ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 2, December,
Localization,
AND BIOPHYSICS pp. 517-524, 1978
Metabolism,
C. LEE ROBINETTE: AND
and Binding of Estrogens ROSANNA MICHAEL
G. MCGRAW, ROBERT G. MAWHINNEY4
Departments of Surgery (Urology) and Pharmacology, West Virginia Morgantown, West Virginia 26506 Received
September
in the Male Rat’, *
19, 1977; revised
July
P. CRICCO,
University Medical
Center,
5, 1978
Estrogen assimilation by male Wistar rats was examined in these studies in several accessory sex organs (seminal vesicles and anterior, dorsal, lateral, and ventral prostates) as well as in a variety of nonaccessory sex organs. When [3H]estradiol was injected into intact 3- to 4-month-old rata in a pulse dose, no selective accumulation of radioactivity recovered as estradiol was found in the accessory sex glands when compared to other organs. This was due at least in part to the metabolism of estradiol to estrone and to the relatively low concentration of high affity estrophilic molecules in the accessory sex organs. The order for the rate of formation of estrone from estradiol in tissues obtained from intact animals was ventral prostate > lateral and dorsal prostate > anterior prostate and seminal vesicles. Steroid specificity studies for cytosol estradiol binding by the ventral prostate and seminal vesicles revealed that estrophilic molecules exist in these organs. Based on Scatchard plot analyses in 24-h castrates, the number of available estradiol binding sites was too low in the ventral prostate to quantify accurately, but the seminal vesicles contained distinctly more estrophilic activity than the ventral prostate. The affinity for the seminal vesicle cytosol estradiol-estrophile binding exceeded that quantified for the seminal vesicle dihydrotestosterone-androphile reaction while the number of estradiol binding sites was less than that quantfied for dihydrotestosterone. In relation to the accessory sex organs of other species, the rat seminal vesicles have a relatively small amount of cytosol estrophile. The findings that the seminal vesicles catabolize less estradiol and contain significantly more estrophilic activity than the ventral prostate is consistent with and offers insight into the noted estrogenic sensitivity of the seminal vesicles and lack thereof in the rat ventral prostate. With aging of the rat from 3-4 months to 22-26 months, the affinity of the seminal vesicle estradiol-estrophile interaction was unchanged but the number of binding sites increased significantly.
Although the influence of androgens on the function of male accessory sex organs has been the subject of much scientific investigation, relatively little effort has been devoted to elucidating the mechanisms which mediate cellular responses to estrogens in these glands. In view of the fact that estrogens may have important physiologi-
cal and pathological ramifications on secondary sex gland function in addition t& their pharmacological use in the treatment of prostatic neoplasia (l), more attention must be directed toward examining their influence in males of several species. Previous investigations have studied some of the characteristics of estrogen assimilation in the male rat (2-7) but have often focused to a large extent on the ventral prostate. Since the ventral prostate has a relative lack of fibromuscular stroma (Ref. 8, and Kingsbury and Edwards, personal communication) which appears to be estrogen-sensitive in some accessory sex organs (1, 9), these studies also examined other rat secondary sex glands in addition to the ventral
’ These studies were supported by Grant ROl-CA13103 from the National Cancer Institute. ’ This paper is the second of a two part series. The other manuscript is: Robinette, C. L., and Mawhinney, M. G. (1978) Arch. B&hem. Biophys. 191, 503-516. ’ Supported by Fellowship NIH-1-T-32 GM07039. 4 Recipient of faculty development award from the Pharmaceutical Manufacturer’s Association Foundation. 517
0003-9861/78/1912-0517$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
518
ROBINETTE
prostate to gain a more comprehensive understanding of estrogen action in this species. The specific parameters investigated were the in vivo distribution, the in vitro metabolism and the cytosol binding of r3H] estradiol-17/? in 3- to 4-month-old rata and the cytosol binding of [3H]estradiol-17/3 in 22- to 26-month-old rats. MATERIALS
AND
METHODS
Animals The animal techniques described elsewhere (10).
employed
in this study
are
Chemicals The chemicals used and the suppliers from which they were obtained are as previously described (10). The only chemical employed which was not previously reported is [2,4,6,7-3H]estradiol-17fi (115 Ci/mol) which was purchased from New England Nuclear Co.
Methodology Procedure
for purification
of PHJestradioLl7t3.
Since the [3H]estradiol decomposed with time, the column chromatography method of Butcher et al. (11) was employed to repurify the steroid. The purity of the [3H]estradiol after this procedure was confirmed using Silica Gel G chromatograms (Methodology, section II). Distribution, uptake, retention, and metabolism of [2,4,6,i’-3H)estradiol-l 7/I in intact rats in vivo. The procedure for these analyses is as previously outlined for the in vivo studies of [3H]testosterone assimilation (10) with the following exceptions: (i) [3H]Estradiol (300 &i/kg) was administered instead of 306 @i/kg [3H]testosterone. (ii) The carrier steroids used for the separation of the various estrogens by Silica Gel G thin-layer chromatography included estriol, estrone, and estradioL17fi. The Rf values obtained using a chloroform:ether (7:3) solvent system were: estrone, 0.81, estradiol-17/I, 0.54, and estriol, 0.08. (iii) Recoveries were based on the use of [3H]estradiol instead of [3H]testosterone. Uptake and metabolism of [2,4,6,7-3H/estradiol176 in vitro. These studies were also performed in an analogous manner to the androgen metabolism studies previously described (10) where tissues were obtained from normal, intact rats that were 3-4 months old. A concentration of 2.6 X 10m9 M [3H]estradiol was employed for these incubations. Although this concentration of r3H]estradiol is higher than the plasma levels previously reported for males of several species (12-14), its use is justified on the basis that concentrations of the hormone that would more closely approximate the physiological level would not allow an accurate detection of 3H-metabolite formation. The Silica Gel G chromatography employed in these investi-
ET AL. gations studies
is the same as that used in the (Methodology, section II).
in vivo estrogen
Scatchardplot analyses of estradiol binding in the cytosol of rat ventral prostate and seminal vesicles: procedures and their verification. The general methods used for these analyses are as described elsewhere (10). The amount of cytosol, the percentage of (w/v) homogenate, and the C3H]estradiol concentrations routinely employed are summarized in Fig. I. In these studies as in the DHT analyses (lo), a 24-h period of castration and a 4-h incubation interval were chosen for the binding assay. Investigations showed that the 4-h incubation produced equilibrium of the binding reaction, and castration intervals of up to 24 h in duration had no significant influences on [3H]estradiol binding. The 24-h period of castration was chosen for routine studies so that the estradiol analyses would be performed under conditions analogous to the [3H] DHT binding studies. When the assays were performed as described, the reactions exhibited a linear protein dependency. Furthermore, no significant metabolic alteration of the [3H]estradiol occurred during the 4-h incubation. Other preliminary studies showed that specific plasma estradiol receptor contamination of the cytosols was insignificant since only nonspecific binding of [3H]estradiol was detectable in isolated plasma samples. Although low concentrations of [3H]estradiol were used to emphasize specific binding, nonspecific interactions accounted for approximately 40% of the total hormone bound in the seminal vesicles and generally more than 60% of the hormone bound in the ventral prostate. The method used to correct for these contri-
FIG. 1. Representative Scatchard plots for estradiol binding in the ventral prostate and seminal vesicles of 3- to I-month-old and 22- to 26-month-old rats. Cytosol aliquots (0.6 ml from a 208, w/v, homogenate) from 24-h castrate rata were incubated for 4 hat 04°C in concentrations of [3H]estradiol ranging from 1.2 x lo-“-3.6 x lo-” M or in [3H]estradiol and a lOO-fold excess of nonradioactive estradiol. At the end of this incubation, the bound 3H-hormone was separated from the free by charcoal precipitation of the free. The bound fraction was then decanted and assessed for radioactivity. Corrections for nonspecific binding were made as described in the text.
