Kjell J. Tveter OF ANDROGEN BY SOME ORGANS OF THE MALE RAT. prostate, the uptake was 205 higher in animals castrated 24 h previously

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1 Institutt for Kirurgisk Forskning, and Institutt for Patologisk Anatomi, Rikshospitalet, Oslo FURTHER STUDIES ON THE UPTAKE OF ANDROGEN BY SOME ORGANS OF THE MALE RAT By Kjell J. Tveter ABSTRACT [1,2-3H]Testosterone with a specific activity of 42.3 Ci/mmole was injected intramuscularly to adult castrated male rats. There was a selective uptake of radioactivity by the prostate, where a high and prolonged accumulation of radioactive material was found, in contrast to the much lower uptake by muscle tissue. The influence of castration on the uptake was investigated. In the ventral % prostate, the uptake was 205 higher in animals castrated 24 h previously than in non-castrated animals. The corresponding values for the lateral prostate, the coagulating glands and the seminal vesicles were 120%, 165 % and 213 % respectively. The uptake by the dorsal prostate was only about 23 % higher one day after orchidectomy. The uptake by muscle was apparently not influenced by castration. Following homogenization of the coagulating glands and the dorsal and ventral prostate, some of the radioactivity in the \m=x\ g supernatant fraction 1 h after the administration of [1,2-3H] testosterone in vivo was associated with macromolecules. In the lateral prostate an interaction between radioactive material and soluble macromolecules was only found in vitro. In a previous study a high and prolonged accumulation of radioactivity was demonstrated in the four lobes of the rat prostate, following the administration of [3H] testosterone in vivo (Tveter 1969). By correlating the uptake of radio activity to the wet weight of the tissues, significant differences were found be tween the various lobes. Seven and a half minutes after administration of the This work was supported by grants from the Norwegian Research Council for Science and the Humanities.

2 labelled hormone, the concentration of radioactivity was almost the same in all lobes. One hour after the injection, however, the uptake by the ventral lobe had increased by about 285 ' /o, while that by the dorsal lobe only by 80 /o. These results indicate that there may be differences between these lobes with regard to their capacity to take up and retain androgen. Similar differences have also been found by correlating the uptake to the amount of total protein (Tveter Sc Aakvaag 1969). The uptake by the seminal vesicles is of the same magnitude as that by the coagulating glands (Tveter Sc Unhjem 1969). Almost all the endogenous androgen is probably removed from these acces sory genital organs one day after orchidectomy (Tveter 1969). Most of the cel lular binding sites for androgen might then be available for the binding of la belled steroid. If one assumes that the greater part of the binding sites in these target organs in non-castrated animals is saturated by endogenous androgen, it is possible that the difference in uptake between non-castrated animals and ani mals castrated one day previously, may give some indication of the capacity of the organs to accumulate androgen. Soluble macromolecules capable of binding androgen have been demonstrat ed in the X g supernatant fraction of seminal vesicles and ventral pro state homogenates (Tveter Sc Unhjem 1969; Unhjem Sc Tveter 1969). Recent studies have shown that the androgen bound is mainly represented by 5a-dihydrotestosterone (5a-androstan-17/j-ol-3-one) (Unhjem et al. 1969). It is now generally agreed that in target tissues this compound is the main metabolite of testosterone (Farnsworth Sc Brown 1963; Shimazaki et al. 1965; Bruchovsky Sc Wilson 1968; Tveter 8c Aakvaag 1969). The present report describes the influence of some factors on the uptake of radioactivity by the prostate gland and the seminal vesicles after administration of [3H] testosterone in vivo. Moreover, evidence will be presented that some of the radioactivity in the high-speed supernatant fraction of homogenized dorsal prostate and coagulating glands is associated with soluble macromolecules in vivo. MATERIALS AND METHODS Adult male rats ol a local strain, weighing approximately 250 g, were used throughout the experiments. [1,2-3H]Testosterone with a specific activity of 42.3 Ci/mmole and a purity of more than 98.5 %> was obtained from the New England Nuclear Corporation. The purity was checked by paper chromatography on Whatman No. 1 paper with hexane:benzene (1:1, v/v) as the mobile phase and formamide as the stationary phase; no radiochemical impurities were found. Uptake studies In these studies, [1,2-3H]testosterone dissolved in propylene-glycole:ethanol:salinc

