The Effect of 5-Bromodeoxyuridine on the Postnatal Development of the Rat Testis JONATHAN M. BARASCH 2 AND ROBERT S. BRESSLER Lkpnrtment of Anatomy, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue at 100th Street, New York, New York 10029
ABSTRACT The effects of 5-bromodeoxyuridine (BrdU) on postnatal testicular development were studied in rats treated on the second, third and fourth days of life. Testes were removed for study at 5, 15 and 35 days of age. Body weights and diameters of seminiferous cords and tubules were significantly less in the treated than control animals. At 15 days of age fewer pachytene sperl matocytes were present in treated animals. At 35 days of age the testes of treated animals contained fewer canalized cords. Spermatids exhibited an altered distribution of chromatin material, contained less agranular endoplasmic reticulum and fewer mitochondria, and had not yet developed tails. Sertoli cells and Leydig cells of treated animals contained less agranular endoplasmic reticulum and more lipid droplets than the normal.
The effects of the thymidine analogue, bromodeoxyuridine (BrdU), on differentiating systems are widely reported. In some cases its action is temporary, preventing expression of certain gene products only while it is present (Rutter et al., '73). In other cases it has been reported to cause a permanent inhibition of synthesis of specific proteins if administered at a critical time (Weintraub et al., '72). While the majority of the investigations have been performed in vitro, the effects of BrdU on development and differentiation in vivo have not been as extensively examined. The dose regimen used in this study has produced a retardation of development of the cerebellum (Yu, '76), submandibular gland (Fukushima and Barka, '76), and skeletal muscle (Ontell, personal communication) in the rat. Preliminary observations have also indicated that BrdU affects pituitary development as well (Yu, unpublished findings). Since observations of the effects of BrdU on the male reproductive system in the mammal are limited to Mukherjee's work ('68) on the drug's action on meiotic chromosomes in Chinese hamsters, the present investigation was undertaken in order to determine if the postnatal differentiation of the male reproductive system is also affected by this drug. This paper reports the findings concerning the testis. J. EXP. ZOOL.,200: 1-8.
MATERIALS AND METHODS
Neonatal Sprague-Dawley rats were injected subcutaneously twice daily for three consecutive days beginning on the second day after birth with 15 mg 5-bromO-2l-deoxyuridine (Sigma Chemical Co.) per 100gram body weight. The BrdU was dissolved in saline, and protected from light. The littermate controls received saline. At the end of the experimental period the animals were weighed and killed by decapitation while under ether anesthesia. The number of experimental and control animals studied was as follows: at 5 days, 2 and 4; at 15 days, 3 and 3; and at 35 days, 2 and 2, respectively. The testes were fixed by immersion into 4 % gluteraldehyde in 0.1 M phosphate buffer (pH 7.3). After one hour the organ was sufficiently firm to allow shaving 1-mm slices from similar locations at the periphery in control and treated testes. The slices were trimmed down to l-mm3 tissue blocks. After an additional four hours in gluteraldehyde the tissue was washed in buffer, fixed further in buffered 1 % osmium tetroxide for one hour, dehydrated through a graded series of ethanol and 1 This investigation was partially supported by Public Health Service Research Grant CA-17038 &om the National Cancer Institute to Dr. T. Barka. 2 This work was performed while serving as a volunteer at Mount Sinai Hospital during his Senior Year at Dalton High School, New York City.
