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Children's Hospital Oakland Research Institute,3 Children's Hospital and Research Center at Oakland, Oakland, California 94609
Department of Internal Medicine,4 University of California Davis, Davis, California 95616
Investigative Developmental Toxicology Labs,5 Pfizer Global Research and Development, Groton, Connecticut 06340
Department of Population Health and Reproduction,6 University of California Davis, Davis, California 95616
Department of Molecular and Cell Biology,7 University of California Berkeley, Berkeley, California 94720
ABSTRACT
Although alcoholism causes changes in hepatic folate metabolism that are aggravated by folate deficiency, male reproductive effects have never been studied. We evaluated changes in folate metabolism in the male reproductive system following chronic ethanol consumption and folate deficiency. Twenty-four juvenile micropigs received folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE or FDE) for 14 wk, and the differences between the groups were determined by ANOVA. Chronic ethanol consumption (FSE and FDE compared with FS and FD groups) reduced testis and epididymis weights, testis sperm concentrations, and total sperm counts and circulating FSH levels. Folate deficiency (FD and FDE compared with FS and FSE groups) reduced circulating testosterone, estradiol and LH levels, and also testicular 17,20-lyase and aromatase activities. There was histological evidence of testicular lesions and incomplete progression of spermatogenesis in all treated groups relative to the FS control, with the FDE group being the most affected. Chronic ethanol consumption increased testis folate concentrations and decreased testis methionine synthase activity, whereas folate deficiency reduced total testis folate levels and increased methionine synthase activity. In all pigs combined, testicular methionine synthase activity was negatively associated with circulating estradiol, LH and FSH, and 17,20-lyase activity after controlling for ethanol, folate deficiency, and their interaction. Thus, while chronic ethanol consumption primarily impairs spermatogenesis, folate deficiency reduces sex hormones, and the two treatments have opposite effects on testicular folate metabolism. Furthermore, methionine synthase may influence the hormonal regulation of spermatogenesis.
male reproductive tract, mechanisms of hormone action, spermatogenesis, steroid hormones, testis
Chronic ethanol consumption causes sexual dysfunction and impaired sperm production in humans and in animal models [1], and it negatively impacts all levels of the male hypothalamic-pituitary-gonadal axis [2, 3]. Early histological studies indicate that the testis may be even more sensitive to the effects of ethanol than the liver [4]. Proposed mechanisms of ethanol-induced testicular damage include oxidative stress, changes in nitric oxide (NO) or opioid metabolism, and/or defective steroid hormone metabolism secondary to altered liver metabolism [3]. Paternal ethanol exposure in rodents has been shown to adversely affect pregnancy outcome [5], including an increased rate of fetal malformations [6].
Though incompletely studied, changes in folate status have been shown to affect the male reproductive system. Primary (i.e., dietary) folate deficiency in the rat triggers a relative conservation of folate in certain tissues, including the testis, at the expense of liver folate [7], and it reduces testis weight and epididymal sperm count [8]. Male reproductive health also can be compromised by secondary folate deficiency from certain antifolate drugs or in conditions where folate is malabsorbed, as shown in humans [9–12] and in animal models [13–15]. We recently showed that nonmethyltetrahydrofolate concentrations (i.e., forms other than 5-methyltetrahydrofolate) in seminal plasma are positively associated with sperm count and density [16]. A clinical study found that subfertile males supplemented with a combination of folic acid and zinc show modest improvements in total normal sperm count [17]. Poor folate status has been implicated as a possible cause of idiopathic male infertility [18].
Chronic alcoholism and its associated liver disease are frequently coincident with circulating and hepatic indicators of folate deficiency [19–23]. In addition to low dietary intake, folate deficiency in chronic alcoholism can be attributed to secondary causes such as decreased intestinal absorption [24, 25], decreased hepatic uptake [26, 27], and increased renal excretion [27, 28].
The effects of chronic ethanol consumption on folate metabolism in the male reproductive system have been almost completely overlooked. Collins et al. reported that folic acid uptake in testis was unchanged following chronic ethanol exposure in rats [7]. Since chronic ethanol exposure adversely affects folate metabolism in hepatic and certain extrahepatic tissues, we hypothesize that the testis may be similarly affected. This is important because the testis critically depends on normal folate metabolism to support continual cell division and attendant DNA synthesis.
To understand the rationale for the endpoints measured in these studies, a brief review of folate biochemistry is useful. Various coenzyme forms of folate are required for cellular purine, thymidylate, and polyamine biosyntheses and for methylation reactions [29] (Fig. 1). The folate and vitamin B12-dependent enzyme methionine synthase (MTR; 5-methyltetrahydrofolate-homocysteine methyltransferase; E.C. 2.1.1.13), plays a central role in these processes by transferring a methyl group from 5-methyltetrahydrofolate (5-methylTHF) for the remethylation of homocysteine. This reaction regenerates methionine which, when activated to S-adenosylmethionine (SAM), participates in polyamine syntheses and methylation reactions. In the process of the methionine synthase reaction, THF is produced from 5-methylTHF, and can then be converted to the coenzyme forms needed for purine and thymidylate syntheses. Changes in intracellular needs can regulate the flow of folate coenzyme forms either toward methionine synthase for methionine synthesis or away from it for nucleic acid and thymidylate syntheses [30, 31]. In the liver, 5,10-methylenetetrahydrofolate reductase (after NADPH and before MTHFR; E.C. 1.5.1.20) activity is allosterically regulated by intracellular SAM concentrations. SAM sufficiency inhibits MTHFR activity, thereby promoting the use of 5,10-methyleneTHF for thymidylate synthesis. Conversely, insufficient SAM levels increase MTHFR activity, which reduces the amount of substrate available for thymidylate synthesis. It is presently unknown whether this level of regulation also occurs in testis.
