Action of Testosterone, Dihydrotestosterone and 5a Androstane 3tr, 17(3 Diol on the Spermatogenesis of Immature Rats

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1 BIOLOGY OF REPRODUCTION 14, (1976) Action of Testosterone, Dihydrotestosterone and 5a Androstane 3tr, 17(3 Diol on the Spermatogenesis of Immature Rats H. E. CHEMES1, E. PODESTA and M. A. RIVAROLA Centro de Investigaciones Endocrinolo gicas (C.E.D.I.E.), Division de Endocrinologia and Division de Patologia, Hospital Municipal de Niios. Gallo Buenos Aires, Argentina ABSTRACT The action of testosterone (T), dihydrotestosterone (DHT) and 5cs-androstane 3a, 1 7 diol (Diol) on the seminiferous epithelium of immature estrogen-treated rats was studied. Quantitative histological evaluation was performed and testicular proteins and androgens contents were determined. Estradiol benzoate (E2B) treatment resulted in a severe testicular atrophy with a reduction of 50 percent in the testicular weight and protein content. In the estrogen treated rats the levels of androgens in the testicular tissue were undetectable. Histologically, the germinal epithelium showed a marked decrease of cell types, particularly the meiotic spermatocytes and young spermatids. In E2Bi-T, E2B+DHT and E2B+Diol-treated animals testicular weight decreased only 10 to 20 percent, with a normal concentration of testicular proteins. In E2B+T-treated animals the three androgens were found in the testis, and in both E2B+DHT and E2B+Diol-treated rats, high levels of these androgens were found, but testosterone was not detectable. In every case the levels of Diol were higher than those of DHT. A diminution of spermatogonia, similar to that found in E2 B-treated animals was noted in all androgen-treated rats. Meiotic spermatocytes showed, however, a significant increase in relation to E2B-treated rats, even though a decrease was observed when compared with the normal controls. Spermatid diminution was only partially prevented in E2B#{247}DHT and E2B+Diol-treated groups, but not in E2B+T treated rats. It is concluded that T, DHT and Diol, exert a meiotic stimulatory effect. In the case of DHT and Diol-treated animals, absence of testicular testosterone indicates that both Sn reduced metabolites are biologically active in the prevention of testicular atrophy induced by E2B at the time of initiation of spermatogenesis. INTRODUCTION It has been shown that seminiferous tubules of maturing rats have both 5tt and 3a-hydroxy reductase activities, which are capable of reducing testosterone (T) to dihydrotestosterone (DHT) and to 5a-androstane, 3a, 1713 diol (Diol). The activities were maximal at the time of development of the first meiotic division (Rivarola et al., 1972; Folinan et al, 1973). These findings were confirmed by an in vivo study showing that the concentration of Diol in seminiferous tubules increased from the 10th to the 20th day of life, remained high up to Day 26 and decreased thereafter. This increment coincided with the initiation and completion of the first meiotic division (Podesta and Rivarola, 1974). Accepted November 19, Received January 30, Present address: Department of Anatomy, Harvard Medical School, 25 Shattuck at. Boston, Massachusetts. Since it has been shown that the reduction of T to DHT is a necessary step in the mechanism of action of T in some androgen-responsive tissues (Bruchowsky and Wilson, 1968; Anderson and Liao, 1968), it was speculated that reduction of T to DHT and Diol might be involved in the stimulation of the first meiotic division. Testosterone (Walsh et al, 1934; Nelson and Gallagher, 1936; Nelson and Merckel, 1937; Cutuli and Cutuli, 1940; Ludwig, 1950; Clermont and Harvey, 1967) and recently DHT (Ahmad et al, 1973) have been shown to maintain spermatogenesis in the adult hypophysectomized rat. However, maintenance of spermatogenesis in adult rats should be differentiated from the initiation of spermatogenesis in immature animals, since the mechanisms involved might be different. In this regard, exogenous T could stimulate spermatogenesis in immature animals (Steinberger and Duckett, 1965, 1967). On the other hand, Steinberger and Steinberger (1972) reported that DHT stimulated the growth of the seminiferous 332

