracellular signal transduction, transcription, heat shock, metabolism, and oxidative stress. Of these genes, over three quarters were not detected in mRNA isolated from Leydig cells of young, aged nonregressed, or regressed testes. A similar number of transcripts was detected in Leydig cells isolated from young rats (56 transcripts), aged rats with nonregressed testes, (63 transcripts), and aged rats with regressed testes (63 transcripts). As shown in Table 1Go, the concentrations of the transcripts of 39 genes did not differ in Leydig cells between young and aged nonregressed testes, whereas the expression of 38 transcripts, many of which were different, did not differ in Leydig cells between old nonregressed and old regressed testes. However, age and testicular regression did result in changes in Leydig cell transcripts. As illustrated in Fig. 2Go, 21 transcripts were increased when Leydig cells from young rats were compared with those from aged nonregressed animals; 23 transcripts were increased when Leydig cells from aged nonregressed testes were compared with those from age-regressed testes. Interestingly, only 3 of these transcripts were in common (Id3, Cox2, and superoxide dismutase 2 (SOD2); Table 2Go). Similarly, none of the transcripts that decreased when comparing Leydig cells from young and aged nonregressed animals (5 transcripts) were the same as those that decreased when comparing aged nonregressed and aged regressed animals (3 transcripts; Table 3Go).


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  		Table 1. Effects of aging and testicular regression on genes having no change in expression (<1.5-fold) in Leydig cells of Brown Norway rats

 


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  		Figure 2. Aging and testicular regression increased (A) or decreased (B) gene expression in Leydig cells. The numbers of genes showing a change in expression between Leydig cells from young and aged nonregressed testes are shown in dark gray circles, whereas those showing a change in expression between Leydig cells from aged nonregressed and aged regressed testes are shown in light gray circles. The intersections highlight genes that are common to both transitions (young to old nonregressed and old nonregressed to old regressed). The number of genes corresponding to the various groups is indicated inside each circle.

 

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  		Table 2. Effects of aging and testicular regression on genes having increased expression in Leydig cells of Brown Norway rats

 

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  		Table 3. Effects of aging and testicular regression on genes having decreased expression in Leydig cells of Brown Norway rats

 
Changes observed by functional families
A major advantage of analyzing a large number of genes at the same time is that it allows the grouping of genes involved in similar functional activities (e.g. signal transduction, DNA synthesis and repair, etc.) and, hence, in this case permits a broad overview of how aging affects cellular pathways or processes. For several functional gene families (e.g. apoptosis), the level of gene expression was minimally altered by age. In contrast, members of other functional gene families (e.g. transcription factors, heat shock proteins (HSP), and oxidative stress markers] were dramatically altered by age and/or testicular regression.

Apoptosis, cell cycle, and intracellular signal transduction genes
A number of genes involved in apoptosis (bcl-2, bcl-x, BAD, BAX, BOK, and caspases 1, 3, and 6) were represented on the membranes. Most were undetectable or revealed only low levels of expression in Leydig cells of young rats; the expression of most of these genes remained undetectable or very low (maximum relative expression level, 0.7) in Leydig cells from both aged nonregressed and aged regressed testes. Likewise, of the 22 genes present on the arrays that belong to the cell cycle family, only Wee 1 tyrosine kinase increased with age (67%). Few changes were observed in the genes encoding intracellular transducers; among 19 genes, only MAPK3 and MAPK kinase 5 increased from young to aged nonregressed (24% and 59%, respectively).

Transcription factors
Leydig cell aging (young to aged nonregressed) was characterized by significantly increased expression of a number of transcription factor genes, including high motility protein group (HMG)2 and DNA binding inhibitor (ID)1, -2, and -3 (Fig. 3Go); the expression of these genes increased by 2.3-fold (ID2) and 1.7-fold (ID3). Both ID1 and HMG2 increased from undetectable levels to values well above the level of detection. Erring on the conservative side and assuming that these genes were at the upper limit of the level of detection (twice background), their increases were, at minimum, 2.7- and 1.8-fold for ID1 and HMG2, respectively. The levels of expression of three of these genes (HMG2, ID1, and ID2) decreased in response to testicular regression. However, ID3 expression increased further, by 2-fold, in Leydig cells isolated from aged nonregressed to aged regressed testes.



