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BOR - Papers in Press, published online ahead of print June 2, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028886
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BIOLOGY OF REPRODUCTION 71, 1088–1095 (2004)
DOI: 10.1095/biolreprod.104.028886
© 2004 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

The Effects of Long-Term Vitamin E Treatment on Gene Expression and Oxidative Stress Damage in the Aging Brown Norway Rat Epididymis1

Kathryn M. Jervis, and Bernard Robaire2

Department of Pharmacology and Therapeutics and of Obstetrics and Gynecology, McGill University, Montreal, Canada H3G 1Y6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The male reproductive tract of the Brown Norway rat is profoundly affected by aging. In the epididymis, the site of sperm maturation and storage, aging results in histological and biochemical changes that are suggestive of oxidative stress. Vitamin E is a potent lipid-soluble antioxidant that ameliorates the oxidative stress load associated with some chronic disease conditions. To determine the effects of long-term (18-mo) vitamin E deficiency and supplementation on aging in the epididymis, we assessed gene expression changes using cDNA microarrays and lipid peroxidation using immunohistochemical detection of 4-hydroxynonenal (4-HNE) in 24-mo-old rats. Plasma vitamin E levels were significantly lower in vitamin E-deficient animals and higher in vitamin E-supplemented animals compared with age-matched controls. Vitamin E deficiency resulted in increased expression of oxidative stress-related transcripts along the epididymis. This effect was most marked in the corpus epididymidis, where expression of glutathione S-transferases pi, 8, and mu, as well as superoxide dismutase, increased by over 50%. The effect of vitamin E supplementation on the expression of oxidative stress-related transcripts was primarily decreased expression; however, the magnitude of the gene expression changes was smaller than that observed for vitamin E deficiency. 4-HNE immunostaining was present throughout the epididymis in control animals. Vitamin E deficiency both increased the intensity and altered the distribution of 4-HNE staining, while vitamin E supplementation had no observable effect. In summary, we found that long-term vitamin E treatment alters the expression of oxidative stress-related transcripts. Moreover, long-term vitamin E deficiency exacerbates the effects of age on the accumulation of oxidative stress damage in the epididymis.

aging, epididymis, male reproductive tract, stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aging profoundly affects the reproductive tract of the male Brown Norway rat. With advancing age, serum testosterone levels and spermatogenesis decline [1, 2] and sperm morphology and motility parameters are altered [3]. We have demonstrated that, in the epididymis, the site of sperm maturation and storage, both the histology and biochemistry of the tissue are affected by age [4, 5].

The epididymis is a complex tissue that is anatomically and histologically separated into four different regions; the initial segment, caput, corpus, and cauda epididymidis. These regions are biochemically distinct and exhibit differential gene expression [6; reviewed in 7, 8]. The four regions of the epididymis also respond to the aging process in a segment-specific manner [4, 5, 9].

Some of the age-related changes, for example, the accumulation of lipofuscin [4], the altered distribution of components of antioxidant defense systems [10], and the decreased expression of gene products involved in antioxidant defenses [9], suggest that oxidative stress may play a role in the aging of the epididymis. Oxidative stress has long been associated with the aging process. Evidence that demonstrates the age-related accumulation of oxidative stress damage is abundant [11], and there are many examples of increased longevity with the attenuation of oxidative stress [12–15].

Vitamin E is a term that refers to a group of lipid-soluble, chain-breaking antioxidant molecules, the most potent of which is {alpha}-tocopherol. Vitamin E is well known for its antioxidant properties [reviewed in 16]; it functions as a chain-breaking antioxidant that prevents the propagation of free radical reactions and thus protects cells from oxidative damage. Vitamin E is particularly important in protecting cells against lipid peroxidation, where free radical attack on fatty acids causes structural damage to membranes and results in the formation of cytotoxic secondary products such as malondialdehyde and 4-hydroxy, 2-nonenal (4-HNE) [17]. These secondary products are abundant and form stable adducts; they are thus often used as indicators of free radical attack on lipids (lipid peroxidation) [17].

