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Morphologic alterations of rat Leydig cells induced by ethanol
Pharmacology Biochemistry & Behavior, Vol. 18, Suppl. 1, pp. 341-347, 1983. " Ankho International. Printed in the U.S.A.
Morphologic Alterations of Rat Leydig Cells Induced by Ethanol J. S. G A V A L E R ,
H . A. P E R E Z , L. E S T E S A N D D. H. V A N T H I E L
University o f Pittsburgh School o f Medicine, Pittsburgh, P A 15261
GAVALER, J. S., H. A. PEREZ, L. ESTES AND D. H. VAN THIEL. Morphoh~gic alterations of rat Leydig cells induced by ethanol. PHARMACOL BIOCHEM BEHAV 18: Suppl. 1,341-347, 1983.--Ultrastructural morphometric studies were employed to compare the morphology of Leydig cells found in the testes of rats fed ethanol, accounting for 36% of total calories for 6 weeks, and those found in the testes of rats isocalorically fed a diet in which ethanol was isocalorically replaced with dextrimaltose. The testes of alcohol-fed rats weighed significantly less (p<0.01) than those of the isocaloric controls. Moreover, plasma testosterone in the alcohol-fed animals was reduced (p<0.01), as compared to the controls. Morphologically, two main differences were encountered between the Leydig cells of alcohol-fed animals and those of the isocalorically-fed controls. These were: (1) an increase in the number of elongated and cup-shaped mitochondria; and (2) an increase in cytoplasmic protrusions in the form of pseudopods. Using morphometric techniques, the Leydig cells of alcohol-fed animals were smaller (p<0.05), had less cytoplasm (p<0.05), larger mitochondria (/9<0.01), and less smooth endoplasmic reticulum IP <0.05) than did those of the isocaloric controls. These morphologic characteristics of Leydig cells of alcohol-fed animals are similar to those reported to occur in the liver and suggest that the biochemical mechanisms responsible for alcohol-induced cellular injury are similar in the testes and the liver. Leydig cells
Alcohol
Testes
Testosterone
Ethanol
Gonadal toxicity
L E Y D I G cells comprise the endocrine compartment of the testes and are responsible for the synthesis and secretion of the male sex hormone, testosterone. Until recently, it was assumed that Leydig cells were relatively resistant to the ravages of systemic disease. With the advent of radioimmunoassay resulting in the availability of plasma testosterone measurements, however, it has become apparent that many systemic diseases are associated with Leydig cells dysfunction [22]. Among the various toxins known to adversely affect Leydig cells, ethanol is by far the most important [21]. Thus, ethanol abuse in the absence of cirrhosis has been shown to be assoicated with reduced libido, loss of potency and hypogonadism in men, as well as in experimental animals [l, 9, 1 l, 13, 18, 25, 32, 34, 36, 37, 40]. Moreover, ethanol has been shown to suppress biosynthesis and secretion of testosterone by Leydig cells in vitro [5-8, 10, 12, 17, 23, 33]. Essentially no data exist describing the morphologic changes that occur at the ultrastructural level in Leydig cells as a result of chronic ethanol exposure. Therefore, we examined the morphology of Leydig cells of rats fed alcohol in order to quantitate ultrastructural changes which might correlate with the findings of reduced testosterone synthesis and secretion by these cells.
were pair-fed either an alcohol-containing liquid diet, in which ethanol accounted for 36% of the total calories, or an identical diet in which the ethanol was isocalorically replaced with dextrimaitose (Bioserve, Diet 71 I-A and 71 I-C, Frenchtown, N J) for 6 weeks, at which time they were sacrificed by exsanguination and autopsied. The testes were removed, decapsulated, weighed and sectioned into cubes l x l mm. Tissue cubes were fixed in 3% glutaraldehyde (Ladd Research Industries, Inc., Burlington, VT) in 0.1 M cacodylate buffer (pH 7.4) for 1 hour at 4°C. After primary aldehyde fixation, samples were washed for 1 hour in 0.1 M cacodylate buffer and post-fixed in I% osmium tetroxide in 0.1 M cacodylate buffer for 2 hours at room temperature. After fixation, the tissue was rinsed, dehydrated in a graded ethanol series, and embedded in Epon-Araldite [24]. Thin sections with a gold interference were cut with glass or diamond knives on a Sorvall MT 1 microtome (Ivan Sorvail, Inc., Norwald, CT), mounted on scored 200 to 300 mesh grids, and stained with 3% uranyl acetate [39] and lead nitrate [26]. Grids were examined and photographed at 60 kV with an electron microscope (Phillips Electronic Instruments, Mount Vernon, NY), using a double condenser system.
METHOD
To guarantee that sampling was random, fields were examined in a predetermined sequence in the four corners and center of the grid squares. From each grid, five photomicrographs of Leydig cells at an original magnification of x2,970, x5,300 and x 10,800 were taken. Prints were made at an enlargement of 2.65. The high power photomicrographs
Morphometric Analysis
Animals Weanling male rats aged 20 days were obtained from Charles River Breeding Laboratories, Wilmington, MA. They were individually caged after pairing for body size, and
341
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GAVALER ET AL.