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  		Figure 3. Effect of age and testicular regression on the expression of transcription factors. Gene expression levels detected in Leydig cells from young adult Brown Norway (3–4 months old; ) are compared with those from old animals (20–22 months old) with nonregressed testes () and those from regressed testes (). Data represent the mean of five experiments and are expressed as the ratio of relative intensity after experiment to experiment normalization using GeneSpring.

 
HSPs
Of the 10 transcripts in this family on the array, 3 members, HSP90-ß, HSP60, and HSP70/90-organizing protein, did not change in expression with aging or testicular regression, whereas transcripts for HSP90-{alpha} and HSC70 were not detected. Although only 1 of the 5 heat shock protein family transcripts increased when Leydig cells from young and aged nonregressed testes were compared, 5 genes of this family were increased with testicular atrophy (aged nonregressed to aged regressed; Fig. 4Go). Thus, with testicular atrophy, HSP27, HSP47, and HSP70-1 were highly induced; these 3 HSPs were among the 4 genes showing the highest increases from aged nonregressed to aged regressed testes (5.6-, 2.7-, and 2.3-fold, respectively).



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  		Figure 4. Effect of age and testicular regression on the expression of genes of the heat shock protein family. Gene expression levels detected in Leydig cells from young adult Brown Norway rats (3–4 months old; ) are compared those from old animals (20–22 months) with nonregressed testes () and from regressed testes (). Data represent the mean of five experiments and are expressed as the ratio of relative intensity after experiment to experiment normalization using GeneSpring.

 
Genes involved in metabolism and oxidative stress
A total of six genes involved in oxidative stress were detectable in Leydig cells, including two SODs (SOD1 and -2), three glutathione-S-transferases (GST12, GST7-7, and GSTM2), and one glutathione peroxidase (GPX1; Fig. 5Go). In Leydig cells from young rats, the copper-zinc SOD (SOD1) was expressed at a level 9.7 times higher than that of manganese SOD (SOD2). SOD1 decreased with aging (young to aged nonregressed) by about one third and did not change further with testicular regression. In contrast, transcripts for SOD2 increased 1.7-fold from young to aged nonregressed Leydig cells and by an additional 1.8-fold from aged nonregressed to aged regressed Leydig cells. Note that the ratio of SOD1 to SOD2 was reduced from 9.7 in Leydig cells of young testes to 2.4 in Leydig cells of aged regressed testes. SOD3 was not detected.



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  		Figure 5. Effect of age and testicular regression on the expression of genes involved in oxidative stress. Gene expression levels detected in Leydig cells from young adult Brown Norway (3–4 months old; ) are compared those from old animals (20–22 months) with nonregressed testes () and from regressed testes (). Data represent the mean of five experiments and are expressed as the ratio of relative intensity after experiment to experiment normalization using GeneSpring.

 
Within the GST family (Fig. 5Go), expression of GST12 was decreased progressively (ultimately by more than 50%) during aging and testicular regression. In contrast, GST7-7 (GST subunit P) expression increased with aging and again with germ cell depletion. Therefore, the ratio of these two GSTs was dramatically reduced from 16.8 in Leydig cells from young testes to only 2.9 in those from aged regressed testes. Interestingly, GSTM2 (also known as GSTYb) expression was reduced with aging, but then increased with testicular regression (data not shown). The transcripts for several other GSTs, including GSTYa and GST5-5, were not detected. GPX1 was expressed at a low level and did not change during aging and testicular regression, whereas GPX3 was not detectable.