ESTROGENS butions of nonspecific binding to the Scatchard plots is outlined in previous publications (15, 16). Chemical methods. DNA and protein were assessed by the methods of Schneider (17) and Lowry et al. (18), respectively. Statistical methods. Duncan’s multiple range test was employed for all statistical analyses (19). RESULTS
In
Vivo Distribution Studies
and
Metabolism
After the injection of [3H]estradiol into intact rata, the concentration of tritium recovered as estradiol-17/?, estrone, hydroxylated metabolites, total water-soluble radioactivity, and total chloroform-ether-soluble radioactivity was assessed for the plasma, liver, pancreas, adrenal, kidney, ileum, gastrocnemius muscle, testes, seminal vesicles, and anterior, dorsal, lateral, and ventral prostates. Data for estradiol and estrone, the predominant radiolabeled steroids recovered from the accessory sex organs, are summarized in Table I. Also found in Table I are the data for all forms of radioactivity recovered from the plasma. Data for the other organs are discussed in text. As with the injection of [3H]testosterone previously reported (lo), a decline in the concentration of detectable tritium in most tissues occurred at 15 and 30 min postinjection, probably reflecting elimination of the steroid from the body. The liver, adrenal, kidney, and, especially, the pancreas retained the highest concentrations of radioactivity detected as water soluble or polar 3H-metabolites when compared to the other tissues examined. In addition, of the radioactivity found in the liver, [3H]estrone was the primary chloroform-ether-soluble steroid found, whereas in the pancreas, adrenal, and kidney, unaltered r3H]estradiol was the predominant radiosteroid recovered. Of the other nonaccessory sex tissues, r3H]estradiol was the major steroid recovered from the gastrocnemius muscle and testes, but r3H]estrone exceeded r3H] estradiol in the ileum. The accessory sex tissues showed no greater ability to retain radioactivity recovered as [3H]estradiol-17/3 at 30 min postinjection than did other tissues such as the gastrocnemius muscle, testes, kidney, ad-
IN
MALE
519
RAT
renal, or pancreas. In addition, the retention of chloroform-ether-soluble radioactivity was lower in accessory sex tissues than in the liver, pancreas, adrenal, or kidney. In general, the accessory sex tissues showed a fairly high ability to form (especially at 5 min) and/or retain cpms recovered as [3H] estrone when compared to their capacity to retain cpms recovered as [3H]estradiol as evidenced by the [3H]estrone/[3H]estradiol ratio (see Table I). Some nonaccessory sex tissues, however, such as liver and ileum showed [3H]estrone/estradio1 ratios similar to the secondary sex glands. Of the accessory sex organs at 30 min postinjection, the ventral prostate had formed and retained the most radioactivity detected as [3H]estrone (Table I). The seminal vesicles and anterior prostate showed the smallest capacity in this regard while the lateral prostate and dorsal prostate were intermediate (Table I). As an overall trend, therefore, the accessory sex organs showed little organ-specific ability to retain r3H]estradiol but tended to exhibit a higher capacity to form [3H]estrone from the r3H]estradiol than several nonaccessory sex tissues.
In Vitro Uptake
and Metabolism
Studies
In the studies dealing with [3H]estradiol17p metabolism in vitro, the only metabolite recovered in any substantial amount was [311]estrone (Fig. 2). Hydroxylated, radiolabeled steroids, including [3H]estriol, were essentially nondetectable. Examining either the slope of the lines in Fig. 2 or the formation of [3H]estrone at a given time period results in the following order for [3H] estrone formation in the various organs: ventral prostate > dorsal prostate r lateral prostate > anterior prostate > seminal vesicles.
Scatchard Plot Analysis of Cytosol Estradiol Receptors in Young and Old Rats In these investigations, using 24-h castrate animals, the seminal vesicles were found to contain high affinity, low capacity binding sites (Fig. 1). The affinity of the estradiol receptor for estradiol was comparable in both age groups, being on the order of 5.33 + 0.56 X 10’ liters/m01 in 3- to 4month-old rats and 4.56 f 0.49 x 10’ li-
520
ET AL.
ROBINETTE TABLE
I
ASSIMILATION OF~H-ESTRADIOL IN Vrvo 5min
15 min
3Omin
Anterior prostate Estradiol-17P Estrone Estrone/estradiol
137 f 22b 353 * 55 2.58
77 * 19 183 k 29 2.38
45 f 15 63 f 15 1.40
Seminal vesicles Estradiol-17/3 Estrone Estrone/estradiol
255 f 33b 303 f 68 1.19
136*27 113* 7 0.83
74 f 10 29 f 6 0.39
Ventral prostate Estradiol-17/3 Estrone Estrone/estradiol
149 f 27b 775 f 111 5.20
16Of19 470 f 53 2.94
90~~8 183 f 23 2.03
Lateral prostate Estradiol-17/I Estrone Estrone/estradiol
298 f 46* 53Of38 1.78
175 f 28 115 f 26 0.66
140 zt 28 9Ozt6 0.64
Dorsal prostate Estradiol-17P Estrone Estrone/estradiol
84 f 4b 263 + 18 3.13
48 + 10 220 -c 35 4.58
70 + 16 108 k 8 1.54
Plasma Hydroxylated steroids’ (origin) Estradiol-17/3 Estrone Total water-soluble radioactivity Total chloroform-ether-soluble Estrone/estradiol
245 f 6Of2 20 f 117 f 123 * 0.33
208 f 35 f 9fl 136 + 68 f 0.26
155 f 18 f 6fl 139 f 39 f 0.33
radioactivity
30’ 1 16 2
21 1 16 1
22 2 6 2
n Radioactivity associated with various estrogens as detected in several tissues of young (3 months old), intact rats after the injection of [4, 5, 6, 7, 3H]estradiol-17/3 (300 &i/kg) in Go. The hormone was injected via the inferior vena cava and at various times postinjection, the tissues were rapidly excised and frozen in liquid nitrogen. The samples were then homogenized in distilled water and the lipid-soluble steroids were extracted into chloroformether (7:3). Aliquots of the extract were subjected to thin-layer chromatography to separate the various lipid-soluble estrogens. (Four to six animals per determination.) * DPM’s f SEM per mg wet tissue weight. ’ Hydroxylated steroids left at origin of the chromatogram which include estriol.