3 (1:1:2, v/v/v), was given intramuscularly in a dose of 40 «Ci per 100 g body weight to rats castrated 3 days previously. Groups of 5 animals were killed by decapitation 1h-1/i-1h-l-2-A-8 and 16 h after the injection. Specimens from the rectus abdominis muscle and from the ventral and dorsal prostate were frozen and kept at -15 C. The specimens were thawed and then blotted to remove surface moisture, and weighed. In one experiment, all the moisture and secretions were expressed from thawed prostatic specimens by finger pressure between two pieces of filter paper. Radioactivity measure ments were performed according to the principles described previously (Tveter 8c Attramadal 1968). Quench correction was obtained by external standardization, and the results calculated as cpm/mg wet weight. The influence of castration on uptake 18 rats were used in these experiments. Of these, six animals were non-castrated; six were castrated one day and six castrated seven days, respectively, before injection of the hormone. Each animal received 11.4,«Ci of [1,2-3H] testosterone dissolved in 15 /o ethanol in saline, per 100 g body weight. The animals were killed 1 h after the in jection, and samples were taken from the various prostatic lobes, the seminal vesicles and from muscle. The samples were homogenized in 0.1 m Tris-HCl buffer, ph 7.4, and aliquots taken for measurements oí radioactivity according to the principles des cribed previously (Unhjem Sc Tveter 1969), and for determination of protein according to the method of Lowry et al. (1951). Gel-filtration of X g supernatant fractions For these experiments, animals castrated one day previously were used. [1,2-3H] Testo sterone was dissolved in 15 /o ethanol in saline, and each animal was injected either with 100 µ of the hormone in some experiments, or with 60.«Ci in others. The prin ciples for homogenization, gel-filtration and determination of radioactivity have been described elsewhere (Unhjem Sc Tveter 1969). The animals were killed 1 h after the injection; in one single experiment gel-filtration was performed on ventral prostate tissue 2 h after the administration of hormone. The gel-column (27 X 1.9 cm) was eluted with Tris-HCl buffer, ph 7.4; ml fractions were collected. Radioactivity was expressed as cpm in 0.5 ml of each fraction. For the in vitro studies, the lateral prostate tissue of five rats was cut into thin slices and incubated in an atmosphere of air for 30 min at 37 C in 3 ml of Eagle's tissue cul ture medium (Eagle 1959) containing µg [1,2-3H] testosterone. RESULTS Fig. 1 shows the concentration of radioactivity in the muscle and in the ven tral and dorsal prostate at different intervals after the injection of [3H] testo sterone into rats castrated 3 days previously. The concentration in the muscle was much lower than that in the prostate. The uptake by the prostate increased after the administration, and reached a maximum after 1 to 2 h. Even after 8 h the uptake by the ventral prostate was almost of the same magnitude as that after 1 h (Fig. 1 A). The uptake by the ventral lobe was about twice as high as that by the dorsal lobe, in accordance with previous results (Tveter 1969). When the secretory products and moisture were removed by pressure after

4 CPM, /MG DORSAL PROSIATE CPM, 'MG 500 « je DORSAL -» PROSTATE Figs. 1 A and 1 B. 1 A: Distribution of radioactivity in the ventral and dorsal prostate, and in skeletal muscle of adult rats orchidectomized 3 days before intramuscular injection of [1,2-3H]- testosterone dissolved in propylene-glycobethanobsaline. 1 B: Almost all the radioactivity is lost from the ventral prostate by expressing the moisture and secretions after thawing of the frozen specimens. thawing of the frozen specimens, the concentration of radioactivity in the dor sal lobe increased by about 100 /o (Fig. 1 B). In the ventral lobe, however, this procedure removed most of the radioactivity from the specimens, except for those examined 7 and 15 min after the injection, when higher values were found. The influence of castration on the uptake of radioactivity by the various tis sues is illustrated in Fig. 2. In the ventral prostate of animals castrated 24 h previously, the uptake was 205 % higher than in non-castrated rats. The corres ponding values for the dorsal lobe, the lateral lobe, the coagulating glands and the seminal vesicles were 23%, 120%, 165% and 213%, respectively. The uptake by the prostate and the seminal vesicles of animals castrated seven days previously was also higher than that in non-castrated animals. The uptake by muscle was not significantly influenced by orchidectomy. Gel-filtration was first performed at room temperature on X g su-

