J. Anat. (1986), 146, pp. 173-183 173 With 13 figures Printed in Great Britain Ultrastructure of Leydig cells in human testes* RICARDO PANIAGUA, PEDRO AMAT, MANUEL NISTAL AND AGUSTIN MARTIN Department ofCytology and Histology, Faculty ofBiology, University of Salamanca, Department ofElectron , University Clinical Hospital, Salamanca and Department ofMorphology, School of Medicine, Autonomous University of Madrid, Spain (Accepted 19 September 1985) INTRODUCTION With advancing years, ageing in somatic tissues is usually associated with a decline in testicular function (Steams, MacDonnel, Kaufman & Padua, 1974; Davidson et al. 1983). Endocrine changes have their counterpart in structural alterations. Mean testis length and volume (Stears et al. 1974) and quality (Schwartz, Mayoux & Spira, 1983) seem to decrease. The seminiferous epithelium shows different degrees of maturation arrest of (Holstein & Hubmann, 1980). The Sertoli cells show several structural alterations (Schulze & Schulze, 1981; Paniagua, Amat, Nistal & Martin, 1985). An increase (Kothari & Gupta, 1974) as well as a decrease (Harbitz, 1973; Kaler & Neaves, 1978) in total Leydig volume and number, together with a diminished response of these cells to human chorionic gonadotropin stimulation (Nankin, Lin, Murono & Osterman, 1983), have been reported in elderly men. The present paper reports several morphologically abnormal types of commonly found in the testes of elderly men which suggest that a process of involution could take place in these cells.

MATERIALS AND METHODS Testicular biopsies were obtained approximately one to two hours after death from 25 young adult (from 21 to 46 years of age) and 42 elderly men (from 65 to 89 years of age) who had not suffered from testicular, endocrine or related pathological diseases including varicocoele. In order to compare hormonal effects and ultra- structural patterns, orchidectomy specimens were obtained from 26 patients (from 66 to 82 years of age) suffering from prostatic carcinoma who had not previously received hormone or chemotherapy treatments nor were suffering from testicular disease. Testosterone, luteinising hormone (LH) and follicle-stimulating hormone (FSH) serum levels were measured by radioimmunoassay in these patients before orchidectomy. All the subjects studied showed proven fertility. Workers dealing with toxic agents were excluded. For light microscopy, the specimens were fixed in Bouin's fluid and embedded in paraffin wax. Sections 6 pm thick were stained with haematoxylin and eosin, periodic acid-Schiff and the van Gieson stain. For electron microscopy, the speci- * Reprint requests to Professor Dr Ricardo Paniagua, Departamento de Citologia e Histologia, Facultad de Biologia, 37008-Salamanca, Spain. 174 R. PANIAGUA AND OTHERS mens were fixed in 4 % cacodylate buffered glutaraldehyde, postfixed in veronal buffered 1% osmium tetroxide, and embedded in Araldite. Sections 1 mm thick were stained with toluidine blue. Ultrathin sections were double stained with uranyl acetate and lead citrate and examined in a Philips 201 electron . The percentage distribution of Leydig cell types was calculated on 20 low magni- fication electron microscopic fields (648 /m2) from each testis. Electron micrographs were taken at a magnification of x 2000, augmented to x 10000. Since individual differences were very marked, only the total mean for each group (young adults and elderly men) was calculated.

