Changes in Male Genital Organs o f Mice Exposed ... male genital organs in laboratory mammals has not yet been studied. ... Some dense clusters of Leydig.
No. 5]
41.
Proc.
Japan
Acad.,
62, Ser. B (1986)
157
Changes in Male Genital Organs of Mice Exposed Neonatally to Tamoxi f en By Taisen IGUdHI and Mariko HIROKAwA Department of Biology, Yokohama City University, Seto 22-2, Kanazawa-ku, Yokohama 236 (Communicated
by Kiyoshi
TAKEWAKI, M. J. A., May 12, 1986)
Tamoxif en, one of the triphenylethylene derivatives, inhibits estrogen action by competing with the hormone in binding the estrogen receptor.1) This compound, however, has different pharmacological actions on various organs in different species of mammals. Tamoxif en acts as an estrogen agonist/antagonist to the adult rat uterus and as an estrogen agonist to the adult mouse uterus.2)-4> Neonatal treatment of female rats and mice with tamoxifen results in vaginal and uterine changes as seen in perinatally estrogen-exposed mice.4~-8 In male mice, neonatal treatments with estrogenic hormones give rise to atrophy of genital organs including testicular lesions, e.g. a long-lasting arrest of spermatogenesis. However, the effect of neonatally administered tamoxifen on male genital organs in laboratory mammals has not yet been studied. In the present investigation, the testis and its accessories were examined in mice given neonatal injections of different doses of tamoxifen. Materials and methods. Three groups of male C57BL/Tw mice were given 5 daily subcutaneous injections of 2, 20 and 100 µg tamoxifen (Sigma, St. Louis, MO), suspended in 0.04 ml saline, respectively, starting within 24 hr after birth. Mice receiving neonatal injections of the vehicle alone served as controls. All mice were killed at 160 days of age. At autopsy, testes, seminal vesicles plus coagulating glands, epididymides and gubernacula were weighed and fixed in Bouin's solution, embedded in paraffin, and serially sectioned at 8µm. Sections were stained with Delafield's hematoxyli.n and eosin. Spermatogenic index (%) was determined by counting tubules containing spermatozoa per 100 crosssectioned tubules. Cell height of epididymal epithelium was measured in randomly chosen five sections. Data were analyzed by Student's t-test. Results. Mice given neonatal injections of 100 µg tamoxifen/day (100-Tx mice) showed a significant reduction in body weight as compared with the controls. Weights of testes and gubernacula in 2-, 20- and 100-Tx mice, seminal vesicles plus coagulating glands and epididymides in 20- and 100-Tx mice were significantly smaller than in the controls (Table I). Testes of control mice consisted of seminiferous tubules with active spermatogenesis and normally developed interstitial tissue (Fig. 1). The mean spermatogenic index was 88.2±1.03% (Table II). Some dense clusters of Leydig cells were seen in interstices among the tubules. Seminal vesicles contained a large amount of eosinophili c secretion in the lumen. The epithelium was composed of a single layer of columnar cells having numerous granules in the supranuclear regions of the cytoplasm. The epididymas contained spermatozoa in the lumen lined with an epithelium consisting of basal cells and ciliated high-columnar cells (Table II). In 9 of 10 2-Tx mice, testes and accessory organs were not different histo-
158
T. IGUCHI
and
M.
logically from those of the controls. The tended intertubular spaces largely occupied genic
index
was
low
Table
Table
II.
(15%) I.
in this
HIROKAWA
[Vol.
62(B),
10th mouse possessed testes with exby proliferated fibroblasts. Spermato-
animal.
Organ weights in 160-day-old male neonatal injections of tamoxifen
mice
given
Spermatogenic index and cell height of epididymal in 160-day-old male mice given neonatal injections of tamoxifen
epithelium
In 20-Tx mice, the mean spermatogenic index was smaller than in the controls, but was greater compared to that in 100-Tx mice. One of 20-Tx mice had testes lacking spermatids and spermatozoa. Two of the remaining 20-Tx mice had testes showing proliferation of fibroblasts in the intertubular spaces (Fig. 2). In the rest of the mice of this group, testicular structure was normal. In half the number of 100-Tx mice, spermatogenic indices were lowered (0-35%, Table II), while in the other half the indices remained unaffected. In all groups of Tx mice, the lumina of seminal vesicles were lined by a single layered columnar epithelium. Colloidal secretions in the lumina were reduced in amount with the increase in daily doses of tamoxifen. Cell height of the epididymal epithelium was significantly lower in 20- and 100-Tx mice than in the controls (Table II). Discussion. In male rats and mice, neonatal injections of estrogen cause a marked atrophy of testes and accessory organs and sustained suppression of spermatogenesis.9)-13) Perinatal exposure of rats and mice to diethylstilbestrol (DES) also brings about similar changes in male genital organs leading to sterility.14>-17) In the present study, a long-lasting suppression of spermato-
No.
5]
Genital
Organs
in Tamoxifenized
159
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Figs. 1-2. 1: Testis of a 160-day-old control mouse. Active spermatogenesis in seminiferous tubules. X97. 2: Testis of a 160-day-old mouse given 5 daily injections of 20 µg tamoxifen/day starting on the day of birth. Spermatogenesis is arrested in seminiferous tubules, some of which contain only Sertoli cells. Intertubular spaces are largely occupied by markedly proliferated fibroblasts. Small culsters of Leydig cells are occasionally seen in the spaces. X97.
genesis was observed in adult mice given neonatal injections of tamoxifen. Seminal vesicles and epididymides in neonatally tamoxif en-treated mice decreased in weight with the rise in tamoxifen dose, suggesting an increasing reduction in androgen secretion from the testis. Previous studies have revealed that the suppressive effect of neonatally injected estrogen on testicular function is nullified or attenuated by simultaneous administration of androgens or gonadotropins.is)-2o) These findings suggest that the decline of spermatogenesis in neonatally estrogenized mice may be due to direct effect of estrogen on the testis and/or estrogen-induced impairment of hypothalamo-hypophysial gonadotropic function. In addition, tamoxifen given to normal adult male rats causes a retardation of the growth of accessory organs, a reduction in testis weight and a suppression of spermatogenesis. In castrated rats, however, tamoxifen had no retarding effect on androgen-stimulated growth of the accessories.2> On the basis of the present and previous results, it may be inferred that tamoxifen exerts an estrogenic action on testes and accessory organs directly and/or indirectly through the hypothalamo-hypophysial system. Acknowledgements. The authors are greatly indebted to Prof. N. Takasugi of Yokohama City University for valuable discussions during the course of the experiments and to Emeritus Prof. K. Takewaki, M. J. A., of the University of Tokyo for his critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research and for Encouragement of Young Scientist from the Ministry of Education, Science and Culture of Japan.
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