The Effects of Interstitial Cell-Stimulating Hormone, Prolactin, and Bovine Growth Hormone on the Transplantable Leydig Cell Tumor in the Mouse

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[CANCER RESEARCH 34, 2440-2450, October 1974] The Effects of Interstitial Cell-stimulating Hormone, Prolactin, and Bovine Growth Hormone on the Transplantable Leydig Cell Tumor in the Mouse

W. H. Yang, A. L. Jones,1 and Choh Hao Li2

Hormone Research Laboratory [W. H. Y., C. H. L] and the Department of Anatomy and Medicine Â¡A.L. J.j, University of California, San Francisco 94143, and Cell Biology Section, Veterans Administration Hospital, San Francisco, California 94121

SUMMARY considerable amount of the agranular endoplasmic reticulum (3). The effects of ovine interstitial cell-stimulating hormone The primary object of the present investigation was to study (ICSH), ovine prolactin, or bovine growth hormone (BGH) on the effects of ICSH, prolactin, and BGH on the growth of the the growth and fine structure of transplantable Leydig cell Leydig cell tumor in the hypophysectomized mouse as well as tumors have been investigated. The Leydig cell tumor was on the fine structure of the individual tumor cells. capable of growth in the hypophysectomized mouse as well as in the intact mouse treated with rabbit antisera to ICSH. The ultrastructure of the Leydig cell was also greatly changed MATERIALS AND METHODS following the malignant transformation. Although the tumor Five- to 6-week-old male mice (C57BL/67) weighing Leydig cell shows growth in the absence of ICSH, daily administrations of 200 ÃŸgICSH, 1 mg prolactin, or l mg BGH between 16 and 18 g were used, and hypophysectomy was were all clearly effective, enhancing the tumor growth to 176, performed by the parapharyngeal approach (23). The Leydig 179, and 185%, respectively, in comparison to those in the cell tumor (M5480/5A) was obtained from Dr. W. F. Dunning control. These hormones also exerted a significant effect on and transplanted successively for the study of testosterone production in this laboratory (27). Ovine ICSH and ovine the growth of seminal vesicles. prolactin4 were prepared according to the procedures of The agranular endoplasmi reticulum, which normally is Papkoff et al. (28) and Li et al. (20), respectively. Bovine BGH abundant in the active Leydig cell, was sparse in the tumor was isolated by the method of Li (18). The antiserum5 to cells growing in the intact mouse. ICSH treatment produced a significant increase in the agranular reticulum of the tumor ICSH was prepared from male New Zealand albino rabbits cells, although similar morphological change was not observed according to the procedures described by Moudgal and Li (25). following prolactin or BGH administration. There was an Preparations for tumor transplantation by cell suspension increase in intracellular ÃŸ-glycogenparticles following prolactin were as follows. Tumors were removed from several donors or BGH treatments, but not with ICSH treatment. about 3 weeks after the last transplantation and were minced into small pieces in a sterilized plastic bottle containing 30 ml Krebs-Ringer solution and a magnetic stirrer. The contents INTRODUCTION were agitated at 60 rpm for 1 hr and then were filtered through a piece of gauze. After centrifugation of the It has been well established that the growth and function of suspension in a graded centrifuge tube at 1000 rpm for 5 min, testicular interstitial or Leydig cells are controlled by pituitary the cellular component was resuspended to make approxi gonadotropins (13). Recent studies demonstrated that ICSH3 mately a 20 to 30% tumor cell suspension. The final but not follicle-stimulating-hormone specifically binds to the proportion of the cell suspension was determined by cen Leydig cell (10, 11). Although isolated Leydig cells from trifugation in the Wintrobe's hematocrit tube at 3000 rpm for malignant tumors in mice were capable of producing small 5 min. The tumor cells were diluted 10 times and placed on a amounts of androgen, ICSH in the medium does enhance hematocytometer for cell counting. The tumor was trans- androgen synthesis (26, 27). Electron microscopic investiga tions showed that active testicular function in the normal 4Ovine ICSH was assayed by the ovarian ascorbic acid depletion test male is characterized by an abundance of agranular endo- and was found to possess an activity of 2 X NIH-LH-S1. Ovine prolactin plasmic reticulum in the interstitial cells (5â€”9). The fine had an activity of 35 lU/mg. When BGH was assayed by the tibia test, it structure of the human Leydig cell tumor also demonstrated a was at least 2 times as active as the NIH preparation. The amino acid sequences of ovine ICSH and ovine prolactin (see Ref. 19) as well as BGH (15) have been elucidated. These 3 hormone preparations were 1Recipient of a grant from the Veterans Administration Hospital. contaminated with no other pituitary hormones when tested at a level 2Recipient of NIH Grant AM-6097. of 0.1% 'The abbreviations used are: ICSH, interstitial cell-stimulating sThe potency of the antiserum was assayed qualitatively by the hormone; BGH, bovine growth hormone. immunodiffusion technique as well as quantitatively by the precipitin Received April 9, 1974; accepted May 30, 1974. test.