FIG. 1. Conspicuous clusters of Leydig cells were located mainly in close contact with blood vessels. Original magnification 2970x; final magnification, 6900×. KEY TO FIGUREABBREVIATIONS AER BV Ch Co FC GC ICS ID IT
Agranular endoplasmic reticulum Blood vessel Chromatin Collagen Fusiform cell Golgi complex Intercellular space Interdigitation Interstitium
were used for determination of volume densities of smooth endoplasmic reticulum and mitochondria. All measurements were made using a Graphic Tablet System ® attached to an Apple II computer (Apple Computer, Inc., Cupertino, CA 95014). A sheet of transparent acetate was placed upon each photomicrograph to prevent electrostatic interference and the detecting pen was carefully passed upon each of the Leydig cells and their organelles. The data thus obtained was interpreted by the computer and translated into square and cubic microns. Prior to use, the graphics tablet was standardized using appropriate test points (standards). To demonstrate that the number of measurements available for any given parameter satisfied requirements of sampling adequacy for sterological analysis, the method of Bolender was used [3]. Using this method, the adequacy of the number of measurements was determined by grouping data from increasing numbers of samples until the standard error of the mean achieved a plateau at a level less than 10% of the mean. For each set of measurements used in the analyses presented here, the number available exceeded the number required to satisfy this criterion.
LD
Lipid droplet
LY
Lysosome Mitochondria Microvilli Nucleus N.ucleolus Nuclear envelope Red blood cell
MV N Nc NE RBC
samples were measured in a single assay. The intra-assay variation for known standards was less than 8%. The detection limit was 0.1 ng. Plasma gonadotropins were measured utilizing specific radioimmunoassays and reagents supplied by the NIAMDD, Rat Pituitary Hormone Distribution Program. NIAMDD Rat FSH-RP-i and NIAMDD Rat LH-RP-I were used as reference preparations. The hormones were iodinated with ~:'I using the chloramine-T method [14]. Iodinated hormones were purified by polyacrylamide gel electrophoresis using 9% acrylamide with 2% cross-linking [27]. Other details of the assays have been described previously [31]. The detection limit for both assays was 4.0 ng with an intra-assay variation of less than 8%. Potency estimates were calculated using the computer program of Rodbard [28]. Statistical Analysis Results were analyzed using the paired t-test. Differences were considered to be significant at a p value <0.05. RESULTS
Hormone Determinations Plasma testosterone [24] was determined in duplicate using a radioimmunoassay established in our laboratory. All
Gross Anatomy The testes of the alcohol-fed animals weighed 1.47-+0.32 g
ETHANOL-INDUCED TESTICULAR ALTERATIONS
343
FIG. 2. Electronmicrograph of rat Leydig cells (control group). There is a single, large, excentric nucleus with a dense rim of chromatin close tothe nuclear envelope, and a prominent nucleolus. Original magnification 2970x; final magnification, 9636x. Low magnification micrographs such as this were used for determinations of surface densities of whole cells and nuclei. Disrupted Leydig cells, such as the one seen on the left half, were not considered for morphometry.
(mean_+ SEM), which was 50% less than that of the isocaioric controls (2.98_+0.17 g) (p<0.01).
Light Microscopy As previously reported, the testes of the alcohol-fed animals contained smaller seminiferous tubules with a reduced number of germ cells, compared to those of the isocaloric controls [34]. The Leydig cells of the alcohol-fed animals appeared to be more numerous per unit area than those of the isocaloric controls. This apparent hyperplasia may reflect the presence of normal or possibly even reduced total numbers of Leydig cells in a reduced testicular volume.
Electron Microscopy Ultrastructural examination of rat interstitial gonadal tissue disclosed the presence of loose connective tissue with a population of polygonal, fusiform, and clear cells, and a rich network of blood vessels (Fig. 1). The large, polygonal cells (Leydig cells) were found to be scattered or in strands or, more often, irregularly grouped in the interstitum around capillaries. Fusiform cells were closely associated with the tubular wall or in the adventitia of the blood vessels. Lymphatic vessels were not conspicuously present, probably due to the fact that fixation of the testes was not accomplished by perfusion. Leydig cells had a single, large, excentric nucleus with a dense rim of chromatin attached to the innter membrane of the nuclear envelope. The prominent nucleolus was composed of a distinct pars amorpha and pars reticularis (Fig. 2). Leydig cells were intimately associated with each other. Contiguous cells were separated by a regular space. Leydig cells from alcohol-fed rats exhibited numerous microvilli varying in length and shape, protruding into the free intercellular space or interdigitating with microvilli of neighboring interstitial cells (Fig. 3). This finding was not encountered in testes of normal or isocalorically-fed rats. Mitochondria, varying in size and shape, were present in
moderate numbers of Leydig cells from control rats. The inner structure of these mitochondria consisted mainly of quite numerous tubular cristae of uniform size. The mitochondrial matrix was moderately dense, and occasionally small, dense granules were encountered (Fig. 4). Cupshaped and elongated mitochondria were most conspicuous in Leydig cells of alcohol-fed rats (Fig. 5). Membranes of the Golgi complex were present as flattened cisternae and associated small vesicles in a single, concentrated form, most often juxtanuclear (Fig. 5). A granular endoplasmic reticulum was characteristically found in all the interstitial cells. Also characteristic was the segregation of this reticulum in areas not occupied by other organelles (Fig. 6).