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Chronic alcoholism reduces hepatic methionine synthase activity in the rat and micropig [32–35]. Folate deficiency limits the availability of substrate 5-methylTHF for hepatic methionine synthase activity, and other disturbances in methionine metabolism, both alone and in conjunction with chronic ethanol treatment, are evident in the micropig model [36, 37]. The effects of ethanol feeding on hepatic methionine synthase activity are attenuated by the presence of the accessory enzyme, betaine-homocysteine methyltransferase (BHMT; E.C. 2.1.1.5), which remethylates homocysteine using the substrate betaine, the oxidation product of choline [35].
The testis has a high rate of cell synthesis, and it is unknown whether a functional BHMT enzyme exists in the testis or any other male reproductive tissue [38–40]. If not, the testis could be especially sensitive to changes in folate levels related to alcohol, dietary folate insufficiency, or both. The present study aimed to determine how chronic ethanol consumption and/or dietary folate deficiency affects reproductive measures and folate metabolism in the sex organs of the male micropig. The reproductive measures of interest included organ weights, sperm counts, circulating sex hormones, and activities of rate-limiting enzymes in testicular testosterone and estrogen synthesis, 17,20-lyase and aromatase. Folate measures of interest included folate levels and folate-dependent methionine synthase activity in reproductive tissues.
The micropig model exhibits a number of features that make it ideal for this type of study. Micropigs voluntarily consume ethanol in intoxicating amounts while being maintained on a diet of solid food [41]. Previously, the micropig has been used successfully to study hepatic methionine metabolism in response to chronic alcohol exposure and/or folate deficiency [34, 36]. Using this model, 14 wk of exposure to either treatment resulted in elevated serum homocysteine levels [36]. Intestinal folate hydrolase (folylpoly-
-glutamate hydrolase) activity in pigs is similar to humans, making this model suitable for natural folate feeding studies [42, 43]. The only known difference in folate metabolism between the two species is that the predominant circulating form of folate in humans is 5-methylTHF, whereas it is tetrahydrofolate in pigs [44]. There is some evidence that reproducing sows may partition folates differently than humans; however, this has not been proven conclusively [45]. The degree of biochemical similarity between the micropig testis and that of the rat or human is not known, but they appear quite similar histologically. To our knowledge, this is the first histopathological study of the interactive effects of ethanol and folate deficiency in the micropig.
The present studies used reproductive tissues of male micropigs that were obtained at the conclusion of a previously described study of the single and combined effects of folate deficiency and ethanol feeding on the liver [36]. Twenty-four 6-mo-old juvenile male Yucatan micropigs were obtained from the Sinclair Research Center (Columbia, MO). All pigs were maintained for a 1-mo adaptation period on a folate-sufficient diet containing 90 kcal/kg body weight, 15% of calories as protein (vitamin-free casein), 30% of kcal as fat (corn oil), 55% as carbohydrate (cornstarch), and a vitamin and mineral mix that was adequate in all micronutrients, including vitamin B12 at 0.46 µg/kg body weight, folic acid at 14.5 µg/kg body weight, choline at 60.3 mg/kg body weight, and methionine at 675 mg/kg body weight [46] (Dyets Inc., Bethlehem, PA). Six micropigs each were then placed on one of four different diets: folate sufficient (FS), folate depleted (FD; complete deletion of folic acid from the vitamin mix), folate sufficient with ethanol (FSE; substitution of 40% of carbohydrate calories with ethanol), and folate depleted with ethanol (FDE). The mean caloric intake per kilogram body weight in the FDE group was used as the basis for dietary provisions to the animals in the remaining groups. The micropigs were housed in facilities approved by the National Institutes of Health and were cared for following standards and procedures outlined in the National Academy of Sciences' Guide for the Care and Use of Laboratory Animals. All procedures were reviewed and approved by the Animal Welfare Committee of University of California Davis. The overnight-fasted micropigs were anesthetized and killed after 14 wk of feeding. Since the published value for the entire spermatogenic process in boars is ~40 days, more than two cycles were covered in this experiment [47]. Reproductive tissues (testis, epididymis, seminal vesicles [including associated fluid], prostate, and bulbourethral glands) were surgically removed under anesthesia, trimmed of visible fat, weighed, and frozen at –70°C for further analysis.
