THE ANATOMICAL RECORD 209:165-176 (1984) Ultrastructure of Immature Leydig Cells in the Human Prepubertal Testis FREDERICK P. PRINCE Department of Pathology, Children’s Hospital, Columbus, OH 43205 ABSTRACT The cellularity of the human prepubertal testicular interstitium has not been well studied a t the ultrastructural level. In this study, testicular biopsies were obtained from 35 boys aged three to nine years and examined by electron microscopy to clarify and quantitate the cell types present during the prepubertal period. The prepubertal testicular interstitium is found to consist of immature Leydig cells (9%),primitive fibroblastic cells (63%)(intertubular in location), and attenuated peritubular fibroblasts (28%).The primitive fibroblastic cells and peritubular fibroblasts appear closely related, being distinguished mainly by shape and location. The immature Leydig cell type contrasts with the fibroblastic cell types by exhibiting a n irregular nucleus with relatively little heterochromatin. The most impressive cytoplasmic feature is the moderate to extensive development of smooth endoplasmic reticulum in the form of anastamosing tubules. In contrast, the rough endoplasmic reticulum is not well developed. Other cytoplasmic characteristics are the highly developed Golgi elements and occasional lipid droplets and lysosomes. Glycogen is also often present and is generally found in those cells that do not contain a welldeveloped smooth endoplasmic reticulum. The ultrastructure of the immature Leydig cell is compared with that of the mature fetal and adult Leydig cells. Although generally found in small clusters between tubules, these cells are often attenuated and closely associated with the seminiferous tubules. Occasional intermediate cell morphologies suggest a relationship between the primitive fibroblasts and immature Leydig cells. The presence of small cells exhibiting a steroid-producing morphology, classified as immature Leydig cells, in the prepubertal testicular interstitium is a n interesting finding and is in accordance with earlier studies on nonhuman mammals. It is unknown whether these cells are remnants of the fetal Leydig cell population or have differentiated neonatally from the primitive fibroblastic cells. It is suggested that the immature Leydig cells are the progenitors of the adult Leydig cell population. In humans, the Leydig cell development follows a biphasic pattern with two temporally discrete mature Leydig cell populations, fetal and adult. A biphasic development of Leydig cells, although often not as distinct as in the human, is found to be a common phenomenon among mammalian species (e.g., mouse-Pehlemann and Lombard, 1978; rat-Lording and de Kretser, 1972; rabbit-Gondos et al., 1976; rhesus monkey-Van Wagenen and Simpson, 1954). The fetal Leydig cell differentiation has been 0 1984 ALAN R. LISS, INC. correlated with the development of the male ductular system (see review by Gondos, 1980) and with “imprinting” of the hypothalamus (e.g., Abramovich and Rowe, 19731, while the adult Leydig cell population is correlated with pubertal development. The morphology of these two populations of Leydig cells has been described in numerous nonhuman mammals (fetal-Black and Christensen, 1969; Russo and de Rosas, 1971; Bjerregaard Received August 15,1983; accepted November 21, 1983. 166 F.P. PRINCE et al. 1974; Gondos et al., 1974, 1976; Pehlemann and Lombard, 1978; adult-e.g., Connell and Christensen, 1975; Gondos et al., 1976; Mori and Christensen, 1980) and humans (fetal-Pelliniemi and Niemi, 1969; adult-Fawcett and Burgos, 1960; de Kretser, 1967; Christensen, 1970). The fetal Leydig cell population is rather transient in its appearance and has been shown in numerous mammalian species to undergo degeneration or regress to less differentiated cells (Moon and Hardy, 1973; Gondos et al., 1976; Tseng et al., 1975; Van Straaten and Wensing, 1978; Pehlemann and Lombard, 1978).The presence of partially differentiated (immature ) Leydig cells during the period between the fetal and adult Leydig cell populations has been demonstrated in