1
2
JONATHAN M. BARASCH AND ROBERT S . BRESSLER
propylene oxide and embedded in Epon weights in control animals were 12.3 gms, (Luft, '61). Control and experimental tis- 40.3 gms, and 147.1 gms, respectively, as sues were processed %imultaneously. Thin compared to those of experimental animals sections, cut at 600 A, with glass knives, which were 12.3 gms, 23.8 gms and 61.9 were stained in 3 % uranyl acetate and gms, respectively. At 15 and 35 days these 0.5% lead citrate (Veneble and Coggeshall, differences were significant (p < 0.005). '65). The sections were examined in a Average tubule diameters of control aniHitachi HS-8 electron microscope. mals at these ages were 102.5 p , 154.75 p One-micron thick sections, cut from the and 410.5 p , respectively, as compared to same tissue blocks and stained with tolui- 85.75, 126.75 and 208 p in treated anidine blue, were used for light microscopic mals, respectively (fig. 1). The differences studies. Diameters of all seminiferous cords are significant at all ages (p < 0.001). Aland tubules exhibiting cross sectioned pro- though initially retarded, the rates of files were measured directly from thick growth in body weight and tubule diameter sections by use of an ocular micrometer. between 5 and 15 days appeared to be the The number of treated and control tubules same in the two groups of animals. After measured was as follows: at 5 days, 21 1 15 days growth is accelerated in the norand 210; at 15 days, 217 and 76; and at mal animal but the rate remains un35 days, 56 and 14, respectively. Calcula- changed in the treated. tions were made of the percentage of tu15 days bules containing primary spermatocytes in the 15-day and lumen formation in 35For the most part, no morphological difday-old animals. ferences were noted at this age. However, relatively few pachytene spermatocytes RESULTS were present in tubules of treated animals. At 5, 15 and 35 days average body They were found in 38 % of the 25 1 tubules
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AGE ( Days) Fig, 1 Growth i n diameter of seminiferous cords and tubules with age. Bromodeoxyuridine was administered on the second, third and fourth day a s described in MATERIALS AND METHODS. The number of tubules measured were (BrdU, control) 5-day: 211, 210; 15-day: 217, 76; and 35-day: 56, 14. Differences in control and treated animals were significant at all ages studied (p < 0.001). (Control: -0-; Experimental: -0-)
EFFECTS OF BrdU ON TESTIS DEVELOPMENT
studied in control testes but only in 2% of the 672 tubules examined in treated testes. 35 days A more pronounced retardation in development between normal and treated animals, is further substantiated by electron and light microscopic observations. Whereas lumen formation was evident in 43.5% of the 33 normal seminiferous tubules studied, it was only seen in 11.3% of 98 tubules observed in treated animals. Furthermore, the chromatin material of the spermatid in the normal animal appeared diffuse and unevenly distributed in comparison with the compact, finely granular appearance observed in the experimental group (figs. 2, 3). In the control, spermatid cytoplasm was more complex, being richer in granular endoplasmic reticulum and mitochondria. Moreover, later stages of spermiogenesis were observed in the testis of the control than in the treated animals, as tails associated with spermatids were only seen in the former. Many disintegrating germ cells were present in the seminiferous tubules of the treated animals but not in the normal. BrdU had an apparent effect on Sertoli cells as well. Smooth endoplasmic reticulum was found to be more abundant in the Sertoli cell cytoplasm of the normal rat, both in apical and in basal regions, than in the treated (figs. 4, 5). Typical Sertoli cell junctions were observed in testes of treated and control animals, independent of lumen formation. The treated also contained more lipid droplets than the normal. Leydig cells follow the pattern of Sertoli cells. In the treated animal they contained less smooth endoplasmic reticulum and more lipid droplets than in the control (figs. 6, 7). DISCUSSION
The present study demonstrates that bromodeoxyuridine (BrdU) given in vivo significantly effects the maturation of the testis. The rapid growth and development of the testis in control animals between 15 and 35 days is consistent with reports of a number of developmental events occurring at this time period. Among these are organization of the tunica propria (Leeson and Leeson, '63; Ross, '67) completion of the first wave of meiosis (Clermont and
3