Cytochromes P450, NADH cytochrome b5, and NADPH-cytochrome P450 reductase
Of the four cytochrome P450s present on these arrays (1A1, VII, 21-hydroxylase, and P450scc), only mitochondrial P450scc was detected. A decline of just over 25% for P450scc was observed in Leydig cells from young to aged nonregressed testes; no further change occurred in aged regressed testes. NADH-cytochrome b5 expression in Leydig cells was not affected by aging, but was increased by 3.0-fold in Leydig cells from aged nonregressed to aged regressed testes. The chain-specific acyl-coenzyme A dehydrogenase precursor decreased by 50% in Leydig cells from aged non- regressed testes and then increased in aged regressed testes. In contrast, the NADPH-cytochrome P450 reductase was increased in Leydig cells from young to aged nonregressed testes (53%) and remained unchanged in aged regressed testes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of cDNA microarrays allows the large scale analysis of gene expression and thus is fast becoming a powerful means by which to obtain global information about tissue and cellular function (23, 24). In the present study we used this approach to obtain global information about the expression of stress response genes in Leydig cells during aging and subsequent testicular regression. The data presented herein provide the first insight into the aging process in Leydig cells at the molecular level and thus provide novel information on the extent to which aging has coordinate effects on specific functions of these cells.

We found that both aging and subsequent testicular regression resulted in changes in the induction or suppression of the expression of a number of Leydig cell genes. Approximately 25% of the 216 genes present on the stress response gene expression arrays were detected in Leydig cells isolated from young rats, whereas about 30% of these 216 genes were expressed in Leydig cells from nonregressed or regressed testes. Of the expressed genes, 29% of those in Leydig cells of aged nonregressed testes had a level of expression greater than that in young cells, whereas 8% decreased in expression. Prominent among the genes that had an expression level that changed with aging were transcription family genes that belong to the basic helix-loop-helix family, including ID1, ID2, and ID3. The expression of all three IDs increased in Leydig cells from young to aged nonregressed testes, but whereas ID1 and ID2 decreased in Leydig cells of regressed testes, the expression of ID3, the most highly expressed ID gene in Leydig cells from young rats, increased by more than 3-fold in Leydig cells from aged, regressed testes. These transcription factors lack DNA-binding domains (25, 26); the ID proteins act as dominant negative regulators of basic helix-loop-helix transcription factors by forming inactive heterodimers with them, inhibiting their DNA-binding and transcriptional activities (25). The expression of ID genes is typically associated with negative regulation of cellular differentiation (25, 26). It is interesting that the expression of these genes was increased during Leydig cell aging, because Leydig cells are considered to be terminally differentiated and are rarely seen to undergo apoptosis. There are, to the best of our knowledge, no reports of changes in ID gene expression in any tissue, but it is interesting to speculate that the observed increased expression during aging may be responsible for some of the decreased cellular functions reported for these cells in the aging process (1, 2, 3, 8, 9, 10, 11, 12, 27).

There is considerable evidence that experimental disruption of spermatogenesis results in morphological and functional changes in Leydig cells. Thus, at first glance it is puzzling that testicular regression in the Brown Norway rat, which results from loss of germ cells, does not alter steady state concentrations of the transcripts for enzymes involved in Leydig cell T production (1, 3, 28). However, there are a number of previous studies that are consistent with this observation. For example, Sprando and Zirkin (28) reported that intratesticular T concentrations did not change after experimental procedures that produced testes with widely differing germ cell contents or with few or no germ cells. Thus, as with testicular regression during aging, these results suggest that the germ cell content of the testis may have little or no effect on Leydig cell steroidogenic function.