ultrastructural studies of the rabbit (Gondos et al., 1976) and mouse (Aoki, 1970)testis. Histologic studies of human material, however, have not reported the presence of either mature or partially differentiated Leydig cells during the period from the first year of life until puberty. These studies generally support fetal Leydig cell degeneration (Sniffen, 1950; Charney et al., 1952; de la Balze et al. 1960; Pelliniemi and Niemi, 1969; Vilar, 1970; Hayashi and Harrison, 1971). The evidence, however, is not always convincing and some authors have suggested regression is present to some degree (Ottowicz, 1963; Mietkiewski, 1966). Ultrastructural studies of fetal Leydig cell development in humans have concluded that the “majority” of fetal Leydig cells degenerate in the late fetal period (Pelliniemi and Niemi, 1969; Pelliniemi et al., 1980). Although there has been much controversy regarding the fate of the fetal Leydig cell population, as well a s concerning the possibility of partially developed Leydig cells being present during the period between the fetal and adult Leydig cell populations, the prepubertal period in humans has been given little attention in the ultrastructural literature. In this study, a large series of testicular biopsies was obtained from boys aged three to nine and examined by electron microscopy to clarify and quantitate the cell types found in the testicular interstitium during the prepubertal years. MATERIALS AND METHODS Bilateral testicular biopsies were obtained at Children’s Hospital (Columbus, Ohio) from prepubertal boys in leukemic remission for the purpose of establishing the presence or absence of leukemic infiltration. In 35 prepubertaI cases found free of leukemic celIs the interstitial components were examined for normal constituents. Sampling and Tissue Preparation Observation of large tissue sections (up to 5 mm x 12 mm) prepared for histologic examination and stained with hematoxylin and eosin reveals a homogeneous distribution of interstitial cells in the prepubertal testis. Due to this homogeneous distribution, it was not difficult to obtain a representative sample. A contiguous segment of the biopsy was prepared for electron microscopy. This specimen was diced and fixed by immersion in 2.5% glutaraldehyde. Following the primary fixation, the tissue was post-fixed in 1%osmium tetroxide, dehydrated in graded alcohols, and embedded in epon 812 or Medcast. In each case, 10 to 20 blocks were thick sectioned and stained with toluidine blue. Of these, two to six blocks were thin sectioned. Thin sections were stained with uranyl acetate followed by lead citrate and viewed with a Hitachi HS-7 (50 kV) electron microscope. Subjects These patients had undergone previous chemotherapy, including vincristine, prednisone, methotrexate, and L-asparaginase. These chemotherapy regimens have been shown, in some instances, to cause variable tubular necrosis, a decrease in the Tubular Fertility Index, thickening of the basal lamina of the tubules, and variable interstitial fibrosis (Lendon et al., 1978; Marboe et al., 1982).No damage has been found to interstitial cells during chemotherapy. Leydig cell function, assessed by luteinizing hormonereleasing hormone and human chorionic gonadotrophin stimulation tests, has been shown to remain normal after chemotherapy (Shalet et al., 1981). Quantitation In 15 cases free of leukemic infiltration and also without obvious fibrosis, tubular necrosis, or tubular basal lamina thickening, the relative percentage of each cell type comprising the interstitial area was quantitated. To determine a relative percentage of each of the three cell types, specimens (two to four representative blocks per subject) were viewed on the screen of the electron microscope, and all interstitial cells present on the 167 LEYDIG CELLS IN HUMAN PREPUBERTAL TESTIS TABLE 1. Distribution of cell types during prepubertal period Age group 3-4 Subjects (N) Mean No. cells/N Primitive fibroblastic cells Peritubular fibroblasts Immature Levdie cells 5-6 5 5 612 65.1 (4.9) 27.0 (4.7) 7.9 (1.9) 568 62.5 (2.4) 27.6 (2.0) 9.8 (1.9) 