Testis and cauda epididymal sperm were prepared and counted by modifying the standard method described by Blazak and coworkers [48]. One-gram frozen sections of testis or cauda epididymis were minced with a razor blade to a fine paste and then quantitatively transferred in a 75-ml volume of 0.9% saline containing 0.01% merthiolate and 0.05% Triton X-100 (SMT; Sigma, St Louis, MO) to a 250-ml stainless steel blender container (Fisher, Pittsburg, PA). The samples were blended for 2 min using a single-speed Waring blender (Waring Products, New York, NY).
Since the homogenates contained considerable unblended material, they were quantitatively filtered through mesh (20-µm pore size; Spectrum Labs, Rancho Dominguez, CA). The nonfilterable material was rinsed with an additional 25 ml SMT. In order to determine whether any experimental treatment affected the amount of nonfilterable material, its dry weight was determined and converted to a wet weight (calculated using the ratio of wet:dry weight of a duplicate, unblended tissue sample). The nonfilterable wet weight was expressed as a percentage of the wet weight of the original tissue sample. In testis, this unblended, nonfilterable material was opaque white, slightly elastic, extraordinarily resistant to both standard blending and Polytron homogenization, and tested positive (by LCMS) for the collagen marker hydroxyproline. We refer to it in subsequent sections of the text as "fibrous material."
Since the testis homogenates contained a substantial number of heteromorphic heads, Rose Bengal (0.08%; Sigma) was added to enhance the contrast for their visualization. The testis sperm homogenate was loaded onto a hemacytometer (Hauser Scientific, Horsham, PA) and counted using a standard light microscope (Zeiss; Precision Instrument Co., Fremont, CA) with phase contrast. Since no abnormal heads were observed in cauda epididymal homogenates, heads from the cauda were counted without dye under bright light conditions. Quadruplicate counts were made for all samples, and a count variation of greater than 20% was considered cause for repeat analysis.
Testis and Epididymis Histology
Specimens from testis and corpus epididymis were taken and fixed in Bouin solution (Sigma) for 2 to 3 days and then rinsed and stored in 50% ethanol. The fixed tissues were rinsed in 70% ethanol prior to embedding in paraffin, sectioning, and staining with periodic acid-Schiff-hematoxylin. Testis and epididymis samples were evaluated by an observer who was blind to treatment. In an attempt to communicate a relative degree of displacement from "normal," testis samples from each group were assigned a subjective grade based on an overall assessment of the quality of structure of the seminiferous epithelium. These grades were 0 (control structure: minimal or scattered focal loss of germ cells); 1 (more than 30% of tubules showed foci of basal epithelial vacuoles and apparent germ cell absence, or columns of mild hypocellularity); 2 (more than 50% of tubules showed some thinning of the epithelium and visible reduction in germ cell number, and 30%–50% of tubules had spaces larger than a pachytene spermatocyte); 3 (as in 2, but with the appearance of some tubules with marked-severe thinning of the epithelium, and very few postspermatogonial germ cells); 4 (approximately 50% of tubules showing marked to severe thinning of the epithelium, with few normal tubules, and the remaining showing some degree of germ cell hypocellularity); and 5 (total atrophy, no postspermatogonial germ cells present).
Serum total testosterone and estradiol concentrations were determined with RIA kits obtained from Diagnostic Products Corp. (Los Angeles, CA). The testosterone kit (Coat-a-Count) had an analytical sensitivity of 4 ng/dl and an intraassay precision of 5% at the concentration range tested. It was highly specific for testosterone, with a cross-reactivity of <1% for most related compounds, except for 5
-dihydrotestosterone (3%), 19-nortestosterone (20%), and 11-ketotestosterone (16%). The estrogen kit (Double Antibody) had an analytical sensitivity of 1.4 pg/ml and an intraassay precision of 5% at the concentration range tested. It was highly specific for estradiol, with a cross-reactivity of <1% for most related compounds, except for d-equilenin (4%), 17-ß-estradiol-3ß-D-glucuronide (6%), and estrone (12%).
Serum luteinizing hormone (LH) concentrations were determined by RIA [49] using anti-porcine LH (AFP 151031194), porcine LH (10714B) for iodination (both provided by Dr. A.F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes & Digestive & Kidney Diseases, Bethesda, MD), and USDA porcine (p)LH-B1 as the reference standard. Serum follicle-stimulating hormone (FSH) concentrations also were determined by RIA [50] using antiporcine FSH (AFP2062096Rb) and porcine FSH (AFP10640B) for both iodination and the reference standard (provided by Dr. A.F. Parlow). All samples were evaluated in a single assay for each hormone. Minimum sensitivities were 0.2 ng/ml for LH and 0.15 ng/ml for FSH assays, respectively.
Testicular tissue was homogenized on ice in buffer (0.1 M potassium phosphate, pH 7.4; 20% glycerol; 5 mM ß-mercaptoethanol; and 0.5 mM phenylmethylethisulfinyl fluoride [PMSF]) at a ratio of approximately 1 ml buffer per 0.1 g tissue. Microsomes were enriched by subcellular fractionation as previously described [51]. Tissue homogenates were sonicated briefly. Cellular debris and mitochondria were removed by centrifugation at 15 000 x g for 10 min. The supernatant was centrifuged again at 100 000 x g for 60 min, and the pellet was resuspended in homogenization buffer containing 1 mM (3-[3-cholamidopropyl-dimethylammonio]-1-propanesulfonate; CHAPS). Protein concentration was determined using the Bicinchoninic Acid Protein Assay Reagent (Pierce, Rockford, IL), and aliquots of 100 µg were saved at –80°C.