Endocrine Studies Plasma testosterone concentrations were reduced in the alcohol-fed animals (2.4_+0.2 ng/ml) compared to those in the isocaloric control animals (4.7+_0.08) (p<0.01). Plasma FSH levels in the alcohol-fed animals (761 _+133 ng/ml) were twice those of the isocaloric controls (378-+57 ng/ml) (p<0.05). In contrast, and despite the marked reduction in testosterone levels present in the alcohol-fed animals, LH levels did not differ between the two groups (alcohol-fed 50.8_+4.6 ng/ml vs. 59.2_+4.1 ng/ml for the isocaloric controls).
Morphometric Analysis When evaluated using morphometric techniques, the Leydig cell volume of the alcohol-fed animals was less (2071.0_+117.0 pt:~) than that of the isocaloric controls (2382.5_+148.5) (/)<0.05). In contrast, no difference in Leydig cell nuclear volume was seen between the two groups (alcohol-fed 786.5_+57.5 p2 vs. 846.5_+65.0 for the isocaioric controls). As a result, the cytoplasmic volume of Leydig cells of alcohol-fed animals was less (1280.5_+71.5 g:3) than that of the isocaloric controls (1535.5_+99.5) (,0<0.05). The volume of smooth endoplasmic reticulum (SER) was re-
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GAVALER ET AL
FIG. 3. Low magnification electron micrograph of rat Leydig cells (alcoholic group). The plane of section does not pass through the nuclei of some cells. The cells are intimately associated with each other, separated in areas by a regular space. At points where the surface of adjacent Leydig cells contact with other or surrounding connective tissue, microvilli of varying length, shape, and number protrude into the surrounding free space or interdigitate with microvilli of adjacent Leydig cells. These cytoplasmic protrusions were included in determinations of cytoplasmic surface densities. Note also the increase in number of mitochondria in Leydig cells of alcohol-fed animals which is apparent, when these cells are compared to those of the controls shown in Figs. I and 2.
FIG. 4. Intermediate magnification electron micrograph of rat Leydig cell (isocaloric control group). Original magnification 4210×; final magnification, 10130×. Micrographs at this higher magnification were used for determinations of surface densities of mitochondria.
ETHANOL-INDUCED
TESTICULAR ALTERATIONS
345
FIG. 5. High magnification electron micrograph of rat Leydig cells (alcoholic group). Original magnification 5150× final magnification, 17510×. Abundant numbers of mitochondria are present, exhibiting different sizes and shapes. The inner mitochondrial membrane exhibits lamellar cristae, tubular projections, and a moderately dense mitochondrial matrix containing numerous dense granules. Note also the juxtanuclear Golgi complex with flattened cisternae and associated small vesicles, as well as the abundant agranular endoplasmic reticulum. The granular endoplasmic reticulum can be seen as loose aggregations of short cisternae with their associated ribosomes, as well as free ribosomes and polysomes.
346
GAVALER ET AL.
FIG. 6. Electron micrograph of rat Leydig cell (alcoholic group). Original magnification 6790×; final magnification, 2237×. This higher magnification permits accurate delineation of closely packed tubules and cisternae of the agranular endoplasmic reticulum.
duced in the Leydig cells of the alcohol-fed animals (422.0_+27.5 p3 as compared to that of the isocaloric controls (497.5_+37.5) (p<0.05); but, when the volume of SER was corrected for the reduced volume of the cytoplasm, no difference existed between the two groups (alcohol-fed 33.15_+0.96% cytoplasmic volume vs. isocaloric control 32.74_+1.05). In contrast, the mitochondria of the Leydig cells of alcohol-fed animals were larger (225.0_+ 15.0 p3) than those of the isocaloric controls (161.0_+13.5) (o<0.005). Moreover, when corrected for the reduced cytoplasmic volume, this increase in mitochondrial volume was even more apparent (alcohol-fed 17.83_+0.77% cytoplasmic volume vs. isocaloric controls 10.28_+0.55) (p<0.005). DISCUSSION The present study confirms earlier reports of (i) testicular atrophy and reduced testosterone levels in alcohol-fed rats compared to isocaloric controls, (2) similar LH concentrations, despite the severe testicular injury present in the alcohol-fed animals and (3) increased FSH levels (p<0.05) in alcohol-fed animals compared to isocaloric controls, as might be expected as a result of the severe germ cell injury manifested by seminiferous tubule atrophy [34,36]. Further, the present study extends earlier studies by providing morphometric data documenting alcohol-induced Leydig cell injury. Thus, compared to isocaloric controls, the Leydig cells of alcohol-fed animals are smaller (p<0.01), have tess cytoplasm (p<0.01), a reduced absolute volume of SER which is appropriate for the reduced cytoplasmic volume, and an enlarged mitochondrial volume (p<0.005), which is even more apparent when corrected for the reduced cytoplasmic
volume of the Leydig cells of the alcohol-fed animals. These morphometric changes in Leydig cells induced by alcohol feeding are similar to those found in hepatocytes of animals fed alcohol and in human alcohol abusers [2, 4, 15, 16, 19, 29, 30]. Thus, the morphometric alterations produced in one organ, the liver, which is a widely accepted target for ethanol toxicity, are produced in yet another target of ethanol's toxicity, the Leydig cells. These data are consistent also with the biochemical data that suggest that the redox state of Leydig cells of alcoholfed animals is reduced as is known to occur in the liver, and that microsomal enzyme activities of the testes of alcoholfed animals are adversely affected as a result of ethanol feeding [5, 17, 23, 33]. Thus, they provide circumstantial evidence in support of a common mechanism for ethanolinduced toxicity within the liver (as reported by others) and the testes (herein reported) at the morphologic level. Relevant to this later issue, it is of interest that both the liver and the testes contain an alcohol dehydrogenase which, in the testes, appears to be located within Leydig cells [35]. Clearly, much remains to be learned about the specific mechanisms responsible for tissue and/or cellular injuries produced by ethanol abuse. The present data, however, would suggest that similar kinds of metabolic injury, at least at the ultrastructural level, are produced in the testes and the liver as a result of such abuse.