2 T, DHT AND DIOL ACTION ON IMMATURE SEMINIFEROUS TUBULES 333 tubules but not to the same degree as T, while androsterone and androstanediol were essentially ineffective in this regard. Therefore, they suggested that T was the active androgen in the seminiferous tubules. In the present paper we wanted to reexamine the question of the induction by androgens of the first steps of spermatogenesis in the maturing rat. We chose the administration of exogenous estrogens as a means of inhibiting the spontaneous development of spermatogenesis. This has been demonstrated in mature as well as immature animals (Ludwig, 1950; Lynch, 1952; Steinberger and Duckett, 1965). The action of estrogens could be mediated by inhibiting gonadotropin production, by a direct effect on the testes, or both. It has also been shown that it can be reversed by the simultaneous administration of T, at least up to the early steps of spermiogenesis (Steinberger and Duckett, 1965, 1967). It is known that in order to achieve the required high concentrations of androgens within the testes from a remote source such as a subcutaneous injection, it is necessary to administer a large dose of T (Ludwig, 1950). The physiological significance of the dose was checked by determining the androgen levels reached in the testes at the time of autopsy. MATERIALS AND METHODS Wistar male rats were divided into five groups of 5 animals each. The protocol of the hormonal administration is stated in Table 1. In every animal the two testes and seminal vesicles were weighed. The seminal vesicles were not emptied before weighing. One of the testes and one of the seminal vesicles were placed in Bouin s fixative, embedded in paraffin and sectioned at 5Mm. The sections were stained with hematoxylin and eosin. In day old rats Clermont and Percy (1957) described four types of tubules on the basis of the presence of different spermatogonia or resting spermatocytes. In 26 day old rats meiotic spermatocytes and young spermatids associate in a definite way with the type of spermatogonia which is present (Chemes et al., 1976). In type 1 there were few type A spermatogonia, the tubules contained mainly intermediate type spermatogonia, zygotene spermatocytes and young spermatids. In type 2 there were again a few type A, abundant type B spermatogonia and pachytene spermatocytes, and scant young spermatids. Type 3 contain a few type A spermatogonia, abundant resting and pachytene primary spermatocytes and occasionally a few spermatids. Type 4 contain exclusively type A spermatogonia and two generations of meiotic spermatocytes at leptotene and dip lotene, although some were at zygotene and secondary spermatocytes could be seen. The germinal cells in ten tubules of each type were counted per animal, Le., a total of 40 tubular sections per animal and 50 tubular sections of each type per experimental group. Only round cross sections of tubules randomly selected from the mid portion of the testis were quantitated. All the seminiferous tubules could be classified into one of the above-described types. This was true even in the case of experimental groups in which pachytene and diplotene spermatocytes were absent or degenerating, since the tubules were classified according to spermatogonia and resting spermatocytes that were always present. Type A spermatogonia and young spermatids were counted only in tubular types 4 and 1, respectively, i.e., in those tubules in which they were predominantly found. Pachytene spermatocytes were recorded only in type 2 tubules. Means and the standard error of the mean were calculated for each group, and after an analysis of variance the statistical significance was evaluated by applying the Tuckey Keul s test (Keuls, 1952). The cellular counts were then corrected for differences in nuclear diameter using Abercrombie s formula (Abercrombie, 1946, T = Cx_S in which T=true counts, S+D C=crude counts, S=thickness of the section, D=nuclear diameter). Ratios between different cellular types were obtained with these counts as a parameter of the yield of pachytene and diplotene spermatocytes. In order to assess the incidence of cellular degenerative changes other than modifications in cellular numbers (e.g., multinucleated spermatocytes, intratubular necrotic cell debris, etc., see results), the percentage of tubules showing these kinds of abnormalities was estimated. Tubules showing only a decreased number of cells in the absence of other degenerative cellular changes, were not considered in this group because their incidence was covered by the cellular counts. Testicular contents of T, DHT and Diol were determined using a competitive binding technique already described (Podesta and Rivarola, 1974). Testicular proteins were determined by the method of I.owryet al. (1951). RESULTS Total body weights and weights of the two testes and the two seminal vesicles are shown in Table 1. Administration of E2B decreased testicular weight by 50 percent while addition of any of the 3 androgens prevented this effect, even though some decrease was observed. Hypertrophy of the muscular wall of the seminal vesicles and atrophy of their glandular epitheliurn were noticed in E2B treated rats. Administration of T resulted in a marked increase in the weight, glandular development and secretory activity of the glands. DHT and Diol treatments resulted in a greater effect in this regard. Microscopic examination of the seminiferous tubules of the control group showed completion of meiosis with the presence of young spermatids in tubules type 1 (Fig. 1). In E2B treated animals, the germ cell population was