In prospective cohort studies, dietary vitamin E intake has been shown to be inversely associated with coronary heart disease risk. Individuals in the top fifth of vitamin E consumption have 30–40% lower risk of cardiovascular disease [reviewed in 18; original studies: 19–22]. This observation, in conjunction with experimental evidence in animals and the recognized potency of vitamin E as an antioxidant, stimulated interest in the potential ability of vitamin E to prevent chronic diseases. In particular, diseases and conditions believed to have an oxidative stress component, such as cardiovascular and neurodegenerative diseases and cancer have been a primary focus of clinical research. Several large-scale clinical trials have been completed and indicate that vitamin E supplementation can decrease the risk of nonfatal myocardial infarction [23], the incidence of prostate cancer [24], and nonfatal myocardial infarction [25]. Other chronic conditions associated with oxidative stress have also been shown to be ameliorated by vitamin E supplementation. For example, chronic hemodialysis and cystic fibrosis patients have reduced acute oxidative stress load and in vivo lipid peroxidation and lung inflammation, respectively [26].

In order to assess the role of oxidative stress in the aging epididymis, we analyzed the effects of long-term vitamin E supplementation and deficiency on gene expression in the epididymis. Because vitamin E is particularly important in protecting cells against lipid peroxidation, we also analyzed the effects of vitamin E on the accumulation of a marker of lipid peroxidation, 4-HNE in the epididymal epithelium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Diets

Six-month-old male Brown Norway rats were randomly assigned to one of three experimental diets: vitamin E deficient (8.36 IU/kg all-rac-{alpha} tocopheryl acetate), control (25.4 IU/kg all-rac-{alpha} tocopheryl acetate) or vitamin E supplemented (106 IU/kg all-rac-{alpha} tocopheryl acetate). All other constituents of the diet were identical. Diet was added to the cage feeders weekly and weighed 7 days later to determine the amount consumed. Access to water was ad libitum. Animals were housed at Harlan Sprague Dawley Inc. (Indianapolis, IN) under controlled light (12L:12D) and temperature (22–24°C). Rats were weighed weekly.

Six-month-old animals (n = 6) were killed by cardiac puncture at the start of the study; blood was collected into heparinized tubes, centrifuged at 1600 x g for 15 min, and the resulting plasma was frozen at –80°C for analysis of vitamin E levels. Epididymides were collected; sectioned into initial segment, caput, corpus, and cauda regions; frozen in liquid nitrogen; and stored at –80°C for subsequent analysis of gene expression at the RNA level. After 18 mo on the experimental diets, at 24 mo of age, six animals for each of the three treatment groups were killed and tissues and blood were collected as described above. No gross abnormalities were observed in any of the tissues examined. All animal studies were conducted in accordance with the principles and procedures outlined in A Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care.

RNA Extraction

Frozen epididymal segments were ground to a powder on dry ice using a mortar and pestle. The resulting powders were then used for RNA extraction according to the RNeasy method (RNeasy Midi Kit; Qiagen, Mississauga, ON) following the manufacturer's instructions. The RNA was then DNase treated using the RNase-Free DNase Set (RNeasy Midi Kit; Qiagen). Concentrations were determined spectrophotometrically (Beckman DU7 spectrophotometer; Beckman, Montreal, PQ), and the quality of the RNA was assessed by denaturing gel electrophoresis on a 1% agarose gel. RNA was extracted individually from each of the four different epididymal segments (n = 5 for each segment); each segment represents a different animal and no tissues were pooled.

cDNA Arrays and Normalization

The cDNA arrays (Atlas Rat 1.2K; Clontech) were used to analyze RNA expression. Five arrays for each epididymal segment for each treatment group and the 6-mo control group were completed following the manufacturer's instructions. Each individual array is referred to as a replicate; it represents the RNA profile of an individual epididymal segment taken from an individual rat. Arrays were exposed to phosphorimager plates (Molecular Dynamics, Sunnyvale, CA) for 24 h before scanning (Storm; Molecular Dynamics). Array images were first analyzed with Atlas Image (version 1.5, Clontech) to quantify the intensity of each cDNA spot, and the raw data for each gene (intensity of each spot on the array minus the background of that array) were imported into GeneSpring (Silicon Genetics, Redwood, CA) for further analysis.

