Tamoxifen inhibits Leydig cell steroidogenesis: In vivo and in vitro studies

Tamoxifen

Inhibits Leydig Cell Steroidogenesis: and In Vitro Studies T. Lin and

Using isolated interstitial and 8-bromo-cyclic could

not be reversed

response incubated

with

to gonadotropin (lo-‘M1)

diminished

adenylate

testosterone

17&estradiol.

stimulation

with pregnenolone

Tamoxifen

The

inhibited

present

testosterone

studies

testosterone

the binding of hCG to receptor

METHODS

Animals Normal mature (60-90 day-old) and hypophysectomized male Sprague-Dawley rats (21-day and 60-day-old) were obtained from Zivic-Miller Laboratory, Allison Park, Pennsylvania. Animals were kept at 12h light/l2 hr dark cycle and fed rat chow and water ad libitum. Hypophysectomized rats were fed rat chow and saline and were used within 4 days after hypophysectomy.

Reagents Collagenase (Type I), human chorionic gonadotropin, adenosineS-triphosphate, creatine phosphate, creatine phosphokinase, Imethyl-3-isobutyl xanthine (MIX), 8-bromo-cyclic AMP and bovine serum albumin (BSA) were from Sigma. Medium 199 with bicarbonate buffer was from Grand Island Biological Company. Human pituitary (LH A-3) was supplied by National Pituitary Agency. ‘*‘I-succinyl cyclic AMP-tyrosine methyl ester (‘*‘ISCAMP-TME) and (1,2,6,7-‘H)-testosterone were obtained from

1982

tamoxifen

directly

rats. When

medium

were

formation.

inhibits

testosterone

interstitial

27.0

inhibits LH

effect of tamoxifen

f

Tamoxifen

cells were

1.9 ng/106

cells.

also significantly

was not affected. These results indicate that

are inhibited by tamoxifen.

B

Metabolrsm, Vol. 3 1, No. 6 (June),

that

levels in the incubation

OTH STEROIDS (androgens, estrogens, glucocorticoids and antiandrogens) and peptide hormones (gonadotropins, prolactin, growth hormone and luteinizing hormone-releasing hormone) have been reported to affect Leydig cell function.“’ Most recently, we and others have reported that antiestrogens (tamoxifen and enclomiphene) have direct inhibitory effects on Leydig cell steroidogenesis.3’4 Using isolated interstitial cells of rats, tamoxifen inhibited LH and 8-bromo-adenosine 3’,5’-monophosphate (8-bromocyclic AMP)-stimulated testosterone synthesis in a dose-dependent manner. Tamoxifen also reduced LHstimulated cyclic AMP formation. Furthermore, the inhibitory effect of tamoxifen could not be reversed with concomitant addition of equimolar concentrations of 17&estradiol.4 In fact, the inhibition produced by tamoxifen was similar to that produced by equimolar amounts of l7@-estradiol. Leydig cells contain high affinity, low capacity receptors for estrogen,5*6 and the effects of tamoxifen and enclomiphene probably are mediated by binding to these estrogen receptors.’ The present study was undertaken to further evaluate the effects of tamoxifen on steroidogenesis and the mechanisms of action. AND

indicate

that tamoxifen

manner. The inhibitory

and mature hypophysectomired

pregnenolone-induced

cyclase activity whereas

several steps of steroidogenesis

rats, we have shown previously

synthesis in a dose-dependent

both in immature

(5~10-~M).

significantly

MATERIALS

E. P. Murono

cells from testes of Sprague-Dawley

AMP stimulated

In Vivo

New England Nuclear. Tamoxifen ceuticals, Wilmington, Delaware. were from Steraloids, Inc.

was a gift from Stuart PharmaPregnenolone and 17&estradiol

The Eflect of Tamoxifen on Testosterone to hCG Stimulation in Hypophysectomized

Resporzse Rats

Hypophysectomized rats (21-day or 60-day-old) were treated with 0, 10 gg, or 100 rg tamoxifen suspended in 0.1 ml corn oil. Twenty four hours later, tail blood samples were obtained from each rat and then 5 units/l00 g body weight of hCG (A.P.L., Ayerst) were given subcutaneously. Two hours after hCG injection, the rats were killed by decapitation and trunk blood collected. Sera were separated and stored at -20°C until assayed for testosterone.