3 334 CHEMES ET AL. TABLE 1. Mean weight of rats and combined weight of the testes and seminal vesicles. Experimental groups Body wt. g ± SEM Testes mg ± SEM S. vesicles mg ± SEM Group 1, Control Group 2, E2B 65.1 ± 2.26a 58.7 ± 2.30a ± 55a 35.3 ± ± ± 72b Group 3, E2B + T 59.6 ± 1.35a ± 2.4a 96.5 ± 71b Group 4, E2B + DI-IT 63.8 ± 0.73a ± 13.2a ± 12.3c Group 5, E2B + Diol 61.2 ± ± 7.0k ± 13.o In the same column, values with different superscript letters are significantly different (P<0.01). Testes in group 3 are different from group 1 (P<0.05). Control group received only vehicle (olive oil). E2B dose was 50 i.ig/100 g body weight, per day. T, DHT and Diol dose was 1.5 mg/100 g body weight per day. The animals were injected daily from day 5 to day 25 of life, and were sacrificed by decapitation at the age of 26 days, i.e., at the end of the first meiotic division (Clermont and Percy, 1957). E2B was administered in an olive oil solution containing 100 Mg/mI. T, DHT and Diol were administered as microcrystals suspended in distilled water at a concentration of 3 mg/mi. All the steroids were injected subcutaneously. The dose and volume of the steroids were changed every 3 days according to the increasing weight of the animals. decreased, particularly the pachytene and the diplotene spermatocytes. Spermatids were practically absent. Several degrees of regressive lesions were observed in the germinal epithelium, mostly in the final stages of meiosis. These included multinuclear meiotic spermatocytes, vesicular nuclei with poor visualization of their chromosomes, intratubular necrotic cell debris, sloughing of degenerating spermatocytes, dilatation of tubular lumen with scarce or absent meiotic spermatocytes. This last type of tubules often showed a focal distribution (Figs. 2, 3, and 4). The animals of the groups treated either with T, DHT, or Diol showed a qualitatively normal appearance with an abundant germ cell population. Some of the abnormalities described for the E2B treated group were also observed in some of the tubules of the androgen treated group, but their number was much smaller (Figs. 5, 6, and 7). Table 2 shows the percentage of qualitatively abnormal tubules in every group. Testosterone, DHT and particularly Diol prevented the regressive changes produced by E2B. The resuits of the crude cell counts in the seminiferous tubules, uncorrected by Abercrombie s formula, are shown in Table 3. In all treated groups, spermatogonia were decreased 20 to 30 percent when compared with the untreated control. Pachytene and diplotene meiotic spermatocytes were markedly decreased in the E2B treated group. This decrease was significantly prevented when T, DHT or Diol were added. The decrease in the number of spermatids observed in the E2B-treated rats, was partially prevented only by DHT and Diol treatments, but their values reached only to percent of the control ones. In all steroid treated animals Leydig cells were found to be atrophic. In Table 4, cellular coefficients obtained after applying Abercrombie s formula show striking changes at the meiotic level. In the E2B treated group two or three times more resting spermatocytes were needed to produce one pachytene or diplotene spermatocyte, when compared with the control group. These coefficients approach their control value in the androgen treated groups, indicating an improve- FIG. 1. Control group. Development of germinal epithelium up to the early steps of spermiogenesis. Type 1 tubule showing a group of young spermatids (arrows). H.E., 480X. FIG. 2. E2B treated group. Meiotic spermatocytes are markedly decreased. No spermatids are present. H.E., 480X. FIG. 3 and 4. E2B treated group. Seminiferous tubules with multinucleated primary spermatocytes, two of them showing a rosette-like disposition of their nuclei surrounding the chromatoid body (Fig. 3). H.E., 640X. FIG. 5. (E2B + T), Fig. 6 (E2B + DHT) and Fig. 7 (E2B + Diol). Numerous meiotic spermatocytes are evident. Note the young spermatids in Figs. 6 and 7 (arrows) and spermatocytes in division in Fig. 7 (bottom tubule). Recovery is not complete in relation to control. Note quantitative and qualitative differences when compared with E2B treated. 1-LE., 480X.