Microsomal enzyme activities.
The 17,20-lyase activity of P450c17 was measured radiometrically as recently described and validated [52, 53]. Briefly, activity was assessed by measuring the release of 3H-acetic acid from [21-3H]-17-OH-pregnenolone (25.9 Ci/mol; a generous gift from Drs. V. Njar and A. Brodie, University of Maryland School of Medicine). Microsomal protein (100 µg) was incubated for 2 h at 37°C in buffer (50 mM KPO4, 1 mM EDTA, 1 mM CHAPS; final volume of 1 ml) in the presence of 10.5 µM 17-OH-pregnenolone (7 µM of the radiolabeled and 3.5 µM of unlabeled 17-OH-pregnenolone; Steraloids, Wilton, NH) with a NADPH-generating system (17 mM glucose-6-phosphate, 1 mM NADPH, 2 mM NADP, and 1 unit of glucose-6-phosphate dehydrogenase [Sigma Chemical Co., St. Louis, MO]). Similar reactions measured aromatase activity by monitoring the incorporation of tritium from [1ß-3H]-androstenedione (24.7 Ci/mmol, New England Nuclear, Wilmington, DE) into 3H2O as previously described [54]. Cold androstenedione (Sigma Chemical Co.) was added to a final concentration of 150 nM (20% labeled, 80% cold). In both assays, the incubations were stopped with 30% trichloroacetic acid, extracted with chloroform, and the aqueous phase combined with a suspension of 8.5% charcoal, 0.85% dextran (17,20-lyase activity), or 5% charcoal, 0.5% dextran (aromatase activity). This was centrifuged at 2000 x g for 30 min, and a portion was removed and counted by liquid scintillation counting.
Tissue Folate and Methionine Synthase Assay
Testis and corpus epididymis samples were homogenized in five volumes of 0.028 M sodium phosphate buffer, pH 7.0, containing 1% ascorbic acid and 2 µg/ml of the protease inhibitors aprotinin and leupeptin. The homogenate was split for separate analyses of folate and methionine synthase activity. For measurements of tissue folate levels, a portion of the homogenate was adjusted to 0.1 M sodium phosphate buffer, pH 6.0, and polyglutamyl folates were hydrolyzed to their monoglutamyl derivatives using purified hog kidney conjugase. Total folates were measured by standard microbiological assay with Lactobacillus casei [55]. The remaining homogenate was centrifuged at 40 000 x g, and the supernatant fraction was used to measure methionine synthase holo-enzyme activity by a published method [56]. Briefly, 4 mg supernatant protein was incubated with 25 µmol potassium phosphate buffer, pH 7.5, 100 µmol sodium chloride, 100 µmol 2-mercaptoethanol, 1.25 µmol L-homocysteine, 50 nmol SAM, 37.5 nmol 5-methylTHF, 7.5 µl 5-[14C]-methylTHF (1233 cmp/nmol; Amersham Phamacia Biotech, Piscataway, NJ), 15 nmol cyanocobalamin, and 50 nmol FADH2 reduced with platinum oxide and hydrogen gas in a total volume of 0.5 ml. After 40 min of incubation at 37°C, samples were boiled 3 min and chilled in ice 5 min, followed by the addition of 500 µl methionine-coated Norit charcoal. After 10 min of incubation at 25°C, samples were centrifuged at 16 000 x g, and 0.5 ml was counted in a liquid scintillation counter.
All values obtained from each group are expressed as mean ± SD. Significant differences among treatments (ethanol feeding, folate deficiency, or their combination) were determined by two-way analysis of variance (ANOVA) using Systat version 9 (SPSS Inc.). Subgroup analyses were performed by one-way ANOVA when significant interactions were found. Since plasma LH and FSH values were not normally distributed, the data were ranked for ANOVA testing. Multiple regression techniques were used to evaluate associations between folate metabolism and hormonal indices, controlling for ethanol treatment, folate deficiency, and their combination. For each hormone variable, a semipartial correlation coefficient (r) and P value were obtained to describe its relationship with a single folate variable (either testis folate concentration or methionine synthase activity). For all tests, P < 0.05 was defined as statistically significant.
Testis and epididymis (total and cauda) weights were reduced by ethanol consumption (FSE and FDE groups), even after adjusting for the reduced body weight of the alcoholic pigs [36] (Table 1). Testis and epididymis weights were not significantly reduced by folate deficiency; however, bulbourethral gland weights were decreased in both folate-deficient groups (FD and FDE).
