Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730–740 day old) rats is decreased 50% when compared to intact, young mature (150–170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 μg testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.     

A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane.

1. The administration of dihydrotestosterone to rats orchidectomized 7 days previously stimulated the synthesis of nuclear receptor in prostatic cells several hours in advance of DNA synthesis and mitosis. 2. The synthesis of nuclear receptor is tightly coupled to cell proliferation; consequently, in resting cells, there is no further net synthesis of nuclear receptor above the maximum of approx. 8000 molecules/cell. 3. After orchidectomy a rapid decline in the concentration of free androgen in the nuceus and a slower decline in the concentration of nuclear receptor are observed. 4. Owing to the apparent scarcity of receptor-inactivating factors in the nucleus, and the inverse relationship between amounts of nuclear and cytoplasmic receptors, it is concluded that the nuclear receptor is discharged into the cytoplasm after orchidectomy. 5. The formation of the cytoplasmic receptor is an early event preceding the onset of cellular autolysis. 6. Regressing prostate develops the capacity to eliminate cytoplasmic receptor, and this capacity is retained by the regenerating prostate for at least 14 days. 7. The synthesis of nuclear receptor in early G1 phase may control the entry of cells into the cell cycle and the prolonged retention of receptor in the nucleus may prevent the activation of autophagic processes.     

Interaction of spironolactone with rat skin androgen receptor.

Some characteristics of the dorsal skin cytoplasmic androgen receptor (AR) have been studied in male rats. The affinity constant, the binding specificity, and the sedimentation profile of the receptor have been found to be similar to the rat prostate AR. The measurement of the number of binding sites in various hormonal conditions (deprivation) led to the conclusion that this receptor was largely occupied by endogeneous hormones from gonadal and (or) adrenal sources. Administration of spironolactone or canrenone to 7-day-castrated rats was accompanied by a rapid and drastic decrease of available binding sites. This diminution was ascribed to the competitive inhibition of canrenone, the active in vivo metabolite of spironolactone. It is postulated that the antiandrogenic action of spironolactone, at the skin level, is mediated by canrenone which inhibits the formation of specific testosterone and (or) 5 alpha-dihydrotestosterone receptor complex in cytoplasm and consequently in nuclei.     

A rapid, specific protocol for determination of available androgen receptor sites in unfractionated rat ventral prostate cytosol preparations

A modification of the dextran-coated charcoal technique has been successfully employed for the measurement of androgen receptor binding of 5α-dihydrotestosterone in unfractionated rat ventral prostate cytoplasmic extracts. The addition of a small amount of ethanol to the dextran-coated charcoal solution during the adsorption of unbound ligand greatly facilitated charcoal adsorption of ligand associated with low affinity, high capacity binding components and reduced the contribution of the latter cytoplasmic binding components to less than 10 percent of the measured binding at near saturating concentrations, 10 nM, of 5α-dihydrotestosterone. The assay is facile, sensitive, and highly reproducible and a complete saturation curve can be obtained with as little as 100 mg of ventral prostate. This protocol therefore represents a unique procedure for the quantitation and characterization of the cytoplasmic androgen receptor of rat ventral prostate. The concentration of available cytoplasmic androgen receptor in ventral prostate from young mature (80–120 day old) albino rats, 24 hours post orchidectomy, was 10,300 ± 1780 sites per cell and the apparent binding constant for 5α-dihydrotestosterone was 6.49 ± 0.35 × 10⁸ M^?1.     

Aging in the rat prostate. Reduction in detectable ventral prostate androgen receptor content.