Nonetheless, given the evidence that Leydig cell structure and function may be regulated at least in part by neighboring seminiferous tubules and that in vitro germ cells may affect Sertoli cell function and Sertoli cell products may affect Leydig cell steroidogenesis, we hypothesized that testicular regression would involve changes in the gene expression profile of Leydig cells. The analyses conducted herein support this hypothesis. A substantial number of genes increased in expression in Leydig cells from aged nonregressed compared with aged regressed testes, and some decreased. Prominent among the genes that changed with testicular regression were those involved in the heat shock response; remarkably few studies have been performed on the presence or regulation of transcripts for this key family of genes using Leydig cells (29, 30). Small increases occurred in HSP10 and HSP70–2 transcripts; these genes are known to be constitutively expressed and developmentally regulated, respectively (31). In contrast, mRNAs for the HSPs specific for the stress response, including HSP27, HSP70-1, and HSP47, increased substantially in Leydig cells from aged nonregressed to aged regressed testes. HSP27 has been reported to be constitutively expressed in most mammalian cells, but its level of expression has been shown to increase in response to heat and other toxicants (32). HSP70-1 and HSP70-3 are well established as stress-inducible proteins (33). Other HSPs have been identified as being stress inducible, such as HSP47 and HSP105 (34, 35). HSP47 is constitutively expressed and heat inducible, specifically in the developing neural plates of rat embryo (36). To our knowledge this study is the first to report the expression of HSP47 within the testis.

Aging of cells is typically characterized by increases in oxidative damage to proteins, mitochondrial and genomic DNA, and lipids (37, 38, 39, 40, 41, 42) resulting from exposure to reactive oxygen species (43, 44). Aerobic organisms have developed an array of antioxidant defenses (45, 46). In the present study we show that the expression of six genes involved in metabolism and oxidative stress responded to both aging and to subsequent testicular regression: SOD1, SOD2, GST12, GST7–7, GSTM2, and GPX1. Consistent with previous reports, these antioxidant defense genes did not exhibit a uniform pattern of age-related changes (46, 47). For example, SOD2 and GST7-7 were induced with both aging and subsequent testicular regression, whereas SOD1, GST12, and GSTM2 declined from young to aged nonregressed.

SOD1, present in both the cytoplasmic and nuclear compartments of cells, and SOD2, present in mitochondria, are the two major intracellular enzymes that inactivate superoxide radicals (48). In Leydig cells from young rats, copper-zinc SOD (SOD1) was expressed in high levels in young Leydig cells (9.7 times higher than SOD2), and decreased with aging (young to aged nonregressed) by about one third. SOD2 significantly increased with aging and then again with testicular regression. Several GSTs are present in Leydig cells (49, 50, 51); GST12, a member of the GST family, decreased, in this case by more than 50%, during aging and testicular regression, whereas GST7-7 increased with aging and again with testicular regression. These data indicate that the ratios of SOD1 to SOD2 and of GST12 to GST7-7 were reduced with aging. The functional significance of the decreases in SOD1 and GST12, the increases in SOD2 and GST7-7, and the decreases in the ratios of SOD1 to SOD2 and of GST12 to GST7-7 with regard to the cell’s ability to prevent damage by free radicals remains to be explored.

As yet, the relationship between reactive oxygen species production and the reduced steroidogenesis that characterizes Leydig cell aging is uncertain. It is noteworthy in this regard that reactive oxygen has been shown to damage critical components of the steroidogenic pathway, and reactive oxygen species have been shown to be produced during steroidogenesis itself (11, 52). Moreover, chronic suppression of steroidogenesis prevented age-related reductions in T production (53). The results presented herein are consistent with the possibility that reactive oxygen might be involved in Leydig cell aging.

In summary, both aging and testicular regression result in an increase in the expression in Leydig cells of a number of stress-related genes, but a decline in the expression of other, although fewer, genes related to stress. It is significant that the gene expression profile in Leydig cells is not a continuum beginning with the consequences of aging and ending with testicular regression. Indeed, these processes affect primarily different genes, with only 3 of 50 genes affected similarly. Thus, our studies of gene expression in Leydig cells have successfully dissociated the consequences of aging from those of regression of the seminiferous tubules; the underlying mechanisms for these fundamentally different processes remain to be elucidated.