In Vitro Studies Using Intact Hypophysectomized Rats

Testes from

After decapitation, testes were quickly removed from above tamoxifen-hCG treated 60-day-old hypophysectomized rats. Testes were carefully decapsulated and washed once in cold Medium 1994.1% BSA solution and then incubated in 4 ml Medium 1994.1% BSA and 0.1 mM MIX. Incubations were carried out for 3 hr at 34OC, under 95% 0,/50/n CO, in a DubnolT shaking incubator. At the end of incubation, medium was centrifuged immediately at 3,OOOxg for I5 min and supernate was saved for testosterone assay.8,9

In Vitro Studies of Enzyme Dispersed Cells from Intact Adult Rats

Interstitial

To investigate the effect of tamoxifen and 17&estradiol on testosterone and cyclic AMP formation, dispersed interstitial cells from intact mature rats were prepared by collagenase digestion according to methods reported previously.8,9 Interstitial cells (1 x 106cells/ml) were incubated in Medium 199-BSA-MIX at 34O, 95% 0,/5% CO,. Varying concentrations of LH. 17@-estradiol (I 0m6 to

From the Medical Service, Wm. Jennings Bryan Dorn Veterans Hospital and Department of Medicine, University of South Carolina School of Medicine, Columbia, South Carolina. Received for publication April 17, 1981. Supported in part by the Veterans Administration and by the National Institute on Aging Grant No. I ROI AG OI217-03-REB. Address reprint requests to Tu Lin. M.D.. Medical Service, Wm. Jennings Bryan Dorn Veterans Hospiial. Columbia, South Carolina 29201. 0 I982 by Grune & Stratton, Inc. 0026-0495/82/3106/0005$01.00/0

543

544

LIN AND MURONO

10e4M), and/or tamoxifen (10e6 to 10m4M) were added simultaneously, testosterone and cyclic AMP levels of medium were measured after 3 hr incubations. To further evaluate the effect of tamoxifen on steroidogenesis, interstitial cells were incubated with pregnenolone (5 x lo-‘M) and with or without tamoxifen (10m5M) for 3 hr. Samples were then purified using the celite chromatography technique of Abraham et al.” with minor modifications before testosterone radioimmunoassay.”

Table 2. Plasma Testosterone Levels in 60-Day-Old Hypophysectomized Rats PlasmaTestosterone(ng/ml) 2 h after hCG Stimulation

Basal

Control (n = 4) Tamoxifen 10 pg (n = 4) Tamoxifen 100 ~(9 (n = 4)

0.14

+ 0.01

1.51 + 0.37

0.12

* 0.02

0.83

* 0.13

0.11

t 0.01

0.48

* 0.14”

The effect of tamoxifen on hCG-stimulated plasma testosterone levels

Measurement of hCG Binding in Testis Homogenates

in 60-day-old

Testicular homogenates were prepared according to the method of Chen et al.” The homogenates were suspended in buffer solution (Medium 199 with 0.1% BSA and 0.25 M sucrose) with a final concentration of 1 g tissue/l0 ml. Testicular homogenates (20 mg) were incubated with ‘*‘I-hCG at 34“C for 2 hr and then 2 ml of ice cold buffer were added and the tubes were centrifuged at 6000xg for 20 min. For nonspecific binding, a lOO-fold excess of unlabelled hCG was added with ‘*‘I-hCG. The supernatant was discarded and the pellet was washed again with 2 ml buffer and centrifuged at the same speed. Final pellet was counted in a gamma scintillation spectrometer.