Aging in the rat is associated with a reduction in the detectable androgen receptor content of the ventral prostate. The reduction in cytoplasmic receptor content did not appear to be attributable to an aging-associated production of a receptor-inactivating factor or to an aging-associated change in the sedimentation properties of the androgen receptor of young and aged animals.Saturation analysis of cytoplasmic extracts prepared from two different breeds of similar albino rats and a genetically distinct strain of inbred brown rats demonstrated quantitative aging-associated reductions in the androgen-receptor content per cell of the ventral prostate. The reduction in receptor content per cell appeared to increase progressively in magnitude with increasing age. The mean value for the cytoplasmic androgen receptor sites per cell for the oldest animals (mean age 884 days) was only 14% of the mean value for the young mature animals (mean age 185 days) of the same breed. The binding affinities of the detectable androgen receptor of the young mature and aged animals were essentially identical. This observation does not eliminate the possibility that the observed reduction results from an aging-associated production of defective receptor. Evaluation of the total DNA content of the ventral prostate did not provide evidence for an aging-associated selective loss of receptor-containing cells. These data in toto were consistent with the interpretation that aging is associated with a mean reduction in the androgen-receptor content per receptor-containing cell.Both cytoplasmic and nuclear androgen retention were evaluated in vivo. These experiments provided qualitative confirmation of the in vitro saturation analyses as there was a highly significant aging-associated reduction in the amount of androgen specifically bound by these prostatic compartments. Total specific androgen retention by the ventral prostate of aging adults was reduced by 55% relative to young mature animals. This result was nearly identical to that obtained for the same breed and age category of animals when evaluated by in vitro saturation analysis.Preliminary in vitro experiments revealed a diminution in the uptake of androgen receptor by purified nuclei from aged animals relative to purified nuclei from young mature animals. The magnitude of the diminution in nuclear acceptor capacity was insufficient to account for the reduction in nuclear retention of androgen determined in vivo. The data were consistent with the interpretation that the cytoplasmic receptor is the major determinant of nuclear androgen retention in the ventral prostate.     

Differential regulation of androgen receptors in the separate rat prostate lobes: androgen independent expression in the lateral lobe

In order to understand the hormonal regulation of androgen receptors (AR) in the separate lobes of the rat prostate gland, the present study examined AR levels in the ventral, dorsal and lateral prostate lobes as a function of androgen withdrawal to complete prostatic regression and subsequent testosterone replacement. In the intact rat, the 3 prostate lobes contained significantly different amounts of androgen binding sites. Mean number of total cellular AR in the ventral, dorsal and lateral lobes was 7370, 1690, and 1015 fm/mg DNA, respectively. These receptors were primarily localized within the nuclear fraction of homogenized tissue: ventral, 86%; dorsal, 83%; and lateral, 100% nuclear localization. Androgen withdrawal was initiated via castration and rats were sacrificed 1, 2, 3, 5, 7, 10 and 14 days thereafter. Nuclear AR levels fell rapidly to 5, 24 and 30% of intact values by 48 h in the ventral, dorsal and lateral lobes, respectively. Levels of nuclear AR continued to decline in the ventral and dorsal lobes to undetectable levels by Day 10. In marked contrast, lateral lobe nuclear AR began to increase on Day 3 postcastration, reaching intact values by Day 7 and 133% intact levels by Day 14. Cytosolic AR in the ventral and dorsal lobes initially increased following castration, but subsequently declined to low levels by Day 14. Cytosolic AR were not detectable in the lateral prostate at any time point following castration. To determine the nuclear AR response to testosterone at this time, 14 day castrate rats were given 2 cm testosterone implants and sacrificed 1, 3, 5, 7, 10 and 14 days thereafter. As expected, nuclear AR rapidly returned in the ventral and dorsal lobes by Day 1 and reached a plateau by Day 5. A short term response to androgen exposure occurred in the lateral lobe where an immediate 9-fold increase in nuclear AR quantity was observed; however, these levels rapidly declined to pre-implant values by Day 5 and remained at that level despite continued exposure to testosterone. These f findings indicate that while nuclear AR levels in the ventral and dorsal prostate are primarily regulated by androgens, a testosterone-independent component exists within the lateral lobe.     