7-8 3-8 5 15 545 63.3 (2.1) 28.1 (1.9) 454 62.3 (4.0) 29.8 (3.5) 7.8 (1.0) 8.6 (0.9) Values are given as mean percentaee with standard error in parenthesis. No significant differences are found between age groups. section were classified and tabulated. Only cells with a nucleus in the plane of section were included. Sections were examined until approximately 500 cells (mean, 545) were counted per subject. Thus the quantitative results are based on examination of 8,200 interstitial cells. Schwann cells, macrophages, and mature lymphocytes were very infrequent and insignificant in terms of the quantitation. The subjects were divided into three age groups (ages three to four, five to six, and seven to eight) with five subjects per group. The results are summarized in Table 1. The data were analyzed by analysis of variance, as well as Student's t-test. RESULTS Histologic examination of the prepubertal testis reveals a n interstitial region containing numerous cells distributed in a homogeneous manner. There appear to be no distinctive cell subtypes with the exception that some cells are attenuated and closely associated with the seminiferous tubules (Fig. 1). Occasional relatively plump cells with prominent nucleoli are also apparent, often being found in small clumps. This histologic pattern is constant in all ages studied, with the exception that larger plump cells are more prominent in the biopsies from the older boys. Plastic embedded tissue processed for electron microscopy presents better histologic detail when thick sectioned and stained with toluidine blue. With this preparation, the cells comprising the clumps, as well as isolated cells, are rather irregular in shape and also exhibit irregular nuclei. Vacuoles are commonly seen in the cytoplasm (Fig. 2). At the ultrastructural level, three cell categories can be distinguished, and are designated as 1) immature Leydig cells, 2) primitive fibroblastic cells (intertubular in location), and 3) peritubular fibroblasts. The immature Leydig cells and the fibroblastic cells (both intertubular and peritubu- lar) are readily distinguished from one another a t low magnification by their contrasting nuclear features (Fig. 3). The nucleus of the immature Leydig cell is very irregular and exhibits relatively little heterochromatin, whereas the nucleus of the primitive fibroblast and peritubular fibroblast is regular in contour and contains a greater amount of heterochromatin. The fibroblastic cells are segregated into two classifications based on location and morphology. The intertubular fibroblasts tend to be rather primitive in morphology, generally containing numerous free ribosomes (Fig. 4).Their shape is variable, with fusiform, rounded, and irregular profiles present. The rough endoplasmic reticulum (RER) is often moderately developed and bundles of filaments are not infrequent. The peritubular fibroblasts are attenuated and in close association with the seminiferous tubules. These cells contain relatively few free ribosomes and are characterized by numerous filaments (Fig. 5), as well as RER. It is apparent that there is a continuum of cell morphologies relating these two fibroblastic cell categories. The immature Leydig cells are variable in shape and contrast with the fibroblastic cells in cytoplasmic characteristics, as well as the nuclear differences. The most impressive cytoplasmic feature is the presence of moderate to extensive elements of smooth endoplasmic reticulum (SER) in the form of anastamosing tubules (Figs. 4,6-9). A vesicular appearance of the SER is rarely observed. The RER is relatively inconspicuous. The Golgi apparatus is well developed, and mitochondria are moderately abundant. Mitochondria1 cristae are of the flattened type. A number of cytoplasmic inclusions are frequently found in the immature Leydig cell, including lipid droplets, myelin figures, and lysosomes. Glycogen is often a prominent feature of these cells, most notably in those that do not contain a well-developed SER (Fig. 6). Reinke 168 F.P. PRINCE Fig. 1. Prepubertal testis at three (A), five (B), and eight (C) years. The interstitial cellularity is similar throughout these years, exhibiting numerous cells of rather diverse shape. Attenuated cells in a peritubular location are observed (arrows). Also, occasional rela- tively plump cells with prominent nucleoli are found (chevrons). These plumper cells are often found in small clumps and are more apparent in the eight and 9-yearold boys. H & E x500. LEYDIG CELLS IN HUMAN PREPUBERTAL TESTIS Fig. 2. Plastic-embedded preparation indicating the presence of vacuoles (arrow) in the cytoplasm of some interstitial cells. Toluidine blue x 530. 