Perey, '57) and maturation of the Sertoli cells. This maturation is characterized by increases in the amount of agranular endoplasmic reticulum and the formation of the specialized Sertoli cell junctions (Flickinger, '67), which are the basis of the blood-testis barrier (Dym and Fawcett, '70; Ross, '70). Concurrent with the establishment of the blood-testis barrier is the canalization of the seminiferous cords and increased secretory activity by the Sertoli cells (Vitale et al., '74). At the same time depletion of stored lipid droplets in Leydig cells (Lording and de Kretser, '72) correlates with increased serum levels of testosterone (Miyachi et al., '73; Gupta et al., '75; Lee et al., '75). Many of these events are pituitary dependent (Steinberger, '71 ; Bressler and Ross, '72; de Kretser and Burger, '72; Hansson et al., '75; Bressler, '76; Fakunding et al., '76; Madhwa Raj and Dym, '76; and Welsh and Weibe, '76) and indeed take place at a time when serum gonadotropin levels are increasing (Miyachi et al., '73; Lee et al., '75) and when testosterone is rapidly being converted to androstanediol (Rivarola et al., '75; Lacroix et al., '76), a characteristic of the immature tubule. In contrast with the control, BrdU treated rats are retarded in the canalization of the seminiferous cords and in the development of agranular endoplasmic reticulum in Sertoli and Leydig cells. The latter contained many lipid droplets, a feature typical of young Leydig cells and which may indicate decreased levels of testosterone (Christensen and Gillim, '69) and its metabolites. This retardation may be a reflection of an overall impairment of growth processes as can be seen by the reduced body weight compared to controls. However, in view of the endocrine relationships involved in testicular development during this time period, and the indications that treatment of these animals affected the pituitary glands as well (Yu, unpublished observations), the possibility exists that the failure of the experimental animals to exhibit the normal acceleration of growth after 15 days is a result of impaired pituitary function. An effect on growth hormone production would also account for the differences in body weight between control and treated animals. The action of BrdU on the testis may
4
JONATHAN M. BARASCH AND ROBERT S. BRESSLER
also be direct. The substitution of BrdU for its analogue thymidine is known to be partially responsible for increased cell death (Packard et al., '74) and for temporary delays of differentiation (Davidson and Horn, '74; Rutter et al., '73) in a variety of organ systems. That this is also true for the testis is seen by the failure of primary spermatocytes to develop in numbers comparable to controls at 15 days, and of flagellar tails to form in spermatids at 35 days in treated rats. The augmentation of cell death by BrdU would result in smaller tubule diameters by reducing the number of stem cells which would otherwise increase in numbers exponentially. In view of the temporary effects of BrdU reported for several systems (Rutter et al., '73) it is difficult to explain why more degenerating cells were observed in the testes of treated animals at 35 days than at 15 days. Under certain circumstances germ cells fail to progress beyond a certain stage of development. For example spermatogonia of rats exposed to X-irradiation during fetal development (Means, '75) appear normal for a short time after birth but then enter into an abortive mitosis and degenerate. In the present study BrdU treatment does not lead to the complete absence of a particular cell type. Nevertheless, the frequency of degenerating cells at 35 days, during the progress of meiosis, may represent the failure of germ cells which have incorporated BrdU to survive this stage of spermatogenesis. This hypothesis is compatible with the finding of abnormal meiotic configurations in testis preparations &om Chinese hamsters (Mukherjee, '68). ACKNOWLEDGMENTS
The authors would like to thank Doctor W. A. Yu for providing the testes from animals used in her experiments and W s . Barbara Park for the secretarial services provided. LITERATURE CITED Bressler, R. S. 1976 Dependence of Sertoli cell maturation on'the pituitary gland in the mouse. Am. J. Anat., 147: 447-456. Bressler, R. S., and M. H. Ross 1972 Differentiation of peritubular myoid cells of the testis: Effects of intratesticular implantation of newborn mouse testes into normal and hypophysectomized adults. Biol. Reprod., 6: 148-159. Christensen, A. K., and S. W. Gillim 1969 The
correlation of fine structure and function in steroid-secreting cells, with emphasis on those of the gonads. In: The Gonads. K. W. McKerns, ed. Appleton-Century-Crofts, New York, pp. 415488. Clermont, Y., and B. Perey 1957 Quantitative study of the cell population of the seminiferous tubules in immature rats. Am. J. Anat., 100: 241-268. Davidson, R. L., and D, Horn 1974 Reversible transformation of bromodeoxvuridine-denendent cells by bromodeoxyuridine: Proc. Nat. Acad. Sci., 71: 3336-3342. de Kretser, D. M., and H. G. Burger 1972 Ultrastructural studies of the human Sertoli cell i n normal men and males with hypogonadotropic hypogonadism before and after gonadotropic treatment. In: Gonadotropins. B. B. Saxena, C. G. Beling and H. M. Gandy, eds. Wiley-Interscience, New York, pp. 640-656. Dym, M., and D. W. Fawcett 1970 The bloodtestis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod., 3: 308-326. Fakunding, J. L., D. J. Tindall, J. R. Dedman, C. R. Mena and A. R. Means 1976 Biochemical actions of follicle-stimulating hormone i n the Sertoli cell of the rat testis. Endocrin., 98: 392402. Flickinger, C. J. 1967 The postnatal development of the Sertoli cell i n the mouse. 