Mechanisms of replenishment of nuclear and rogen receptor in rat ventral prostate

1. The concentration of androgen receptor in the nucleus of the prostatic cell is rapidly elevated by the administration in vivo of 2μg of [³H]testosterone to 1-day-castrated rats. From a concentration of 2300 receptors/nucleus at 5min after intravenous injection of hormone, there is an increase to 21000 receptors/nucleus at 60min. At the same time, the amount of binding of androgen in the cytoplasm remains constant at a relatively low value. 2. An identical dose of [³H]testosterone administered to 7-day-castrated rats produces a much smaller change in the concentration of nuclear receptor, from 700 receptors/nucleus at 5min to only 4300 receptors/nucleus at 60min. Thus the reservoir from which nuclear receptor is replenished is considerably smaller in regressed prostatic cells. Again, the amount of binding of androgen in the cytoplasm remains unchanged at a low value over the experimental time course of 60min. 3. In contrast with the scant labelling of cytoplasmic receptor achieved by injecting animals with [³H]testosterone, labelling in vitro, by incubation of tissue slices with radioisotope, indicates that prostate of 1-day-castrated animals actually contains 21400 receptors/cell in the cytoplasmic compartment, and prostate of 7-day-castrated animals 3000 receptors/cell. 4. Owing to the similarity between the concentration of nuclear receptor measured in vivo and the concentration of cytoplasmic receptor measured in vitro, the labelling techniques in vivo and in vitro were used in sequence to demonstrate the movement of most of the cytoplasmic receptor into the nucleus. In the 5–60min interval after the administration of [³H]testosterone to 1-day-castrated rats, a decrease of 17400 receptor molecules in the cytoplasm is exactly mirrored by an increase of 17200 receptor molecules in the nucleus. 5. These results imply that, in prostate of 1-day-castrated rats, nuclear receptor is replenished exclusively by translocation of cytoplasmic receptor. However, in the regressed prostate of 7-day-castrated rats, only about 25% of the nuclear receptor is replenished through translocation of existing cytoplasmic receptor. The remainder is ultimately synthesized during new rounds of cell division induced by hormone.     

Autoregulation of androgen receptor expression in rodent prostate: immunohistochemical and in situ hybridization analysis

Autoregulation of androgen receptor mRNA and protein was investigated by immunohistochemical and in situ hybridization techniques. In both mouse and rat prostate, the epithelial cell nuclei were stained with the monoclonal or polyclonal antibodies raised against human androgen receptor. It was observed that 3 days after castration, nuclear staining of the epithelium was greatly reduced, while androgen treatment restored the staining intensity to a normal level. In situ hybridization using an androgen receptor cDNA fragment as probe demonstrated that the change in androgen receptor mRNA level correlated with the change in antibody staining intensity. These data suggested an up-regulation of androgen receptor expression by androgen.     

Regulation of androgen receptor mRNA and protein level by steroid hormones in human mammary cancer cells.

The regulation of the human androgen receptor (AR) by steroid hormones in human mammary cancer cells was investigated using immunocytochemical and ligand binding assays for its protein and Northern blot analyses for the corresponding mRNA. MFM-223 cells contain high levels of ARs and are growth-inhibited by dihydrotestosterone (DHT). The AR protein is down-regulated to 57% of the control by 10 nM DHT after 24 h, and the corresponding mRNA is also reduced. The nonsteroidal antiandrogen hydroxyflutamide had no effect on the AR level, whereas after incubation with 1 microM cyproterone acetate a slight down-regulation was observed. The AR level was restored completely after release from a 7 day treatment with DHT. However, only 60% of the control level was restored, if the cells wer grown in the presence of DHT for 6 weeks. In androgen-pretreated cells the proliferation rate remained decreased even after the withdrawal of DHT. Concomitantly the distinct growth inhibition was lost. Transfection experiments demonstrated a reduced activity of the residual androgen receptor in these pretreated cells. In addition to the AR, EFM-19 cells also contain significant amounts of estrogen and progesterone receptors. EFM-19 cells are not growth inhibited by physiological concentrations of DHT. Autoregulation of AR was also found in this cell line. Additionally, reduced levels of AR protein and mRNA were found in EFM-19 cells after treatment with the synthetic progestin R5020. The maximum effect of R5020 was observed at the high concentration of 1 microM. Estrogen treatment with 10 nM 17 beta-estradiol for 3 days reduced the AR level only by 25%.     