4 T, DHT AND DIOL ACTION ON IMMATURE SEMINIFEROUS TUBULES 335 #{149} T :i k-

5 336 CHEMES ETAL. TABLE 2. Percentage of tubules with qualitative abnormalities. *1*1*1+1*1,_ Experimental groups No. of tubules examined % - U s Group 1, Control Group 2, E2B Group3.E2B+T Group4,E2B+DHT Group5.E2B+Diol *t*i*i*i+!.1! U.5 -o >S ment in the yield of these meiotic cellular types. Table 5 shows the contents of proteins, T, DHT and Diol in the five groups studied. It can be seen that E2B administration resulted in a sharp decrease of protein contents in the testis that was prevented by either T, DHT or Diol. These changes were parallel to changes in total testicular weight and cellular contents. Androgens could not be detected in the E2B treated group, but were similar to the control when T was added. No T was detected after administration of DHT or Diol, the latter being the predominant androgen in all androgen treated rats. -. * ite.i +1+14*1+ 5 o roo 5 -. E -,s 5.O-c. U-O,.. - SJ V E *1 +1 U.C z tm O t-.wseo,r, - U 0 0. DISCUSSION The effect of the administration of sex steroids on the testis could be mediated in part by the inhibition of gonadotropin production. On the other hand they can have a direct action upon the gonad itself. A 30 percent decrease in the number of spermatogonia has been reported after hypophysectomy by Clermont and Harvey (1967). Davies (1971) and Mills and Means (1972) have reported that FSH stimulates spermatogonial proliferation in immature animals, and Huckins et al. (1973) showed that the incidence of spermatogonial degeneration in the immature testis was strikingly decreased shortly after the administration of FSH. The inhibition at the spermatogonial step of spermatogenesis which we observed in our animals (approximately percent) could be secondary to FSH inhibition produced by E2B. There are similarities between our findings on the effect of hormonal inhibition at this step of spermatogenesis and those of Clermont and Harvey (1967), even though their results refer to hypophysectomized adult rats, a situation that 5 5 cc V o..f ce Ii a 5. o +1 *1 *1 *1 +1 b y,.5 r i.e u - U.5 S. o *e-sr-,s U *1 +1 *1 *1 t 0 t- 0 o. -,, 0 C C - 5 C,s C.C.., o..2- b e >.. - E- E C V - UOV 5 (.4 *.5 j U,d V < ;.si C c

6 T, DHT AND DIOL ACTION ON IMMATURE SEMINIFEROUS TUBULES 337 TABLE 4. Cellular coefficients between different cell types in the five groups studied. Experimental groups R:P R:D Group 1, Control 2.9:1 14.7:1 Group 2, E2B 9:1 24.8:1 Group3,E2B+T 3.6:1 9.1:1 Group4,E2B+DHT 4.4:1 20.5:1 Group 5, E2B + Diol 4.5:1 12.9:1 Ratios between different cell types: R:P, resting primary: pachytene spermatocytes, R:D, resting primary: diplotene spermatocytes. may be different to the estrogen treatment of immature rats. On the other hand, Matsuyama et al. (1971) have reported quantitatively normal spermatogonial numbers in hypophysectomized adult rats treated with