Data were normalized by array normalization and gene normalization. Array normalization controls for array-wide variations in intensity that may be due to technical issues such as inconsistent washing or inconsistent sample preparation. In array normalization, the expression of each gene on an array is normalized to the median expression on that array. Only gene intensities that are above a specified cutoff value are included in the determination of the median expression of an array. This was done for every single array individually. Gene normalization accounts for the difference in detection efficiency between spots. In gene normalization, the signal strength of a gene is divided by the median expression of every measurement taken for that gene throughout the experiment. If the median of the gene's measurements is below the specified cutoff value, the cutoff is used instead. This is done for every gene on every array. The resulting value of each spot on all five replicate arrays was calculated and averaged, and this average is referred to as the relative intensity for any given gene. A gene was only considered to be expressed if its relative intensity was at least twofold the average background of all the arrays in that treatment group. Replication is important in gene-array studies [27] and, therefore, changes in the relative intensity of a gene were considered only when consistent in at least three out of five replicate experiments [6].

Vitamin E Analysis

Plasma vitamin E (n = 6 per group) levels were assessed using HPLC by a modification of the procedure by Widicus and Kirk [28]. Analyses were conducted by Laboratory Services (Guelph, ON).

Perfusion and 4-Hydroxy-2-Nonenal Immunohistochemistry

Animals (six animals/treatment group and the 6-mo control group) were perfused for immunohistochemical analysis of the presence of 4-HNE. Briefly, animals were anesthetized with a cocktail (20:10:1) of Vetalar (ketamine hydrochloride 115.4 mg/ml; Vetrepharm, London, ON), Anased (xylazine hydrochloride 20 mg/ml; Novopharm, Toronto, ON), and Atravet (Acepromazine Maleate 10 mg/ml; Ayerst, Montreal, PQ). Epididymides were then fixed with Bouin solution via perfusion through the abdominal aorta. After perfusion, epididymides were post fixed for 24 h with the same fixative, dehydrated, and embedded in paraffin. Thin sections (5µm) were cut on a microtome and mounted on glass slides.

For immunohistochemical detection of 4-HNE, sections were deparaffinized with xylene and then rehydrated in graded alcohol solutions. Endogenous peroxidase activity was neutralized by a 15-min incubation in 70% alcohol containing 1% hydrogen peroxide. After hydration, free aldehydes were blocked by incubation in 300 nM glycine for 5 min. In order to minimize nonspecific binding, sections were blocked with 10% normal horse serum for 30 min at room temperature prior to incubation with the primary antibody (mouse anti-4-hydroxy-2-nonenal antibody; OXIS International Inc., Portland, OR). Immunohistochemical staining was done using a Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). Sections were incubated for 1.5 h at room temperature with the primary antibody (12.5 µg/ml or 8.3 µg/ml) in the blocking serum. Bound antibodies were observed through the use of biotinylated goat anti-rabbit IgG secondary antibodies and the avidin:biotinylated horseradish peroxidase complex. Sections were counterstained with a 0.075% methylene blue solution. Negative controls were processed in exactly the same manner except for the omission of the primary antibody. The immunohistochemical results were analyzed by light microscopy. Results demonstrate representative sections of three experiments, with three animals per experiment.

Statistics

The effects of vitamin E on body weight, tissue weights, and plasma vitamin E levels were analyzed by Bonferroni adjusted t-tests. For all analyses, values were considered statistically significantly different at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Vitamin E on Body and Tissue Weights

Vitamin E deficiency or supplementation did not result in any significant change in body weight (range 508–515 g) or in the testis (range 1.4–1.7 g), or epididymis weight (range 0.62–0.71 g) (P > 0.5, n = 6).