ACKNOWLEDGMENT The authors are grateful to Mr. Robert G. Florida for his valuable technical assistance on electron microscopic preparations.
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347
REFERENCES
1. Baker, H. M. G., H. G. Burger, D. M. DeKretsen, A. Dulmanis, B. Hudson, S. O'Connor, C. A. Paulsen, N. Purcell, G. C. Dennie, C. S. Scali, H. P. Taft and C. Wang. A study of the endocrine manifestations of hepatic cirrhosis. C J Med 45: 145178, 1976. 2. Bianchi, L. W. and M. Mihatsch. Mallory bodies and giant mitochondria--two different structures in liver biopsies from alcoholics. Beitr Pathol 150: 298-310, 1973. 3. Bolender, R. P. Correlation of morphometry and sterology with biochemical analysis of cell fractions, lnt Rev Cytol 55: 247-252, 1978. 4. Bruguera, M., A. Bertran, J. A. Bombi and J. Rodec. Giant mitochondria in hepatocytes. Gastroenterology 73: 1383-1387, 1977. 5. Chiao, Y.-B., D. E. Johnson, J. S. Gavaler and D. H. Van Thiel. Effect of chronic ethanol feeding on testicular content of enzymes required for testosterogenesis. Alcoholism: Clin Exp Res 5: 230-236, 1981. 6. Cicero, T. J. and R. D. Bell. Effects of ethanol and acetaldehyde on the biosynthesis of testosterone in the rodent testes. Biochem Biophys Res Commun 94: 814-819, 1980. 7. Cicero, T. J., J. D. Bell, E. R. Meyer and T. M. Badger. Ethanol and acetaldehyde directly inhibit testicular steroidogenesis. J Pharmacol I:\rp Ther 213: 228-233, 1980. 8. Cobb, C. F.. M. F. Ennis, D. H. Van Thiel, J. S. Gavaler and R. Lester. Isolated testes perfusion. Metabolism 29: 71-75, 1980. 9. Distiller, L. A., J. Sagel, B. Dubowitz, G. Kay, P. J. Carr, M. Katz and M. C. Kew. Pituitary gonadal function in men with alcoholic cirrhosis of the liver. Horm Metab Res 8: 461-465, 1976. 10. Ellingboe, J. and C. G. Varanelli. Ethanol inhibits testosterone biosynthesis by direct action on Leydig cells. Res Commun Chem Pathol Pharmacol 24: 87-102, 1979. I I. Galvao-Teles, A., D. C. Anderson, C. W. Burke, J. C. Marshall, C. S. Corker, R. C. Brown and M. L. Clark. Biologically active androgens and estrogens in men with chronic liver disease. Lancet 1: 173-177, 1973. 12. Gordon, G. G., J. Vittek, A. C. Southren, P. Murrary and C. S. Lieber. Effects of chronic ethanol ingestion on the biosynthesis of steroids in rat testicular homogenate in vitro. Endocrinology 106: 1880-1885, 1980. 13. Green, J. R. B. Mechanism of hypogonadism in cirrhotic males. Gut 18: 843-853, 1977. 14. Greenwood, R. C., W. M. Hunter and J. S. Glover. The preparation of t:~q-labeled human growth hormone of high specific activity. Biochern J 89:114-118, 1963. 15. Horvath, E., K. Kovacs and R. C. Ross. Alcoholic liver lesion. Frequency and diagnostic value of fine structural alterations in hepatocytes. Beitr Pathol 148: 67-85, 1975. 16. Iseri, O. A. and L. S. Gottlieb. Alcoholic hyalin and megamitochondria as separate and distinct entities in liver disease associated with alcoholism. Gastroenterology 60: 10271035, 1971. 17. Johnson, D. E., Y.-B. Chiao, J. S. Gavaler and D. H. Van Thiel. Inhibition of testosterone synthesis by ethanol and acetaldehyde. Biochem Pharmacol 60: 1827-1831, 1981. 18. Kent, J. E., R. J. Scaramuzzi, W. Lauwers, A. Parlow, M. Hill, R. Pendari and J. HiUiard. Plasma testosterone, estradiol and gonadotropins in hepatic insufficiency. Gastroenterology 64: 111-115, 1973. 19. Kiessling, K. H., L. Pilstrom and B. Strandberg. Ethanol and the human liver; correlation between mitochondrial size and degree of ethanol abuse. Acta Med Scand 178: 533-535, 1965.