before and 2 hr after hCG (5 u/lOOg body weight) injection. Results are

The Eflect of 17P-Estradiol and Tamoxifen on Adenylate Cyclase Activity Testicular tissue from normal mature rats was placed in 5 vol of ice cold 10 mM Tris with 1 mM EDTA (TE-buffer) and 27% sucrose solution (pH 7.5). Tissue was homogenized by polytron PTlO for 10 set with the dial set at 5. The homogenate was centrifuged at 500xg for 5 min and the pellet (tissue fragments, nuclei and major cell debris) was discarded. The supernatant was centrifuged again at 12,OOOxg for 15 min. The final pellet was resuspended in 2 ml TE-buffer (pH 7.5) and used immediately for adenylate cyclase assay.” All procedures were carried out at 4OC. Protein was measured by the method of Lowry et a1.15 using bovine serum albumin as a standard. For assaying adenylate cyclase, the method of Brooker et al. was used with slight modifications.“.” The reaction mixture contained 50 mM MIX, 20 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, 1 mM ATP, 0.4 mM EDTA, 0.4 mM EGTA, 25-50 pg membrane protein preparation and varying amounts of tamoxifen and 17@-estradiol in a total volume of 100 ul. The mixtures were incubated at 37°C and the reactions were terminated by the addition of 900 ul of 50 mM sodium acetate buffer, pH 4.0. Blanks were prepared by adding sodium acetate buffer, pH 4.0, prior to the addition of the membrane preparation. After incubation, the mixtures were heated at 100% for three minutes and centrifuged. The supernatant fractions were assayed for cyclic AMP content by radioimmunoassay.8.9

hypophysactomized rats. Rats were treated with corn oil,

10 pg or 100 pg tamoxifen. The next day, blood samples were obtained the mean + SE. ‘p < 0.05 vs control.

RESULTS

In response to hCG stimulation, mean plasma testosterone levels of 2 1-day-old hypophysectomized rats were 0.74 + 0.05 ng/ml (x + SE, n = 5). Tamoxifen in a dose of 10 pg had no significant effect on hCG stimulated testosterone formation, however, 100 pg of tamoxifen reduced hCG stimulated testosterone response to a mean of 0.54 + 0.02 ng/ml (p < 0.025) (Table 1). Basal testosterone levels of 60-day-old hypophysectomized rats were 0.14 -+ 0.01 ng/ml. Tamoxifen (10 pg and 100 pg) had no significant effect on basal testosterone levels. Two hours after hCG stimulation, plasma testosterone levels of the control group increased to 1.51 2 0.37 ng/ml. In response to hCG stimulation, there was a 46% reduction in mean plasma testosterone levels in rats treated with 10 pg tamoxifen and 69% reduction in rats treated with 100 pg tamoxifen (p < 0.05) compared to control rats (Table 2). When decapsulated testes from these rats were incubated in Medium 199-BSA-MIX for 3h, rats treated with 100 pg tamoxifen produced significantly less testosterone when compared with control rats or rats receiving a lower dose of tamoxifen (10 pg) (Table 3). These results suggest that tamoxifen treatment directly inhibits testosterone response to gonadotropin stimulation both in immature and mature Table 3. Testosterone Formation of Decapsulated Whole Tastes lncubatlonMedium Testosterone(ng/ml)

Table 1. Plasma Testosterone Levels in 21 -Day-Old Hypophysectomized Rats PlasmaTestosterone(ng/ml) Control group (n = 5)

0.74

& 0.05

Tamoxifen 10 fig (n = 3)

0.72

+ 0.13

Tamoxifen 100 pg (n = 3)

0.54

* 0.02’

The effect of tamoxifen on hCG-stimulated plasma testosterone levels in 21-day-old

hypophysectomized rats. Rats were divided into three

Control (n = 6)

5.86

+ 0.21

Tamoxifen 10 pg (n = 31

6.86

* 1.32

Tamoxifen 100 pg (n = 3)

3.76

? 0.32.