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Testis sperm concentrations were decreased by 19% in the ethanol-fed groups (Table 2). The testis samples from the ethanol-fed pigs contained 3-fold higher amounts of fibrous material (8.4% ± 4.8%) compared with non-ethanol-fed pigs (2.7% ± 0.5%; P < 0.0001). After adjusting the sperm concentration data for this fibrous content, the statistical significance of the ethanol effect was eliminated (P = 0.06). The total testis sperm count was decreased by 45% with ethanol treatment, whereas the fraction of morphologically abnormal sperm was not affected by either treatment. Cauda epididymal sperm concentrations were not affected by ethanol feeding or folate deficiency. The adjusted sperm concentrations were not calculated, since the fibrous material in epididymis (38% of tissue wet weight on average) was not significantly altered by the treatment regimens. Total sperm count per cauda was not affected by ethanol or folate deficiency.
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The testes of the control (FS) pigs had generally thick strands of connective tissue separating lobes of tubules and two to five confluent layers of interstitial cells between adjacent tubules (Fig. 2a). The interstitial cells had abundant, evenly stained, smooth cytoplasm (three to eight times the nuclear area). There were very few spaces and sinusoids. Although there was significant animal-to-animal variation, most tubules were packed with regular stacked layers of germ cells. Tubules around the stage of sperm release routinely had some basal vacuoles in Sertoli cells. Although we did not quantify apoptosis, cell death appeared to be infrequent, with only 2%–8% of the tubules having one fewer germ cell layer or too few cells in one generation or another. Round luminal cells were seen in some pigs (5%–20% of tubules), but not in every animal. Overall, the testis lesion grade for the control pigs was 0.75 on a scale of 0 (perfect condition) to 5 (total atrophy). The epididymis (corpus) showed two to five round luminal cells per tissue section, with the remaining cells being packed sperm (Fig. 3a). The principal cells of the epididymal epithelium were stacked closely together and were very narrow. The interstitial tissue was variably thick textured or thin textured, interlaced with sinuous blood vessels, and plentiful.
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In pigs fed just the folate-deficient diet (FD), the testis of one animal appeared normal, three had mild-moderate thinning of the seminiferous epithelium or focal hypospermatogenesis, and two had moderately affected spermatogenesis (Fig. 2b). In affected pigs, the lesions were characterized by a variable reduction in germ cells, ranging from one spot in a tubule (focal) to involving most or all tubules to some degree. Overall, the testis lesion grade for the FD group was 2.5 of 5. The epididymides contained sperm and occasionally testicular debris (dying cells or parts thereof; Fig. 3b). Aside from testicular debris in the folate-deficient epididymides, there was no evidence of pathologic lesions in the epididymal cells.
The testes from pigs fed ethanol only (FSE) had many basal vacuoles, and only a few sections contained a complete progression of spermatogenesis (Fig. 2c). Most testes in this group had fewer long spermatids and had incomplete populations of the various cell ages. The interstitial cell populations ranged from large cells with abundant cytoplasm, numerous and thick between tubules (three animals), to markedly thin with reduced cytoplasm (two animals). Overall, the qualitative lesion grade for the FSE group was 3.5 of 5. In epididymides from this group, sperm density was greatly reduced in some animals and apparently normal in others (Fig. 3c). The number of round luminal cells in the epididymis was increased in many animals, a clear reflection of testis efflux of dead/dying germ cells.
Among the testes from pigs fed the combined folate-deficient and ethanol diet (FDE), one appeared normal, and the remaining five animals were heavily affected, as evidenced by basal epithelial vacuoles with significant reductions in all germ cell populations (Fig. 2d). Most testes showed abundant and plentiful interstitium. The qualitative lesion grade for the FDE group was 4 of 5. In the epididymides it was difficult to appreciate a reduction in sperm density, but there was a distinct increase in luminal debris from the testis (Fig. 3d).
Serum testosterone and estradiol levels were reduced by 52% and 74%, respectively, in the folate-deficient pigs (FD and FDE groups; Table 3). Circulating LH concentrations were independently reduced by 33% by folate deficiency, whereas FSH concentrations were reduced by 43% by ethanol treatment (FSE and FDE groups). The ratios of estradiol to testosterone were unaffected by the different diet treatments (data not shown). Testicular 17,20-lyase and aromatase activities were reduced by 23% and 40%, respectively, in the folate-deficient pigs.
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The concentrations of testis folate were increased by ethanol feeding (FSE and FDE groups), whereas total testis folate content was reduced by folate-deficient diets (FD and FDE; Table 4). Testis methionine synthase activity was decreased by ethanol feeding but increased by folate deficiency. Folate deficiency had no effect on folate concentrations in corpus epididymis, whereas total folate content was reduced. There was an interaction between ethanol and folate deficiency for corpus epididymis methionine synthase, such that activity increased in both the FD and FSE pigs relative to the FS control. Overall, methionine synthase activity was higher in the epididymis than in the testis or as previously shown in the liver [36].