ters/mol in 22-26-month-old rats. Although no age-related changes in receptor affinity were observed, the concentration of receptor sites showed a statistically significant increase of approximately 2-fold with advancing age (young rats, 8.4 +- 1.9 X lo-l4 sites per mg DNA or 18.7 & 4.2 x lo-l7 sites per mg wet tissue weight; old rats, 18.6 f 2.2 X lo-l4 sites per mg DNA or 36.2 + 4.2 x lo-l7 sites per mg wet tissue weight). An age-associated trend for an increase which was not statistically significant was also
noted when the receptors were expressed on the basis of milligrams cytosol protein (young rats, 0.73 + 0.13 X lo-l4 sites per mg cytosol protein, old rats, 0.93 + 0.10-14sites per mg cytosol protein). Scatchard plots which would indicate that specific, high affinity receptor sites were present in the ventral prostate were not producible (Fig. 1) even though our competitor studies (see below) indicated that specific estradiol receptors were present in this gland. It is believed that the high
ESTROGENS
IN
MALE
521
RAT
20.000-
17.500~
w
15.000~
2 !c IP
12.500
2 z E
10,000
-
ki t? 5
7.500-
In i :
5.000-
ANTERIOR
2.500-
5
IO
I5 TIME
20 OF
5
INCUBATION
IO
15
20
,MIN”TES)
FIG. 2. Reaction rates for [3H]estrone formation in various accessory sex organs of young (3month-old) rats. Chopped tissue samples (40 mg) were incubated for varying periods of time in 2 ml of Krebs-Ringer bicarbonate buffer maintained at 37’C and pH 7.4 under an atmosphere of 95% h-5% CO,. The concentration of [3H]estradiol used was 2.6 X IO-’ M. Two milliliters of ice cold chlorofornrether (7:3) were added to each sample to terminate the reaction and to extract the lipid-soluble 3H-steroids. Aliouots of the extract were subjected to thin-layer chromatography to separate the various lipid-soluble ‘H-estrogens.
levels of nonspecific binding in relation to specific binding found in the ventral prostate probably masked the presence of the saturable binding moieties. That such an explanation is plausible is evidenced by the findings of other investigators (20, 21).
In Vitro Binding
Competition
Studies
Figure 3a demonstrates that in the ventral prostate, estradiol, DHT, and testosterone were approximately equally effective as competitors for [3H]estradiol binding with estrone being a much less effective competitor. In the seminal vesicles, estradiol and estrone were more effective competitors for r3H]estradiol binding than DHT or testosterone. Since in all cases, estradiol was at least equally effective or more so in competing for r3H]estradiol binding than any of the other steroids tested, the presence of a specific estradiol receptor is indicated. In the ventral prostate where androgens
were more effective competitors for r3H] estradiol binding than estrone, possibly relatively large amounts of [3H]estradiol were bound to the androgen receptor. Certainly, in our previous study (lo), estradiol was shown to compete for androgen binding, presumably by binding to the androgen receptor. Although [3H]estradiol binding to the androgen receptor appeared to occur in the ventral prostate in the present studies, nonradioactive DHT would have been a better competitor than unlabeled estradiol if the [3H]estradiol were binding only to the DHT receptor. This was not observed and, therefore, specific estradiol receptors appeared to be present. DISCUSSION
Retie and Bruchovsky (5) have previously indicated that the rat ventral prostate can form [3H]estrone from C3H]estradiol in vivo. The present investigations confirmed
522
ROBINETTE
FIG. 3. (a) The effects of nonradioactive steroids on [3H]estradiol binding in rat ventral prostate cytosol preparations from 24-h castrates. Aliquota (0.6 ml from a 20%, w/v, homogenate) of cytosol were incubated for 4 h at 0-4°C in the presence of 2.4 X 10-l’ M [3H] estradiol to which various amounts of unlabeled competitors were sometimes added. At the end of the incubation, the bound 3H-hormone was separated from the free ‘H-hormone by charcoal precipitation of the free. The bound fraction was then decanted and assessed for radioactivity. Values represent the mean f S.E.M. of four or more observations. The data are expressed as the percentages of control values where the control contains no unlabeled competitors. (b) The effects of nonradioactive steroids on [3H]estradiol binding in rat seminal vesicle cytosol preparations from 24-h castrates. Aliquota (0.6 ml. from a 20’%, w/v, homogenate) of cytosol were incubated for 4 h at 0-4’C in the presence of 2.4 X lo-” M [3H]estradiol to which various amounts of unlabeled competitors were sometimes added. At the end of the incubation, the bound ‘H-hormone was separated from the free 3Hhormone by charcoal precipitation of the free. The bound fraction was then decanted and assessed for radioactivity. Values represent the mean f SEM of four or more observations. The data are expressed as the percentages of control values where the control contains no unlabeled competitors.
ET
AL.