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Table 1 The effect of ICSH on the growth of the transplan table Leydig cell tumor0 in the hypophysectomized mouse

wtSeminaland organ

dose wt vesicles AnimalsH6 (Mg)200 (mg)1091 (g)19. (mg)130.3 (mg)131.1 Â±182c-d 7 Â±0.9 Â±5.5e Â±4Af HNDaily 0 621 Â±1181287 18.0 + 0.5 103.9 Â±4.7 125.6 + 3.3 0Tumor Â±200Body 24.0 Â±1.0Body 194.5 Â±12.3Testis 182.0 Â±5.6 0 0.2 ml, 25% tumor cell suspension (3.3 X IO6 cells). b H, hypophysectomized; N, normal; 10 animals/group; injections for 14 days. c Mean Â±S.E. d Significant difference by r test (f = 2.17; p < 0.05) when compared with the hypophysectomized control group. e No significant difference by t test when compared with the hypophysectomized control group. 'Significant difference by f test (t = 3.65; p < 0.01) when compared with the hypophysectomized control group. planted by s.c. injection of 0.1 ml tumor cell suspension with a rapid dehydration in a graded series of cold ethanols, the tuberculin syringe and a 25-gauge needle on the back of the tissues were embedded in the low-viscosity embedding media recipient animal. according to Spurr (33). Following polymerization, the blocks For examination of the hormonal effect on tumor growth, were sectioned with a Porter-Blum MT-2 ultramicrotome. For experiments were designed to compare the tumor weight light microscopy, sections (l Â¿im)were stained with toluidine obtained in the hormone-treated hypophysectomized mice blue (35). Adjacent thin sections were stained with uranium with those observed in the hypophysectomized or intact acetate solution for 15 min (34) and with lead citrate solution control groups. Each group consisted of about 10 animals for 3 min (31). Electron micrographs were taken at an original randomly selected from about 30 animals of the same age. magnification of 4,6000 to 16,000 X with a Philips EM 300 About 3 days after the hypophysectomy, all the animals were electron microscope. transplanted with the same amount of tumor cell suspension from the same preparation. To a group of hypophysectomized mice, daily injections of either 200 jug ICSH, 1 mg prolactin, RESULTS or l mg BGH were given s.c. for 14 days immediately after transplantation of the tumor suspension. Both the hypophy Daily administrations of 200 /ag ICSH to hypophysecto sectomized control and the intact control groups were treated mized mice bearing transplantable Leydig cell tumors were daily with the hormone vehicle (0.1 ml distilled water adjusted very effective in enhancing tumor weight (Table 1). Con comitant with the growth of the Leydig cell tumor, an to approximately pH 9 with sodium hydroxide) for the same increase6 in the weight of testes and seminal vesicles was also time period. All animals were autopsied 24 hr after the last injection, and the tumors, testes, and seminal vesicles were observed. When normal mice were treated daily with a dose of weighed and submitted to microscopic examinations. antiserum to ICSH capable of neutralizing 20 Â¿/govine ICSH For the experiments with rabbit antisera to ICSH, a group (36), a retardation in the growth of the tumor, testes, and seminal vesicles was seen. The tumor weight in the antiserum- of intact animals was given 0.4 ml of the antiserum while another group of intact mice and a group of hypophysecto treated animal was almost identical to that in the hypophy sectomized control (Table 2). mized mice were treated daily with 0.4 ml normal rabbit Daily administrations of 1 mg prolactin7 significantly serum. In another experiment, some hypophysectomized- castrated mice with and without the transplanted tumor were increased tumor growth in hypophysectomized mice (Table 3). treated daily with 1 mg prolactin or l mg BGH in order to An increase in the testicular and seminal vesicle weight was examine the possible synergism between the pituitary hor also observed following the prolactin injection. When hypoph mones and the testosterone-producing tumor for growth of the ysectomized mice were given daily doses of l mg BGH, the seminal vesicles. tumor, testicle, and seminal vesicle weight were also signifi In order to obtain fresh tissue fragments for electron cantly increased when compared to those of the hypophysec microscopic examination, a portion of the tumor was resected tomized control (Table 4). from some recipients in each group under Nembutal anes Electron microscopic examinations of the Leydig tumor cell thesia. The tumor fragments were immersed in 0.1 M disclosed a characteristic epithelial cell with a moderate phosphate buffer containing 2.4% glutaraldehyde (pH 7.4) and 'The increase in the weight of testes and seminal vesicles was 0.8% paraformaldehyde fixative (17). The tissues were trimmed in the fixative to 1- or 2-mm blocks, fixed for 2 hr in probably caused by ICSH stimulation of Leydig cells to produce androgenic steroids. a refrigerator, rinsed several times in the buffer, and postfixed 7Prolactin caused slight but significant growth in body weight (Table for 2 hr in cold phosphate buffer containing 2% OsO4. After 3), whereas BGH markedly increased body weight (Table 4).