The effect of tamoxifen (in viva treatment) on testosterone formation of &capsulated

whole testes. Hypophysectomized rats, 60 days old.

were treated with plain vehicle, 10 or 100 pg of tamoxifen. HCG 5 U/lOOg

was given the next day, and 2 hr later, rats were killed and

groups and treated with either corn oil vehicle, or with vehicle plus 10 fig

testes

or 100 pg tamoxifen. The next day, 5 U/lOOg

incubated in Medium 199-O.

body weight hcG was

were removed and decapsulated.

Decapsulated

testes ware

1% BSA-0.1 mM MIX. Testosterone levels

given and rats were killed 2 hr later. Plasma testosterone was measured

were measured in the incubation medium after 3h incubations. Results

by radioimmunoassay. Results are the mean + SE.

are the mean + SE.

lp < 0.025

vs control.

lp < 0.001

vs control.

TAMOXIFEN

INHIBITS LEYDIG CELL FUNCTION

545

Table 4. Conversion of Pregnenolone to Testosterone Testosterone

Pregnenolone (5 x lo-’

M)

Table 6. hCG Binding

(na/ 10’ Cells1

hCG Bindlno

27.0 + 1.9

Pregnenolone(5 x 10-r M) +

15.9 k 1.0

Control

p < 0.01

100

Tamoxifen lo-’

Tamoxifen (10-s M)

+

5.2

M

109 + 8.8

Tamoxifen 10-s M

113 -t 2.0

The effect of tamoxifen on conversion of pragnenolone to testoster-

Tamoxifen 10-s M

116 + 4.5

one. Interstitial cells were incubated with pregnenolone with or without

Tamoxifen 1O-’ M

126 + 3.2

tamoxifen and testosterone levels were measured after 3 hr incubation.

The effect of tamoxifen on specific binding of hCG to testis homo-

The extracted steroids were separated using celite chromatography

genate. The results are the mean (*SE)

before radioimmunoassay. Results are the mean k SE.

effect of tamoxifen was expressed as a percent of ‘251-hCG binding to

of triplicate incubations. The

homogenate in control tubes. The differences were not statistically

hypophysectomized rats. Previously we have shown that using interstitial cell populations, tamoxifen can directly inhibit LH-stimulated testosterone formation.4 To further evaluate the steps of steroidogenesis affected by tamoxifen, interstitial cells were incubated with tamoxifen (lo-‘M) and pregnenolone (5 x 1O-‘M) and testosterone levels were measured after 3h incubation. As shown in Table 4, when cells were incubated with pregnenolone alone, testosterone levels in the incubation medium were 27.0 * 1.9 ng/ lo6 cells. When pregnenolone was added with tamoxifen, there was a marked reduction of testosterone production by interstitial cells. This indicates that the conversion of pregnenolone to testosterone is blocked by tamoxifen. We next investigated the effects of 17/3-estradiol and tamoxifen on cyclic AMP responses to LH stimulation. Similar to that reported previously,4 a low dose of tamoxifen ( 10e6M) had no effect on LH-stimulated cyclic AMP formation, while 10e5 and 10-4M tamoxifen significantly blocked LH-stimulated cyclic AMP levels. In contrast, the effect of 17P-estradiol on LHinduced cyclic AMP formation was biphasic. 17@estradiol at a low dose ( 10e6M) slightly increased while a high dose (10m4M) inhibited cyclic AMP responses to LH (Table 5). Thus, even though both estrogen and tamoxifen inhibited LH-stimulated testosterone formation, the pattern of inhibition of these two agents were not identical. Since tamoxifen inhibited LH-induced cyclic AMP

significant.

and testosterone formation, it may affect the binding of gonadotropin to receptors or the adenylate cyclase activity. Accordingly, the effect of tamoxifen on ‘*‘IhCG binding to the gonadotropin receptor was studied. As shown in Table 6, tamoxifen had no effect on hCG binding to gonadotropin receptors. Next, the effect of tamoxifen on the adenylate cyclase activity was investigated. Control levels of adenylate cyclase activity were 3.54 + 0.20 pmole/mg - min. With the addition of hCG (1 pg/ml), adenylate cyclase activity increased to 9.03 + 0.27 pmole/mg - min. Tamoxifen, 10m6M, had no effect on adenylate cyclase activity; however, lo-’ and 10e4M of tamoxifen significantly reduced adenylate cyclase activity 11% and 48% respectively. In contrast, estradiol only at the highest dose, 10m4M, reduced adenylate cyclase 16%, while 10e5 and 10m6M of estradiol had no appreciable effect. Both tamoxifen and estradiol had no significant effect on basal activity of adenylate cyclase (data not shown). DISCUSSION