this finding both in viva and in vitro for a variety of accessory sex organs. Of particular interest was the close correlation found between the in vivo and in vitro studies concerning the wide range in the ability of the various secondary sex organs to produce C3H]estrone. The findings of Mangan et al. (3) who reported a selective accumulation of [3H] diethylstilbestrol by the prostate are in conflict with the present studies and the findings of Eisenfeld and Axehod (2) and Tveter (4), all of whom could not demonstrate a selective accumulation of estrogens by rat accessory sex organs. This discrepancy may relate in part to the use of diethylstilbestrol uersus estradiol. In comparing the studies of [3H]estradiol distribution in the rat, it must be noted that in previous investigations (2,4), only total tritiated material was quantified. It is reasonable to expect that the ventral prostate which contains minute amounts of cytosol estrophile and rapidly catabolizes estradiol to estrone would not be capable of concentrating and retaining significant amounts of plasma estradiol. Even though the rat seminal vesicles contained far more estrogen receptor and metabolized much less estradiol to estrone than the ventral prostate, it, like the ventral prostate, did not concentrate and retain injected estradiol. On the other hand, the muscle layer of the guinea pig seminal vesicles which has a great avidity for injected r3H]estradiol, metabolizes no [3H]estradiol and contains far more cytosol estrophile than the rat seminal vesicles (25) where no attempt was made to separate the individual cell lines. It is possible, then, that although the rat seminal vesicles do contain modest levels of cytosol estrophiles and low estradiol catalytic activity, conditions are still unfavorable for demonstrating the accumulation of injected [3H]estradiol when no separation of cell types was achieved. Knowledge of endogenous estradiol in plasma and in the individual cell lines of the various rat accessory sex organs will aid in interpretation of the significance of the estrophilic activity of the rat seminal vesicle cytosol. In the work of Van Beurden-Lamers et al. (7), higher levels of saturable cytosol
ESTROGENS
IN MALE
RAT
523
estradiol binding were found in the young vesicles in this study may confer an inrat prostate (presumably ventral prostate) creased sensitivity of the fibromuscular than in the seminal vesicles which is in stroma to estradiol. The relative lack of contrast to the present findings. The steroid fibromuscular stroma and of estrogen respecificity of the binding was not deter- ceptors in the ventral prostate is consistent mined in the earlier study, however, and at with the lack of growth of the ventral prosthe concentration of [3H]estradiol used (4 tate in response to exogenously adminisx lo-’ M), their results may represent a tered estradiol in relation to the seminal significant binding of the hormone to an- vesicles. Another factor which must be recdrogen receptors in the prostate in relation ognized, however, as possibly contributing to the seminal vesicles. to the lack of ventral prostate sensitivity to Armstrong and Bashirelahi (6) identified estradiol is its pronounced conversion of a specific cytosol estradiol receptor in the estradiol to estrone. As far as androgen ventral prostate of intact retired breeder receptor localization is concerned, the venrats. This observation was made by show- tral prostate which is composed largely of ing that nonradioactive DHT and estradiol epithelial cells contained the highest level were preferential inhibitors of specific [3H] of androphile of any organ examined in our DHT and [3H]estradiol binding, respec- previous study (10). Therefore, the evitively, as determined by sucrose density dence indicates that in the rat as in the gradient analyses. guinea pig, the estrophiles may be primarily The presence of estrogen receptors in localized in the fibromuscular tissue of the male accessory sex glands is not a novel accessory sex organs. This hypothesis must discovery in that specific estrophiles dis- be regarded as only speculative, however, tinct from androgen binding proteins have until the epithelium and muscle of accesbeen established in the calf prostate (221, in sory sex glands from various embryological pig seminal vesicles (22), in the human origins and from various species can be prostate (23,24), in the guinea pig prostate, separately analyzed as in the guinea pig and in the separated epithelium and muscle seminal vesicles. of the guinea pig seminal vesicles (1, 25). Estradiol-17P is generally considered to The muscle of the guinea pig seminal vesi- be a poor antagonist of exogenously admincles contained the highest level of cytosol istered testosterone in the ventral prostate estradiol binding, indicating that the recep- of young, sexually mature castrate rats (1). tor may be primarily associated with the Such findings may be related to a lack of muscular portion of the glands. Androgen physiological antagonism of androgen acreceptors, on the other hand, were located tion mediated by estradiol/estrophile interlargely in the epithelium (1, 25). actions due to the low levels of estradiol Although the rat accessory sex organs receptors found in this prostatic lobe. Conhave not as yet been separated into epithetributing to this phenomenon would be the lial and muscular components, perhaps es- pronounced conversion of estradiol-17fi to trogen receptors are predominantly located estrone. On the other hand, if the antiin the muscle cell lines and androgen recep- androgenic effect of estradiol-17/3 is metors are primarily located in the epithelial diated primarily by a pharmacological comcell lines as in the guinea pig seminal vesi- petition for the binding of DHT to its recles. In support of this interpretation is ceptor, then the formation of estrone would histological evidence that the fibromuscufunction to attenuate this response since in lar portions of young, sexually mature rat our previously reported (10) competitor seminal vesicles were selectively stimulated studies, estrone had a lower capacity to to grow after estrogen treatment to cas- reduce r3H]DHT binding than estradiol. trated adrenalectomized rats (1,9), perhaps In contrast to the rat ventral prostate, reflecting a consequence of an estro- the effects of exogenously administered tesgen/estrogen receptor interaction. It is in- tosterone on the prostate of castrate dogs teresting to speculate that the increased can be effectively antagonized by estradiol concentration of estradiol receptors de- treatment (1). These differences between tected with advancing age in the seminal the two species may be accounted for in
524
ROBINETTE
part by the fact that relatively high levels of specific estradiol receptors have been found in the dog prostate (15) and by preliminary evidence that the dog prostate has a limited capacity to form estrone from estradiol. In conclusion, the presence of estrogen responsiveness or the lack thereof in the various male rat accessory sex organs correlates well with the concentration of estradiol specific molecules found in these glands. Circumstantial evidence favors the hypothesis that the estrophiles may be primarily associated with fibromuscular cells. Although estradiol receptors have been identified in the rat accessory sex organs, the low concentrations of these binding moieties in the rat in relation to other species may explain the relative lack of antiandrogenic effects of estrogens in the rat. Furthermore, the ability of the rat secondary sex glands to effect the rapid conversion of estradiol to estrone may also contribute to this lack of anti-androgenic activity. The significance of the age-associated changes in estradiol receptor concentration on responsiveness to steroids in the seminal vesicles remains to be elucidated.
wish to acknowledge the expert techof Lynne Schwartz and Brenda Lyons. REFERENCES
1. MAWHINNEY, M. G., AND BELIS, J. A. (1976) in Advances in Sex Hormone Research (Thomas, J. A., and Singhai, R. L., eds), Vol. 2, pp. 141-209, University Park Press, Baltimore. 2. EISENFELD, A. J., AND AXELROD, J. (1966) Endo-
crinology 19,30-42. 3. MANGAN, F. R., NEAL, G. E. AND WILLIAMS, D. C. (1967) Biochem. J. 104, 1075-1081. 4. TVETER, K. J. (1970) Acta Endocrinol (Copenhagen) 5. RENNIE,
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Chem. 240,3286-3297. 6. ARMSTRONG, E. G., AND BASHIRELAHI, N. (1974) B&hem. Biophys. Res. Commun. 61,578-584. 7. VAN BEURDEN-LAMERS, W. M. O., BRINKMAN, A. O., MULDER, E., AND VAN DER MOLEN, H. J. (1974) Biochem. J. 140,495~502. 8. PRICE, D. AND WILLIAMS-ASHMAN, H. G. (1961), in Sex and Internal Secretions (W. C. Young, ed.), Vol. 1, 3rd ed, WiUiams & Wilkins Co., Baltimore. (Copen9. TISELL, L.-E. (1971) Acta Endocrinol. hagen) 68,485-501. C. L., AND MAWHINNEY, M. G. (1978) 10. ROBINETTE, Arch. Biochem. Biophys. 191, 503-516. 11. BUTCHER, R. L., COLLINS, W. E., AND FUGO, N. W. (1974) Endocrinology 94,1704-1708. 12. DEJONG, F., HEY, S., AND VAN DER MOLEN, J. (1974) J. Endocrinol. 60,409-419. 13. HAWKINS, R. A., AND OAKEY, R. E. (1974) J.
Endocrinol. 60,3-17. 14. LLOYD, J. W., THOMAS, J. A., AND MAWHINNEY, M. G. (1975) Invest. Ural. l&220-222. 15. ROBINETTE, NEY, M. 16. CHAMNESS,
C. L., BLUME,
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G. (1978) Invest. Ural. l&425-431. C. G., AND MCGUIRE, W. L. (1975) Steroids 26,538-542. 17. SCHNEIDER, W. C. (1957), in Methods in Enxy-
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ACKNOWLEDGMENTS The authors nical assistance
ET AL.