2. Zellforsch., 78: 92-113. Fukushima, M., and T. Barka 1976 The effects of 5-bromodeoxyuridine and isoproterenol on the postnatal differentiation of rat submandibular gland. Am. J. Anat., 147: 159-182. Gupta, D., K. Rager, J. Zaraycki and M. Eichner 1975 Levels of luteinizing hormone, testosterone, and dihydrotestosterone i n the circulation of sexually maturing intact male rats and after orchidectomy and experimental bilateral cryptorchidism. J. Endocr., 66: 183-193. Hansson, V., S. C. Weddington, 0. Naess, A. Attramadal, F. S. French, N. Kotite, S. N. Neyfeh, E. M. Ritzen and L. Hagenas 1975 Testicular androgen binding protein (ABP) A parameter of Sertoli cell secretory function. In: Hormonal Regulation of Spermatogenesis. F. S. French, V. Hansson, E. M. Ritzen and S. N. Neyfeh, eds. Plenum Press, New York, pp. 323-336. Lacroix, E., W. Eechaute and I. Leusen 1975 Influence of age on the formation of Ba-androstanediol and 7a-hydroxytestosterone by incubated rat testes. Steroids, 25: 64-61. Lee, V. W. K., D. M. de Kretser, B. Hudson and C. Wang 1975 Variations in serum FSH, LH and testosterone levels i n male rats from birth to sexual maturity. J. Reprod. Fert., 42: 121-126. Leeson, C. R., and T. S. Leeson 1963 The postnatal development and differentiation of the boundary tissue of the seminiferous tubule of the rat. Anat. Rec., 147: 243-259. Lording, D. W., and D. M. de Kretser 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. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 11:736-739. Madhwa Raj, H. G., and M. Dym 1976 The
-
EFFECTS OF BrdU ON TESTIS DEVELOPMENT effects of selective withdrawal of FSH or LH on spermatogenesis in the immature rat. Biol. Reprod., 14: 489-494. Means, A. R. 1975 Early sequence of biochemical events in the action of follicle stimulating hormone on the testis. Life Sci., 15: 371-389. Miyachi, Y., E. Nieschlag and M. B. Lipsett 1973 The secretion of gonadotropins and testosterone by the neonatal male rat. Endocrin., 92: 1-5. Mukherjee, A. B. 1968 Effect of 5-bromodeoxyuridine on the male meiosis i n Chinese hamsters (Cricetus griseus). Mutat. Res., 6: 173-174. Packard, D. S., R. G. Skalko and R. A. Menzies 1974 Growth retardation and cell death i n mouse embryos following exposure to the teratogen bromodeoxyuridine. Exp. Mol. Path., 21 : 351-362. Rivarola, M. A., E. J. Podesta, H. E. Chemes and S. Cigorraga 1975 Androgen metabolism i n the seminiferous tubule. In: Hormonal Regulation of Spermatogenesis. F. S. French, V. Hansson, E. M. Ritzen and S. N. Neyfeh, eds. Plenum Press, New York, pp. 25-35. Ross, M. H. 1967 The fine structure and development of the peritubular contractile cell com-
5
ponent i n the seminiferous tubules of the mouse. Am. J. Anat., 121: 523-528. 1970 The Sertoli cell and the blood-testicular barrier: An electron-microscopic study. Fortschritte der Andrologie, Morphological Aspects of andrology, 1 : 83-86. Rutter, W. J., R. L. Pictet and P. W. Morris 1973 Toward molecular mechanisms of developmental processes. Ann. Rev. Biochem., 42: 601-646. Steinberger, E. 1971 Hormonal control of mammalian spermatogenesis Physiol. Rev., 51: 1-22. Veneble, J. H., and R. Coggeshall 1965 A simplified lead citrate stain for use i n electron microscopy. J. Cell Biol., 25: 407408. Walsh, M. J., and J. P. Wiebe 1976 Sertoli cells from immature rats: i n vitro stimulation of steroid metabolism by FSH. Biochem. Biophys. Res. Comm., 69: 936941. Weintraub, H., G. Campbell and H. Holtzer 1972 Identification of a developmental program using bromodeoxyuridine. J. Mol. Biol., 70: 337-350. Yu, W. A. 1976 The effect of 5-bromodeoxyuridine on the postnatal development of the rat cerebellum: A biochemical study. Brain Res., 1 1 8 : 281-291.
PLATE 1 EXPLANATION OF FIGURES
Figs. 2 , 3
Nuclei of spermatids from 35-day-old animals.
2
Spermatid from control animal showing irregular distribution of granular nuclear contents. X 5,300.
3
Spermatid from treated animal showing even distribution of granular nuclear contents. X 3,800.
4
Sertoli cell from control animal showing abundance of dilated profiles of agranular endoplasmic reticulum. X 9,100.
5
Sertoli cell from treated animal showing scant presence of agranular endoplasmic reticulum. X 5,800.
Figs. 4 , 5
Sertoli cell cytoplasm from 35-day-old animals.
Figs. 6, 7
6
Leydig cells from 35-day-old animals.
6
Leydig cell from control animal showing abundance of profiles of agranular endoplasmic reticulum. X 10,000.
7
Leydig cell from treated animal showing less development of agranular endoplasmic reticulum. X 10,000.
