Visualization of X-chromosome inactivation mosaicism of Tfm gene in XTfm/X+ heterozygous female mice

The testicular feminization (Tfm) locus, which produces a deficiency in androgen receptors, is located on the X-chromosome. Steroid autoradiographic techniques were used to demonstrate the mosaicism of the X-chromosome inactivation in two androgen target tissues of XTfm/X+ heterozygous female mice. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), about half of the cells were receptor positive (Tfm gene inactive). Statistical analysis of coherent clone size was applied to the heterozygous mesenchyme of the urogenital sinus and the coherent clone size of receptor-positive cells was estimated to be two or three cells per clone. This small clone size suggests that considerable cell mixing occurred in the tissue during embryonic development. Androgen binding in the mammary gland rudiments was restricted to the mesenchymal cells only in close vicinity to the epithelial mammary bud. In the wild-type rudiments most of the mesenchymal cells beneath the epithelium were receptor positive, while in heterozygous rudiments, receptor-positive and -negative cells intermingled. This observation suggests that in the wild-type mammary gland rudiments the epithelial bud may induce the formation of androgen receptors in adjacent mesenchymal cells rather than attract pre-existing receptor-rich mesenchymal cells.     

Tissue interaction in androgen response of embryonic mammary rudiment of mouse: identification of target tissue for testosterone.

In the androgen response of the embryonic mammary rudiment of the mouse, both gland epithelium and surrounding mesenchyme are visibly involved. The question whether this is due to a direct action of testosterone on both tissues was investigated in experimental combination of mammary epithelium and mammary mesenchyme, derived either from normal or from androgen-insensitive (XTfm/Y) embryos. A typical androgen response occurred in combinations of androgen-insensitive epithelium with normal mesenchyme, whereas all combinations of normal epithelium with androgen-insensitive mesenchyme failed to respond. It is therefore concluded that only the mesenchyme of the mammary rudiment is the target tissue for testosterone, and that all changes in the gland epithelium, including its necrosis, are secondarily caused by testosterone-activated mesenchymal cells.     

Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate

Binding sites of [3H]testosterone and [3H]dihydrotestosterone in the rat fetal urogenital sinus and postnatal prostate and vagina grown in vitro were examined by steroid autoradiography. Distinct nuclear incorporation of both androgens appeared between 14.5 and 16.5 days of gestation in rat fetuses. Nuclear labelling in the sinus was restricted to the mesenchyme surrounding the epithelium which showed no nuclear labelling. A similar distribution of labelled cells was observed in male and female sinuses up to 18.5 days of gestation. By 20.5 days of gestation, the labelling in the ventral mesenchyme of female urogenital sinuses became less intense but persisted in the mesenchyme of the dorsal sinus wall from which the vagina is formed. In the postnatal prostate, the epithelium showed nuclear [3H]testosterone labelling at 10 days coinciding with the onset of its functional differentiation. Epithelial labelling became more intensive at 4 weeks post partum while that of the mesenchyme declined. The results suggest two phases of androgen action: formation of the prostatic buds mediated by the androgen-activated mesenchyme of the fetal urogenital sinus and the differentiation of the postnatal prostatic epithelium directly stimulated by androgens.     

Change of mosaic pattern by androgens during prostatic bud formation in XTfm/X+ heterozygous female mice

The testicular feminization (Tfm) gene, which is characterized by a deficiency in androgen receptors, is located on the X-chromosome. Using steroid autoradiography, the mosaicism of the Tfm gene has been demonstrated in the androgen target tissues of XTfm/X+ heterozygous female mouse fetuses and the effects of androgens on the mosaic pattern analysed. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), only half of the cells were receptor positive (Tfm gene inactive), and receptor-positive cells and -negative cells formed small irregular patches. When the heterozygous sinuses were cultured in vitro in the presence of androgens, the sinuses underwent male sexual development and formed epithelial buds (prostate gland rudiments) projecting into the surrounding mesenchyme. Autoradiographic analysis revealed that the mosaicism of the mesenchyme disappeared around the developing epithelial buds: almost all the mesenchymal cells in close vicinity to the buds were receptor positive while in the outer layers receptor-positive and -negative cells coexisted. The proportion of receptor-positive cells was greatly increased in the mesenchyme beneath the non-budding area of the sinus epithelium. This androgen-induced increase was observed before the onset of bud formation. The results obtained in the thymidine incorporation experiments suggest that the increase of receptor-positive cells beneath the sinus epithelium might be explained by the migratory behaviour of the androgen-incorporating cells rather than by their selective proliferation.     