   Footnotes
 
This work was supported by a program project grant from the NIA, NIH (AG-08321).

1 Recipient of a France-Québec Exchange Fellowship. Back

2 P.S. and H.C. contributed equally to this work. Back

Abbreviations: GPX, Glutathione peroxidase; GST, glutathione-S-transferase; HMG, high motility protein group; HSP, heat shock protein; ID, DNA binding inhibitor; SOD, superoxide dismutase.

Received April 10, 2001.

Accepted for publication August 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zirkin BR, Santulli R, Strandberg JD, Wright WW, Ewing LL 1993 Testicular steroidogenesis in the aging brown Norway rat. J Androl 14:118–123[Abstract/Free Full Text]
  2. Gruenewald DA, Naai MA, Hess DL, Matsumoto AM 1994 The Brown Norway rat as a model of male reproductive aging: evidence for both primary and secondary testicular failure. J Gerontol 49:B42–B50
  3. Wang C, Leung A, Sinha-Hikim AP 1993 Reproductive aging in the male Brown-Norway rat: a model for the human. Endocrinology 133:2773–2781[Abstract]
  4. Tenover JL 1999 Testosterone replacement therapy in older adult men. Int J Androl 22:300–306[CrossRef][Medline]
  5. Swerdloff RS, Wang C 2000 Androgens, estrogens, and bone in men. Ann Intern Med 133:1002–1004[Free Full Text]
  6. Tenenhouse A, Joseph L, Kreiger N, Poliquin S, Murray TM, Blondeau L, Berger C, Hanley DA, Prior JC 2000 Estimation of the prevalence of low bone density in Canadian women and men using a population-specific DXA reference standard: the Canadian Multicentre Osteoporosis Study (CaMos). Osteop Int 11:897–904[CrossRef][Medline]
  7. Lamberts SW, van den Beld AW, van der Lely AJ 1997 The endocrinology of aging. Science 278:419–424[Abstract/Free Full Text]
  8. Chen H, Hardy MP, Huhtaniemi I, Zirkin BR 1994 Age-related decreased Leydig cell testosterone production in the brown Norway rat. J Androl 15:551–557[Abstract/Free Full Text]
  9. Luo L, Chen H, Zirkin BR 1996 Are Leydig cell steroidogenic enzymes differentially regulated with aging? J Androl 17:509–515[Abstract/Free Full Text]
  10. Zirkin BR, Chen H, Luo L 1997 Leydig cell steroidogenesis in aging rats. Exp Gerontol 32:529–537[CrossRef][Medline]
  11. Zirkin BR, Chen H 2000 Regulation of Leydig cell steroidogenic function during aging. Biol Reprod 63:977–981[Abstract/Free Full Text]
  12. Leers-Sucheta S, Stocco DM, Azhar S 1999 Down-regulation of steroidogenic acute regulatory (StAR) protein in rat Leydig cells: implications for regulation of testosterone production during aging. Mech Ageing Dev 107:197–203[CrossRef][Medline]
  13. Wright WW, Fiore C, Zirkin BR 1993 The effect of aging on the seminiferous epithelium of the brown Norway rat. J Androl 14:110–117[Abstract/Free Full Text]
  14. Holstein AF 1996 Spermatogenesis in the aged–a borderland between normal and pathologic anatomy. Urologe A 25:130–137
  15. Barnes CJ, Covington BW, Lee M 1999 Effect of aging and dietary restriction on rat testicular germ cell apoptosis. J Gerontol A Biol Sci Med Sci 54:B199–B204
  16. Wang C, Sinha Hikim AP, Lue YH, Leung A, Baravarian S, Swerdloff RS 1999 Reproductive aging in the Brown Norway rat is characterized by accelerated germ cell apoptosis and is not altered by luteinizing hormone replacement. J Androl 20:509–518[Abstract/Free Full Text]