Actin and creatine kinase mRNAs in rat levator ani and vastus muscles as a function of androgen status

The plasticity of two selected mRNAs was studied in two typical fast-twitch muscles at different time intervals after orchiectomy (GDX). The levator ani muscle of the rat (LA) is exquisitely sensitive to androgens, whereas the superficial vastus lateralis (SVL) lacks such sensitivity. In vitro translation of RNA isolated from both tissues indicated that actin was among the most repressed proteins of the LA at day 10 postsurgery (GDX-10 days), whereas the template activity of the SVL mRNAs remains virtually unmodified. We used an available actin cDNA and demonstrated that the expression of the LA actin message is reduced by 85% in GDX-10 days and can be recovered after testosterone propionate (TP) injections (GDX + TP). In contrast, the actin expression in SVL remains constant up to day 20 postsurgery. In the LA, the expression of creatine kinase (CK) mRNA was increased 140% in GDX-5 days and decreased 34 and 17% in GDX-10 days and GDX-20 days, respectively, although the measured CK activity, as well as the in vitro translation of the message, remained elevated in those two latter groups. Control level of the CK mRNA expression was recovered in the GDX + TP group. Again, the expression of the message was unchanged in SVL, suggesting that the protein synthesis of this skeletal muscle is far less sensitive to androgen deprivation than that of the LA muscle.     

Characterization of estrogen binding in the developing rat testis. Ontogeny of the testicular cytoplasmic estrogen receptor.

The properties and physical characteristics of a steroid binding component present in the immature (7 to 35 day) rat were investigated and found to be different from those of the 17 beta-estradiol receptor in the mature rat testis. These properties include a binding capacity of 483 fmol estradiol/mg protein, a Ka at equilibrium of 4.23 x 10(7)M-1, and broad steroid specificity as shown by interaction with several steroids; no binding was observed with diethylstilbestrol. The component, found in blood and several tissues including the testis, migrated as a 4.6S peak on sucrose gradients. This 4.6S component, which interacted with an anti-alphafetoprotein antiserum, decreased with age and was not detectable in the testis after day 21 or in the serum after day 25. These data suggest that this component is alphafetoprotein. Ontogenic appearance of the testicular cytoplasmic 17 beta-estradiol receptor in the developing rat was further elucidated. Sucrose gradient sedimentation analysis of cytosols revealed an 8S binding component that was first detectable at 23 days. Specific binding (fmol [3H]-estradiol/testis) was relatively low in neonates, rose to 59 fmol during the third week, and increased dramatically to 333 fmol at 7 weeks; binding rose only gradually after maturity. The receptor was tissue specific and steroid specificity studies demonstrated that only diethylstilbestrol and other estrogens were effective in competing with 17 beta-estradiol for binding sites. The Ka at equilibrium was determined as 3 x 10(10)M-1 and the binding sites were saturable in an in vitro system. The receptor did not interact with anti-alphafetoprotein antiserum as indicated by sucrose gradient studies. These data demonstrate the developmental appearance of the testicular cytoplasmic estradiol receptor in the immature rat.     