169 Fig. 3. Low-power electron micrograph demonstrating the nuclear differences between the primitive fibroblastic cells (three upper cells) and the immature Leydig cells (three lower cells.) ~7,800. Fig. 4. A primitive fibroblast (top) and an immature Leydig cell in the interstitium. Free ribosomes (chevron) are often a prominent feature of the primitive fibroblasts. Rough endoplasmic reticulum (wide arrow) is also a common cytoplasmic component. The oustanding cytoplasmic feature of the immature Leydig cell type is the extensive network of tubular elements of smooth endoplasmic reticulum (long arrow). x 14,400. Fig. 5. An attenuated peritubular fibroblast exhibiting a dense accumulation of filaments (wide arrow). Cytoplasmic extensions from other peritubular fibroblasts are present, and exhibit rough endoplasmic reticulum. large arrowhead-basal lamina of seminiferous tubule. x 11,900. Fig. 6. Two immature Leydig cells of prepubertal testis. A prominent nucleolus (N) is present. The cytoplasm exhibits numerous profiles of smooth endoplasmic reticulum (arrowheads)and weI1-developed Golgi regions (G). Glycogen deposits (large arrowhead) are also present. Glycogen is generally associated with those cells that do not contain an extensive development of the smooth endoplasmic reticulum. x 13,900. Fig. 7. High magnification of cytoplasm of an immature Leydig cell illustrating the typical cytoplasmic features of this cell type. Arrowhead, profile of smooth endoplasmic reticulum; G, Golgi region; L, lysosome; Li, lipid droplet. x28,200. 172 F.P. PRINCE Fig. 8. A cell classified as a n immature Leydig cell due to the presence of smooth endoplasmic reticulum Oong arrows), but that also exhibits features more typical of the primitive fibroblastic cell type, e.g., numerous ribosomes (chevron), a relatively prominent development of the rough endoplasmic reticulum (wide arrow) and relatively prominent heterochromatin. X 13,700. crystals are absent. The majority of the immature Leydig cells are found in the “intertubular” areas, often in clusters; however, many are attenuated and closely associated with the seminiferous tubules pig. 9). It is apparent that the more prominent immature Leydig cells correspond to the plump cells seen in the histologic preparations. Cell profiles that possess nuclear and cytoplasmic features intermediate between the primitive fibroblastic cell and immature Leydig cell are encountered (Fig. 8). These cells are included in the immature Leydig cell group based on the presence of elements of SER in their cytoplasm. The quantitative results indicate stable cell populations through the ages of three to eight with the primitive fibroblastic cells comprising approximately 63%of the interstitial cell population, peritubular fibroblasts 28%, and immature Leydig cells 9%(Table 1).Schwann cells and mature lymphocytes are infrequently found and are insignificant in terms of cell quantitation. DISCUSSION The consensus of the histologic literature on human testicular development is that Leydig cells, either mature or partially developed, do not exist during the prepubertal period. In the late prepubertal period, an in- LEYDIG CELLS IN HUMAN PREPUBERTAL TESTIS Fig. 9. An attenuated immature Leydig cell closely associated with a seminiferous tubule (ST).Large arrowhead, basal lamina of seminiferous tubule; arrowheads, 173 profiles of smooth endoplasmic reticulum; L, lipid. ~21,200. 