The present results confirm our previous finding that tamoxifen has direct inhibitory effects on testicular steroidogenesis4 It is apparent that several steps of steroidogenesis are affected by tamoxifen. Tamoxifen significantly reduced LH-induced cyclic AMP and testosterone formation, inhibited adenylate cyclase Table 7. Adenylate Cyclase Activity

Table 5. Cyclic AMP Response to LH Stimulation

Adenylate

(pmol 10’ Cells)

Cyclase

pmol mg . min

Cyclic AMP p Value

Control

Control

2.87

+ 0.14

KG

LH (5 mllJ/ml)

6.73

? 0.21

3.54 &ml)

+ 0.20

9.03

f 0.27

hCG + Tamoxifen 10m6 M

9.05

i- 0.26

10.05

hCG + Tamoxifen 10e5 M

8.06

+ 0.09’

NS

hCG + Tamoxifen 10m4 M

4.74

+ 0.16t

(1

LH + E, 10m6 M

7.31 + 0.02

LH + E, 10m5 M

6.50

c 0.12

LH + E, 10m4 M

5.58

2 0.15

<0.005

hCG + E, lo-= M

9.60

t 1.00

LH + Tamoxifen 10m6 M

7.12 t 0.10

NS

hCG + E, 1O-5 M

8.84

+ 0.09

LH + Tamoxifen 10m5 M

5.13

t 0.44

<0.01

hCG + E, 1O-4 M

7.57

+ 0.17’

LH + Tamoxifen lo-*

3.96

k 0.14

<0.005

The effect of tamorifen

and 17@-estradiol on adenylate cyclase

M

The effect of tamoxifen and 17&estradiol

on cyclic AMP response to

LH stimulation. Interstitial cells (1 x 10s cells/ml) were incubated with LH. 17&estradiol

or tamoxifen and cyclic AMP levels were measured

after 3h incubations. Results are the mean + SE of seven incubations.

activity. Results are the mean c SE of triplicate incubabons. Adenylate cyclase activny is expressed as pmol/mg

lp <

0.005.

tp < 0.001

YS hCG only.

.

min cyclic AMP formed.

546

LIN AND MURONO

activity and the conversion of pregnenolone to testosterone, whereas the binding of hCG to its receptor was not affected. The effect of LH is mediated by binding to its high affinity receptors on the cell membrane surface, and the activation of adenylate cyclase resulting in increased cyclic AMP and ultimately in testosterone formation. However, using rat testis homogenates or with Leydig cell membrane particles, it has been difficult to demonstrate the response of adenylate cyclase to hCG/LH stimulation. We recently reported that using a higher incubation temperature, 37O, hCG consistently increased adenylate cyclase activity twofold above controLi In the present study we have demonstrated for the first time that tamoxifen reduced LH-induced cyclic AMP formation by directly inhibiting adenylate cyclase activity whereas the binding of hCG to its receptors remained intact. Antiestrogens have been used for evaluation of hypothalamic-pituitary-gonadal axis in the human,‘6.‘7 to treat delayed puberty and to enhance spermatogenesis presumably by blocking the negative feedback have exerted by estrogen.‘8,‘9 However, antiestrogens also been reported to have direct effects on the ovary and testes. Watson and Howson reported that tamoxifen can directly inhibit FSH-stimulated ovarian 17/3estradiol synthesis2’ In a short communication, Bartke et al. found that plasma concentrations of testosterone and LH in rats were significantly lower after five days of treatment with tamoxifen (2 or 10 mg).2’ Reductions in the plasma levels of testosterone and gonadotropin have also been reported after treatment of male rats with another anti-estrogen, 19-norspiroxenone.22 Most recently, Wang et al. reported that clomiphene in low doses can inhibit 17a-hydroxylase and 17,20-lyase activities whereas high doses decrease testicular hCG/ LH receptor content of testes in hypophysectomized rats.3 In five eugonadal men, Smals et al. studied the effect of tamoxifen on hCG-stimulated testosterone and 17a-hydroxyprogesterone formation.23 They reported that tamoxifen (20 mg twice daily) almost completely abolished the hCG-induced steroidogenic block localized between 17a-hydroxyprogesterone and testosterone. However, their data suggested that