Intracellular inhibition of chromatin binding and transformation of androgen receptor by 3'-deoxyadenosine

Incubation of minced rat ventral prostate with 3'-deoxyadenosine (3'-dA) prior to labeling with the androgen, tritiated 7 alpha, 17 alpha-dimethyl-19-nortestosterone, reduced the level of androgen receptor bound to chromatin and increased the level of cytosolic androgen receptor and the fraction of cytosolic androgen receptor that did not bind to DNA. This effect was specific for 3'-dA and not mimicked by adenosine, 2'-deoxy-adenosine, cytidine, guanosine, or uridine. Adenosine was a competitive inhibitor of the 3'-dA effect. Labeled cytosolic androgen receptor from 3'-dA-treated prostate had properties that were similar to those exhibited by untransformed androgen receptor from prostate cytosol prepared in the presence of Na2MoO4, an inhibitor of receptor transformation in cell-free systems. Both androgen receptors had sedimentation coefficients of 8-9 S in low-salt gradients, did not bind to DNA tightly, and had a high affinity for DEAE-cellulose. The 3'-dA effect on these properties was not observed if androgen receptor from 3'-dA-treated prostate was isolated on high-salt gradients. These findings show that androgen receptor transformation does take place in intact prostate cells and suggest that 3'-dA inhibits chromatin binding of androgen receptor by interfering with androgen receptor transformation. The transformation process appears to involve removal of components from androgen receptor. Since 3'-dA is a potent inhibitor of the synthesis, polyadenylation, and nucleocytoplasmic transport of RNA, the 3'-dA effect may indicate a role for RNA in the mechanism of receptor transformation in intact target cells.     

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone.

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.     

The effect of pituitary homografts on the accessory sex organs and serum hormonal levels with or without androgen in mature male rats

The effect of pituitary homografts on the accessory sex organs and hormonal levels were studied in Wistar mature male rats. Grafted rats were further divided into four experiments: rats were bled once daily via a jugular vein cannula for seven days to investigate when serum prolactin began to rise after transplantation. rats were decapitated on the seventh day after transplantation to test whether 7 days were long enough to show the effect of pituitary grafts on the weight of prostate and seminal vesicles. rats were orchiectomized or orchiectomized and adrenalectomized on the seventh day after transplantation and then decapitated 4 weeks later to test a long term action of pituitary grafts and hormonal levels on the accessory sex organs without androgen. Rats grafted with several pieces of muscle were used as controls in each experiment. The initial rise in serum prolactin level was observed on the fourth day after pituitary transplantation, and then a higher serum prolactin level was maintained thereafter. Despite the higher prolactin level in the pituitary-grafted rat than in the control, no significant differences from the control in the weight of prostates and seminal vesicles and adrenal gland and the concentrations of serum luteinizing hormone (LH) and follicular stimulating hormone (FSH) were measured. This result showed that the weight of accessory sex organs was not affected by a higher serum prolactin within seven days.(ABSTRACT TRUNCATED AT 250 WORDS)     

PRPF6 promotes androgen receptor/androgen receptor-variant 7 actions in castration-resistant prostate cancer cells

Androgen receptor (AR) and its variants play vital roles in development and progression of prostate cancer. To clarify the mechanisms involved in the enhancement of their actions would be crucial for understanding the process in prostate cancer and castration-resistant prostate cancer transformation. Here, we provided the evidence to show that pre-mRNA processing factor 6 (PRPF6) acts as a key regulator for action of both AR full length (AR-FL) and AR variant 7 (AR-V7), thereby participating in the enhancement of AR-FL and AR-V7-induced transactivation in prostate cancer. In addition, PRPF6 is recruited to cis-regulatory elements in AR target genes and associates with JMJD1A to enhance AR-induced transactivation. PRPF6 also promotes expression of AR-FL and AR-V7. Moreover, PRPF6 depletion reduces tumor growth in prostate cancer-derived cell lines and results in significant suppression of xenograft tumors even under castration condition in mouse model. Furthermore, PRPF6 is obviously highly expressed in human prostate cancer samples. Collectively, our results suggest PRPF6 is involved in enhancement of oncogenic AR signaling, which support a previously unknown role of PRPF6 during progression of prostate cancer and castration-resistant prostate cancers.     