testosterone. Steinberger and Duckett (1965, 1967) noted that the simultaneous administration of T protected the testes of immature rats from the depresive effects of E2B. Administration of either T, DHT or Diol along with E2B in our animals produced a similar effect, particularly stimulation of meiosis. A quantitative improvement of this process was evident as shown by the number of pachytene and diplotene spermatocytes formed per each resting spermatocyte. In a previous study we found a similar action of T in immature intact animals (Chemes et al., 1976). Therefore, these steps of the meiotic prophase are probably under androgen control. However, in the androgen-treated rats restitution was not complete, probably because of the spermatogonial inhibition that decreases the number of cells which are required for the production of the more mature elements. We could not detect major differences in the effect of the three androgens. This biological stimulation of the first meiotic division can be correlated with the high contents of T and Diol in the seminiferous tubules that we reported previously (Podesta and Rivarola, 1974) as well as with the stimulation of 5a-reductase activity found at this stage of development (Rivarola et al., 1972). The levels of T, DHT and Diol in the testes of the E2B#{247}Ttreated group were similar to the control group, suggesting that the dose of T was close to the physiological range for the testis. The dose was obviously excessive for seminal vesicle function. These levels demonstrate that both 5a and 3ct hydroxy reductases were fully active in the presence of the administration of high doses of E2B and androgens which presumably suppress endogenous gonadotrophic secretion. In the E2B+DHT and E2B+Diol treated groups, however, no T could be detected, indicating absence of oxidation of the 4-5 bond. This is not surprising since oxidation of 5ct reduced steroids is very poor in biological fluids. DHT and Diol can therefore stimulate meiosis in the absence of T. Whether T can act by itself or after reduction to DHT or to Diol, can not be determined at present. Therefore, we do not know which androgen stimulates the first meiotic division in developing male rats. However, at this time of development, Diol is quantitatively the main androgen present in the seminiferous tubules (Podesta and Rivarola, 1974), and also in the testes of our T, DI-IT or Diol treated rats. Finally, these data provide further evidence for the major role played by androgens in the stimulation of meiosis in the testis. REFERENCES Abercrombie, M. (1946). Estimation of nuclear population from microtome sections. Anat. Rec. 94, TABLE 5. Contents of proteins and various androgens per testis. Experimental groups Proteins T DHT Diol Group 1, Control Group 2, E2B 1.12 ND ND ND Group 3, E2B + T Group 4, E2B + DHT 4.87 ND Group 5, E2B + Diol 5.03 ND mg per testis. ng per testis. ND: non detectable.