20. Lieber, C. S. and L. M. DeCarli. Quantitative relationship between amount of dietary fat and severity of alcohol fatty liver. Am J Clin Nutr 25: 414-428, 1970. 21. Lipsett, M. B. Physiology and pathology of the Leydig cell. N Engl J Med 303: 682-688, 1980. 22. Morley, J. E. and S. Melmed. Gonadal dysfunction in systemic disorders. Metabolism 28: 1051-1073, 1979. 23. Murono, E. P., T. Lin, J. Osterman and H. R. Nankin. Direct inhibition of testosterone synthesis in rat testis interstitial cells by ethanol. Steroids 36" 615-631, 1980. 24. Neischlag, E. and D. L. Loriaux. Radioimmunoassay for plasma testosterone. Z Klin Chem Klin Bioehem 10: 164-168, 1972. 25. Pentikainen, P. J., L. A. Pentikainen, D. L. Azarnoffand C. A. Dujovne. Plasma levels and excretion of estrogen in urine in chronic liver disease. Gastroenterology 69: 20-27, 1975. 26. Reynolds, E. S. The use of lead nitrate of high pH on an electron opaque stain in electron microscopy. J Cell Biol 17: 208--212, 1963. 27. Rodbard, D. and A. Chrambach. Estimation of molecular radius free motility and valence using polyacrylamide gel electrophoresis. Ann Bioehem 40: 95-105, 1971. 28. Rodbard, D. and J. E. Leward. Computer analysis of radio ligand assay and radioimmunoassay data. In: Proceedings of the Second Symposium on Steroid Assay By Protein Binding. Stockholm: Karolinska Institutet, 1970, pp. 75-103. 29. Rubin, E. The spectrum of alcoholic liver injury, lnt Acad Pathol Monogr 13: 19%127, 1973. 30. Rubin, E. and C. S. Lieber. Alcohol induced hepatic injury in non-alcoholic vonunteers. N Engl J Med 278: 86%876, 1968. 31. Sherins, R. J., J. Vaitukaitus and A. Chrambach. Physical characteristics of hFSH and its desalization products by isoelectric focusing and electrophoresis in polyacrylamide gel. Endocrinology 92: 1135-1140, 1972. 32. Symons, A. M. and V. Marks. The effect of alcohol on weight gain and the hypothalamic-pituitary-gonadotropin axis in the maturing rat. Biochem Pharmacol 24: 955-958, 1975. 33. Van Thiel, D. H., C. F. Cobb, G. B. Herman, H. A. Perez, L. Estes and J. S. Gavaler. An examination of various mechanisms for ethanol-induced testicular injury. Endocrinology 109: 20092015, 1981. 34. Van Thiel, D. H., J. S. Gavaler, C. F. Cobb, R. J. Sherins and R. Lester. Alcohol-induced testicular atrophy in the adult male rat. Endocrinology 105: 888-895, 1979. 35. Van Thiel, D. H., J. S. Gavaler and R. Lester. Ethanol inhibition of vitamin A metabolism in the testes: possible mechanism for sterility in alcoholics. Science 186: 941-942, 1974. 36. Van Thiel, C. H., J. S. Gavaler, R. Lester and M. D. Goodman. Alcohol-induced testicular atrophy. Gastroenterology 69: 326332, 1975. 37. Van Thiel, D. H., R. Lester and R. J. Sherins. Hypogonadism in alcoholic liver disease: evidence for a double defect. Gastroenterology 67:1188-1199, 1974. 38. Van Thiel, D. H., W. D. Williams, J. S. Gavaler, J. M. Little, L. W. Estes and B. S. Rabin. Ethanol: its nephrotoxic effect in the rat. Am J Pathol 89: 67-73, 1977. 39. Watson, M. L. Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem ~),tol 4: 475-478, 1958. 40. Ylikahri, R., M. Huttanen, M. Harkonen, U. Senderling, S. Onikki and S. L. Karonen and H. Aldercreutz. Low plasma testosterone value in men during hangover. J Steroid Biochem 5: 655-658, 1974.