EFFECTS OF BrdU ON TESTIS DEVELOPMENT Jonathan M. Barasch and Robert S . Bressler
PLATE 1
ABSTRACT The effects of 5-bromodeoxyuridine (BrdU) on postnatal testicular development were studied in rats treated on the second, third and fourth days of life. Testes were removed for study at 5, 15 and 35 days of age. Body weights and diameters of seminiferous cords and tubules were significantly less in the treated than control animals. At 15 days of age fewer pachytene sperl matocytes were present in treated animals. At 35 days of age the testes of treated animals contained fewer canalized cords. Spermatids exhibited an altered distribution of chromatin material, contained less agranular endoplasmic reticulum and fewer mitochondria, and had not yet developed tails. Sertoli cells and Leydig cells of treated animals contained less agranular endoplasmic reticulum and more lipid droplets than the normal.
The effects of the thymidine analogue, bromodeoxyuridine (BrdU), on differentiating systems are widely reported. In some cases its action is temporary, preventing expression of certain gene products only while it is present (Rutter et al., '73). In other cases it has been reported to cause a permanent inhibition of synthesis of specific proteins if administered at a critical time (Weintraub et al., '72). While the majority of the investigations have been performed in vitro, the effects of BrdU on development and differentiation in vivo have not been as extensively examined. The dose regimen used in this study has produced a retardation of development of the cerebellum (Yu, '76), submandibular gland (Fukushima and Barka, '76), and skeletal muscle (Ontell, personal communication) in the rat. Preliminary observations have also indicated that BrdU affects pituitary development as well (Yu, unpublished findings). Since observations of the effects of BrdU on the male reproductive system in the mammal are limited to Mukherjee's work ('68) on the drug's action on meiotic chromosomes in Chinese hamsters, the present investigation was undertaken in order to determine if the postnatal differentiation of the male reproductive system is also affected by this drug. This paper reports the findings concerning the testis. J. EXP. ZOOL.,200: 1-8.
MATERIALS AND METHODS
Neonatal Sprague-Dawley rats were injected subcutaneously twice daily for three consecutive days beginning on the second day after birth with 15 mg 5-bromO-2l-deoxyuridine (Sigma Chemical Co.) per 100gram body weight. The BrdU was dissolved in saline, and protected from light. The littermate controls received saline. At the end of the experimental period the animals were weighed and killed by decapitation while under ether anesthesia. The number of experimental and control animals studied was as follows: at 5 days, 2 and 4; at 15 days, 3 and 3; and at 35 days, 2 and 2, respectively. The testes were fixed by immersion into 4 % gluteraldehyde in 0.1 M phosphate buffer (pH 7.3). After one hour the organ was sufficiently firm to allow shaving 1-mm slices from similar locations at the periphery in control and treated testes. The slices were trimmed down to l-mm3 tissue blocks. After an additional four hours in gluteraldehyde the tissue was washed in buffer, fixed further in buffered 1 % osmium tetroxide for one hour, dehydrated through a graded series of ethanol and 1 This investigation was partially supported by Public Health Service Research Grant CA-17038 &om the National Cancer Institute to Dr. T. Barka. 2 This work was performed while serving as a volunteer at Mount Sinai Hospital during his Senior Year at Dalton High School, New York City.