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19. HARTER, L. H. (1960) Biometrics 16,671-685. 20. WITTLIFFE, J. L., HILF, R., BROOKS, W. F., SAVLOV, E. D., HALL, T. C., AND ORLANDO, R. A. (1972) Cancer Res. 32, 1983-1992. 21. BRAUNSBERG, H. (1975) Eur. J. Cancer 11, 499-507. 22. JUNGBLUT, P. W., HUGHES, S. F., GGRLICH, L., GOWERS, U., AND R~~DIGER, K. W. (1971) Hoppe-Seyler’s Z. Physiol. Chem. 362, 16031610. N., O’TOOLE, J. H., AND YOUNG, J. 23. BASHIRELAHI, D. (1976) Biochem. Med. l&254-261. 24. BASHIRELAHI, N., AND YOUNG, J. D. (1976) Urology 8,553-558. M. 25. BELIS, J. A., BLUME, C. D., AND MAWHINNEY, G. (1977) Endocrinology 101, 726-740.
Localization,
AND BIOPHYSICS pp. 517-524, 1978
Metabolism,
C. LEE ROBINETTE: AND
and Binding of Estrogens ROSANNA MICHAEL
G. MCGRAW, ROBERT G. MAWHINNEY4
Departments of Surgery (Urology) and Pharmacology, West Virginia Morgantown, West Virginia 26506 Received
September
in the Male Rat’, *
19, 1977; revised
July
P. CRICCO,
University Medical
Center,
5, 1978
Estrogen assimilation by male Wistar rats was examined in these studies in several accessory sex organs (seminal vesicles and anterior, dorsal, lateral, and ventral prostates) as well as in a variety of nonaccessory sex organs. When [3H]estradiol was injected into intact 3- to 4-month-old rata in a pulse dose, no selective accumulation of radioactivity recovered as estradiol was found in the accessory sex glands when compared to other organs. This was due at least in part to the metabolism of estradiol to estrone and to the relatively low concentration of high affity estrophilic molecules in the accessory sex organs. The order for the rate of formation of estrone from estradiol in tissues obtained from intact animals was ventral prostate > lateral and dorsal prostate > anterior prostate and seminal vesicles. Steroid specificity studies for cytosol estradiol binding by the ventral prostate and seminal vesicles revealed that estrophilic molecules exist in these organs. Based on Scatchard plot analyses in 24-h castrates, the number of available estradiol binding sites was too low in the ventral prostate to quantify accurately, but the seminal vesicles contained distinctly more estrophilic activity than the ventral prostate. The affinity for the seminal vesicle cytosol estradiol-estrophile binding exceeded that quantified for the seminal vesicle dihydrotestosterone-androphile reaction while the number of estradiol binding sites was less than that quantfied for dihydrotestosterone. In relation to the accessory sex organs of other species, the rat seminal vesicles have a relatively small amount of cytosol estrophile. The findings that the seminal vesicles catabolize less estradiol and contain significantly more estrophilic activity than the ventral prostate is consistent with and offers insight into the noted estrogenic sensitivity of the seminal vesicles and lack thereof in the rat ventral prostate. With aging of the rat from 3-4 months to 22-26 months, the affinity of the seminal vesicle estradiol-estrophile interaction was unchanged but the number of binding sites increased significantly.
Although the influence of androgens on the function of male accessory sex organs has been the subject of much scientific investigation, relatively little effort has been devoted to elucidating the mechanisms which mediate cellular responses to estrogens in these glands. In view of the fact that estrogens may have important physiologi-
cal and pathological ramifications on secondary sex gland function in addition t& their pharmacological use in the treatment of prostatic neoplasia (l), more attention must be directed toward examining their influence in males of several species. Previous investigations have studied some of the characteristics of estrogen assimilation in the male rat (2-7) but have often focused to a large extent on the ventral prostate. Since the ventral prostate has a relative lack of fibromuscular stroma (Ref. 8, and Kingsbury and Edwards, personal communication) which appears to be estrogen-sensitive in some accessory sex organs (1, 9), these studies also examined other rat secondary sex glands in addition to the ventral
’ These studies were supported by Grant ROl-CA13103 from the National Cancer Institute. ’ This paper is the second of a two part series. The other manuscript is: Robinette, C. L., and Mawhinney, M. G. (1978) Arch. B&hem. Biophys. 191, 503-516. ’ Supported by Fellowship NIH-1-T-32 GM07039. 4 Recipient of faculty development award from the Pharmaceutical Manufacturer’s Association Foundation. 517
0003-9861/78/1912-0517$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
518
ROBINETTE
prostate to gain a more comprehensive understanding of estrogen action in this species. The specific parameters investigated were the in vivo distribution, the in vitro metabolism and the cytosol binding of r3H] estradiol-17/? in 3- to 4-month-old rata and the cytosol binding of [3H]estradiol-17/3 in 22- to 26-month-old rats. MATERIALS
AND
METHODS
Animals The animal techniques described elsewhere (10).
employed
in this study
are
Chemicals The chemicals used and the suppliers from which they were obtained are as previously described (10). The only chemical employed which was not previously reported is [2,4,6,7-3H]estradiol-17fi (115 Ci/mol) which was purchased from New England Nuclear Co.
Methodology Procedure
for purification
of PHJestradioLl7t3.
Since the [3H]estradiol decomposed with time, the column chromatography method of Butcher et al. (11) was employed to repurify the steroid. The purity of the [3H]estradiol after this procedure was confirmed using Silica Gel G chromatograms (Methodology, section II). Distribution, uptake, retention, and metabolism of [2,4,6,i’-3H)estradiol-l 7/I in intact rats in vivo. The procedure for these analyses is as previously outlined for the in vivo studies of [3H]testosterone assimilation (10) with the following exceptions: (i) [3H]Estradiol (300 &i/kg) was administered instead of 306 @i/kg [3H]testosterone. (ii) The carrier steroids used for the separation of the various estrogens by Silica Gel G thin-layer chromatography included estriol, estrone, and estradioL17fi. The Rf values obtained using a chloroform:ether (7:3) solvent system were: estrone, 0.81, estradiol-17/I, 0.54, and estriol, 0.08. (iii) Recoveries were based on the use of [3H]estradiol instead of [3H]testosterone. Uptake and metabolism of [2,4,6,7-3H/estradiol176 in vitro. These studies were also performed in an analogous manner to the androgen metabolism studies previously described (10) where tissues were obtained from normal, intact rats that were 3-4 months old. A concentration of 2.6 X 10m9 M [3H]estradiol was employed for these incubations. Although this concentration of r3H]estradiol is higher than the plasma levels previously reported for males of several species (12-14), its use is justified on the basis that concentrations of the hormone that would more closely approximate the physiological level would not allow an accurate detection of 3H-metabolite formation. The Silica Gel G chromatography employed in these investi-
ET AL. gations studies
is the same as that used in the (Methodology, section II).