Effects of Vitamin E on Plasma Vitamin E Levels

Age had no significant effect on plasma vitamin E levels in animals fed the control diet (P = 0.066) (Fig. 1). In contrast, at 24 mo of age, plasma vitamin E levels were 52% lower in animals on the vitamin E-deficient diet (P = 0.001) and 106% higher (P = 0.003) in animals on the supplemented diet than animals on the control diet.



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  		FIG. 1. Effects of vitamin E deficiency or supplementation on plasma vitamin E levels. Plasma vitamin E (in µg/ml) in animals on control diets at 6 mo of age and vitamin E-deficient, control, or vitamin E-supplemented diets at 24 mo of age. Results are presented as mean of six animals. Error bars represent ± SEM. White bar, control at 6 mo of age; horizontal striped bar, deficient at 24 mo of age; black bar, control at 24 mo of age; diagonal striped bar, supplemented at 24 mo of age

Effects of Vitamin E on Gene Expression in the Epididymis

The predominant effect of vitamin E deficiency on gene expression in the epididymis was an increase in the relative intensity of gene expression compared with that in control animals (Tables 1–2). The initial segment was the only segment where there were more genes with decreased relative intensity in vitamin E-deficient animals than in controls. The identity of these genes is diverse (Table 2B) and includes several growth factors/growth factor receptors (basic fibroblast growth factor receptor, platelet-derived growth factor, heparin-binding growth factor, f-spondin) and channels/ transporters (P2X purinoceptor 1, voltage-gated potassium channel protein 2.1, antigen peptide transporter 1). Interestingly, particularly in the caput and cauda epididymidis, the expression of many ribosomal proteins increased with vitamin E deficiency (Table 2). In the corpus epididymidis, vitamin E deficiency increased the expression of copper-zinc superoxide dismutase 1 (Cu-Zn SOD1) by 77%.


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  		TABLE 1. Number of genes that increase ({uparrow}) or decrease ({downarrow}) in relative expression with vitamin E deficiency (D) or supplementation (S) compared with controls


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  		TABLE 2B. Gene expression decreases with vitamin E treatment.*


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  		TABLE 2B. Continued


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  		TABLE 2A. Gene expression increases with vitamin E treatment.*

The effects of vitamin E supplementation on gene expression in the epididymis were more variable. In the proximal regions of the tissue (initial segment and caput), few genes increased or decreased in vitamin E-supplemented animals as compared with controls (Tables 1–2). In the initial segment of the epididymis, the expression of several proto-oncogenes (c-ros, sky, p21) decreased with vitamin E supplementation. In the cauda epididymidis, however, vitamin E supplementation resulted only in increased gene expression. Gene families with increased expression in the cauda epididymidis include proteases (amonipeptidase B, cathepsin L, presenilin-1), ATPases (brain calcium-transporting ATPase, sodium/potassium-transporting ATPase ß1), and translation-associated transcripts (elongation factor 2, elongation initiation factor 2{alpha}). Gene expression in the corpus epididymidis was largely unaffected by vitamin E supplementation.

Effects of Vitamin E on Oxidative Stress-Related Genes

Of the oxidative stress genes on the array, only four (glutathione-s-transferase pi, glutathione-s-transferase 8, glutathione-s-transferase mu, and superoxide dismutase) were expressed above the level of detection in all four segments under all three conditions (Fig. 2). Vitamin E deficiency predominantly increased the relative intensity of expression of oxidative stress-related genes in the epididymis. This effect was most pronounced in the corpus epididymidis, where the relative intensity of expression of all four transcripts increased in vitamin E-deficient animals as compared with controls. Superoxide dismutase expression was the most dramatically affected of all four transcripts; the relative intensity of expression increased by over 200% in the corpus and cauda epididymidis. Interestingly, the initial segment was the only region of the tissue where the expression of oxidative stress-related genes was largely unaffected (<30% change in either direction) by vitamin E deficiency. In contrast, vitamin E supplementation had a variable effect on the expression of oxidative stress-related transcripts in the epididymis. Gene expression in the caput epididymidis was the most profoundly affected; expression of three of the four transcripts decreased by more than 50% as compared with controls. The magnitude of gene expression changes in vitamin E-supplemented animals was also much lower than that observed in vitamin E-deficient animals. The largest percent change in expression was 63% (GSTpi in the caput epididymidis) in supplemented animals, compared with 334% (SOD in the cauda epididymidis) in deficient animals.