174 F.P. PRINCE creasing heterogeneity in cellularity has been described, prompting classification schemes for different “types” of fibroblasts (e.g., Tillinger et al., 1955; de la Balze et al., 1960; Vilar, 1970). The majority of these studies report that Leydig cells begin development a t approximately 11 to 14 years of age (Sniffen, 1950; Charney et al., 1952; Mancini et al., 1952; de la Balze et al., 1960; Vilar, 1970). The earliest evidence of the “reappearance” of small Leydig cells has been a t approximately eight years of age (Hayashi and Harrison, 1971). In the present study, infrequent, relatively plump cells (although quite small compared with a mature Leydig cell) with prominent nucleoli are apparent in the histologic material throughout the prepubertal ages studied. The prepubertal testis has been given little attention by electron microscopists. Previous ultrastructural studies are not in agreement as to the presence or absence of partially developed Leydig cells during this period. Leeson (1966) and Vilar (1970) report only primitive fibroblastic cells during the prepubertal years. Nistal and Paniagua (1979) give brief comment to the presence of SER in cells from four prepubertal controls in a manuscript that emphasizes the effects of human chorionic gonadotropin on two 26-year-old patients with hypogonadotropic hypogonadism. Hadziselimovic (1977) reported remnants of the fetal Leydig cell population in a one year old and also described Leydig cells in a six year old. The Leydig cells of the six year old are reported to be unlike those of the neonatal specimen, being smaller and having a more prominent nucleolus. His figure also shows a more highly irregular nucleus. Both of these latter studies also report fibroblasts to be present. Nistal and Paniagua (1979)indicate those in association with the tubules are myofibroblastic (not the myoid cell of the adult). The current study of a large series of prepubertal biopsies has clarified the diversity in cell morphology present in the prepubertal interstitiurn and demonstrated a constant cell constituency during this period. The cellularity has been classified into three general types: 1)immature Leydig cells (9%), 2) primitive fibroblastic cells (intertubular location) (63%), and 3) peritubular fibroblasts (28%). The occurrence of partially differentiated Leydig cells in humans during the period between the fetal and adult Leydig cell populations is the most impressive finding and is in accordance with previous studies on nonhuman mammals (Aoki, 1970; Gondos et al., 1977). The major cytoplasmic feature of the immature Leydig cell is the widespread network of tubular elements of SER. The SER has been shown to be of major involvement in the synthesis of steroids (Murota et al., 1965; Christensen, 1969) and is the predominate fine structural feature of the fetal and adult Leydig cell populations (see review by Christensen, 1975). The report of Hadziselimovic (1977) differs from the current study in that it reports a vesicular appearance of the SER. The material in that study was obtained postmortem, however. In studies of the adult Leydig cell it has been shown that vesicular SER is a n artifact of nonoptimal fixation (Christensen, 1975). The morphological description of partially differentiated Leydig cells in the human prepubertal testis is supported by the histochemical findings of Wolfe and Cohen (1964). They examined the activity of glucose-6-phosphate-dehydrogenase (G-6-PD),a n enzyme of the hexose phosphate shunt that has been shown to be important in the production of steroid hormone by supplying NADH. Their findings revealed the presence, during the prepubertal period, of “fibrocyte-like cells in the testicular interstitium which are metabolically related to the mature Leydig cell.” The immature Leydig cells found during the prepubertal years differ from the mature fetal and adult Leydig cells in a number of cytoplasmic features, as well a s by their small size. The irregularity of the nucleus is quite impressive. In contrast, the fetal and adult Leydig cells generally exhibit circular nuclear profiles (Christensen, 1975; Pelliniemi et al., 1980),although a certain degree of irregularity is