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Table 2 The effect of rabbit antiserum to ICSH on the growth of the transplantable0 Leydig cell tumor in normal mice

Body and organ wt

wt(mg)589 vesicles(mg)126.6 AnimalsNbHNRabbitserumAntiserumNormalNormalTumor Â±84c-d654 Â±0.6e18.4 Â±6.\f101.7 Â±4.5*129.7 Â±941175 Â±0.622.5 Â±7.1158.3 +5.5182.3 Â±185Body(g)21.2 Â±0.4Seminal Â±3.2Testis(mg)165.7Â±1.2 a 0.2 ml, 25% tumor cell suspension (4.3 X IO6 cells). b N, normal; H, hypophysectomized; 10 animals/group; each animal given injections of 0.4 ml of antiserum or normal serum for 14 days. c Mean Â±S.E. d No significant difference by t test when compared with the hypophysectomized control group. e Significant difference by t test (t = 3.3; p < 0.01) when compared with the hypophysectomized control group. 'Significant difference by t test (f = 2.6; p < 0.02) when compared with the hypophysectomized control group. g Significant difference by t test (/ = 5.1; p < 0.001) when compared with the hypophysectomized control group.

Table 3 The effect ofprolactin on the growth of the transplantable Leydig cell tumor" in hypophysectomized mice