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Associations Between Folate Metabolism and Sex Hormone Indices
Associations between testis methionine synthase activity and several hormonal variables are shown in Figure 4. Methionine synthase activity was inversely associated with circulating estradiol (semipartial correlation coefficient (r) = –0.51; P < 0.05; Fig. 4a), but not with testosterone. Methionine synthase activity was inversely associated with circulating LH and FSH (semipartial r = –0.56; P < 0.01 and semipartial r = –0.81; P < 0.0001, respectively; Fig. 4, b and c). The FSH finding is particularly strong and suggests that in our statistical model 66% of the variation in FSH can be explained by changes in methionine synthase activity. Methionine synthase activity also was inversely associated with testicular 17,20-lyase (semipartial r = –0.64; P < 0.01; Fig. 4d), but not with aromatase activity. Testis folate concentration was not associated with any hormonal indices
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We describe here the effects of ethanol consumption or folate deficiency in the male reproductive system of micropigs. In general, we discovered that the effects of ethanol consumption and folate deficiency were independent of each other. Hence, they will be discussed separately below. Furthermore, we discovered that testicular methionine synthase activity is inversely associated with various hormonal indices.
All animals gained weight and were therefore in positive energy balance during the feeding experiments. While there was no effect of folate deficiency on weight gain, the ethanol-fed groups gained half as much as the control group [36]. These findings are consistent with a prior body composition study that showed that lesser weight gain in ethanol-fed micropigs is due to a decrease in body fat with no differences in lean body mass [57]. In addition, there were no changes in either group in hemoglobin or serum albumin levels, which is consistent with adequate nutritional status [36].
Organ weights, sperm assessments, and histology. We found that chronic ethanol consumption reduced testis and epididymis (whole-organ and cauda) weights (Table 1). These data conform to reports of smaller testes in rats fed alcohol starting peripubertally [58], but are in only partial agreement with results in pubertal mice [59]. Concomitant with the reduction in testis weights in the alcoholic pigs, testis sperm concentrations (unadjusted) and total testis sperm numbers were reduced with ethanol consumption (Table 2). The abolition of the ethanol effect after adjusting for excessive fibrous material implies that the cellularity of the seminiferous epithelium was replaced with fibrous interstitial tissue. This has been reported in the alcoholic human testis [60].
Overall, this scenario is consistent with the histology picture. Both ethanol-fed groups exhibited a generally reduced proportion of normal tubules and increased degree of damage to the seminiferous epithelium compared with their respective controls (Fig. 3), although each group was heterogeneous in terms of the degree of histological change. This finding is in general agreement with previously published reports [61, 62]. The FDE group was clearly worse than all others in terms of testicular epithelial damage and poorly developed spermatogenesis.
Hormone indices. While the histology clearly showed an effect on the tubular compartment of the testis, the lack of a concomitant rise in FSH (indeed, FSH levels fell in these animals) also shows an effect on the central mechanisms that normally respond to peripheral insult. This agrees with previous reports [63], but the lack of any other significant hormonal effect is somewhat inconsistent with the literature [2, 3]. Selective suppression of FSH could result from direct effects of ethanol on pituitary levels of activin, which are known to be positively correlated with FSH levels in pigs [64]. Also, ethanol consumption induces hepatic cytochrome P450 enzymes, and porcine testes express substantial levels of nonsteroidogenic P450s that may be induced by ethanol, inciting local cell and tissue damage [53, 65].
Folate metabolism. We found that ethanol consumption increased folate concentrations in the testis, but not in the epididymis (Table 4). The observed increase in testis folate concentrations with chronic ethanol feeding could simply reflect a shift in the cell population (i.e., a reduction in germ cells) as testis size becomes smaller. There is precedent for this in the toxicology literature, as rats exposed to 2,5-hexanedione have an increase in the relative activity of various Sertoli cell enzymes as germ cell number decreases [66]. This explanation implies that folate is mainly compartmentalized in Sertoli cells. However, data on the folate content of the various cell types in the testis are currently lacking.
The increase in testicular folate concentration could reflect an impact on one or more aspects of folate metabolism. Besides a report showing that testicular folate uptake is unaffected by chronic ethanol feeding in rats [7], we found no other reproductive studies that could shed light on our findings. Whole-animal studies found that chronic ethanol feeding increases folate turnover and urine excretion in monkeys [27] but had no effect on folate catabolism in mice [67]. However, the metabolism of folate in the testis may not reflect its whole-body metabolism.
Elevated folate concentrations in the testis of ethanol-fed micropigs may be linked to the concomitant reduction in methionine synthase activity (Table 4). When methionine synthase is inhibited, its substrate 5-methylTHF becomes trapped, because the MTHFR reaction leading to its production is irreversible (Fig. 1). The buildup of 5-methylTHF occurs at the expense of all other folate forms, effectively limiting the availability of folates for other critical functions. This scenario is known as the methyl folate trap hypothesis [68], and it has been documented previously in the alcoholic liver [69]. This could presumably happen in the testis if there were reduced SAM concentrations secondary to reduced methionine synthase activity, which would cause an allosteric activation of MTHFR activity and a consequent reduction in 5,10-methyleneTHF as well as all other non-5-methylTHF forms. This would result in inhibited thymidylate or purine synthesis with consequent reductions in the availability of DNA nucleotides for spermatogenesis. Further, the reduced production of methionine and SAM would influence both polyamine synthesis and DNA methylation reactions, affecting spermatogenic events and germ cell development. In essence, the reduction in testis methionine synthase activity with ethanol feeding could be causing a functional folate deficiency in the testis by limiting the availability of any one of several folate forms needed for proper spermatogenesis.