in vivo estrogen
Scatchardplot analyses of estradiol binding in the cytosol of rat ventral prostate and seminal vesicles: procedures and their verification. The general methods used for these analyses are as described elsewhere (10). The amount of cytosol, the percentage of (w/v) homogenate, and the C3H]estradiol concentrations routinely employed are summarized in Fig. I. In these studies as in the DHT analyses (lo), a 24-h period of castration and a 4-h incubation interval were chosen for the binding assay. Investigations showed that the 4-h incubation produced equilibrium of the binding reaction, and castration intervals of up to 24 h in duration had no significant influences on [3H]estradiol binding. The 24-h period of castration was chosen for routine studies so that the estradiol analyses would be performed under conditions analogous to the [3H] DHT binding studies. When the assays were performed as described, the reactions exhibited a linear protein dependency. Furthermore, no significant metabolic alteration of the [3H]estradiol occurred during the 4-h incubation. Other preliminary studies showed that specific plasma estradiol receptor contamination of the cytosols was insignificant since only nonspecific binding of [3H]estradiol was detectable in isolated plasma samples. Although low concentrations of [3H]estradiol were used to emphasize specific binding, nonspecific interactions accounted for approximately 40% of the total hormone bound in the seminal vesicles and generally more than 60% of the hormone bound in the ventral prostate. The method used to correct for these contri-
FIG. 1. Representative Scatchard plots for estradiol binding in the ventral prostate and seminal vesicles of 3- to I-month-old and 22- to 26-month-old rats. Cytosol aliquots (0.6 ml from a 208, w/v, homogenate) from 24-h castrate rata were incubated for 4 hat 04°C in concentrations of [3H]estradiol ranging from 1.2 x lo-“-3.6 x lo-” M or in [3H]estradiol and a lOO-fold excess of nonradioactive estradiol. At the end of this incubation, the bound 3H-hormone was separated from the free by charcoal precipitation of the free. The bound fraction was then decanted and assessed for radioactivity. Corrections for nonspecific binding were made as described in the text.
ESTROGENS butions of nonspecific binding to the Scatchard plots is outlined in previous publications (15, 16). Chemical methods. DNA and protein were assessed by the methods of Schneider (17) and Lowry et al. (18), respectively. Statistical methods. Duncan’s multiple range test was employed for all statistical analyses (19). RESULTS
In
Vivo Distribution Studies
and
Metabolism
After the injection of [3H]estradiol into intact rata, the concentration of tritium recovered as estradiol-17/?, estrone, hydroxylated metabolites, total water-soluble radioactivity, and total chloroform-ether-soluble radioactivity was assessed for the plasma, liver, pancreas, adrenal, kidney, ileum, gastrocnemius muscle, testes, seminal vesicles, and anterior, dorsal, lateral, and ventral prostates. Data for estradiol and estrone, the predominant radiolabeled steroids recovered from the accessory sex organs, are summarized in Table I. Also found in Table I are the data for all forms of radioactivity recovered from the plasma. Data for the other organs are discussed in text. As with the injection of [3H]testosterone previously reported (lo), a decline in the concentration of detectable tritium in most tissues occurred at 15 and 30 min postinjection, probably reflecting elimination of the steroid from the body. The liver, adrenal, kidney, and, especially, the pancreas retained the highest concentrations of radioactivity detected as water soluble or polar 3H-metabolites when compared to the other tissues examined. In addition, of the radioactivity found in the liver, [3H]estrone was the primary chloroform-ether-soluble steroid found, whereas in the pancreas, adrenal, and kidney, unaltered r3H]estradiol was the predominant radiosteroid recovered. Of the other nonaccessory sex tissues, r3H]estradiol was the major steroid recovered from the gastrocnemius muscle and testes, but r3H]estrone exceeded r3H] estradiol in the ileum. The accessory sex tissues showed no greater ability to retain radioactivity recovered as [3H]estradiol-17/3 at 30 min postinjection than did other tissues such as the gastrocnemius muscle, testes, kidney, ad-
IN
MALE
519
RAT
renal, or pancreas. In addition, the retention of chloroform-ether-soluble radioactivity was lower in accessory sex tissues than in the liver, pancreas, adrenal, or kidney. In general, the accessory sex tissues showed a fairly high ability to form (especially at 5 min) and/or retain cpms recovered as [3H] estrone when compared to their capacity to retain cpms recovered as [3H]estradiol as evidenced by the [3H]estrone/[3H]estradiol ratio (see Table I). Some nonaccessory sex tissues, however, such as liver and ileum showed [3H]estrone/estradio1 ratios similar to the secondary sex glands. Of the accessory sex organs at 30 min postinjection, the ventral prostate had formed and retained the most radioactivity detected as [3H]estrone (Table I). The seminal vesicles and anterior prostate showed the smallest capacity in this regard while the lateral prostate and dorsal prostate were intermediate (Table I). As an overall trend, therefore, the accessory sex organs showed little organ-specific ability to retain r3H]estradiol but tended to exhibit a higher capacity to form [3H]estrone from the r3H]estradiol than several nonaccessory sex tissues.
In Vitro Uptake
and Metabolism
Studies
In the studies dealing with [3H]estradiol17p metabolism in vitro, the only metabolite recovered in any substantial amount was [311]estrone (Fig. 2). Hydroxylated, radiolabeled steroids, including [3H]estriol, were essentially nondetectable. Examining either the slope of the lines in Fig. 2 or the formation of [3H]estrone at a given time period results in the following order for [3H] estrone formation in the various organs: ventral prostate > dorsal prostate r lateral prostate > anterior prostate > seminal vesicles.
Scatchard Plot Analysis of Cytosol Estradiol Receptors in Young and Old Rats In these investigations, using 24-h castrate animals, the seminal vesicles were found to contain high affinity, low capacity binding sites (Fig. 1). The affinity of the estradiol receptor for estradiol was comparable in both age groups, being on the order of 5.33 + 0.56 X 10’ liters/m01 in 3- to 4month-old rats and 4.56 f 0.49 x 10’ li-
520
ET AL.