plasma testosterone and 17a-hydroxyprogesterone levels were lower when tamoxifen was given simultaneously with hCG as compared to hCG alone.23 In the present study, we have used hypophysectomized rats to assess the direct effect of an antiestrogen on testicular function and to eliminate the possible feedback action of estrogen on gonadotropin secretion by the pituitary. Extremely low basal testosterone levels in these rats indicated that hypophysectomy was complete. Tamoxifen inhibited hCG-stimulated testosterone formation in immature and mature hypophysectomized rats both in vivo and in vitro. Likewise, Donaldson et al. reported that tamoxifen in doses of 100 pg significantly diminished testosterone and 17a-hydroxyprogesterone response to hCG in rats.24 These data therefore confirm our previous finding that tamoxifen does have direct deleterious effects on testicular steroidogenesis. The dosages used in this study were comparable to those reported in the treatment of breast carcinoma.24S26 Very low doses of tamoxifen (less than 5 pg) do not depress steroidogenic function and/or responses of the Leydig ceil of rats, but in fact, prevent the steroidogenie block induced by estrogen during hCG stimulation.27.28 These actions of very low concentration of tamoxifen are probably mediated by the estrogen receptor. Recently polar metabolites of tamoxifen were identified in plasma, liver, uterus and oviduct after in vivo administration of 3H-tamoxifen.29 The major metabolite is 4-hydroxy-tamoxifen which is more potent than tamoxifen itself in binding to the estrogen receptor. It has been suggested that hydroxylated metabolites of tamoxifen instead of tamoxifen are responsible for its in vivo effect. However, both in our interstitial cells and membrane particle preparations, tamoxifen inhibited adenylate cyclase activity and reduced cyclic AMP response to LH stimulation. These results suggest that tamoxifen probably has a direct effect on the Leydig cell and metabolism to 4-hydroxy-tamoxifen is not required. The effects of tamoxifen we observed in this study may not be mediated by an estrogen receptor mechanism. Whether metabolites of tamoxifen are more potent than tamoxifen itself in our system remains to be investigated.

REFERENCES I. Dufau ML, Catt KH: Gonadotropin receptors and regulation of steroidogenesis in the testis and ovary. Vit Horm 36:461-592, 1978 2. Purvis K, Clausen OPF, Hansson V: Regulation of Leydig cell sensitivity and responsiveness of LH/hCG. Int J Andrology Suppl 2~247-263, 1978 3. Wang C, Erickson GF, Hopper B, et al: Direct inhibitory effect of enclomiphene citrate and estradiof on testis functions in hypophysectomized rats. Biol Reprod 22:645-653, 1980

4. Lin T, Murono EP, Osterman J, et al: Direct inhibition of rat Leydig cell function by tamoxifen. Metabolism 30: 156-l 59, 198 I 5. Brinkmann AO, Mulder E, Lamers-Stahlhofen GJM et al: An estradiol receptor in rat testis interstitial tissue. FEBS Lett. 26:301305, 1972 6. Kato J, Onouchi T, Okinaga testis. Endocrinology 94902-907, 7. Capony