Morphologic Alterations of Rat Leydig Cells Induced by Ethanol J. S. G A V A L E R ,
H . A. P E R E Z , L. E S T E S A N D D. H. V A N T H I E L
University o f Pittsburgh School o f Medicine, Pittsburgh, P A 15261
GAVALER, J. S., H. A. PEREZ, L. ESTES AND D. H. VAN THIEL. Morphoh~gic alterations of rat Leydig cells induced by ethanol. PHARMACOL BIOCHEM BEHAV 18: Suppl. 1,341-347, 1983.--Ultrastructural morphometric studies were employed to compare the morphology of Leydig cells found in the testes of rats fed ethanol, accounting for 36% of total calories for 6 weeks, and those found in the testes of rats isocalorically fed a diet in which ethanol was isocalorically replaced with dextrimaltose. The testes of alcohol-fed rats weighed significantly less (p<0.01) than those of the isocaloric controls. Moreover, plasma testosterone in the alcohol-fed animals was reduced (p<0.01), as compared to the controls. Morphologically, two main differences were encountered between the Leydig cells of alcohol-fed animals and those of the isocalorically-fed controls. These were: (1) an increase in the number of elongated and cup-shaped mitochondria; and (2) an increase in cytoplasmic protrusions in the form of pseudopods. Using morphometric techniques, the Leydig cells of alcohol-fed animals were smaller (p<0.05), had less cytoplasm (p<0.05), larger mitochondria (/9<0.01), and less smooth endoplasmic reticulum IP <0.05) than did those of the isocaloric controls. These morphologic characteristics of Leydig cells of alcohol-fed animals are similar to those reported to occur in the liver and suggest that the biochemical mechanisms responsible for alcohol-induced cellular injury are similar in the testes and the liver. Leydig cells
Alcohol
Testes
Testosterone
Ethanol
Gonadal toxicity
L E Y D I G cells comprise the endocrine compartment of the testes and are responsible for the synthesis and secretion of the male sex hormone, testosterone. Until recently, it was assumed that Leydig cells were relatively resistant to the ravages of systemic disease. With the advent of radioimmunoassay resulting in the availability of plasma testosterone measurements, however, it has become apparent that many systemic diseases are associated with Leydig cells dysfunction [22]. Among the various toxins known to adversely affect Leydig cells, ethanol is by far the most important [21]. Thus, ethanol abuse in the absence of cirrhosis has been shown to be assoicated with reduced libido, loss of potency and hypogonadism in men, as well as in experimental animals [l, 9, 1 l, 13, 18, 25, 32, 34, 36, 37, 40]. Moreover, ethanol has been shown to suppress biosynthesis and secretion of testosterone by Leydig cells in vitro [5-8, 10, 12, 17, 23, 33]. Essentially no data exist describing the morphologic changes that occur at the ultrastructural level in Leydig cells as a result of chronic ethanol exposure. Therefore, we examined the morphology of Leydig cells of rats fed alcohol in order to quantitate ultrastructural changes which might correlate with the findings of reduced testosterone synthesis and secretion by these cells.
were pair-fed either an alcohol-containing liquid diet, in which ethanol accounted for 36% of the total calories, or an identical diet in which the ethanol was isocalorically replaced with dextrimaitose (Bioserve, Diet 71 I-A and 71 I-C, Frenchtown, N J) for 6 weeks, at which time they were sacrificed by exsanguination and autopsied. The testes were removed, decapsulated, weighed and sectioned into cubes l x l mm. Tissue cubes were fixed in 3% glutaraldehyde (Ladd Research Industries, Inc., Burlington, VT) in 0.1 M cacodylate buffer (pH 7.4) for 1 hour at 4°C. After primary aldehyde fixation, samples were washed for 1 hour in 0.1 M cacodylate buffer and post-fixed in I% osmium tetroxide in 0.1 M cacodylate buffer for 2 hours at room temperature. After fixation, the tissue was rinsed, dehydrated in a graded ethanol series, and embedded in Epon-Araldite [24]. Thin sections with a gold interference were cut with glass or diamond knives on a Sorvall MT 1 microtome (Ivan Sorvail, Inc., Norwald, CT), mounted on scored 200 to 300 mesh grids, and stained with 3% uranyl acetate [39] and lead nitrate [26]. Grids were examined and photographed at 60 kV with an electron microscope (Phillips Electronic Instruments, Mount Vernon, NY), using a double condenser system.
METHOD
To guarantee that sampling was random, fields were examined in a predetermined sequence in the four corners and center of the grid squares. From each grid, five photomicrographs of Leydig cells at an original magnification of x2,970, x5,300 and x 10,800 were taken. Prints were made at an enlargement of 2.65. The high power photomicrographs
Morphometric Analysis
Animals Weanling male rats aged 20 days were obtained from Charles River Breeding Laboratories, Wilmington, MA. They were individually caged after pairing for body size, and
341
342
GAVALER ET AL.
FIG. 1. Conspicuous clusters of Leydig cells were located mainly in close contact with blood vessels. Original magnification 2970x; final magnification, 6900×. KEY TO FIGUREABBREVIATIONS AER BV Ch Co FC GC ICS ID IT
Agranular endoplasmic reticulum Blood vessel Chromatin Collagen Fusiform cell Golgi complex Intercellular space Interdigitation Interstitium
were used for determination of volume densities of smooth endoplasmic reticulum and mitochondria. All measurements were made using a Graphic Tablet System ® attached to an Apple II computer (Apple Computer, Inc., Cupertino, CA 95014). A sheet of transparent acetate was placed upon each photomicrograph to prevent electrostatic interference and the detecting pen was carefully passed upon each of the Leydig cells and their organelles. The data thus obtained was interpreted by the computer and translated into square and cubic microns. Prior to use, the graphics tablet was standardized using appropriate test points (standards). To demonstrate that the number of measurements available for any given parameter satisfied requirements of sampling adequacy for sterological analysis, the method of Bolender was used [3]. Using this method, the adequacy of the number of measurements was determined by grouping data from increasing numbers of samples until the standard error of the mean achieved a plateau at a level less than 10% of the mean. For each set of measurements used in the analyses presented here, the number available exceeded the number required to satisfy this criterion.
