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Certain Effects of Irradiation, Nitrogen Mustard, Urethane and Colchicine on the Testis
THE JOURNAL OF UROLOGY
Vol. 85, No. 4 April 1961 Copyright © 1961 by The Williams & Wilkins Co. Printed in U.S.A.
CERTAIK EFFECTS OF IRRADIATION, NITROGEN ::VIUSTARD, URETHANE AND COLCHICINE ON THE TESTIS :'\ATHAN B. FRIEDMAN
AND
EILEEN DRUTZ, B.A.
From the Division of Laboratories, Cedars of Lebanon Hospital and the Department of Patholo(ly, University of Southern California, Los Angeles, Cal.
It is well known that irradiation and many chemotherapeutic agents, particularly those used in the treatment of neoplastic disease, damage the testicular tubules and result in atrophy of the seminiferous epithelium. 1 It has been demonstratecl2 in other tissues that irradiation and the administration of nitrogen mustard need not interfere with the differentiation of certain postmitotic elements although they do inhibit mitosis in the precursor cells. On the other hand, agents like colchicine and urethane appear to inhibit differentiation as well as division. In view of the extreme sensitivity of germinal epithelium to irradiation and chemical agents 3 •4 and the striking differentiation which takes place in the elements composing the epithelial membrane of the testicular tubules it was decided to compare the effects of irradiation and the administration of nitrogen mustard, of colchicine and of urethane on the testes of rats and mice. The rats were treated with irradiation and colchicine and the mice with nitrogen mustard, urethane and colchicine. Young adult mice (2-3 months old) of the C3H and A strains and Accepted for publication July 17, 1960. This study was supported by Research Grant C2490 from the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. Histologic preparations by Eugene Rogers, Ph.D. and photomicrographs by Maurice LeCover. 1 Fogg, L. C. and Cowing, R. F.: The changes in cell morphology and histochemistry of the testis following irradiation and their relation to other induced testicular changes: I. Quantitative random sampling of germinal cells at intervals following direct irradiation. Cancer Res., 11: 23-28, 1951. 2 Friedman, N. B., Sargent, J. A. and Drutz, E.: Certain effects of irradiation and chemotherapy on cellular division and differentiation. Cancer Res., 15: 479-484, 1955. 3 Goldberg, G. M., Pfau A. and Ungar, H.: Testicular lesions following ingestion of DLethionine studied by a quantitative cytologic method. Am. J. Path., 35: 383-397, 1959. 4 Suomalainen, H. 0. T.: Effects of colchicine on spermatogenesis in certain grasshoppers. Exper. Cell Res., 7: 563-564, 1954. 609
young adult rats (3-6 months old) derived frorn a VVistar strain were used. Irradiation therapy was administered through the courtesy of Dr. Henry Jaffe. The physical factors were the same as those detailed in a previous publication. 5 Nitrogen mustard, urethane and colchicine, diluted in distilled water or saline, were administered daily by intraperitoneal injection, except for 1 or 2 day weekend intervals. The rats' testes were irradiated with from one to three daily doses of 500 roentgens and were studied at 5 day intervals during the early postirradiation period and at one to two week intervals thereafter for up to 24 weeks. The rats treated with colchicine received a variety of dosage schedules, which ranged from a single injection of 2 mg. per kg. to 25 injections of 0.5 mg. per kg. The mice treated with colchicine received a variety of progressively increasing doses. The courses administered ranged up to a maximum of 20 injections, 10 of 0.5 mg. ger kg., 5 of 0.75 mg. per kg. and 5 of 1.0 mg. per kg. The courses of treatment with urethane went to a maximum of 20 injections of 500 mg. per kg. In the case of nitrogen mustard the maximum course attained consisted of 28 doses of 0 ..5 mg. per kg. Tissues were obtained at regular intervals during the course of treatment, usually at least weekly. The testes were fixed in Bouin's and Zenker's solutions. Paraffin sections were stained with hematoxylin and eosin, the periodic acid Schiff reaction with and without diastase pretreatment and counterstained with methyl green, the Feulgen reaction and a diluted Hucker-Conn acid fast stain. It is not our intention to detail the well known degenerative and atrophic alterations seen in testicular tubules damaged by irradiation and chemical agents. The observations which we wish to emphasize are concerned, on the one 5 Friedman, N. B. and Drutz, E.: The effects of chemotherapy and irradiation therapy on the differentiation of experimental tumors, Cancer, 11: 1060-1069, 1958.
610
FRIEDMAN AND DRUTZ
FIG. 1. Rat testis 5 weeks after irradiation with 500 r. Remaining germinal epithelium consists of spermatogonia and sperm without intermediate forms.
FIG. 2. Mouse testis following 23 doses of nitrogen mustard (0.5 mg. per kg.). Sperm are present as in irradiated testis.