1
2
JONATHAN M. BARASCH AND ROBERT S . BRESSLER
propylene oxide and embedded in Epon weights in control animals were 12.3 gms, (Luft, '61). Control and experimental tis- 40.3 gms, and 147.1 gms, respectively, as sues were processed %imultaneously. Thin compared to those of experimental animals sections, cut at 600 A, with glass knives, which were 12.3 gms, 23.8 gms and 61.9 were stained in 3 % uranyl acetate and gms, respectively. At 15 and 35 days these 0.5% lead citrate (Veneble and Coggeshall, differences were significant (p < 0.005). '65). The sections were examined in a Average tubule diameters of control aniHitachi HS-8 electron microscope. mals at these ages were 102.5 p , 154.75 p One-micron thick sections, cut from the and 410.5 p , respectively, as compared to same tissue blocks and stained with tolui- 85.75, 126.75 and 208 p in treated anidine blue, were used for light microscopic mals, respectively (fig. 1). The differences studies. Diameters of all seminiferous cords are significant at all ages (p < 0.001). Aland tubules exhibiting cross sectioned pro- though initially retarded, the rates of files were measured directly from thick growth in body weight and tubule diameter sections by use of an ocular micrometer. between 5 and 15 days appeared to be the The number of treated and control tubules same in the two groups of animals. After measured was as follows: at 5 days, 21 1 15 days growth is accelerated in the norand 210; at 15 days, 217 and 76; and at mal animal but the rate remains un35 days, 56 and 14, respectively. Calcula- changed in the treated. tions were made of the percentage of tu15 days bules containing primary spermatocytes in the 15-day and lumen formation in 35For the most part, no morphological difday-old animals. ferences were noted at this age. However, relatively few pachytene spermatocytes RESULTS were present in tubules of treated animals. At 5, 15 and 35 days average body They were found in 38 % of the 25 1 tubules
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r
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I
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I
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1600
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4
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16 20 24 28 32 36
AGE ( Days) Fig, 1 Growth i n diameter of seminiferous cords and tubules with age. Bromodeoxyuridine was administered on the second, third and fourth day a s described in MATERIALS AND METHODS. The number of tubules measured were (BrdU, control) 5-day: 211, 210; 15-day: 217, 76; and 35-day: 56, 14. Differences in control and treated animals were significant at all ages studied (p < 0.001). (Control: -0-; Experimental: -0-)
EFFECTS OF BrdU ON TESTIS DEVELOPMENT
studied in control testes but only in 2% of the 672 tubules examined in treated testes. 35 days A more pronounced retardation in development between normal and treated animals, is further substantiated by electron and light microscopic observations. Whereas lumen formation was evident in 43.5% of the 33 normal seminiferous tubules studied, it was only seen in 11.3% of 98 tubules observed in treated animals. Furthermore, the chromatin material of the spermatid in the normal animal appeared diffuse and unevenly distributed in comparison with the compact, finely granular appearance observed in the experimental group (figs. 2, 3). In the control, spermatid cytoplasm was more complex, being richer in granular endoplasmic reticulum and mitochondria. Moreover, later stages of spermiogenesis were observed in the testis of the control than in the treated animals, as tails associated with spermatids were only seen in the former. Many disintegrating germ cells were present in the seminiferous tubules of the treated animals but not in the normal. BrdU had an apparent effect on Sertoli cells as well. Smooth endoplasmic reticulum was found to be more abundant in the Sertoli cell cytoplasm of the normal rat, both in apical and in basal regions, than in the treated (figs. 4, 5). Typical Sertoli cell junctions were observed in testes of treated and control animals, independent of lumen formation. The treated also contained more lipid droplets than the normal. Leydig cells follow the pattern of Sertoli cells. In the treated animal they contained less smooth endoplasmic reticulum and more lipid droplets than in the control (figs. 6, 7). DISCUSSION
The present study demonstrates that bromodeoxyuridine (BrdU) given in vivo significantly effects the maturation of the testis. The rapid growth and development of the testis in control animals between 15 and 35 days is consistent with reports of a number of developmental events occurring at this time period. Among these are organization of the tunica propria (Leeson and Leeson, '63; Ross, '67) completion of the first wave of meiosis (Clermont and
3