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  		FIG. 2. Effects of vitamin E on the expression of oxidative stress-related genes. Percent change from control in the relative intensity of expression in the four segments of the epididymis of vitamin E deficient (A) and supplemented animals (B) at 24 mo of age. GSTpi, glutathione-s-transferase pi; GST8, glutathione-s-transferase 8; GSTmu, glutathione-s-transferase mu; SOD, superoxide dismutase. Black bar, initial segment; white bar, caput epididymidis; dark grey bar, corpus epididymidis; light grey bar, cauda epididymidis

Effects of Vitamin E on 4-Hydroxy-2-Nonenal Immunoreactivity

All regions of tissues from control animals at 24 mo of age exhibited 4-HNE immunoreactivity distributed throughout the cytoplasm of principal cells (Fig. 3, A and D). The intensity of the immunoreactivity was light throughout the cytoplasm but increased toward the apical plasma membrane and appeared to form a dark line just under the plasma membrane (arrow, Fig. 3). Other epididymal cell types (basal and clear cells) were unstained (arrowhead, Fig. 4D).



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  		FIG. 3. Representative epididymal sections demonstrating anti-4-HNE immunostaining. A–C) Proximal corpus epididymidis. D–F) Caput epididymidis. A and D) Tissue sections from animals on the control diet at 24 mo of age. B and E) Tissue sections from animals on the vitamin E-deficient diet at 24 mo of age. C and F) Tissue sections from animals on the vitamin E-supplemented diet at 24 mo of age. Original magnification x400. Inset (A–C), representative epididymal sections demonstrating anti-4-HNE immunostaining of vacuoles in the corpus epididymidis. A) Tissue section from animals on the control diet at 24 mo of age. B) Tissue section from animals on the vitamin E-deficient diet at 24 mo of age. C) Tissue section from animals on the vitamin E-supplemented diet at 24 mo of age. Original magnification x1000. Int, interstitium; Lu, lumen; vac, vacuole; arrowhead, basal cell; arrows indicate anti-4-HNE immunoreactivity



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  		FIG. 4. Negative control for 4-HNE immunoreactivity. Representative epididymal section demonstrating the absence of 4-HNE immunoreactivity in the caput epididymidis when the primary antibody is omitted. Original magnification x400

The pattern of 4-HNE immunoreactivity was maintained in tissues from both vitamin E-deficient and vitamin E-supplemented animals. However, in vitamin E-deficient tissues, the intensity of immunoreactivity in the cytoplasm of principal cells was increased along the epididymis (Fig. 3, B and E, and data not shown). This effect was most dramatic in the corpus epididymidis (Fig. 3, B and E) where the 4-HNE immunoreactivity was much more intense. In addition, vacuoles in the corpus epididymidis from vitamin E-deficient animals exhibited intense 4-HNE immunoreactivity along their periphery (Fig. 3B inset). This staining of the inside aspect of vacuoles was not observed in tissues from the control or vitamin E-supplemented group (Fig. 3, A and C inset). The number of vacuoles was not quantified; however, vitamin E treatment did not appear to effect the number of vacuoles in the epithelium. In tissues from vitamin E-supplemented animals, 4-HNE immunoreactivity was equivalent in intensity to that observed in control animals (Fig. 3, C and F). When the primary antibody was omitted, there was an absence of immunoreactivity (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In addition to its well-documented role as an antioxidant, vitamin E exhibits biological functions not related to its antioxidant properties. The effects of vitamin E on gene transcription and translation are known to result in the inhibition of cell proliferation, platelet aggregation, and monocyte adhesion [29]. Vitamin E is thought to exert a number of its effects on gene expression by acting on protein kinase C activity [30]. Protein phosphatase 2A, which dephosphorylates and inhibits protein kinase C, is activated by vitamin E [31]. Other effects of vitamin E on gene expression, such as the expression of the adhesion molecule ICAM-1 [32] and some integrins [33], are protein kinase C independent [34]. For example, vitamin E is known to activate gene expression via the pregnane X receptor, a nuclear receptor that regulates the expression of a variety of drug metabolizing enzymes [35]. In the epididymis, vitamin E treatment affected the expression of known regulators of protein kinase C activity, such as of phosphatase 2A{alpha} and 14-3-3 proteins [31, 36]. We also found that epididymal expression of a number of cell adhesion molecules decreased and expression of cytochrome P450's increased in response to vitamin E treatment. This suggests that both protein kinase C-dependent and protein kinase C-independent mechanisms are mediating the effects of vitamin E on gene expression in the epididymis.