present in a segment of the mature populations. Although the SER is abundant, it is not as extensive as in the mature Leydig cells. The Golgi apparatus is well developed, as is the case in the mature Leydig cells. Mitochondria of the immature Leydig cells are less pleomorphic than the adult Leydig cells and exhibit flattened cristae, as opposed to tubular cristae. The RER is not found segregated in the cytoplasm, as is the case in the fetal and adult populations. Cellular inclusions, such as lysosomes and lipid are also less apparent. The presence of glycogen is interesting in that it is generally not a feature of the adult Leydig cell population (human-Fawcett and Burgos, 1960; de LEYDIG CELLS IN HUMAN PREPUBERTAL TESTIS Kretser, 1967; Christensen, 1975; non-human-Ichihara, 1970; Gondos et al, 19761, but has been reported in immature Leydig cells found during the prepubertal period in the mouse (Ichihara, 1970). Reinke crystals are not present. These inclusions are a distinctive feature of the adult Leydig cells, but are not found in the fetal Leydig cell population. The description of partially developed Leydig cells during the prepubertal years will stimulate interest into their physiological significance. Although these cells are generally found in small clumps in the intertubular region many are attenuated and intimately associated with the seminiferous tubules, suggesting a functional relationship. The low level of testosterone found in the serum throughout the prepubertal years (August et al., 1972; Winter et al., 1972) may be indicative of production from these cells or perhaps they produce other steroids. The majority of the cells in the prepubertal interstitium do not exhibit a steroid-producing morphology. These cells have been subdivided by location and morphology into two groups, peritubular fibroblasts and primitive fibroblasts (intertubular location). There is a continuum of cell morphologies that relates the intertubular cells to those surrounding the tubules. The rather primitive nature of the intertubular fibroblastic cells of the prepubertal testis has been noted by previous studies. An early histologic study (de la Bake et al., 1960) distinguished a “type a” fibroblast, intertubular in location, of the prepubertal period as different from the inner peritubular fibroblasts and also from the fibroblasts of the adult testis. Previous ultrastructural studies also recognized the primitive nature of these cells and used terminologies such as “primitive fibroblasts” (Leeson, 1966) and ‘Ijuvenile fibroblasts” (Vilar, 1970) to describe them. It is interesting that the [‘undifferentiated mesenchymal” cells described in the eight-week human fetus by Pelliniemi and Niemi (1969) differ from the primitive fibroblasts of the prepubertal period. The fetal cells are more irregular in shape and exhibit less heterochromatin in the nucleus. The peritubular fibroblasts have a more typical fibroblast shape (elongate) and generally do not contain as many free ribosomes as the primitive fibroblasts. These peritubular cells are characterized by RER and numerous filaments. They do not show, however, the extensive filaments and dense 175 plaques of the myoid cells found in the adult peritubular region in humans (personal obervation; Bustos-Obregon and Holstein, 1973; Hermo et al, 1977). The occurrence of cells with a morphology intermediate between the primitive fibroblasts and immature Leydig cells raises the possibility of a relationship between these cell types. This, however, is speculative and will require further study to substantiate. At present, it is best to maintain the terminology “primitive fibroblasts” to describe the primitive cells of the prepubertal interstitium. A cause of confusion in the literature is the presumptive terminology that is occasionally applied to this primitive cell population. Hadziselimovic (1977) described these cells as a “second type of Leydig cell . . . the so-called ‘precursor’ Leydig cell,” referring to the description by Fawcett and Burgos (1960) of fusiform interstitial cells in the testis of mature men. This study