Perey, '57) and maturation of the Sertoli cells. This maturation is characterized by increases in the amount of agranular endoplasmic reticulum and the formation of the specialized Sertoli cell junctions (Flickinger, '67), which are the basis of the blood-testis barrier (Dym and Fawcett, '70; Ross, '70). Concurrent with the establishment of the blood-testis barrier is the canalization of the seminiferous cords and increased secretory activity by the Sertoli cells (Vitale et al., '74). At the same time depletion of stored lipid droplets in Leydig cells (Lording and de Kretser, '72) correlates with increased serum levels of testosterone (Miyachi et al., '73; Gupta et al., '75; Lee et al., '75). Many of these events are pituitary dependent (Steinberger, '71 ; Bressler and Ross, '72; de Kretser and Burger, '72; Hansson et al., '75; Bressler, '76; Fakunding et al., '76; Madhwa Raj and Dym, '76; and Welsh and Weibe, '76) and indeed take place at a time when serum gonadotropin levels are increasing (Miyachi et al., '73; Lee et al., '75) and when testosterone is rapidly being converted to androstanediol (Rivarola et al., '75; Lacroix et al., '76), a characteristic of the immature tubule. In contrast with the control, BrdU treated rats are retarded in the canalization of the seminiferous cords and in the development of agranular endoplasmic reticulum in Sertoli and Leydig cells. The latter contained many lipid droplets, a feature typical of young Leydig cells and which may indicate decreased levels of testosterone (Christensen and Gillim, '69) and its metabolites. This retardation may be a reflection of an overall impairment of growth processes as can be seen by the reduced body weight compared to controls. However, in view of the endocrine relationships involved in testicular development during this time period, and the indications that treatment of these animals affected the pituitary glands as well (Yu, unpublished observations), the possibility exists that the failure of the experimental animals to exhibit the normal acceleration of growth after 15 days is a result of impaired pituitary function. An effect on growth hormone production would also account for the differences in body weight between control and treated animals. The action of BrdU on the testis may
4
JONATHAN M. BARASCH AND ROBERT S. BRESSLER
also be direct. The substitution of BrdU for its analogue thymidine is known to be partially responsible for increased cell death (Packard et al., '74) and for temporary delays of differentiation (Davidson and Horn, '74; Rutter et al., '73) in a variety of organ systems. That this is also true for the testis is seen by the failure of primary spermatocytes to develop in numbers comparable to controls at 15 days, and of flagellar tails to form in spermatids at 35 days in treated rats. The augmentation of cell death by BrdU would result in smaller tubule diameters by reducing the number of stem cells which would otherwise increase in numbers exponentially. In view of the temporary effects of BrdU reported for several systems (Rutter et al., '73) it is difficult to explain why more degenerating cells were observed in the testes of treated animals at 35 days than at 15 days. Under certain circumstances germ cells fail to progress beyond a certain stage of development. For example spermatogonia of rats exposed to X-irradiation during fetal development (Means, '75) appear normal for a short time after birth but then enter into an abortive mitosis and degenerate. In the present study BrdU treatment does not lead to the complete absence of a particular cell type. Nevertheless, the frequency of degenerating cells at 35 days, during the progress of meiosis, may represent the failure of germ cells which have incorporated BrdU to survive this stage of spermatogenesis. This hypothesis is compatible with the finding of abnormal meiotic configurations in testis preparations &om Chinese hamsters (Mukherjee, '68). ACKNOWLEDGMENTS
The authors would like to thank Doctor W. A. Yu for providing the testes from animals used in her experiments and W s . Barbara Park for the secretarial services provided. LITERATURE CITED Bressler, R. S. 1976 Dependence of Sertoli cell maturation on'the pituitary gland in the mouse. Am. J. Anat., 147: 447-456. Bressler, R. S., and M. H. Ross 1972 Differentiation of peritubular myoid cells of the testis: Effects of intratesticular implantation of newborn mouse testes into normal and hypophysectomized adults. Biol. Reprod., 6: 148-159. Christensen, A. K., and S. W. Gillim 1969 The
correlation of fine structure and function in steroid-secreting cells, with emphasis on those of the gonads. In: The Gonads. K. W. McKerns, ed. Appleton-Century-Crofts, New York, pp. 415488. Clermont, Y., and B. Perey 1957 Quantitative study of the cell population of the seminiferous tubules in immature rats. Am. J. Anat., 100: 241-268. Davidson, R. L., and D, Horn 1974 Reversible transformation of bromodeoxvuridine-denendent cells by bromodeoxyuridine: Proc. Nat. Acad. Sci., 71: 3336-3342. de Kretser, D. M., and H. G. Burger 1972 Ultrastructural studies of the human Sertoli cell i n normal men and males with hypogonadotropic hypogonadism before and after gonadotropic treatment. In: Gonadotropins. B. B. Saxena, C. G. Beling and H. M. Gandy, eds. Wiley-Interscience, New York, pp. 640-656. Dym, M., and D. W. Fawcett 1970 The bloodtestis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod., 3: 308-326. Fakunding, J. L., D. J. Tindall, J. R. Dedman, C. R. Mena and A. R. Means 1976 Biochemical actions of follicle-stimulating hormone i n the Sertoli cell of the rat testis. Endocrin., 98: 392402. Flickinger, C. J. 1967 The postnatal development of the Sertoli cell i n the mouse. 