FIG. 3. Rat testis following 4 doses of colchicine (0.75 mg. per kg.). Note sloughing.
EFFECTS OF IRRADIATION AND CHEMOTHERAPEUTIC AGENTS ON TESTIS
611
Frn. 4. A, rat testis 20 days after one dose of colchicine (2 mg. per kg.). Note absence of maturing sperm. B, rat testis following 20 doses of colchicine (0.5 mg. per kg.). Note absence of maturing sperm. Giant cells abundant.
Frn. 5. Mouse testis following 20 doses of urethane (500 mg. per kg.). Note absence of niaturing sperm.
hand, with the continued maturation of sperm despite severe damage to the germinal epithelium by irradiation and nitrogen mustard and, on the other, ,Yith the inhibition of differentiation of sperm by colchicine and urethane in the face of considerably less damage to the seminiferous epithelium. Figure 1 illustrates the appearance of the tubules in a rat after irradiation has resulted
in practically complete disappearance of the lining epithelial membrane. 1Yhat remains consists only of sperm in the lumen and an almost single layered basilar epithelium. Although spermatogonia are present in this layer, the intermediate forms between them and the sperm are missing. This pattern indicates that the differentiation of prespermatic elements and the maturation of sperm had continued despite
612
FRIEDMAN AND DRUTZ
inhibition of division at the level of the precursor spermatogonia. Our observations are in agreement with this long held interpretation. a-s One of the effects of nitrogen mustard on the testes of mice is illustrated by the pattern demonstrated in figure 2. Despite the disappearance of most of the germinal epithelium, sperm have been able to differentiate as in the irradiated testis of the rat. One of the reactions of the rats' testis to colchicine is illustrated in figure 3. The cleavage9 and sloughing of the germinal epithelium result in the appearance of some sperm in the lumen. However, the response to colchicine (fig. 4) emphasizes the differences from the results of irradiation in that no sperm form despite the persistence of much of the epithelial membrane and most of the precursor elements. Although giant cells are frequently encountered in testes after irradiation or damage by other antimitotic agents, multinucleated elements were particularly striking in the colchicine treated animals. The effects of urethane on the testis of the mouse are illustrated in figure 5. The similarities to the colchicine reaction and the divergences from the mustard and irradiation reactions are evident. Although a fairly well developed ger6 Eschenbrenner, A. B. and Miller, E.: Effect of roentgen rays on the testis: Quantitative histolo~ical analysis following whole body exposure of rmce. A.M.A. Arch. Path., 60: 736-749, 1950. • 7 Shaver, _S. L.: ;c-ir~adiation injury and repair m the germmal ep1thehum of male rats. I. Injury and repair in adult rats. Am. J. Anat., 92: 391-431
1953.
.
'
Oakberg, E. F.: Sensitivity and time of degeneration of spermatogenic cells irradiated in various stages of maturation in the mouse. Rad. Res., 2: 369-391, 1955. 9 Roosen-Runge, E. C.: Quantitative studies on spermatogenesis in the albino rat: II. The duration of spermatogenesis and some effects of colchicine. Am. J. Anat., 88: 163-176, 1951. 8
minal epithelial layer still exists, the differentiation to mature sperm has been inhibited. It must be emphasized that the tubular patterns illustrated were not present throughout the testis. There is considerable variation in the constellations encountered in different tubules in normal testes. 10 The abnormal patterns described were generally encountered focally but were more disseminated in the irradiated testes than in those treated with the chemical agents. The tendency for the altered tubules to be seen at the periphery of the testis might be related to the injection of the drugs by the intraperitoneal route but external irradiation has also been noted as affecting mostly the marginal tubules.11 SUMMARY
Under certain circumstances the effectiveness of a number of antitumor agents depends more upon their ability to alter the dynamics of cellular cycles than on direct destruction of cells. Irradiation, nitrogen mustard, urethane and colchicine all cause degeneration and atrophy of the testicular germinal epithelium in rats and mice. However, irradiation and nitrogen mustard permit the continued differentiation of intermediate germinal cells and the progressive maturation of sperm despite inhibition of cellular division and proliferation among the precursor elements. Urethane and colchicine, on the other hand, interfere with the differentiation of germinal epithelial cells into sperm. These divergences are consistent with the variable effects of these agents on differentiation in other tissues, both normal and neoplastic. 10 Le Blond, C. P. and Clermont, Y.: Spermiogenesis of rat, mouse, hamster and guinea pig as revealed by the "periodic acid-fuchsin sulfurous acid" technique. Am. J. Anat., 90: 167-215, 1952. 11 Heller, M.: The Testis. In: Histopathology of Irradiation from External and Internal Sources, edited by W. Bloom, New York: McGraw-Hill Book Co., 1948, pp. 550-597.