wtSeminaland organ

dose wt vesicles AnimalsH6HNDaily(mg)1 (mg)1174Â± (g)19.1 (mg)174.2 (mg)139.5Â± 114c'd Â±0.3e Â±6.3' 2.8* 0 656 Â±113 17.0 Â±0.7 94.4 Â±2.6 128.5 Â±4.5 0Tumor 1176Â±203Body 24.6 Â±0.8Body 158.1 + 7.1Testis 179.3 Â±4.5 " 0.2 ml, 28.5% tumor cell suspension (7.4 X IO6 cells) 6 H, hypophysectomized; N, normal; 9 animals/group; injections for 14 days. c Mean Â±S.E. d Significant difference by i test (f = 3.22; p < 0.01) when compared with the hypophysectomized control group. "Significant difference by t test (t = 2.75; p < 0.02) when compared with the hypophysectomized control group. f Significant difference by / test (f = 11.7; p < 0.001) when compared with the hypophysectomized control group. K No significant difference by t test when compared with the hypophysectomized control group. amount of cytoplasmic vacuoles that were shown to be lipid of the controls, and with the administration of prolactin or droplets (Figs. 3, 5, 8, 11, and 14). Neither ICSH, nor BGH, produced an effect resulting in an augmentation of prolactin, nor BGH treatment inhibited replication of tumor seminal vesicle weight by 2 or 3 times (Table 5). cells since mitotic figures were frequently found following Electron microscopy of tumor cells growing in the intact such treatment (Figs. 8, 11, and 14), although no transforma mice showed a sparse amount of agranular endoplasmic tional change of cell shape was noted after hormone reticulum (Figs. 1, 2, and 4). The granular reticulum was administration. moderately developed and occasionally formed whorls around In the castrated and hypophysectomized mice without the mitochondria (Fig. 2). Features considered characteristic hormonal supplementation or tumor transplantation, the of Leydig cells, such as the formation of whorls, double-walled average seminal vesicle weight was about 4.5 mg (Table 5). tubules, and concentric membranes made up of agranular Daily treatment with prolactin for 14 days significantly reticulum (7), were not observed in the malignant tumor cell increased the animal's organ weight to twice that of the from normal recipients. A similar scarcity of agranular control. Similarly, BGH treatment produced an increase in reticulum was also observed in the hypophysectomized control weight of this organ, although that increase was less than after (Fig. 4). Daily administration of 200 /ig ICSH to the the prolactin treatment. On the other hand, mice bearing the hypophysectomized mice, however, induced increases to transplantable tumor had a seminal vesicle weight 6 times that various degrees in the amount of agranular reticulum in the

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Table 4 The effect of BGH on the growth of the transplantable Leydig cell tumor" in the hypophysectomized mouse

Body and organ wt

dose(mg)100No.ofanimals131210Tumorwt(mg)1234Â±666 AnimalsHbHNijaiiy

Â±16.9 Â±1021 Â±22.3 Â±110c'd102138BodySeminal(g)22.9Â±0.3e0.20.512170157vesicles(mg).1.0.5Â±3.2'Â±4.5Â±4.8Testis(mg)134.9126.1185.1Â±3.2*Â±2.0Â±3.7 " 0.1 ml 20.5% tumor cell suspension (3.0 X 10' cells). b H. hypophysectomized; N, normal. Each animal given injections for 14 days. c Mean Â±S.E. d Significant difference by t test (t = 3.76; p < 0.01) when compared with the hypophysectomized control. e Significant difference by i test (/ = 18.7;p < 0.001) when compared with the hypophysectomized control. ' Significant difference by t test (t = 9.46; p < 0.001) when compared with the hypophysectomized control. g Significant difference by t test (t = 2.68; p < 0.02) when compared with the hypophysectomized control.