indicates a constant cell constituency in the interstitium during the years three to eight. Whether the immature Leydig cell population is a remnant of the fetal Leydig cell population or has developed neonatally from the primitive fibroblastic cells is unknown. A study of early neonatal tissue is necessary to resolve this question. The cell or origin of the adult Leydig cell has been a topic of controversy. Although the consensus of the histologic literature has been that the adult Leydig cells arise from primitive mesenchymal (fibroblastic) cells, the exact nature of these cells has been unknown. The described immature Leydig cells of the prepubertal testis are very likely precursors of the adult Leydig cell population. Pubertal biopsies are being examined to elucidate this maturation. ACKNOWLEDGMENTS Discussion with Dr. Nigel Palmer is appreciated. Also appreciated is the excellent technical assistance of Mrs. Melinda Little in EM preparation and the members of the histology lab (Mr. Louis Wensloff, Mrs. Vera Small, and Mrs. Flora Jaynes) in histologic preparation. LITERATURE CITED Abramovich, D., and P. Rowe (1973) Foetal plasma testosterone levels a t mid-pregnancy and at term: Relationship to foetal sex. J. Endocrinol. 56:621-622. 176 F.P. PRINCE Aoki, A. (1970)Hormonal control of Leydig cell differentiation. Protoplasma 71:209-225. August, G., M. Grumbach, and S. Kaplan (1972) Hormonal changes in puberty: 111. Correlation of plasma testosterone, LH, FSH, testicular size, and bone age with male pubertal development. J. Clin. Endocrinol. 34.319-326, Bjerregaard, B., F. Bro-Rasmussen, and T. Reumert (1974) Ultrastructural development of fetal rabbit testis. 2. Zellforsch., 147:401-413. Black, V., and A. Christensen (1969) Differentiation of interstitital cells and Sertoli cells in fetal guinea pig testes. Am. J. Anat., 124:211-238. Bustos-Obregon, E., and A. Holstein (1973)On structural patterns of the lamina propria of human seminiferous tubules. 2. Zellforsch., 141t413-425. Charny, C., A. Conston, and D. Meranze (1952)Development of the testis. Fert. Steril. 3.461-479. Christensen, A., and S. Gillim (1969) The correlation of tine structure and function in steroid-secreting cells, with emphasis on those of the gonads. In: The Gonads. K. McKerns, ed. Appleton-Century-Crofts, New York, pp. 415-487. Christensen, A. (1970)Fine structure of testicular interstitial cells in humans. In: The Human Testis. E. Rosemberg and c . Paulsen, eds. Plenum Press, New Sork, London, pp. 75-92. Christensen, A. (1975) Leydig Cells. In: Handbook of Physiology, vol. 5, Am. Physiological SOC.,pp. 57-94. Connell, C., and A. Christensen (1975) The ultrastructure of the canine testicular interstitial tissue. Biol. Reprod., 12t368-382. De la Balze, F., R. Mancini, F. Arrillaga, J. Andrada, 0. Vilar, A. Gurtman, and 0. Davidson (1960) Pubertal maturation of the normal human testis: A histologic study. J. Clin. Endocrinol., 20,266-285, De Kretser, D.(1967)The fine structure of the testicular interstitial cells in men of normal androgenic status. 2. Zellforsch., 80:594-609. Fawcett, D., and M. Burgos (1960) Studies on the fine structure of the mammalian testis. 11. The human interstitial tissue. Am. J. Anat., 107:245-269. Gondos, B., D. Paup, J. Ross, and R. Gorski (1974)Ultrastructural differentiation of Leydig cells in the fetal and postnatal hamster testis. Anat. Rec., 178r551-566. Gondos, B., R. Renston, and D. Goldstein (1976) Postnatal differentiation of Leydig cells in the rabbit testis. Am. J. Anat., 145167-182. Gondos, B., K. Morrison, and R. Renston (1977) Leydig cell differentiation in the prepubertal rabbit testis. Biol. Reprod., 17,745-748. Gondos, B. (1980)Development and differentiation of the testis and male reproductive tract. In: Testicular Development, Structure, and Function. A. Steinberger and E. Steinberger, eds. Raven Press, New Sork, pp. 3-20. Hadziselimovic, F. (1977) Cryptorchidism: Ultrastructure of normal and cryptorchid testis development. Adv. Anat. Embryol. Cell Biol., 53:l-71. Hayashi, H., and R. Harrison (1971)The development of the interstitial tissue of the human testis. Fert. Steril. 