ROBINETTE TABLE
I
ASSIMILATION OF~H-ESTRADIOL IN Vrvo 5min
15 min
3Omin
Anterior prostate Estradiol-17P Estrone Estrone/estradiol
137 f 22b 353 * 55 2.58
77 * 19 183 k 29 2.38
45 f 15 63 f 15 1.40
Seminal vesicles Estradiol-17/3 Estrone Estrone/estradiol
255 f 33b 303 f 68 1.19
136*27 113* 7 0.83
74 f 10 29 f 6 0.39
Ventral prostate Estradiol-17/3 Estrone Estrone/estradiol
149 f 27b 775 f 111 5.20
16Of19 470 f 53 2.94
90~~8 183 f 23 2.03
Lateral prostate Estradiol-17/I Estrone Estrone/estradiol
298 f 46* 53Of38 1.78
175 f 28 115 f 26 0.66
140 zt 28 9Ozt6 0.64
Dorsal prostate Estradiol-17P Estrone Estrone/estradiol
84 f 4b 263 + 18 3.13
48 + 10 220 -c 35 4.58
70 + 16 108 k 8 1.54
Plasma Hydroxylated steroids’ (origin) Estradiol-17/3 Estrone Total water-soluble radioactivity Total chloroform-ether-soluble Estrone/estradiol
245 f 6Of2 20 f 117 f 123 * 0.33
208 f 35 f 9fl 136 + 68 f 0.26
155 f 18 f 6fl 139 f 39 f 0.33
radioactivity
30’ 1 16 2
21 1 16 1
22 2 6 2
n Radioactivity associated with various estrogens as detected in several tissues of young (3 months old), intact rats after the injection of [4, 5, 6, 7, 3H]estradiol-17/3 (300 &i/kg) in Go. The hormone was injected via the inferior vena cava and at various times postinjection, the tissues were rapidly excised and frozen in liquid nitrogen. The samples were then homogenized in distilled water and the lipid-soluble steroids were extracted into chloroformether (7:3). Aliquots of the extract were subjected to thin-layer chromatography to separate the various lipid-soluble estrogens. (Four to six animals per determination.) * DPM’s f SEM per mg wet tissue weight. ’ Hydroxylated steroids left at origin of the chromatogram which include estriol.
ters/mol in 22-26-month-old rats. Although no age-related changes in receptor affinity were observed, the concentration of receptor sites showed a statistically significant increase of approximately 2-fold with advancing age (young rats, 8.4 +- 1.9 X lo-l4 sites per mg DNA or 18.7 & 4.2 x lo-l7 sites per mg wet tissue weight; old rats, 18.6 f 2.2 X lo-l4 sites per mg DNA or 36.2 + 4.2 x lo-l7 sites per mg wet tissue weight). An age-associated trend for an increase which was not statistically significant was also
noted when the receptors were expressed on the basis of milligrams cytosol protein (young rats, 0.73 + 0.13 X lo-l4 sites per mg cytosol protein, old rats, 0.93 + 0.10-14sites per mg cytosol protein). Scatchard plots which would indicate that specific, high affinity receptor sites were present in the ventral prostate were not producible (Fig. 1) even though our competitor studies (see below) indicated that specific estradiol receptors were present in this gland. It is believed that the high
ESTROGENS
IN
MALE
521
RAT
20.000-
17.500~
w
15.000~
2 !c IP
12.500
2 z E
10,000
-
ki t? 5
7.500-
In i :
5.000-
ANTERIOR
2.500-
5
IO
I5 TIME
20 OF
5
INCUBATION
IO
15
20
,MIN”TES)
FIG. 2. Reaction rates for [3H]estrone formation in various accessory sex organs of young (3month-old) rats. Chopped tissue samples (40 mg) were incubated for varying periods of time in 2 ml of Krebs-Ringer bicarbonate buffer maintained at 37’C and pH 7.4 under an atmosphere of 95% h-5% CO,. The concentration of [3H]estradiol used was 2.6 X IO-’ M. Two milliliters of ice cold chlorofornrether (7:3) were added to each sample to terminate the reaction and to extract the lipid-soluble 3H-steroids. Aliouots of the extract were subjected to thin-layer chromatography to separate the various lipid-soluble ‘H-estrogens.
levels of nonspecific binding in relation to specific binding found in the ventral prostate probably masked the presence of the saturable binding moieties. That such an explanation is plausible is evidenced by the findings of other investigators (20, 21).
In Vitro Binding
Competition
Studies
Figure 3a demonstrates that in the ventral prostate, estradiol, DHT, and testosterone were approximately equally effective as competitors for [3H]estradiol binding with estrone being a much less effective competitor. In the seminal vesicles, estradiol and estrone were more effective competitors for r3H]estradiol binding than DHT or testosterone. Since in all cases, estradiol was at least equally effective or more so in competing for r3H]estradiol binding than any of the other steroids tested, the presence of a specific estradiol receptor is indicated. In the ventral prostate where androgens
were more effective competitors for r3H] estradiol binding than estrone, possibly relatively large amounts of [3H]estradiol were bound to the androgen receptor. Certainly, in our previous study (lo), estradiol was shown to compete for androgen binding, presumably by binding to the androgen receptor. Although [3H]estradiol binding to the androgen receptor appeared to occur in the ventral prostate in the present studies, nonradioactive DHT would have been a better competitor than unlabeled estradiol if the [3H]estradiol were binding only to the DHT receptor. This was not observed and, therefore, specific estradiol receptors appeared to be present. DISCUSSION
Retie and Bruchovsky (5) have previously indicated that the rat ventral prostate can form [3H]estrone from C3H]estradiol in vivo. The present investigations confirmed
522
ROBINETTE
FIG. 3. (a) The effects of nonradioactive steroids on [3H]estradiol binding in rat ventral prostate cytosol preparations from 24-h castrates. Aliquota (0.6 ml from a 20%, w/v, homogenate) of cytosol were incubated for 4 h at 0-4°C in the presence of 2.4 X 10-l’ M [3H] estradiol to which various amounts of unlabeled competitors were sometimes added. At the end of the incubation, the bound 3H-hormone was separated from the free ‘H-hormone by charcoal precipitation of the free. The bound fraction was then decanted and assessed for radioactivity. Values represent the mean f S.E.M. of four or more observations. The data are expressed as the percentages of control values where the control contains no unlabeled competitors. (b) The effects of nonradioactive steroids on [3H]estradiol binding in rat seminal vesicle cytosol preparations from 24-h castrates. Aliquota (0.6 ml. from a 20’%, w/v, homogenate) of cytosol were incubated for 4 h at 0-4’C in the presence of 2.4 X lo-” M [3H]estradiol to which various amounts of unlabeled competitors were sometimes added. At the end of the incubation, the bound ‘H-hormone was separated from the free 3Hhormone by charcoal precipitation of the free. The bound fraction was then decanted and assessed for radioactivity. Values represent the mean f SEM of four or more observations. The data are expressed as the percentages of control values where the control contains no unlabeled competitors.