Table 5 mouse (Table 2), although tumor weights in these animals The effect oj prolactin, BGH, and Leydig cell tumors on the growth of were reduced to about 37 to 51% of those in the intact seminal vesicles in the castrated and hypophysectomized mouse controls. It is possible that the genetic character of the cell Seminal vesicles may have been changed following transformation of the Leydig cells so that the tumor cell became capable of growing Treatment" Tumor-free Tumor-bearing in the absence of ICSH. Shin (32) has shown that cells from Â±l.O6 Leydig cell tumor in culture are capable of growth and None Â±4.2 androgen production in the absence of ICSH; however, the Prolactin 8.9 Â±1.1 83. 3 Â±14.3 BGHa 6.5Â±0.81 12.5BGH69.2 Â± addition of ICSH would enhance the tumor growth and androgen production. Our in vivo experiments confirm these Daily dose of mg prolactin or27.3 for 14 days;10 observations. It may be seen in Table 1 and Fig. 8 that ICSH animals/group. b Mean Â±S.E.4.5 increased a mean tumor weight from 621 to 1091 mg, and mitotic figures were also seen in the tumor cells of the treated hypophysectomized animals. Likewise, daily administrations tumor cell (Figs. 6 and 7). In some cells, the increase resulted of either prolactin or BGH were also shown to enhance tumor in agranular reticulum literally filling the cytoplasm. Similar growth (Tables 3 and 4). treatment with 1 mg prolactin or l mg BGH appeared to have Following ICSH, prolactin, or BGH treatment, the growth no effect on the amount of agranular reticulum (Figs. 9, 10, of the seminal vesicles was also increased; the growth of testes, 12, and 13). Nevertheless, an increase in the deposition of 0-glycogen granules was frequently observed in the tumor cell however, was less markedly affected by the hormones. Since the growth-promoting effect of prolactin on the seminal following daily administration of prolactin (Fig. 10). Deposi vesicles was also seen in the hypophysectomized-castrated tion of the glycogen granules in the cytoplasm was less frequently observed following the BGH treatment and was not male in the absence of the transplantable tumor (Table 5), it is observed at all after the administration of ICSH. Tumor cell clear that prolactin could exert its effect directly on the mitochondria did not appear to be changed in structural seminal vesicle without the synergistic action with androgen characteristics by any of the above treatments. from either the testis or the tumor. In all probability, the Leydig cell tumors were producing testosterone in the absence of ICSH, since the transplantation of the tumor into the DISCUSSION doubly operated animal was effective in increasing the seminal vesicle weight to 6 times that of the control group (Table 5). Malignant transformation of the normal Leydig cell to the The growth-promoting effect of the tumor Leydig cell on the tumor cell in vivo usually requires an excessive amount of seminal vesicles is enhanced by additional treatments with exogenous gonadotropins for stimulation (14, 21). Previous prolactin or BGH (Tables 3 and 4). Earlier studies have shown studies have shown that inhibition of endogenous gonado- that prolactin (4) or BGH (16) increased the growth of seminal tropic secretion either by estrogen or testosterone (22) or by vesicles and/or ventral prostates in the immature hypophysec gonadotropin antiserum (12) failed to induce the Leydig cell tomized-castrated rat. The synergistic action between prolactin tumor. In the present study, the Leydig cell tumor was able to or BGH and testosterone has also been observed by various both grow and show mitosis in either the hypophysectomized investigators (1,4, 16,24, 29). mouse (Tables 1, 3, and 5; Fig. 5) or the antiserum-treated Christensen and Fawcett (6) first suggested that one of the