  17. Grzywacz FW, Chen H, Allegretti J, Zirkin BR 1998 Does age-associated reduced Leydig cell testosterone production in Brown Norway rats result from under-stimulation by luteinizing hormone? J Androl 19:625–630[Abstract/Free Full Text]
  18. Klinfelter GR, Hall PF, Ewing LL 1987 Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36:769–783[Abstract]
  19. Payne AH, Dowing JR, Wong KL 1980 Luteinizing hormone receptors and testosterone synthesis in two distinct populations of Leydig cells. Endocrinology 106:1424–1429[Abstract]
  20. Chomczynshi P, Sacchi N 1987 Single-step method of RNA isolation by guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  21. Lockhart DJ, Winzeler EA 2000 Genomics, gene expression and DNA arrays. Nature 405:827–836[CrossRef][Medline]
  22. Aguilar-Mahecha A, Hales BF, Robaire B 2001 Expression of stress response genes in germ cells during spermatogenesis. Biol Reprod 65:119–127[Abstract/Free Full Text]
  23. van Hal NL, Vorst O, van Houwelingen AM, Kok EJ, Peijnenburg A, Aharoni A, van Tunen AJ, Keijer J 2000 The application of DNA microarrays in gene expression analysis. J Biotechnol 78:271–280[CrossRef][Medline]
  24. Lee CK, Klopp RG, Weindruch R, Prolla TA 1999 Gene expression profile of aging and its retardation by caloric restriction. Science 285:1390–1393[Abstract/Free Full Text]
  25. Norton JD, Deed RW, Craggs G, Sablitzky F 1998 Id helix-loop-helix proteins in cell growth and differentiation. Trends Cell Biol 8:58–65[CrossRef][Medline]
  26. Stablitzky F, Moore A, Bromley M, Deed RW, Newton JS, Notton JD 1998 Stage- and subcellular-specific expression of Id proteins in male germ and Sertoli cells implicates distinctive regulatory roles for Id proteins during meiosis, spermatogenesis and Sertoli cell function. Cell Growth Diff 9:1015–1024[Abstract]
  27. Nagata T, Ito M, Liang Y, Gao F 2000 Study of the effects of aging on macromolecular synthesis in mouse steroid secreting cells using microscopic radioautography. Methods Find Exp Clin Pharmacol 22:5–18[CrossRef][Medline]
  28. Sprando RL, Zirkin BR 1997 Is Leydig cell steroidogenic function affected by the germ cell content of the seminiferous tubules. J Androl 18:424–430[Abstract/Free Full Text]
  29. Meinhardt A, Seitz J, Arslan M, Aumuller G, Weinbauer GF 1998 Hormonal regulation and germ cell-specific expression of heat shock protein 60 (hsp60) in the testis of macaque monkeys (Macaca mulatta and M. fascicularis). Int J Androl 21:301–307[CrossRef][Medline]
  30. Itoh H, Tashima Y 1991 Different expression time of the 105-kDa protein and 90-kDa heat-shock protein in rat testis. FEBS Lett 289:110–112[CrossRef][Medline]
  31. Dix DJ, Hong RL 1998 Protective mechanism in germ cells: stress proteins in spermatogenesis. Adv Exp Med Biol 444:137–144[Medline]
  32. Mirkes PE, Littles SA, Cornel L, Welsh MJ, Laney TN, Wright FH 1996 Induction of heat shock protein 27 in rat embryos exposed to hyperthermia. Mol Reprod Dev 25:276–284[CrossRef]
  33. Mirkes PE, Doggett B, Cornel L 1994 Induction of a heat shock response in (HSP72) in rat embryos exposed to selected chemical teratogens. Teratology 49:135–142[CrossRef][Medline]
  34. Turner CP, Panter SS, Sharp FR 1999 Anti-oxidants prevent focal rat brain injury as assessed by induction of heat shock proteins (HSP70, HO-1/HSP32, HSP47) following subarachnoid injections of lysed blood. Brain Res Mol Brain Res 65:87–102[Medline]