Studies on the kinetics of the RNA metabolism in the prostate of normal and castrated rats (author's transl)]

In the prostate of adult Wistar rats the RNA/DNA quotient of the whole organ as well as the amount of RNA and DNA in the nucleus was measured at different times after castration. Furthermore the half-life time for the turnover of the RNA in the nucleus and the cytoplasm was determined for normal and castrated rats with the aid of pulse labelling using [5(-3)H]uridine. A mathematical model was developed to analyze the experimental results. This model enabled us to make differentiated statements on the heterogeneous nuclear RNA (hmRNA) and the remaining RNA in the nucleus. The evaluation of the experimental values gave the following results: 1. By deprivation of androgens the uptake of [3H]uridine into the prostate is lowered. 2. The amount of DNA in the morphologically intact nucleus remains constant at least up to the 12th day after castration. 3. 6 days after castration the amount of hmRNA decreases to 1/10 and that of cytoplasmic RNA to 1/4. 4. The half-life time for the decrease of the whole nuclear RNA is 3.7 d and that of the cytoplasmic RNA 1.7 d. 5. The half-life time for the turnover of hmRNA is 16 min and that of cytoplasmic RNA about 2 days. 6 days after castration the half-life times are unchanged. The experimental results suggest that the observed decrease of nuclear RNA following castration can mainly be attributed to a reduced synthesis of hnRNA, while the decrease of cytoplasmic RNA is first of all caused by an increase in RNA degradation.     

Case scenarios in androgen deficiency

Deciding whether to choose androgen replacement for a particular patient is one of the many tasks facing the urologist. Factors including androgen levels, medical history, symptom profile, current medications, and prostate cancer risk all need to be considered when making this decision. However, the role each of these factors plays in arguing for or against androgen replacement remains controversial and more research is needed in many of these areas before the outstanding issues can be resolved. This article presents three cases involving patients who may require androgen supplementation. The cases describe (1) partial androgen deficiency syndrome, (2) testosterone deficiency in an anorchic man after bilateral orchiectomy for seminoma, and (3) a patient with sildenafil-refractory erectile dysfunction following treatment of localized prostate cancer with radiation therapy and androgen ablation. These cases illustrate some of the dilemmas and controversies surrounding androgen replacement that face the practicing urologist.     

Effects of an LHRH agonist on endocrine function, LHRH receptor and LH/hCG receptor in the pituitary-gonadal axis of male rats

The effects of an LHRH agonist (LHRHa), [D-Ser (tBu)]6 des-Gly-NH210) ethylamide, on endocrine function and the LHRH and LH/hCG receptors in the pituitary-gonadal axis were examined. The LHRHa was injected at 100 ng/100 g body weight into male rats once a day for 4 weeks and its effects were observed until 2 weeks after the end of treatment. Due to LHRHa treatment, the plasma LH concentration began to increase on day 3, reached a peak on day 7, and then decreased, although it remained above the control level during the treatment. The pituitary LH content decreased on day 1, reached a minimum (about 40% of the control) between days 3 and 7, and then was maintained at 60% of the control level until week 4. In contrast, the pituitary LHRH receptor concentration increased only on day 3, and the association constant (Ka) remained unchanged during the observation period. The testis weight and plasma testosterone concentration began to decrease on day 3, reached the minimum on day 7 and remained at this level until week 4, and their levels were not completely restored to normal 2 weeks after cessation of treatment. The testicular LH/hCG receptor concentration was decreased on day 1, and markedly decreased to 10-15% of the control value between day 7 and week 4, but the Ka value was slightly increased during the treatment. However, these values had completely recovered 2 weeks after the cessation of treatment. The testicular LHRH receptor concentration increased between days 1 and 7, returned to the control level in weeks 2 and 4, and then decreased 2 weeks after cessation of treatment. Its Ka value was reduced in weeks 2 and 4. These data suggest that the inhibitory effect of LHRHa on the gonad in male rats is not due to reduced pituitary LH release, but to changes in the number and Ka values of gonadal receptors for LH/hCG and LHRH.