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1974 American Association for Cancer Research. W. H. Yang et al. principal sites of hormone production in the steroid-producing 9. Christensen, A. K., and Gillim, S. W. in: K. W. McKerns (ed.). The cell is in the agranular reticulum. This organelle is particularly Gonads, p. 415. Appleton-Century-Crofts, Inc., New York: 1969. abundant in the testicular interstitial cells of mice (7), guinea 10. de Kretser, D. M., Catt, K. J., and Paulsen, C. A. Studies on the pigs (5), rats (9), and humans (8). In this respect the Leydig in vitro Testicular Binding of lodinated Luteinizing Hormone in Rats. Endocrinology,*Â«: 332-337, 1971. tumor cells are remarkable, since those growing in either 11. de Krester, D. M., Catt, K. J., and Smith, G. Radioautographic normal (Figs. 1 and 2) or hypophysectomized (Fig. 4) mice Studies on the Localization of '2 51-labelled Human Luteinizing contained only a small quantity of agranular reticulum. ICSH, and Growth Hormone in Immature Male Rats. J. Endocrino!., 43: however, induced a marked increase in the amount of 105-111, 1969. agranular reticulum in the tumor Leydig cells (Figs. 6 and 7). 12. Ely, C. A. Inhibition of Tumor Formation in Ovarian Splenic Although it is apparent that the Leydig cells in the normal Implants after Gonadotropic Antiserum. Cancer Res., 19: 37-45, state are more responsive to this treatment than are cells in the 1959. tumor state, it is likely that the capability of the Leydig cells 13. Evans, H. M., and Simpson, M. E. Physiology of the Gonado- to produce steroids might have been greatly reduced following tropins. Hormones, 2: 351-398, 1950. the malignant transformation. 14. Furth, J., and Sobel, H. J. Nati. Cancer Inst.,#: 7, 1947. The growth-promoting effect of either prolactin or BGH on 15. Graf, L., and Li, C. H. On the Primary Structure of Pituitary Bovine Growth Hormone. Biochem. Biophys. Res. Commun., 56: the Leydig cell tumor (Tables 3 and 4) apparently was not 168-176,1974. associated with an enhancement of steroid production, since 16. Huggins, C., Parsons, F. M., and Jensen, E. V. Promotion of there was no increase of agranular reticulum following the Growth of Preputial Glands by Steroids and the Pituitary Growth administration of these hormones (Figs. 9, 10, 12, and 13). An Hormone. Endocrinology, 57: 25-32, 1955. earlier worker also observed no increase in testosterone 17. Karnovsky,M. J. J. Cell Biol., 27: 137A, 1965. production following treatment of the Leydig tumor cell with 18. Li, C. H. A Simplified Procedure for the Isolation of Hypophyseal prolactin (27). The role of prolactin and ICSH in the Growth Hormone. J. Biol. Chem., 211: 555-558, 1954. cholesterol metabolism appeared to be counteractive. Prolactin 19. Li, C. H. Hormones of the Adenohypophysis. Proc. Am. Phil. Soc., promoted the acumulation of cholesterol esters in the Leydig 116: 365-382, 1972. cells (2), whereas ICSH appeared to deplete the cells of 20. Li, C. H., Dixon, J. S., Lo, T. B., Schmidt, K. D., and Pankov, Y. cholesterol esters (30). Nevertheless, following ICSH, pro A. Studies on Pituitary Lactogenic Hormone. XXX. The Primary Structure of the Sheep Hormone. Arch. Biochem. Biophys., 141: lactin, or BGH treatment, the number of lipid droplets in the 705-737, 1970. tumor cell did not appear to be markedly changed (Figs. 6,9, 21. Li, M. H., and Gardner, W. V. Tumors in Intrasplenic Ovarian 