RESULTS The biopsies from the young adults revealed a normal testicular pattern with seminiferous showing complete spermatogenesis and a normal collagen content of the tunica propria and interstitium. The Leydig cells were found in small clusters and most of them exhibited the normal ultrastructural pattern. Each had a spherical euchromatic nucleus with a prominent nucleolus and a peripherally placed band of heterochromatin. Nuclear pores were numerous. The was almost filled by a tubulovesicular meshwork of agranular . The mitochondria were numerous and had characteristic tubular cristae. Both shape and outline, as well as the electron density of the matrix, varied widely. Small, granular, electron-dense mitochondrial inclusions were frequent. The Golgi complex was inconspicuous and the granular endoplasmic reticulum sparse. Primary and second- ary lysosomes, principally lipofuscin granules, were a frequent finding. inclusions were usually present. Many but not all cells contained a single large Reinke crystal, which in some cells consisted of either a single or several subunits. In a few cells without Reinke crystals paracrystalline inclusions were seen. Some ultra- structurally abnormal Leydig cells, like those that will be described in ageing testes, could be seen in small numbers (Table 1). The appearance of seminiferous tubules from elderly men varied widely among testes in both postmortem biopsies and orchidectomy specimens and, thus, these differences were independent of the source of the material. Whereas some testes showed complete spermatogenesis, others exhibited maturation arrest at the level of , or spermatogonia. Tubules lined only by Sertoli cells were also seen. The tunica propria appeared either normal or thickened. Areas of sclerosed tubules were occasionally encountered. Individual differences with regard to the ultrastructural pattern of Leydig cells were also marked. The Leydig cell types most frequently found were (1) Leydig cells which had a normal, mature ultra- structural appearance, like those reported in young adult testes (Fig. 1). (2) Leydig cells with abnormalities in the number or location of crystalline inclusions. These cells showed a normal cytoplasm and differed from the afore- mentioned normal Leydig cells only in the presence of multiple cytoplasmic Reinke crystals (Fig. 2), intranuclear Reinke crystals (Fig. 3), or numerous cytoplasmic (Fig. 4) or intranuclear (Fig. 5) paracrystalline inclusions corresponding to the types reported by Payer (1980). (3) Multivacuolated Leydig cells with the cytoplasm almost filled by lipid droplets (Fig. 6). Cytoplasmic organelles, including agranular endoplasmic reticulum, were scarce. The mitochondria were small and decreased in number. The nuclei were more irregular and darker than those of normal Leydig cells. Some multivacuolated Leydig cells in human ageing testes 175 !Et110g |W SE ii 2 iig S ! «i > >1

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Leydig cells exhibited numerous Reinke crystals or paracrystalline inclusions (Fig. 7). (4) Leydig cells were also present which had poorly developed agranular endo- plasmic reticulum and mitochondria (Figs. 8, 9). These cells usually showed numer- ous lipofuscin granules. In the most advanced stages of dedifferentiation, the nucleolus was small or apparently absent (Fig. 9). Some cells exhibited numerous paracrystalline inclusions or Reinke crystals (Fig. 8). (5) Bi- or trinucleate Leydig cells. Although some of these cells exhibited a normal ultrastructural appearance (Fig. 10), most of them showed poorly developed agranular endoplasmic reticulum and small mitochondria, and were similar to the dedifferentiated Leydig cells. Reinke crystals or paracrystalline inclusions were frequent (Figs. 11, 12). The percentage distribution of Leydig cell types in young adults and elderly men is shown in Table 1. The percentages were similar in both postmortem biopsies and orchidectomy specimens. Hormone assays performed in orchidectomised patients revealed normal (14 patients), slightly increased (4 patients) or elevated (8 patients) LH levels. FSH levels were increased in 14 patients who also exhibited increased LH levels. Testo- sterone levels were normal in 17 patients, and diminished in 9 patients who also showed elevated gonadotropin levels. These 9 patients showed a larger number of altered Leydig cells compared with those with normal testosterone and gonadotropin levels. Leydig cells in human ageing testes 181