ET
AL.
this finding both in viva and in vitro for a variety of accessory sex organs. Of particular interest was the close correlation found between the in vivo and in vitro studies concerning the wide range in the ability of the various secondary sex organs to produce C3H]estrone. The findings of Mangan et al. (3) who reported a selective accumulation of [3H] diethylstilbestrol by the prostate are in conflict with the present studies and the findings of Eisenfeld and Axehod (2) and Tveter (4), all of whom could not demonstrate a selective accumulation of estrogens by rat accessory sex organs. This discrepancy may relate in part to the use of diethylstilbestrol uersus estradiol. In comparing the studies of [3H]estradiol distribution in the rat, it must be noted that in previous investigations (2,4), only total tritiated material was quantified. It is reasonable to expect that the ventral prostate which contains minute amounts of cytosol estrophile and rapidly catabolizes estradiol to estrone would not be capable of concentrating and retaining significant amounts of plasma estradiol. Even though the rat seminal vesicles contained far more estrogen receptor and metabolized much less estradiol to estrone than the ventral prostate, it, like the ventral prostate, did not concentrate and retain injected estradiol. On the other hand, the muscle layer of the guinea pig seminal vesicles which has a great avidity for injected r3H]estradiol, metabolizes no [3H]estradiol and contains far more cytosol estrophile than the rat seminal vesicles (25) where no attempt was made to separate the individual cell lines. It is possible, then, that although the rat seminal vesicles do contain modest levels of cytosol estrophiles and low estradiol catalytic activity, conditions are still unfavorable for demonstrating the accumulation of injected [3H]estradiol when no separation of cell types was achieved. Knowledge of endogenous estradiol in plasma and in the individual cell lines of the various rat accessory sex organs will aid in interpretation of the significance of the estrophilic activity of the rat seminal vesicle cytosol. In the work of Van Beurden-Lamers et al. (7), higher levels of saturable cytosol
ESTROGENS
IN MALE
RAT
523
estradiol binding were found in the young vesicles in this study may confer an inrat prostate (presumably ventral prostate) creased sensitivity of the fibromuscular than in the seminal vesicles which is in stroma to estradiol. The relative lack of contrast to the present findings. The steroid fibromuscular stroma and of estrogen respecificity of the binding was not deter- ceptors in the ventral prostate is consistent mined in the earlier study, however, and at with the lack of growth of the ventral prosthe concentration of [3H]estradiol used (4 tate in response to exogenously adminisx lo-’ M), their results may represent a tered estradiol in relation to the seminal significant binding of the hormone to an- vesicles. Another factor which must be recdrogen receptors in the prostate in relation ognized, however, as possibly contributing to the seminal vesicles. to the lack of ventral prostate sensitivity to Armstrong and Bashirelahi (6) identified estradiol is its pronounced conversion of a specific cytosol estradiol receptor in the estradiol to estrone. As far as androgen ventral prostate of intact retired breeder receptor localization is concerned, the venrats. This observation was made by show- tral prostate which is composed largely of ing that nonradioactive DHT and estradiol epithelial cells contained the highest level were preferential inhibitors of specific [3H] of androphile of any organ examined in our DHT and [3H]estradiol binding, respec- previous study (10). Therefore, the evitively, as determined by sucrose density dence indicates that in the rat as in the gradient analyses. guinea pig, the estrophiles may be primarily The presence of estrogen receptors in localized in the fibromuscular tissue of the male accessory sex glands is not a novel accessory sex organs. This hypothesis must discovery in that specific estrophiles dis- be regarded as only speculative, however, tinct from androgen binding proteins have until the epithelium and muscle of accesbeen established in the calf prostate (221, in sory sex glands from various embryological pig seminal vesicles (22), in the human origins and from various species can be prostate (23,24), in the guinea pig prostate, separately analyzed as in the guinea pig and in the separated epithelium and muscle seminal vesicles. of the guinea pig seminal vesicles (1, 25). Estradiol-17P is generally considered to The muscle of the guinea pig seminal vesi- be a poor antagonist of exogenously admincles contained the highest level of cytosol istered testosterone in the ventral prostate estradiol binding, indicating that the recep- of young, sexually mature castrate rats (1). tor may be primarily associated with the Such findings may be related to a lack of muscular portion of the glands. Androgen physiological antagonism of androgen acreceptors, on the other hand, were located tion mediated by estradiol/estrophile interlargely in the epithelium (1, 25). actions due to the low levels of estradiol Although the rat accessory sex organs receptors found in this prostatic lobe. Conhave not as yet been separated into epithetributing to this phenomenon would be the lial and muscular components, perhaps es- pronounced conversion of estradiol-17fi to trogen receptors are predominantly located estrone. On the other hand, if the antiin the muscle cell lines and androgen recep- androgenic effect of estradiol-17/3 is metors are primarily located in the epithelial diated primarily by a pharmacological comcell lines as in the guinea pig seminal vesi- petition for the binding of DHT to its recles. In support of this interpretation is ceptor, then the formation of estrone would histological evidence that the fibromuscufunction to attenuate this response since in lar portions of young, sexually mature rat our previously reported (10) competitor seminal vesicles were selectively stimulated studies, estrone had a lower capacity to to grow after estrogen treatment to cas- reduce r3H]DHT binding than estradiol. trated adrenalectomized rats (1,9), perhaps In contrast to the rat ventral prostate, reflecting a consequence of an estro- the effects of exogenously administered tesgen/estrogen receptor interaction. It is in- tosterone on the prostate of castrate dogs teresting to speculate that the increased can be effectively antagonized by estradiol concentration of estradiol receptors de- treatment (1). These differences between tected with advancing age in the seminal the two species may be accounted for in
524
ROBINETTE
part by the fact that relatively high levels of specific estradiol receptors have been found in the dog prostate (15) and by preliminary evidence that the dog prostate has a limited capacity to form estrone from estradiol. In conclusion, the presence of estrogen responsiveness or the lack thereof in the various male rat accessory sex organs correlates well with the concentration of estradiol specific molecules found in these glands. Circumstantial evidence favors the hypothesis that the estrophiles may be primarily associated with fibromuscular cells. Although estradiol receptors have been identified in the rat accessory sex organs, the low concentrations of these binding moieties in the rat in relation to other species may explain the relative lack of antiandrogenic effects of estrogens in the rat. Furthermore, the ability of the rat secondary sex glands to effect the rapid conversion of estradiol to estrone may also contribute to this lack of anti-androgenic activity. The significance of the age-associated changes in estradiol receptor concentration on responsiveness to steroids in the seminal vesicles remains to be elucidated.
wish to acknowledge the expert techof Lynne Schwartz and Brenda Lyons. REFERENCES
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ACKNOWLEDGMENTS The authors nical assistance
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19. HARTER, L. H. (1960) Biometrics 16,671-685. 20. WITTLIFFE, J. L., HILF, R., BROOKS, W. F., SAVLOV, E. D., HALL, T. C., AND ORLANDO, R. A. (1972) Cancer Res. 32, 1983-1992. 21. BRAUNSBERG, H. (1975) Eur. J. Cancer 11, 499-507. 22. JUNGBLUT, P. W., HUGHES, S. F., GGRLICH, L., GOWERS, U., AND R~~DIGER, K. W. (1971) Hoppe-Seyler’s Z. Physiol. Chem. 362, 16031610. N., O’TOOLE, J. H., AND YOUNG, J. 23. BASHIRELAHI, D. (1976) Biochem. Med. l&254-261. 24. BASHIRELAHI, N., AND YOUNG, J. D. (1976) Urology 8,553-558. M. 25. BELIS, J. A., BLUME, C. D., AND MAWHINNEY, G. (1977) Endocrinology 101, 726-740.