10, 12, and 13). It is of interest to note the increased Transplants in Castra ted Mice. Science, 105: 13-15, 1947. accumulation of 0-glycogen particles in the tumor cell 22. Li, M. H., and Gardner, W. V. Further Studies on the Pathogenesis following prolactin treatment (Fig. 10). Such findings in the of Ovarian Tumors in Mice. Cancer Res., 9: 35-41, 1949. tumor cell were less frequently observed following BGH 23. Lostroh, A. J., and Jordan, C. W. Improved Procedure for treatment and were not seen after ICSH treatment. Hypophysectomy of the Mouse. Proc. Soc. Exptl. Biol. Med., 90: 267-269, 1955. ACKNOWLEDGMENTS 24. Lostroh, A. J., and Li, C. H. The Effect of Pituitary Growth and Lactogenic Hormones on the Sex Accessories of the Hypophy- We thank J. D. Nelson and J. Quan for their technical assistance. sectomized-Castrated Male Rat. Rev. Arg. Endocrinol. Metal., 2: 213-219, 1956. REFERENCES 25. Moudgal, N. R., and Li, C. H. An Immunochemical Study of Sheep Pituitary Interstitial Cell-stimulating Hormone. Arch Biochem. 1. Antliff, H. R., Prasad, M. R. N., and Meyer, R. K. Action of Biophys., 95: 93-98,1961. Prolactin on Seminal Vesicles of the Guinea Pig. Proc. Soc. Exptl. 26. Moyle, W. R., Moudgal, N. R., and Creep, R. O. Cessation of Biol. Med., 103: 77-80, 1960. Steroidogenesis in Leydig Cell Tumors after Removal of Luteiniz 2. Bartke, A. Effects of Prolactin on Spermatogenesis in Hypophysec ing Hormone and Adenosine Cyclic 3',5' Monophosphate. J. Biol. tomized Mice. J. Endocrino!., 49: 311-316, 1971. Chem., 246: 4978-4982, 1971. 3. Cervos-Navarro, J., Tonutti, E., and Bayer, J. M. Elektronen 27. Moyle, W. R., and Ramachandran, J. Effect of LH on Steroidogen mikroskopische Untersuchung eines androgenbildenden Leydigzell- esis and Cyclic AMP Accumulation in Rat Leydig Cell Preparations tumors. Endokrinologie, 47: 23-51, 1964. and Mouse Tumor Leydig Cells. Endocrinology, 93: 127-134, 4. Chase, M. D., Geschwind, 1. I., and Bern, H. A. Synergistic Role of 1973. Prolactin in Response of Male Rat Sex Accessories to Androgen. 28. Papkoff, H., Gospodarowicz, D., Condiotti, A., and Li, C. H. Proc. Soc. Exptl. Biol. Med., 94: 680-683, 1957. Preparation of Ovine Interstitial Cell-stimulating Hormone in High 5. Christensen. A. K. The Fine Structure of Testicular Interstitial Yield. Arch. Biochem. Biophys., ///: 431-438, 1965. Cells in Guinea Pigs. J. Cell Biol., 26: 911-935, 1965. 29. Pasqualini, R. Q. Prensa Med. Arg., 40: 2658, 1953. 6. Christensen, A. K., and Fawcett, D. W. The Normal Fine Structures 30. PÃ¶kel, J. D., Moyle, W. R., and Creep, R. O. Depletion of of Opossum Testicular Interstitial Cells. J. Biophys. Biochem. Esterified Cholesterol in Mouse Testes and Leydig Cell Tumors by Cytol., 9: 653-670,^1961. Luteinizing Hormone. Endocrinology, 91: 323-325, 1972. 7. Christensen, A. K., and Fawcett, D. W. Am. J. Anat., 118: 551, 31. Reynolds, E.S.J. Cell Biol., 77:208, 1963. 1966. 32. Shin, S. I. Studies on Interstitial Cells in Tissue Culture: Steroid 8. Christensen, A. K., and Fawcett, D. W. Fine Structure of Testicular Biosynthesis in Monolayers of Mouse Testicular Interstitial Cells. Interstitial Cells in Humans. In: E. Rosenberg and C. A. Paulsen Endocrinology, 81: 440-444, 1967. (eds.), The Human Testis, pp. 75-111. New York: Plenum Press, 33. Spurr, A. R. A Low-Viscosity Epoxy Resin Embedding Medium for Inc., 1970. Electron Microscopy. J. Ultrastructure Res., 26: 31-43, 1969.