7 338 CFIEMES ET AL Ahmad, N., Haltmeyer, C. G. and Eik-Nes, K. B. (1973). Maintenance of spermatogenesis in rats with intratesticular implants containing Testosterone or Dihydrotestosterone (DHT). Biol. Reprod. 8, Anderson, K. M. and Liao, 5. (1968). Selective retention of dihydrotestosrerone by prostatic nuclei. Nature (London) 219, Bruchowsky, N. and Wilson, J. D. (1968). The intranuclear binding of testosterone and 5o-Androstan-17p-ol-3-one by rat prostate. J. Biol. Chem. 243, Chemes, H. E., Rivarola, M. A. and Bergada, C. (1976). Action of gonadotropins and testosterone on the seminiferous tubules of the immature rat. J. Reprod. Fertil. (in press). Clermont, V. and Harvey, C. S. (1967). Effects of hormones on spermatogenesis in the rat. Ciba Found. Colloquia on Endocrinology of the testis. 16, Clermont, V. and Perey, B. (1957). Quantitative study of the cell population of the seminiferous tubules in immature rats. Am. J. Anat. 100, Cutuli, E. and Cutuli, E. C. (1940). Observations on spermatogenesis in the rat. Endocrinology 26, Davies, A. C. (1971). Histological changes in the seminiferous tubules of immature mice following administration of gonadotropins. J. Reprod. Fertil. 25, Folman, V., Ahmad, N., Sowell, J. G. and Eik-Nes, K. B. (1973). Formation in vitro of 5cs-dihydrotesrosterone and other 5cs-reduced metabolites of H- Testosterone by the seminiferous tubules and interstitial tissue from immature and mature rat testis. Endocrinology 92, Huckins, C., Mills, N., Besch, P. and Means, A. R. (1973). Spermatogonial differentiation in immature testis: regulation by FSH. Endocrinology 92, A-94. Keuls, M. (1952). The use of the studentized range in connection with an analysis of variance. Euphysical, Lowry, 0. H., Rosenbrough, N. J., Fan, A. L and Randall, R. J. (1951). Protein Measurement with the Folin phenol reagent. J. Biol. Chem. 193, Ludwig, D. J. (1950). The effect of androgens on spermatogenesis. Endocrinology 46, Lynch, K. M. (1952). Recovery of the rat testis following estrogen therapy. Ann. N.Y. Acad. Sci. 55, Matsuyama, S., Voyoki, V. and Ogasa, A. (1971). A quantitative study of spermatogenesis in androgen treated hypophysectomized rats. Nat. Inst. Anim. HIth. Quart. 11, Mills, N. C. and Means, A. R. (1972). Sorbitol dehydrogenase of rat testis: Changes of activity during development, after hypophysectomy and following gonadotropic hormone administration. Endocrinology 91, Nelson, W. 0. and Gallagher, T. F. (1936). Some effects of androgenic substances in the rat. Science 84, Nelson, W. 0. and Merckel, C. (1937). Maintenance of spermatogenesis in testis of the hypophysectomized rat with sterol derivatives. Proc. Soc. Exp. Biol, Med. 36, Podesta, E. and Rivarola, M. A. (1974). Concentration of androgens in whole testis, seminiferous tubules and interstitial tissue of rats at different stages of development. Endocrinology 95, Rivarola, M. A., Podesta, E. and Chemes, H. E. (1972). In vitro testosterone 14C metabolism by rat seminiferous tubules at different stages of development. Formation of 5n-androstandiol at meiosis. Endocrinology 91, Steinberger, E. and Duckett, G. E. (1965). Effect of estrogens and testosterone on initiation and maintenance of spermatogenesis in the rat. Endocrinology 76, Steinberger, E. and Duckett, G. E. (1967). Hormonal control of spermatogenesis. J. Reprod. Fertil. Suppl. 2, Steinberger, E. and Steinberger, A. (1972). The testis: Growth versus function. In: Regulation of Organ and Tissue Growth, R. J. Goss (Ed.) pp Acad. Press, Inc. N.V.-I.ondon. Walsh, E. L., Cuyer, W. K. and McCullag, D. R. (1934). The physiologic maintenance of the male sex glands. Amer. J. Physiol. 107, RECOMMENDED REVIEW Steinberger, E. and Steinberger, A. (1975). Hormonal control of testicular function in mammals. In: Handbook of Physiology. Section 7; Endocrinology. VoL IV, part 2, pp Greep, R. 0. and Astwood, E. R., Eds. Williams & Wilkins Co., Baltimore. ADDENDUM While this paper was being considered for publication, a work by Chowdhury, A. K. and Steinberger, E. (Biol. Reprod. 12: , 1975) appeared. Our results and conclusions in relation to the action of T and two So reduced androgens on meiosis coincide with theirs, but we have not observed any delay in the completion of meiosis in our E2B + DHT and E2B + Diol treated animals, even though the number of spermatids was reduced when compared with normal rats of the same age.