Vitamin E treatment also affected the expression of oxidative stress-related transcripts in the epididymis. In particular, vitamin E deficiency resulted in increased relative intensity of glutathione S-transferases and superoxide dismutase along the tissue, most notably in the corpus epididymidis. The expression of most oxidative stress defense-related transcripts is inducible in response to oxidative stress [37]. Therefore, the increased expression of oxidative stress-related transcripts may be suggestive of increased oxidative stress in the corpus epididymidis. Interestingly, the corpus epididymidis was also the most profoundly affected region of the tissue at the level of 4-HNE immunoreactivity, exhibiting both increased intensity and a differential staining pattern in vitamin E-deficient animals as compared with age-matched animals on the control or vitamin E-supplemented diet. 4-HNE, the most abundant product of lipid peroxidation, is a highly reactive molecule that forms stable adducts with cellular proteins [38]. The stability and abundance of 4-HNE make it a commonly used indicator of oxidative stress in tissues. Increased immunoreactivity toward 4-HNE in the corpus epididymidis of vitamin E-deficient animals is thus suggestive of increased oxidative stress in this region of the tissue. Vacuoles are an indicator of oxidative stress, and the accumulation of oxidatively damaged cellular components around the inside aspect of vacuoles has been reported for other oxidative stress markers in other tissues [39, 40].

The increase in 4-HNE immunostaining in the corpus epididymidis occurs in conjunction with dramatically increased expression of oxidative stress-related transcripts. Together, these data suggest that the corpus epididymidis is the most sensitive region of the epididymis to the effects of vitamin E deficiency. This is in keeping with our previous results that demonstrate that the corpus epididymidis is the region that is the most dramatically affected with age at the histological and gene expression level [4, 9].

The effects of vitamin E supplementation on the expression of oxidative stress-related transcripts and 4-HNE immunoreactivity were less pronounced than vitamin E deficiency. In the caput epididymidis, vitamin E supplementation did result in decreased expression of oxidative stress transcripts; however, the effects on the rest of the tissue were more variable. While the reasons for this are unclear, it is possible that the dose used for supplementation was not large enough to generate an effect of great magnitude.

In conclusion, these data show that vitamin E treatment has a profound effect on epididymal gene expression. Moreover, we show that vitamin E deficiency impacts oxidative stress in the epididymis, with a pronounced effect on the corpus epididymidis, suggesting that this region of the tissue is the most vulnerable to oxidative stress with age.


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  		TABLE 2A. Cauda


   ACKNOWLEDGMENTS
 
The authors would like to acknowledge Dr. R.R. Manimaran and Sandra Robaire for their help with experiments.


   FOOTNOTES
 
1 Supported by grants from the NIH (NIA-AG08321) and a doctoral fellowship award from the Canadian Institutes of Health Research to K.M.J. Back

2 Correspondence: B. Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montréal, Québec, Canada H3G 1Y6. FAX: 514 398 7120; bernard.robaire{at}mcgill.ca Back

Received: 24 February 2004.

First decision: 15 March 2004.

Accepted: 7 May 2004.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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