Vol. 85, No. 4 April 1961 Copyright © 1961 by The Williams & Wilkins Co. Printed in U.S.A.
CERTAIK EFFECTS OF IRRADIATION, NITROGEN ::VIUSTARD, URETHANE AND COLCHICINE ON THE TESTIS :'\ATHAN B. FRIEDMAN
AND
EILEEN DRUTZ, B.A.
From the Division of Laboratories, Cedars of Lebanon Hospital and the Department of Patholo(ly, University of Southern California, Los Angeles, Cal.
It is well known that irradiation and many chemotherapeutic agents, particularly those used in the treatment of neoplastic disease, damage the testicular tubules and result in atrophy of the seminiferous epithelium. 1 It has been demonstratecl2 in other tissues that irradiation and the administration of nitrogen mustard need not interfere with the differentiation of certain postmitotic elements although they do inhibit mitosis in the precursor cells. On the other hand, agents like colchicine and urethane appear to inhibit differentiation as well as division. In view of the extreme sensitivity of germinal epithelium to irradiation and chemical agents 3 •4 and the striking differentiation which takes place in the elements composing the epithelial membrane of the testicular tubules it was decided to compare the effects of irradiation and the administration of nitrogen mustard, of colchicine and of urethane on the testes of rats and mice. The rats were treated with irradiation and colchicine and the mice with nitrogen mustard, urethane and colchicine. Young adult mice (2-3 months old) of the C3H and A strains and Accepted for publication July 17, 1960. This study was supported by Research Grant C2490 from the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. Histologic preparations by Eugene Rogers, Ph.D. and photomicrographs by Maurice LeCover. 1 Fogg, L. C. and Cowing, R. F.: The changes in cell morphology and histochemistry of the testis following irradiation and their relation to other induced testicular changes: I. Quantitative random sampling of germinal cells at intervals following direct irradiation. Cancer Res., 11: 23-28, 1951. 2 Friedman, N. B., Sargent, J. A. and Drutz, E.: Certain effects of irradiation and chemotherapy on cellular division and differentiation. Cancer Res., 15: 479-484, 1955. 3 Goldberg, G. M., Pfau A. and Ungar, H.: Testicular lesions following ingestion of DLethionine studied by a quantitative cytologic method. Am. J. Path., 35: 383-397, 1959. 4 Suomalainen, H. 0. T.: Effects of colchicine on spermatogenesis in certain grasshoppers. Exper. Cell Res., 7: 563-564, 1954. 609
young adult rats (3-6 months old) derived frorn a VVistar strain were used. Irradiation therapy was administered through the courtesy of Dr. Henry Jaffe. The physical factors were the same as those detailed in a previous publication. 5 Nitrogen mustard, urethane and colchicine, diluted in distilled water or saline, were administered daily by intraperitoneal injection, except for 1 or 2 day weekend intervals. The rats' testes were irradiated with from one to three daily doses of 500 roentgens and were studied at 5 day intervals during the early postirradiation period and at one to two week intervals thereafter for up to 24 weeks. The rats treated with colchicine received a variety of dosage schedules, which ranged from a single injection of 2 mg. per kg. to 25 injections of 0.5 mg. per kg. The mice treated with colchicine received a variety of progressively increasing doses. The courses administered ranged up to a maximum of 20 injections, 10 of 0.5 mg. ger kg., 5 of 0.75 mg. per kg. and 5 of 1.0 mg. per kg. The courses of treatment with urethane went to a maximum of 20 injections of 500 mg. per kg. In the case of nitrogen mustard the maximum course attained consisted of 28 doses of 0 ..5 mg. per kg. Tissues were obtained at regular intervals during the course of treatment, usually at least weekly. The testes were fixed in Bouin's and Zenker's solutions. Paraffin sections were stained with hematoxylin and eosin, the periodic acid Schiff reaction with and without diastase pretreatment and counterstained with methyl green, the Feulgen reaction and a diluted Hucker-Conn acid fast stain. It is not our intention to detail the well known degenerative and atrophic alterations seen in testicular tubules damaged by irradiation and chemical agents. The observations which we wish to emphasize are concerned, on the one 5 Friedman, N. B. and Drutz, E.: The effects of chemotherapy and irradiation therapy on the differentiation of experimental tumors, Cancer, 11: 1060-1069, 1958.
610
FRIEDMAN AND DRUTZ
FIG. 1. Rat testis 5 weeks after irradiation with 500 r. Remaining germinal epithelium consists of spermatogonia and sperm without intermediate forms.
FIG. 2. Mouse testis following 23 doses of nitrogen mustard (0.5 mg. per kg.). Sperm are present as in irradiated testis.