22:351-355. Hermo, L., M. Lalli, and S. Clermont (1977) Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey. Am. J. Anat. 148:433-446. Ichihara, I. (1970) The fine structure of testicular interstitial cells in mice during postnatal development. 2. Zellforsch., 108t475-486. Leeson, C. (1966) An electron microscopic study of cryptorchid and scrota1 human testes, with special reference to pubertal maturation. Invest. Urol., 3:498-511. Lendon, M., I. Hann, M. Palmer, S. Shalet, and P. Morris-Jones (1978)Testicular histology after combination chemotherapy in childhood for acute lymphoblastic leukemia. Lancet, August 26, pp. 439-441. Lording, D., and D. DeKrestser (1972) Comparative ultrastructural and histochemical studies of the interstitial cells of the rat testis during fetal and postnatal development J. Reprod. Fert., 29:261-269. Mancini, R., J. Nolazco, and F. de la Balze (1952) Histochemical study of normal adult human testes. Anat. Rec., 114t127-142. Marboe, C., T. Hensle, and H. Wigger (1982) Testicular biopsies following therapy for acute leukemia in childhood. Lab. Invest., 46;lOP-llP. Mietkiewski, K., Z. Cymerys, and M. Walczak (1966) Histologie et histochimie du testicule foetal humain. Arch. Anat. Microscop., 55t23-36. Moon, Y. and M. Hardy (1973) The early diffferentiation of the testis and interstitial cells in the fetal pig, and its duplication in organ culture. Am. J. Anat., 138:253268. Mori, H., and A. Christensen (1980)Morphometric analysis of Leydig cells in the normal rat testis. J. Cell Biol., 84t340-354. Murota, S., M. Shikita, and B. Tamaoki (1965) Intracellular distribution of the enzymes related to androgen formation in mouse testes. Steroids, 5t409-413. Nistal, M. and R. Paniagua (1979) Leydig cell differentiation induced by stimulation with HCG and HMG in two patients affected with hypogonadotropic hypogonadism. Andrologia, 11:211-222. Ottowicz, J. (1963) The stadia1 development of Leydig cells. Acta Med. Pol., 1:l-14. Pehlemann, F., and M. Lombard (1978) Differentiation of ovarian and testicular interstitial cells during embryonic and post-embryonic development in mice. Cell Tiss. Res., 188:465-480. Pelliniemi, L., and M. Niemi (1969) Fine structure of the human feotal testis. Z. Zellforsch., 99507-522. Pelliniemi, L., M. Dym, J. Crigler, A. Retik, and D. Fawcett (1980) Development of Leydig cells in human fetuses and in patients with androgen insensitivity. In: Testicular Development, Structure, and Function. A. Steinberger and E. Steinberger, eds. Raven Press, New York, pp. 49-54. Russo, J., and J. de Rosas (1971) Differentiation of the Leydig cell of the mouse testis during the fetal period -An ultrastructural study. Am. J. Anat., 13Ot461-480. Shalet, S., I. Hann, M. Lendon, P. Morris-Jones, and C. Beardwell (1981)Testicular function after combination chemotherapy in childhood for acute lymphoblastic leukaemia. Arch. Disease Childhood 56:275-278. Sniffin, R. (1950) The testis. I. The normal testis. Arch. Pathol. 50:259-284. Tillinger, K., G. Birke, C. Franksson and L.-0. Plantin (1955) The steroid production of the testicles and its relation to number and morphology of Leydig cells. Acta Endocrinol. 19:340-348. Tseng, M., N. Alexander, and G. Kittinger (1975) Effects of fetal decapitation on the structure and function of Leydig cells in Rhesus monkeys (Macaca mulatta). Am. J. Anat., 143:349-362. Van Straaten, H., and C. Wensing (1978) Leydig cell development in the testis of the pig. Biol. Reprod., 18t86-93. Van Wagenen, G., and M. Simpson (1954) Testicular development in the rhesus monkey. Anat. Rec., 118:231251. Vilar, 0. (1970)Histology of the human testis from neonatal period to adolescence. In: The Human Testis. E. Rosemberg and C. Paulsen, eds. Plenum Press, New York, London, pp. 95-111. Winter, J. and C. Faiman (1972) Pituitary-gonadal relations in male children and adolescents. Fediatr. Res., 6:126-135. Wolfe, H. and R. Cohen (1964) Glucose-6-phosphate-dehydrogenase activity in the human fetal and prepubertal testis: A histochemical study. J. Clin. Endocrinol., 24~616-620.