5 - cpm/mg protein VENTRAL PROSTATE NON-CASTRATED m là SEMINAL VESICLE m COAG. GLAND CASTRATED LATERAL PROSTATE ID Fig. 2. The uptake of radioactivity by some organs of the male rat 1 DORSAL PROSTATE CASTRATED 7D nr h after administration of [1,2-3H] testosterone in vivo. The left columns indicate the values for non-castrated before the in animals; the uptake in animals castrated 1 day and 7 days, respectively, jection of hormone is illustrated in the median and right columns. The vertical bars indicate the standard error of the mean. pernatant fractions from frozen tissue which was homogenized after thawing. Under these circumstances no radioactivity was recovered with soluble macro molecules from the dorsal and lateral prostate and the coagulating glands. In the next experiment fresh tissue was used, and the gel-filtration carried out at room temperature. Even under these conditions no radioactivity was associated with macromolecules. Since much more radioactivity is associated with soluble macromolecules when the gel-filtration is performed at 4 C than at room tem perature (Tveter Sc Unhjem 1969), care was taken in the next experiments to keep the tissue ice-cold from the moment of sacrifice and throughout all proce dures. Gel-filtration was therefore carried out at 4 C, and some of the radio activity in the X g supernatant fraction was then associated with so luble macromolecules both in the dorsal prostate and the coagulating glands (Figs. 3 and 4). In the lateral prostate, no association of radioactivity with so luble macromolecules could be demonstrated in vivo (Fig. 5). Fig. 6 shows that when gel-filtration is performed at 4 C, almost all the ra dioactivity in the supernatant fraction prepared from ventral prostate homo genates is associated with macromolecules 1 h after the administration of 3Hlabelled hormone; quite identical results were obtained 2 h after the injection. The in vitro experiments indicated that some radioactive material was asso ciated with Tris-HCl soluble macromolecules of lateral prostate homogenates after incubation with [3H] testosterone for one half hour (Fig. 7).

6 - 100 CPM 80 DORSAL PROSTATE OD 280 mu Á FRACTION Fig. 3. Dotted line shows the elution pattern obtained when subjecting the X g super natant fraction ot dorsal prostate homogenate of 5 rats to gel filtration (Sephadex G-100), determined at 280 µ. Solid line represents radioactivity 1 h after administra tion of [1,2-3H] testosterone in vivo. NO FRACTION NO. Fig. 4. Dotted line shows the elution pattern obtained when subjecting the X g super natant fraction of coagulating gland homogenate of 2 rats to gel filtration; solid line shows radioactivity 1 h after administration of [1,2-3H] testosterone in vivo.

7 CPM OD FRACTION NO. Fig. 5. Dotted line shows the elution pattern obtained when subjecting the X g super natant fraction of lateral prostate homogenate of 7 rats to gel filtration; solid line re presents radioactivity 1 h after administration of [1,2-3H] testosterone in vivo FRACTION NO. Fig. 6. Dotted line shows the elution pattern obtained when subjecting the X g super natant fraction of the ventral prostate homogenate of 1 rat to gel filtration; solid line shows radioactivity 1 h after administration of [1,2-3H] testosterone in vivo.

8 OD 280 mi FRACTION Fig. 7. Dotted line shows the elution pattern obtained when subjecting the X g super natant fraction of lateral prostate homogenate of 4 rats to gel filtration. Solid line re presents radioactivity after incubation with ßg [1.2-3H] testosterone in vitro for 30 min. NO. DISCUSSION The first part of the present study is concerned with the uptake of radioactivity by the ventral and dorsal prostate in adult castrated male rats injected with [1,2-8H] testosterone. The results indicate that there is a selective uptake by the prostate, where the radioactivity is accumulated in concentrations far higher than in the muscle. An almost constant level of radioactivity in the ventral prostate was observed between 1 and 8 h after the administration of [3H] testo sterone (Fig. 1 A). This rather prolonged accumulation and retention is pro bably due to the use of propylene-glycol as the hormone solvent, since a much more rapid decrease in radioactivity is observed when the hormone is only dis solved in ethanol:saline (Tveter Sc Attramadal 1968; Tveter 1969). Most of the radioactivity in the ventral prostate can be removed by pressure after freezing and thawing of the specimens (Fig. 1 B), in contrast to the findings obtained by using fresh tissue (Tveter 1969). It thus appears that the freezing-thawing pro cedure may, under certain conditions, represent a source of error in studies of this kind. The second part of the present report describes the effect of castration on the uptake of [3H] testosterone and/or testosterone radiometabolites in vivo. Ac-