FIG. 3. Rat testis following 4 doses of colchicine (0.75 mg. per kg.). Note sloughing.
EFFECTS OF IRRADIATION AND CHEMOTHERAPEUTIC AGENTS ON TESTIS
611
Frn. 4. A, rat testis 20 days after one dose of colchicine (2 mg. per kg.). Note absence of maturing sperm. B, rat testis following 20 doses of colchicine (0.5 mg. per kg.). Note absence of maturing sperm. Giant cells abundant.
Frn. 5. Mouse testis following 20 doses of urethane (500 mg. per kg.). Note absence of niaturing sperm.
hand, with the continued maturation of sperm despite severe damage to the germinal epithelium by irradiation and nitrogen mustard and, on the other, ,Yith the inhibition of differentiation of sperm by colchicine and urethane in the face of considerably less damage to the seminiferous epithelium. Figure 1 illustrates the appearance of the tubules in a rat after irradiation has resulted
in practically complete disappearance of the lining epithelial membrane. 1Yhat remains consists only of sperm in the lumen and an almost single layered basilar epithelium. Although spermatogonia are present in this layer, the intermediate forms between them and the sperm are missing. This pattern indicates that the differentiation of prespermatic elements and the maturation of sperm had continued despite
612
FRIEDMAN AND DRUTZ
inhibition of division at the level of the precursor spermatogonia. Our observations are in agreement with this long held interpretation. a-s One of the effects of nitrogen mustard on the testes of mice is illustrated by the pattern demonstrated in figure 2. Despite the disappearance of most of the germinal epithelium, sperm have been able to differentiate as in the irradiated testis of the rat. One of the reactions of the rats' testis to colchicine is illustrated in figure 3. The cleavage9 and sloughing of the germinal epithelium result in the appearance of some sperm in the lumen. However, the response to colchicine (fig. 4) emphasizes the differences from the results of irradiation in that no sperm form despite the persistence of much of the epithelial membrane and most of the precursor elements. Although giant cells are frequently encountered in testes after irradiation or damage by other antimitotic agents, multinucleated elements were particularly striking in the colchicine treated animals. The effects of urethane on the testis of the mouse are illustrated in figure 5. The similarities to the colchicine reaction and the divergences from the mustard and irradiation reactions are evident. Although a fairly well developed ger6 Eschenbrenner, A. B. and Miller, E.: Effect of roentgen rays on the testis: Quantitative histolo~ical analysis following whole body exposure of rmce. A.M.A. Arch. Path., 60: 736-749, 1950. • 7 Shaver, _S. L.: ;c-ir~adiation injury and repair m the germmal ep1thehum of male rats. I. Injury and repair in adult rats. Am. J. Anat., 92: 391-431
1953.
.
'
Oakberg, E. F.: Sensitivity and time of degeneration of spermatogenic cells irradiated in various stages of maturation in the mouse. Rad. Res., 2: 369-391, 1955. 9 Roosen-Runge, E. C.: Quantitative studies on spermatogenesis in the albino rat: II. The duration of spermatogenesis and some effects of colchicine. Am. J. Anat., 88: 163-176, 1951. 8
minal epithelial layer still exists, the differentiation to mature sperm has been inhibited. It must be emphasized that the tubular patterns illustrated were not present throughout the testis. There is considerable variation in the constellations encountered in different tubules in normal testes. 10 The abnormal patterns described were generally encountered focally but were more disseminated in the irradiated testes than in those treated with the chemical agents. The tendency for the altered tubules to be seen at the periphery of the testis might be related to the injection of the drugs by the intraperitoneal route but external irradiation has also been noted as affecting mostly the marginal tubules.11 SUMMARY
Under certain circumstances the effectiveness of a number of antitumor agents depends more upon their ability to alter the dynamics of cellular cycles than on direct destruction of cells. Irradiation, nitrogen mustard, urethane and colchicine all cause degeneration and atrophy of the testicular germinal epithelium in rats and mice. However, irradiation and nitrogen mustard permit the continued differentiation of intermediate germinal cells and the progressive maturation of sperm despite inhibition of cellular division and proliferation among the precursor elements. Urethane and colchicine, on the other hand, interfere with the differentiation of germinal epithelial cells into sperm. These divergences are consistent with the variable effects of these agents on differentiation in other tissues, both normal and neoplastic. 10 Le Blond, C. P. and Clermont, Y.: Spermiogenesis of rat, mouse, hamster and guinea pig as revealed by the "periodic acid-fuchsin sulfurous acid" technique. Am. J. Anat., 90: 167-215, 1952. 11 Heller, M.: The Testis. In: Histopathology of Irradiation from External and Internal Sources, edited by W. Bloom, New York: McGraw-Hill Book Co., 1948, pp. 550-597.