  35. Wakatsuki T, Hatayama T 1998 Characteristic expression of 105-kDa heat shock protein (HSP105) in various tissues of nonstressed and heat-stressed rats. Biol Pharm Bull 21:905–910[Medline]
  36. Walsh D, Li Z, Wu Y, Nagata K 1997 Heat shock and the role of the HSPs during neural plate induction in early mammalian CNS and brain development. Cell Mol Life Sci 53:198–211[CrossRef][Medline]
  37. Stadtman ER 1982 Protein oxidation and aging. Science 28:257:1220–1224
  38. Sastre J, Pallardo FV, Garcia de la Asuncion J, Vina J2000 Mitochondria, oxidative stress and aging. Free Radic Res 32:189–198
  39. Barja G 2000 The flux of free radical attack through mitochondrial DNA is related to aging rate. Aging 12:342–355[Medline]
  40. Cadenas E, Davies KJ 2000 Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 29:222–230[CrossRef][Medline]
  41. Bohr VA, Dianov GL 1999 Oxidative DNA damage processing in nuclear and mitochondrial DNA. Biochimie 81:155–160[Medline]
  42. Miro O, Casademont J, Casals E, Perea M, Urbano-Marquez A, Rustin P, Cardellach F 2000 Aging is associated with increased lipid peroxidation in human hearts, but not with mitochondrial respiratory chain enzyme defects. Cardiovasc Res 47:624–631[CrossRef][Medline]
  43. Adelman R, Saul RL, Ames BN 1988 Oxidative damage to DNA: relation to species metabolic rate life span. Proc Natl Acad Sci USA 85:2706–2708[Abstract/Free Full Text]
  44. Brunauer LS, Clarke S 1986 Age-dependent accumulation of protein residues which can be hydrolyzed to D-aspartic acid in human erythrocytes. J Biol Chem 261:12538–12543[Abstract/Free Full Text]
  45. Davies KJA 1986 Intracellular proteolitic systems may function as secondary antioxidant defenses: a hypothesis. J Free Radic Biol Med 2:155–173[CrossRef][Medline]
  46. Kukucha MA, Misra HP 1993 The antioxidant defense system of isolated guinea pig Leydig cells. Mol Cell Biochem 126:1–7[CrossRef][Medline]
  47. Sohal RS, Agrawal S, Sohal BH 1995 Oxidative stress and aging in the Mongolian gerbol (Merion unguicaltus). Mech Ageing Dev 30:15–25
  48. Marklund SL 1986 Superoxide dismutase in human tissues, cells and extracellular fluids: clinical implications. In: Johnson JE, Walford R, Harman D, Miguel J, eds. Free radicals, aging, and degenerative diseases. New York: Liss; 509–526
  49. Mueller A, Hermo L, Robaire B 1998 The effects of aging on the expression of glutathione S-transferases in the rat testis and epididymis of the Brown Norway rat. J Androl 19:450–465[Abstract/Free Full Text]
  50. Papp S, Robaire B, Hermo L 1995 Immunocytochemical localization of the Ya, Yc, Yb1, and Yb2 subunits of glutathione S-transferases in the testis and epididymis of adult rats. Microsc Res Technol 30:1–23[CrossRef][Medline]
  51. Kelce WR 1994 Buthionine sulfoximine protects the viability of adult rat Leydig cells exposed to ethane dimethanesulfonate. Toxicol Appl Pharmacol 125:237–246[CrossRef][Medline]
  52. Hochberg RB, Ladany S, Lieberman S 1976 Conversion of a C-20-deoxy-C21, steroid, 5-pregnen-3ß-ol, into testosterone by rat testicular microsomes. J Biol Chem 251:3320–3325[Abstract/Free Full Text]
  53. Chen H, Zirkin BR 1999 Long-term suppression of Leydig cell steroidogenesis prevents Leydig cell aging. Proc Natl Acad Sci USA 96:14877–14881[Abstract/Free Full Text]



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