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34. Stempak, S., and Waid, R. An Improved Staining Method for 36. Yang, W. H., and Papkoff, H. Effect of Desialylation on Electron Microscopy. J. Cell. Biology, 22: 697-701, 1964. Ovulation-inducing Activity of Ovine Interstitial Cell-stimulating 35. Trump, B. F., Smackler, E. A., and Benditt, E. P. A Method for Hormone, Ovine Follicle-stimulating Hormone, Pregnant Mare's Staining Epoxy Sections for Light Microscopy. J. Ultrastruct. Res., Serum Gonadotropin, and Human Chorionic Gonadotropin in the 5: 343-348, 1961. Hamster. Fertility Sterility, 24: 633-639, 1973.

Fig. 1. Electron micrograph of a Leydig tumor cell grown in an intact control mouse for 2 weeks. Unlike normal Leydig cells obtained from the intact animal, an abundance of agranular reticulum was not observed in the cytoplasm. However, large lipid droplets (L) were frequently seen as in the normal steroid-producing cell. A', nucleus; GR, granular reticulum; M, mitochondria. X 36,800. Fig. 2. Electron micrograph of a tumor cell grown in the same recipient mouse as in Fig. 1. Note the formation of a whorl of granular reticulum (GR) encircling a mitochondria (M). Whorl formations of agranular reticulum have been frequently observed in normal Leydig cells in the intact mouse. X 36,800. Fig. 3. Photomicrograph of toluidine blue-stained tumor cells in an adjacent thick section from the tissue block used in Fig. 1. Larger tumor cells of epithelial cell type with few smaller reticulocytes and lymphocytes were seen. Lipid droplets seen as vacuoles were abundant in the tumor cells. Mitotic figures of tumor cells were frequently encountered (arrows). X 1236. Fig. 4. lilectron micrograph of portions of 2 Leydig tumor cells grown in a hypophysectomized control mouse for 2 weeks. Note the lack of agranular reticulum. The nature of the granules (S) found in a few of these cells is unknown, although the possibility that they are virus particles cannot be ruled out. Similar granules were also seen in other tumor cells in this study. G, Golgi apparatus; M, mitochondria; W, nucleus; GR, granular reticulum. X 30,900. Fig. 5. Thick sections of Leydig tumor cells shown in Fig. 4. Epithelial-type cells with fewer liquid droplets were seen as compared with Fig. 3. Mitotic figures were scarce (arrow). X 1236. Fig. 6. A marked increase of fenestrated and tubular agranular reticulum (AR) characteristic of normal Leydig cells was obtained following transplantation of Leydig tumor cells in a mouse that was hypophysectomized and was treated daily with 200 jugICSH for 2 weeks. N, nucleus; M, mitochondria;5, granules. X 35,200. Fig. 7. Less prominent increase of agranular reticulum was also observed in some tumor cells following similar treatment as described in Fig. 6. Â¿,lipid droplets;A7, nucleus;A/, mitochondria. X 30,900. F'ig. 8. Photomicrograph thick sections of tumor cells seen in Fig. 6. Epithelial tumor cells showed hyperchromatic nuclei with abundant cytoplasmic lipid droplets. Mitotic figures of tumor cells were frequently encountered (arrows). X 1236. F'ig. 9. Electron micrograph of a tumor cell grown for 2 weeks in a hypophysectomized mouse that was treated daily with 1 mg prolactin for 2 weeks. Although the growth of the tumor was markedly enhanced by the hormone treatment as demonstrated in Table 4, no increase of agranular reticulum, suggesting enhancement of steroid synthesis, was observed. M, mitochondria; Â¿,lipid droplets; N, nucleus. X 30,900. Fig. 10. Deposition of glycogen granules (Gl) in the cytoplasm was occasionally seen in Leydig cells following the treatment with prolactin. L, lipid droplets;^, nucleus. X 75,300. Fig. 11. Thick section of tumor cells seen in Figs. 9 and 10. Cytoplasm of the tumor cell is abundant with large lipid droplets seen as vacuoles. In general, the tumor cells appear to be epithelial cells; however, some cells appear to be intermediate between epithelial cells and fibroblasts. A mitotic figure of a tumor cell was seen (arrow). X 1236. Fig. 12. Electron micrograph of 2 tumor cells grown for 2 weeks in a hypophysectomized mouse treated daily with l mg BGH for 2 weeks. The tumor growth was considerably enhanced by the hormone treatment; however, no increase of agranular reticulum was noted. C, centriole; G, Golgi apparatus;M, mitochondria; A', nucleus. X 30,900. Fig. 13. Electron micrograph of a tumor cell grown in the same animal described in F'ig. 12. The Golgi apparatus (G) was well developed. M, mitochondria^, nucleus; GR, granular reticulum. X 36,050. Fig. 14. Photomicrograph of thick sections of tumor cells seen in Fig. 12. The tumor cells are epithelial cell type with vacuoles formed by lipid droplets. A mitotic figure of a tumor cell was seen (arrow). X 1236.

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2448 CANCER RESEARCH VOL. 34

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1974 American Association for Cancer Research. The Effects of Interstitial Cell-stimulating Hormone, Prolactin, and Bovine Growth Hormone on the Transplantable Leydig Cell Tumor in the Mouse

W. H. Yang, A. L. Jones and Choh Hao Li

Cancer Res 1974;34:2440-2450.

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