F, Rochefort

S, et al: Estradiol 1974

H: High-affinity

binding

receptors

in rat

of the anties-

TAMOXIFEN

INHIBITS

LEYDIG CELL FUNCTION

trogen (‘H) tamoxifen to the 8s estradiol receptor. Mol Cell Endocrinol 11:18l-198, 1978 8. Lin T, Murono E, Osterman J, et al: The effects of verapamil on interstitial cell steroidogenesis. Int J Andrology 25499558, 1979 9. Lin T, Murono E, Osterman J, et al: The effects of calcium ionophore A23 187 on interstitial cell steroidogenesis. Biochim Biophys Acta 627: 157-l 64, 1980 10. Abraham GE, Swerdloff R, Tulchinsky 0, et al: Radioimmunoassay of plasma progesterone. J Clin Endocrinol Metab 32:619624, 1971 II Murono E, Nankin HR. Lin T, et al: The Aging Leydig Cell V. Diurnal Rhythms in Aging Men. Acta Endocrinol (in press) 12. Chen CJH, Lindeman JG, Trowbridge CG, et al: Gonadal receptors 1. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone. Biochim Biophys Acta 584:407435, 1979 13. Brooker G, Harper JF, Terasaki WL, et al: Radioimmunoassay of Cyclic AMP and Cyclic GMP. Adv Cycl Nucl Res lO:l-35, 1979 14. Lin T, Chen GCC, Murono E, et al: The Aging Leydig Cell IV. Adenylate cyclase activity. (in press) 15. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 16. Bardin CW. Ross GT, Lipsett MB: Site of action of clomiphene citrate in man: A study of the pituitary-Leydig cell axis. J Clin Endocrinol Metab 27: I55881 564, 1967 17. Sante” RJ. Leonard JM, Sherins RJ, et al: Short and long-term effects of clomiphene citrate on the pituitary-testicular axis. J Clin Endocrinol Metab 33:97&979, 197 I 18. Jungck EC, Roy J, Greenblatt RB, et al: Effect of clomiphene citrate on spermatogenesis in the human. Fertil Steril 15:40-43, 1964

547

19. Paulson OF, Wacksman J, Hammond CB, et al: Hypofertility and clomiphene citrate therapy. Fertil Steril 26:982-987, 1975 20. Watson J. Howson JWH: Inhibition by tamoxifen of the stimulatory action of FSH on estradiol-I 7 synthesis by rat ovaries in vitro. J Reprod Fert 491315-376, 1977 21. Bartke A, Mason M, Dalterio S, et al: Effects of tamoxifen on plasma concentrations of testosterone and gonadotropins in the male rat. J Endocrinol79:239-240, 1978 22. Goldman AS, Shapiro BH, Root AW: Effects of new multisite hormone blockers on the fertility of male rats. J Endocrinol 69:l I-21, 1976 23. Smals AGH, Pieters FRRM, Draywer JIM, et al: Tamoxifen suppresses gonadotropin-induced 17a-hydroxyprogesterone accumulation in normal men. J Clin Endocrinol Metab 51:1026-1029, 1980 24. Donaldson MDC, Saez JM, Forest MG: Effects of the aromatase inhibitor 1.4.6 Androstatriene-3,17-dione and the antiestrogen tamoxifen on rat testicular function. J Steroid Biochem 14:823-828, 1981 25. Jordan VC, Koerner S: Tamoxifen (ICI 46, 474) and the human carcinoma 8s oestrogen receptor. Eur J Cancer 11:205-206, 1975 26. Adam HK, Gay MA, Moore RH: Measurement of tamoxifen in serum by thin-layer densitometry. J Endocrinol 84:35-42, 1980 27. Cigorraga SB, Sorrel1 S, Bator J et al: Estrogen dependence of a gonadotropin-induced steroidogenic lesion in rat testicular Leydig cells. J Clin lnvest 65:699-705, 1980 28. Nozu K, Dufau ML, Catt KJ: Estrogen Receptor-mediated Regulation of Steroidogenesis in Gonadotropin-desensitized Leydig Cells. J Biol Chem 256:1915-1922, 1981 29. Borgna J-L, Rochefort H: Hydroxylated metabolites of tamoxifen are formed in viva and bound to estrogen receptor in target tissues. J Biol Chem 256:859-868, 198 I