LD
Lipid droplet
LY
Lysosome Mitochondria Microvilli Nucleus N.ucleolus Nuclear envelope Red blood cell
MV N Nc NE RBC
samples were measured in a single assay. The intra-assay variation for known standards was less than 8%. The detection limit was 0.1 ng. Plasma gonadotropins were measured utilizing specific radioimmunoassays and reagents supplied by the NIAMDD, Rat Pituitary Hormone Distribution Program. NIAMDD Rat FSH-RP-i and NIAMDD Rat LH-RP-I were used as reference preparations. The hormones were iodinated with ~:'I using the chloramine-T method [14]. Iodinated hormones were purified by polyacrylamide gel electrophoresis using 9% acrylamide with 2% cross-linking [27]. Other details of the assays have been described previously [31]. The detection limit for both assays was 4.0 ng with an intra-assay variation of less than 8%. Potency estimates were calculated using the computer program of Rodbard [28]. Statistical Analysis Results were analyzed using the paired t-test. Differences were considered to be significant at a p value <0.05. RESULTS
Hormone Determinations Plasma testosterone [24] was determined in duplicate using a radioimmunoassay established in our laboratory. All
Gross Anatomy The testes of the alcohol-fed animals weighed 1.47-+0.32 g
ETHANOL-INDUCED TESTICULAR ALTERATIONS
343
FIG. 2. Electronmicrograph of rat Leydig cells (control group). There is a single, large, excentric nucleus with a dense rim of chromatin close tothe nuclear envelope, and a prominent nucleolus. Original magnification 2970x; final magnification, 9636x. Low magnification micrographs such as this were used for determinations of surface densities of whole cells and nuclei. Disrupted Leydig cells, such as the one seen on the left half, were not considered for morphometry.
(mean_+ SEM), which was 50% less than that of the isocaioric controls (2.98_+0.17 g) (p<0.01).
Light Microscopy As previously reported, the testes of the alcohol-fed animals contained smaller seminiferous tubules with a reduced number of germ cells, compared to those of the isocaloric controls [34]. The Leydig cells of the alcohol-fed animals appeared to be more numerous per unit area than those of the isocaloric controls. This apparent hyperplasia may reflect the presence of normal or possibly even reduced total numbers of Leydig cells in a reduced testicular volume.
Electron Microscopy Ultrastructural examination of rat interstitial gonadal tissue disclosed the presence of loose connective tissue with a population of polygonal, fusiform, and clear cells, and a rich network of blood vessels (Fig. 1). The large, polygonal cells (Leydig cells) were found to be scattered or in strands or, more often, irregularly grouped in the interstitum around capillaries. Fusiform cells were closely associated with the tubular wall or in the adventitia of the blood vessels. Lymphatic vessels were not conspicuously present, probably due to the fact that fixation of the testes was not accomplished by perfusion. Leydig cells had a single, large, excentric nucleus with a dense rim of chromatin attached to the innter membrane of the nuclear envelope. The prominent nucleolus was composed of a distinct pars amorpha and pars reticularis (Fig. 2). Leydig cells were intimately associated with each other. Contiguous cells were separated by a regular space. Leydig cells from alcohol-fed rats exhibited numerous microvilli varying in length and shape, protruding into the free intercellular space or interdigitating with microvilli of neighboring interstitial cells (Fig. 3). This finding was not encountered in testes of normal or isocalorically-fed rats. Mitochondria, varying in size and shape, were present in
moderate numbers of Leydig cells from control rats. The inner structure of these mitochondria consisted mainly of quite numerous tubular cristae of uniform size. The mitochondrial matrix was moderately dense, and occasionally small, dense granules were encountered (Fig. 4). Cupshaped and elongated mitochondria were most conspicuous in Leydig cells of alcohol-fed rats (Fig. 5). Membranes of the Golgi complex were present as flattened cisternae and associated small vesicles in a single, concentrated form, most often juxtanuclear (Fig. 5). A granular endoplasmic reticulum was characteristically found in all the interstitial cells. Also characteristic was the segregation of this reticulum in areas not occupied by other organelles (Fig. 6).
Endocrine Studies Plasma testosterone concentrations were reduced in the alcohol-fed animals (2.4_+0.2 ng/ml) compared to those in the isocaloric control animals (4.7+_0.08) (p<0.01). Plasma FSH levels in the alcohol-fed animals (761 _+133 ng/ml) were twice those of the isocaloric controls (378-+57 ng/ml) (p<0.05). In contrast, and despite the marked reduction in testosterone levels present in the alcohol-fed animals, LH levels did not differ between the two groups (alcohol-fed 50.8_+4.6 ng/ml vs. 59.2_+4.1 ng/ml for the isocaloric controls).