DISCUSSION The present results indicate that with advancing age the Leydig cells undergo alterations and that, in addition to structurally normal Leydig cells, other abnormal Leydig cell types appear. Although individual differences are marked, there is a correlation between the proportion of altered Leydig cells and the decrease in testosterone and increase in LH levels. The most frequent Leydig cell alteration consists of poor development of the endoplasmic reticulum and mitochondria. These cells differ from the immature Leydig cells found in normal testes and in several pathological conditions (Paniagua, Nistal & Bravo, 1984), and show the characteristic shape and nuclear pattern of mature Leydig cells, though the organelles principally involved in steroid bio- synthesis are sparse. In addition, many of these cells show increased amounts of lipofuscin granules characteristic of aged cells. These cells may be regarded as dedifferentiated cells in which the steroidogenic function is diminished. Such a dedifferentiation differs from that observed after long term oestrogen or stil- boestrol administration (Schulze, 1984), since many characteristics of Leydig cells which are seen after these treatments, such as infolded nuclei, dilated cisterae of agranular endoplasmic reticulum and numerous lipid droplets, are lacking in this group ofageing Leydig cells. Another abundant cell type was the multivacuolated, lipid-loaded Leydig cell. These cells have been observed in mammalian testes undergoing seasonal involution (Belt & Cavazos, 1971), mouse testes after drug-induced suppression of gonado- tropins (Aoki & Massa, 1975), rat testes after exposure to heat (Damber, Bergh & Hanson, 1980), and in human testes in normal or pathological conditions (Paniagua et al. 1984). Lipid accumulation has been explained in terms of free cholesterol, not utilised for hormone synthesis, leaving the agranular endoplasmic reticulum and, after esterification, being stored in lipid droplets (Aoki & Massa, 1975). Such an increase in is considered as a sign of functional impairment (Schulze, 1984), and multivacuolated Leydig cells are probably a form of cell involution. The presence of a considerable proportion of bi- or trinucleate Leydig cells is another characteristic of ageing testes. The appearance of these cells varied from those with normal cytoplasm to those which were multivacuolated or dedifferentiated. The proportion of normal altered Leydig cells was higher in mononucleate than in multinucleate Leydig cells, suggesting that occurrence of several nuclei accompanies Leydig cell involution with age. Multinucleation in ageing testes is not exclusive to Leydig cells, since multinucleate Sertoli (Schulze & Schulze, 1981) and germ cells (Holstein & Hubmann, 1980) have been reported in elderly men. The causes are still unknown and no evidence points to the occurrence either of karyokinesis without cytokinesis or of cell fusion, since both increase (Kothari & Gupta, 1974) as well as decrease (Kaler & Neaves, 1978) in the Leydig cell population with advancing age has been reported. Abnormalities in crystalline inclusions are not distinctive features of ageing testes. Cytoplasmic or intranuclear paracrystalline inclusions are abundant in some primary testicular disorders, principally in Klinefelter's syndrome (Nistal & Paniagua, 1984), though they have also been reported in normal Leydig cells (Payer, 1980). The importance of crystalline inclusions in ageing testes is due to their elevated number - including both Reinke crystals and paracrystalline inclusions - in comparison to young adult testes. This agrees with Mori et al. (1978) who observed an age-related 182 R. PANIAGUA AND OTHERS increase in the size of Reinke crystals and suggested that crystalline inclusions may be degenerative products rather than constituents necessary for testosterone production. The ultrastructurally abnormal Leydig cells of elderly men differ from those with primary testicular disorders (Nistal & Paniagua, 1984), and from those found after prolonged human chorionic gonadotropin stimulation (De Kretser, 1967). The abnormal Leydig cells of elderly men seem to be involuting cells which have lost the profuse cytoplasmic organelles necessary for testosterone production and which accumulate degenerative products (lipofuscin pigment and, perhaps, crystalline inclusions) and/or unused precursors (lipids). Associated with cell dedifferentiation, aged Leydig cells either undergo or fuse with each other giving rise to multinucleate cells. The decrease in total testosterone levels with advancing age, principally in free testosterone (Royer et al. 1984), could be involved in the involution of the seminiferous epithelium, the increase in gonadotropins and the partial regression ofsecondary characteristics in aged men.

SUMMARY Ultrastructural study of Leydig cells in elderly men revealed the following Leydig cell types: (1) ultrastructurally normal Leydig cells (46-2 %); (2) Leydig cells either with multiple cytoplasmic or intranuclear Reinke crystals or with numerous para- crystalline inclusions (6-1 %); (3) multivacuolated Leydig cells with the cytoplasm almost filled by lipid droplets (16-7 %; (4) dedifferentiated Leydig cells with poor development of agranular endoplasmic reticulum and mitochondria, and increased amounts of lipofuscin granules (22-3 %); and (5) bi- or trinucleate Leydig cells (8-7 %) showing either a normal (2-8 %) or dedifferentiated (5-9 %) cytoplasm. These results suggest an involution of Leydig cells with advancing age. A correlation between the proportion of altered Leydig cells and the decrease in testosterone and increase in luteinising hormone levels could be observed.

This work was supported by a grant from the Comision Asesora de Investigacion Cientifica y T6cnica, Madrid, Spain.

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