2. Zellforsch., 78: 92-113. Fukushima, M., and T. Barka 1976 The effects of 5-bromodeoxyuridine and isoproterenol on the postnatal differentiation of rat submandibular gland. Am. J. Anat., 147: 159-182. Gupta, D., K. Rager, J. Zaraycki and M. Eichner 1975 Levels of luteinizing hormone, testosterone, and dihydrotestosterone i n the circulation of sexually maturing intact male rats and after orchidectomy and experimental bilateral cryptorchidism. J. Endocr., 66: 183-193. Hansson, V., S. C. Weddington, 0. Naess, A. Attramadal, F. S. French, N. Kotite, S. N. Neyfeh, E. M. Ritzen and L. Hagenas 1975 Testicular androgen binding protein (ABP) A parameter of Sertoli cell secretory function. In: Hormonal Regulation of Spermatogenesis. F. S. French, V. Hansson, E. M. Ritzen and S. N. Neyfeh, eds. Plenum Press, New York, pp. 323-336. Lacroix, E., W. Eechaute and I. Leusen 1975 Influence of age on the formation of Ba-androstanediol and 7a-hydroxytestosterone by incubated rat testes. Steroids, 25: 64-61. Lee, V. W. K., D. M. de Kretser, B. Hudson and C. Wang 1975 Variations in serum FSH, LH and testosterone levels i n male rats from birth to sexual maturity. J. Reprod. Fert., 42: 121-126. Leeson, C. R., and T. S. Leeson 1963 The postnatal development and differentiation of the boundary tissue of the seminiferous tubule of the rat. Anat. Rec., 147: 243-259. Lording, D. W., and D. M. de Kretser 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. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 11:736-739. Madhwa Raj, H. G., and M. Dym 1976 The
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EFFECTS OF BrdU ON TESTIS DEVELOPMENT effects of selective withdrawal of FSH or LH on spermatogenesis in the immature rat. Biol. Reprod., 14: 489-494. Means, A. R. 1975 Early sequence of biochemical events in the action of follicle stimulating hormone on the testis. Life Sci., 15: 371-389. Miyachi, Y., E. Nieschlag and M. B. Lipsett 1973 The secretion of gonadotropins and testosterone by the neonatal male rat. Endocrin., 92: 1-5. Mukherjee, A. B. 1968 Effect of 5-bromodeoxyuridine on the male meiosis i n Chinese hamsters (Cricetus griseus). Mutat. Res., 6: 173-174. Packard, D. S., R. G. Skalko and R. A. Menzies 1974 Growth retardation and cell death i n mouse embryos following exposure to the teratogen bromodeoxyuridine. Exp. Mol. Path., 21 : 351-362. Rivarola, M. A., E. J. Podesta, H. E. Chemes and S. Cigorraga 1975 Androgen metabolism i n the seminiferous tubule. In: Hormonal Regulation of Spermatogenesis. F. S. French, V. Hansson, E. M. Ritzen and S. N. Neyfeh, eds. Plenum Press, New York, pp. 25-35. Ross, M. H. 1967 The fine structure and development of the peritubular contractile cell com-
5
ponent i n the seminiferous tubules of the mouse. Am. J. Anat., 121: 523-528. 1970 The Sertoli cell and the blood-testicular barrier: An electron-microscopic study. Fortschritte der Andrologie, Morphological Aspects of andrology, 1 : 83-86. Rutter, W. J., R. L. Pictet and P. W. Morris 1973 Toward molecular mechanisms of developmental processes. Ann. Rev. Biochem., 42: 601-646. Steinberger, E. 1971 Hormonal control of mammalian spermatogenesis Physiol. Rev., 51: 1-22. Veneble, J. H., and R. Coggeshall 1965 A simplified lead citrate stain for use i n electron microscopy. J. Cell Biol., 25: 407408. Walsh, M. J., and J. P. Wiebe 1976 Sertoli cells from immature rats: i n vitro stimulation of steroid metabolism by FSH. Biochem. Biophys. Res. Comm., 69: 936941. Weintraub, H., G. Campbell and H. Holtzer 1972 Identification of a developmental program using bromodeoxyuridine. J. Mol. Biol., 70: 337-350. Yu, W. A. 1976 The effect of 5-bromodeoxyuridine on the postnatal development of the rat cerebellum: A biochemical study. Brain Res., 1 1 8 : 281-291.
PLATE 1 EXPLANATION OF FIGURES
Figs. 2 , 3
Nuclei of spermatids from 35-day-old animals.
2
Spermatid from control animal showing irregular distribution of granular nuclear contents. X 5,300.
3
Spermatid from treated animal showing even distribution of granular nuclear contents. X 3,800.
4
Sertoli cell from control animal showing abundance of dilated profiles of agranular endoplasmic reticulum. X 9,100.
5
Sertoli cell from treated animal showing scant presence of agranular endoplasmic reticulum. X 5,800.
Figs. 4 , 5
Sertoli cell cytoplasm from 35-day-old animals.
Figs. 6, 7
6
Leydig cells from 35-day-old animals.
6
Leydig cell from control animal showing abundance of profiles of agranular endoplasmic reticulum. X 10,000.
7
Leydig cell from treated animal showing less development of agranular endoplasmic reticulum. X 10,000.
EFFECTS OF BrdU ON TESTIS DEVELOPMENT Jonathan M. Barasch and Robert S . Bressler
PLATE 1