Morphometric Analysis When evaluated using morphometric techniques, the Leydig cell volume of the alcohol-fed animals was less (2071.0_+117.0 pt:~) than that of the isocaloric controls (2382.5_+148.5) (/)<0.05). In contrast, no difference in Leydig cell nuclear volume was seen between the two groups (alcohol-fed 786.5_+57.5 p2 vs. 846.5_+65.0 for the isocaioric controls). As a result, the cytoplasmic volume of Leydig cells of alcohol-fed animals was less (1280.5_+71.5 g:3) than that of the isocaloric controls (1535.5_+99.5) (,0<0.05). The volume of smooth endoplasmic reticulum (SER) was re-
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FIG. 3. Low magnification electron micrograph of rat Leydig cells (alcoholic group). The plane of section does not pass through the nuclei of some cells. The cells are intimately associated with each other, separated in areas by a regular space. At points where the surface of adjacent Leydig cells contact with other or surrounding connective tissue, microvilli of varying length, shape, and number protrude into the surrounding free space or interdigitate with microvilli of adjacent Leydig cells. These cytoplasmic protrusions were included in determinations of cytoplasmic surface densities. Note also the increase in number of mitochondria in Leydig cells of alcohol-fed animals which is apparent, when these cells are compared to those of the controls shown in Figs. I and 2.
FIG. 4. Intermediate magnification electron micrograph of rat Leydig cell (isocaloric control group). Original magnification 4210×; final magnification, 10130×. Micrographs at this higher magnification were used for determinations of surface densities of mitochondria.
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FIG. 5. High magnification electron micrograph of rat Leydig cells (alcoholic group). Original magnification 5150× final magnification, 17510×. Abundant numbers of mitochondria are present, exhibiting different sizes and shapes. The inner mitochondrial membrane exhibits lamellar cristae, tubular projections, and a moderately dense mitochondrial matrix containing numerous dense granules. Note also the juxtanuclear Golgi complex with flattened cisternae and associated small vesicles, as well as the abundant agranular endoplasmic reticulum. The granular endoplasmic reticulum can be seen as loose aggregations of short cisternae with their associated ribosomes, as well as free ribosomes and polysomes.
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FIG. 6. Electron micrograph of rat Leydig cell (alcoholic group). Original magnification 6790×; final magnification, 2237×. This higher magnification permits accurate delineation of closely packed tubules and cisternae of the agranular endoplasmic reticulum.
duced in the Leydig cells of the alcohol-fed animals (422.0_+27.5 p3 as compared to that of the isocaloric controls (497.5_+37.5) (p<0.05); but, when the volume of SER was corrected for the reduced volume of the cytoplasm, no difference existed between the two groups (alcohol-fed 33.15_+0.96% cytoplasmic volume vs. isocaloric control 32.74_+1.05). In contrast, the mitochondria of the Leydig cells of alcohol-fed animals were larger (225.0_+ 15.0 p3) than those of the isocaloric controls (161.0_+13.5) (o<0.005). Moreover, when corrected for the reduced cytoplasmic volume, this increase in mitochondrial volume was even more apparent (alcohol-fed 17.83_+0.77% cytoplasmic volume vs. isocaloric controls 10.28_+0.55) (p<0.005). DISCUSSION The present study confirms earlier reports of (i) testicular atrophy and reduced testosterone levels in alcohol-fed rats compared to isocaloric controls, (2) similar LH concentrations, despite the severe testicular injury present in the alcohol-fed animals and (3) increased FSH levels (p<0.05) in alcohol-fed animals compared to isocaloric controls, as might be expected as a result of the severe germ cell injury manifested by seminiferous tubule atrophy [34,36]. Further, the present study extends earlier studies by providing morphometric data documenting alcohol-induced Leydig cell injury. Thus, compared to isocaloric controls, the Leydig cells of alcohol-fed animals are smaller (p<0.01), have tess cytoplasm (p<0.01), a reduced absolute volume of SER which is appropriate for the reduced cytoplasmic volume, and an enlarged mitochondrial volume (p<0.005), which is even more apparent when corrected for the reduced cytoplasmic
volume of the Leydig cells of the alcohol-fed animals. These morphometric changes in Leydig cells induced by alcohol feeding are similar to those found in hepatocytes of animals fed alcohol and in human alcohol abusers [2, 4, 15, 16, 19, 29, 30]. Thus, the morphometric alterations produced in one organ, the liver, which is a widely accepted target for ethanol toxicity, are produced in yet another target of ethanol's toxicity, the Leydig cells. These data are consistent also with the biochemical data that suggest that the redox state of Leydig cells of alcoholfed animals is reduced as is known to occur in the liver, and that microsomal enzyme activities of the testes of alcoholfed animals are adversely affected as a result of ethanol feeding [5, 17, 23, 33]. Thus, they provide circumstantial evidence in support of a common mechanism for ethanolinduced toxicity within the liver (as reported by others) and the testes (herein reported) at the morphologic level. Relevant to this later issue, it is of interest that both the liver and the testes contain an alcohol dehydrogenase which, in the testes, appears to be located within Leydig cells [35]. Clearly, much remains to be learned about the specific mechanisms responsible for tissue and/or cellular injuries produced by ethanol abuse. The present data, however, would suggest that similar kinds of metabolic injury, at least at the ultrastructural level, are produced in the testes and the liver as a result of such abuse.
ACKNOWLEDGMENT The authors are grateful to Mr. Robert G. Florida for his valuable technical assistance on electron microscopic preparations.
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