The argument for the methyl folate trap hypothesis is weakened by the fact that methionine synthase activity was increased by folate deficiency, which resulted in the FDE animals having much greater activity than controls. Furthermore, the hepatic response to ethanol in the present study does not agree with previous reports of ethanol-induced folate trapping such that folate concentrations were not increased in the ethanol-treated groups [36]. However, ethanol could be causing some other secondary defect in folate metabolism. The antifolate drugs sulphasalazine and pyrimethamine, which cause secondary folate deficiency by a different mechanism, also reduce sperm numbers in humans and in animal models [70, 71].
It is presently unknown how ethanol consumption interferes with testicular methionine synthase. The ethanol metabolite, acetaldehyde, can inhibit liver methionine synthase; however, the concentrations reported on so far have been supraphysiological [72]. It also is possible that ethanol-induced inflammatory events in the testis could interfere with methionine synthase activity. Inhibition of NO synthase reverses the endocrine suppression seen in the alcoholic testis [73, 74]. NO inhibits methionine synthase activity in liver and brain [68], inactivates the essential cofactor for methionine synthase, methylcobalamin (methylB12), and limits purine nucleotide and methionine synthesis [75]. Vitamin B12-deficient rats show evidence of reproductive impairment, including reduced testis weight and atrophy of the seminiferous tubules [76]. Another nitrogen oxide, nitrous oxide, reduces testicular methionine synthase activity [77] and mature sperm number and increases abnormal multinucleated sperm in rats [78]. Nitrous oxide exposure in males is linked to an increased spontaneous abortion rate among their spouses [79].
Organ weights, sperm assessments, and histology. We observed no change in testis and epididymis weight with folate deficiency. In rodents, testis weight declines with folate deficiency [80]. The absence of an effect on sperm counts (Table 2) disagrees with reports of reduced testis weight and testis sperm in mice with disturbed folate metabolism due to a deficiency in the MTHFR enzyme [81]. They also disagree with our unpublished observation of reduced testis weight and epididymal sperm in folate-deficient rats. Reduced testis weight may be important for the gross reduction in spermatogenesis seen in folate deficiency, and boars may be more resistant to this effect than rodents. Despite there being no gross differences in organ weights or sperm counts, the FD group had moderate and variable reductions in germ cells (Fig. 3), which agrees reasonably well with a recent finding that MTHFR-deficient mice have a variable lack of elongating spermatids [81].
Hormone indices. Folate deficiency reduced LH, testosterone, and estradiol secretion (Table 3), suggesting a targeting of folate deficiency on the brain and the interstitial compartment of the testis. The decline in both testosterone and estradiol secretion was concomitant with reduced bulbourethral gland weight (Table 1). This also is consistent with observations that both androgen and estrogen are required to maintain seminal fluid volume [82], and that the inhibition of estradiol synthesis in boars significantly reduces accessory sex gland weights (Berger and Conley, unpublished observations).
Parallel decreases were observed in the folate-deficient groups for 17,20-lyase and aromatase activities, each of which results from P450 enzyme expression in Leydig cells of the interstitial compartment of the porcine testis [83]. Aromatase activity was more severely depressed than 17,20-lyase, which is reminiscent of the effects of suppression of the hypothalamic-pituitary axis by GnRH antagonist treatment of neonatal pigs [53]. Chronic GnRH antagonist treatment also decreases LH without affecting FSH concentrations, similar to the effects of folate deficiency noted here [84].
Data on the relationship of folate deficiency to male hormone metabolism are sparse. However, very early literature (prior to the discovery of the individual B vitamins) demonstrated that combined B-complex deficiency reduced accessory sex gland size and function by disturbing sex hormone metabolism [85, 86]. Defective testosterone metabolism is common in men with celiac disease, a condition associated with reduced folate status [12]. In large population studies, circulating estrogen and homocysteine levels were inversely associated [87]. Serum homocysteine levels were increased in the pigs fed folate-deficient diets only (FD), and were further increased in the ethanol-treated groups [36, 37]. Since estrogen levels were reduced only by folate deficiency and not ethanol treatment, it is difficult to link these findings together. However, serum homocysteine may not accurately reflect homocysteine metabolism in the testis. Folate may play a role in the synthesis of catechol estrogens [88] and in the regulation of the expression of the estrogen receptor gene, both of which involve methylation reactions [89].
Folate metabolism. Folate deficiency caused a reduction in total folate levels in both testis and epididymis (Table 4). This could reflect a cumulative effect of slight, nonsignificant reductions in organ weights and folate concentrations. These data agree with a previous report in weanling rats fed a folate-deficient diet for 25 days [80]. In longer deficiency studies, folate-deficient rats preferentially incorporate folates into the testis, probably at the expense of liver folate [90]. This suggests that any effects observed in the testis may be variable, depending on length of deficiency (e.g., acute vs. chronic).