9 cording to the present results, the influence of castration on the uptake is dif ferent in various organs. The uptake by the ventral prostate and the seminal vesicles is about three times higher one day after castration than in intact noncastrated animals. In the lateral prostate and the coagulating glands, the response is somewhat less. In the dorsal prostate, on the other hand, the aug mentation in uptake due to castration is much lower. If an accumulation of a hormone and/or metabolite(s) in a tissue is related to its metabolic effects, these findings might indicate that the dorsal prostate is less androgen dependent than the other target organs investigated. If this suggestion is correct, one would expect that the metabolic effects of administration of testosterone would be less in this lobe. It may therefore be interesting to note that Awapara (1952) reported that after castration the weight of the dorsal prostate does not de crease to the same extent as that of the ventral prostate. According to the present study, the prostate gland and the seminal vesicles also have the capacity to accumulate androgen seven days after castration. Te stosterone has likewise a stimulating effect on the accessory genital organs of male animals castrated even many weeks before injection of the hormone (Price Sc Williams-Ashman 1961). The uptake by muscle tissue was apparently not influenced by castration (see Fig. 2). In a previous report, it has furthermore been found that after admini stration of [3H] testosterone in vivo, the uptake of radioactivity by muscle was not influenced by the simultaneous administration of hormonal compounds capable of reducing the uptake by target organs (Tveter Sc Aakvaag 1969). The third part of this study describes the association of some 3H-labelled com pound^) with soluble macromolecules in the X g supernatant fractions of homogenates obtained from the various prostatic lobes after the administra tion of [3H] testosterone. While almost all the radioactivity in the supernatant fraction of the ventral prostate is associated with macromolecules (Fig. 6), only part of the radioactivity in the dorsal prostate (Fig. 3), the coagulating glands (Fig. 4) and the seminal vesicles (Tveter Sc Unhjem 1969), is associated with macromolecules. No similar association has been detected in the lateral prostate in repeated investigations in vivo; the present experiments in vitro indicate, however, that such binding may take place even in the lateral prostate. The possible physiological significance of this binding, and of the possible differen ces between various organs, is not understood. The capacity of these organs to accumulate androgen might, however, be contingent upon the presence of these androphilic macromolecules since no similar binding has been detected in»nontarget«organs such as muscle, kidney and liver (Unhjem Sc Tveter 1969). The recent report of Fang Sc Liao (1969) describes a cytoplasmic fraction which is necessary for the association of 5«-dihydrotestosterone with androphilic sub stances in isolated prostatic nuclei. Their results suggest a two-step mechanism for the interaction of 5a-dihydrotestosterone with the ventral prostate.

10 REFERENCES Awapara ].: Endocrinology 51 (1952) 75. Bruchovsky. & Wilson J. D.: J. biol. Chem. 243 (1968) Eagle H.: Science 130 (1959) 432. Fang S. 8c Liao S.: Fed. Proc. 28 (1969) 846. Farnsworth W. W. 8c Brown J. R.: Nat. Cancer Inst, Monograph No. 12, Bethesda, Maryland (1963) p Lowry 0. H.. Rosebrough N. J.. Fair A. L. 8- Randall R. J.: J. biol. Chem. 193 (1951) 265. Price D. 8 Williams-Ashman H. G. In: Sex and Internal Secretions. Yound W. C, ed., vol. 1. Williams & Wilkins, Baltimore (1961) 366. Shimazaki J.. Kurihara IL, lio Y & Snida.: Gunma J. med. Sci. 14 (1965) 313. Tveter K. J.: Acta endocr. (Kbh.) 60 (1969) 60. Tveter K. J. & Aakvaag.: Endocrinology 85 (1969) 683. Tveter K. J. 8- Attramadal.: Acta endocr. (Kbh.) 59 (1968) 218. Tveter K. J. & Unhjem O.: Endocrinology 84 (1969) 963. Unhjem O. 8c Tveter K. J.: Acta endocr. (Kbh.) 60 (1969) 571. Unhjem O., Tveter K. J. 8- Aakvaag.: Acta endocr. (Kbh.) 62 (1969) 153. Received on June 18th, 1969.