Testis methionine synthase activity was increased in both folate-deficient groups (Table 4), which is likely a compensatory response to the reduction in total tissue folate. An increase in methionine synthase activity would be expected to enhance methylation reactions and polyamine synthesis, with probable end results being gene silencing and reduced cell cycle activity. Although published data in this area are extremely sparse, a single report demonstrates that combined methyl deficiency (including low folate and B12) for up to 5 wk decreases testicular SAM:SAH ratios in immature rats, a change suggestive of a reduced supply of methyl groups [91]. This contrasts with our results and may mean that the immature testis responds differently to changes in dietary methyl supply or that the effects are time dependant. We previously reported that hepatic folate concentrations were reduced and methionine synthase activity was unchanged by folate deficiency [36]. This scenario contrasts with our findings in the testis, suggesting that compared with liver, the testis responds differently to folate deficiency. Synthesizing this information with our hormone data, we postulate that either methylation processes or polyamine syntheses in the folate-deficient testis are altered in such a way that interferes with the hormonal regulation of spermatogenesis.
Epididymal methionine synthase activity was substantially higher than that observed in testis (Table 4) and liver [36], suggesting that methionine metabolism may be especially important in this tissue. Since methionine metabolism is linked to sulfur-amino acid metabolism via the disposal of homocysteine, it may be involved with glutathione-mediated antioxidant protection to epididymal spermatozoa [92, 93].
Possible Roles for Folate in Male Reproductive Function
The regulation of folate metabolism via changes in methionine synthase activity will alter the flow of folate coenzymes either toward methylation reactions and polyamine synthesis (high methionine synthase activity) or toward thymidylate and purine syntheses (low methionine synthase activity). The inverse associations between testis methionine synthase activity and several hormonal variables imply that either relative reductions in methylation and polyamine synthesis or increases in thymidylate and purine biosynthesis are important for sex hormone metabolism. This scenario is not entirely consistent with evidence on methylation and normal steroid hormone metabolism and spermatogenesis [88, 89, 94], and it suggests that the role of folate in polyamine synthesis could be more critical for reproductive function. Moreover, these data suggest that the regulation of testicular folate metabolism may impact various points along the pituitary-hypothalamic-gonadal axis, including estrogen synthesis within the Leydig cell, as well as some aspects of LH and FSH metabolism. Integrating this with our findings on the independent effects of ethanol and folate deficiency on spermatogenesis, we postulate that methionine synthase activity may be influencing sex hormone profiles which, in turn, influence spermatogenesis.
In summary, our findings on the effects of ethanol consumption on male reproductive function are generally in agreement with data from other species and demonstrate that the alcoholic micropig can be a suitable model for examining male reproductive defects induced by ethanol. We have expanded this literature by showing evidence of disturbed folate metabolism in this alcoholic animal model, such that testis folate concentration was increased and methionine synthase activity was reduced. These effects may be linked to the reduction in spermatogenesis seen in alcoholics. Future studies of ethanol effects on folate metabolism should include a search for the primary defect in testicular folate metabolism (e.g., measuring the distribution of coenzyme forms, nucleotide pools, uracil misincorporation, polyamine levels, and DNA methylation status). We also found that folate deficiency decreased total testis folate, increased testis methionine synthase activity, reduced circulating sex hormones, and impaired testicular steroid hormone biosyntheses. Although the FDE group had the worst tubule histology due to a lack of supporting data from any of our other endpoints, we are not able to conclude that ethanol and folate deficiency exacerbate each other in the testis. In all animals combined, we found evidence to suggest that methionine synthase activity influences the hormonal regulation of spermatogenesis. Our observed reproductive findings suggest that chronic ethanol targets the tubular compartment of the testis, whereas folate deficiency targets the interstitial compartment. This may be related to the type of disturbance in folate status invoked by the two treatments. Specifically, the effects of folate deficiency are reflective of a primary deficiency, whereas our evidence suggests ethanol exposure may be causing a secondary or functional disturbance in folate metabolism in the testis. This contrasts with findings in the liver [36] and suggests that folate metabolism and/or its regulation in the male reproductive system is somewhat different. Our data illustrate the variable impacts that these exposures may have not only on functionally distinct tissues but also within the male reproductive system. Finally, these data open up a new appreciation of folate biology in the endocrine system and suggest that an elucidation of the role for folate in male sex hormone metabolism should be pursued.
ACKNOWLEDGMENTS
We thank Jan Peerson for statistical advice. We are indebted to Dr. Joe Ford from the U.S. Meat Animal Research Center (Clay Center, NE) for conducting the gonadotropin assays and also for valuable input on interpreting the reproductive data.
FOOTNOTES
1Supported by grants from the National Foundation for Cancer Research (M2661) and the National Institutes of Health (K05-AT 001323 to B.N.A., and RO1-AA 14145 and P-30-DK 35737 to C.H.H). 
Correspondence: 2Lynn M. Montelius, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609. FAX: 510 597 7151; e-mail: lmontelius{at}chori.org
Received: 30 May 2006.
First decision: 5 July 2006.
Accepted: 20 November 2006.
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