Serum bioavailability and tissue metabolism of testosterone and estradiol in rat salivary gland

The concentration of testosterone in saliva is probably in equilibrium with the concentration of cellular exchangeable testosterone in salivary gland, and these pools are a function of hormone transplant from the plasma compartment, and hormone metabolism in salivary gland cells. Both of these processes were examined in the present study using the carotid injection technique in normal and pilocarpine-stimulated ketamine-anesthetized rats. Both testosterone and estradiol were rapidly transported across salivary gland capillaries in vivo from the circulating albumin-bound pool. Estradiol, but not testosterone, was also rapidly transported into salivary gland from the circulating human sex hormone-binding globulin-bound pool. Hormone transport was several-fold greater than the capillary transport of [3H]bovine albumin, indicating that bound hormone was available for transport across salivary gland capillaries via an enhanced dissociation mechanism, with the plasma protein primarily residing in the plasma compartment. This result was confirmed by thaw-mount autoradiography, which showed diffuse distribution of [3H]testosterone in salivary gland, but vascular retention of [3H]bovine albumin. The concentration of exchangeable cellular testosterone in rat saliva was less than 4% of the total or plasma exchangeable testosterone in the rat. This marked discrepancy between the concentration of plasma and cellular exchangeable hormone suggested that there was rapid metabolism of androgen by salivary gland in vivo. This was confirmed by chromatographic separation of [3H] testosterone and labeled metabolites in homogenates of salivary gland. By 60 sec after injection, approximately 30% of the radioactivity in the salivary gland was in the form of androgen metabolites, which primarily comigrated with an androstenedione standard. The data indicate that albumin-bound testosterone, albumin-bound estradiol, and sex hormone-binding globulin-bound estradiol are all exchangeable in salivary gland capillaries. The low concentration of cellular exchangeable testosterone in salivary gland appears to be due to rapid tissue metabolism of this hormone. Thus, changes in androgen metabolism may alter salivary gland hormone concentrations independent of any change in the concentration of biologically active hormone in plasma.     

Ontogeny of mesenchymal androgen receptors in the embryonic mouse mammary gland

We demonstrate the presence of a high affinity androgen-binding site in the embryonic mouse mammary gland, and describe its appearance and development from the initial formation of the gland bud (day 12) through the androgen-responsive stage (day 14) until term (day 19). Binding assays were done on entire organ rudiments exposed to the ligand [( 3H] testosterone, [3H]5 alpha-dihydrotestosterone, or [3H]methyltrienolone) in vitro, and receptor levels are expressed per gland. In competition experiments, the binding site shows a ligand specificity of a typical rodent androgen receptor. Scatchard analysis yielded a figure of 90-100 million binding sites for one 14-day gland, or approximately 30,000 per target cell. The apparent dissociation constant Kd for ligand binding by the intact tissue was between 0.55 and 0.75 X 10(-9) M. The first binding sites become detectable at the time of mammary bud formation (day 12); their number increases about 20-fold towards the responsive stage (day 14), and they persist at high levels at least until birth. The loss of androgen responsiveness on day 15 is neither accompanied by a loss of receptors nor by a change of their binding affinity.     

Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone

Testosterone and dihydrotestosterone are believed to exert their androgenic effects by interacting with a single intracellular receptor protein in androgen target tissues. During fetal life, however, testosterone mediates the virilization of the Wolffian ducts into the epididymis, vas deferens, and seminal vesicles, whereas the urogenital sinus and external genitalia require the in situ conversion of testosterone to dihydrotestosterone to undergo male development. The reason why the signal provided by testosterone needs to be amplified in some androgen target tissues but not in others remains an enigma. To provide insight into the different actions of these androgens we studied their interaction with the human androgen receptor in fibroblasts cultured from the genital skin of a patient with 5 alpha-reductase deficiency. Dihydrotestosterone was formed in negligible amounts in these cells, and in some experiments the residual 5 alpha-reductase activity was further blocked with the 5 alpha-reductase inhibitor finasteride. Saturation analysis in fibroblast monolayers disclosed similar amounts of binding with testosterone and dihydrotestosterone, and the affinity of binding of dihydrotestosterone was, on the average, about 2-fold greater than that of testosterone. [3H]Testosterone also exhibited a 5-fold faster dissociation rate from the receptor than [3H]dihydrotestosterone. In thermolability experiments the [3H]testosterone-receptor complex displayed marked instability at 42 C with 2 nM [3H] testosterone, whereas with 20 nM [3H]testosterone, receptor stability was similar to that seen with [3H]dihydrotestosterone. In up-regulation experiments, 2 nM [3H]testosterone produced a 34% increase in specific androgen receptor binding after 24 h, whereas 20 nM [3H]testosterone produced an average increase of 64%. Our results suggest that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone resides in its weaker interaction with the androgen receptor, most clearly demonstrable as an increase in the dissociation rate of testosterone from the receptor. When present in relatively high concentrations, however, testosterone overcomes this defect by mass action.(ABSTRACT TRUNCATED AT 400 WORDS)     

De novo induction of a gene product during heterologous epithelial-mesenchymal interactions in vitro

Mesenchymal specification of epithelial cytodifferentiation and morphogenesis has been considered to be a general feature of various epithelial-mesenchymal interacting systems (e.g., salivary gland, mammary gland, feather, hair, and tooth morphogenesis). In contrast, we have demonstrated that a mesenchyme can be induced by a heterologous epithelium to synthesize in quantity a specific gene product(s) unorthodox to the organ from which the mesenchyme was taken. Stage 22-23 avian limb bud epithelium induced 17-day embryonic mouse tooth mesenchyme to differentiate into cartilage. Peptide analysis (cyanogen bromide cleavage after purification of extracted collagen chains) demonstrated that heterologous tissue recombinations produced type II collagen [alpha(II)](3) (i.e., cartilage-type) in addition to type I collagen [alpha(I)](2)alpha(2). Intact or reconstituted mouse molar tooth organs synthesized type I collagen and type I trimer [alpha(I)](3) collagen. Immunohistochemical criteria using anti-type II collagen antibodies identified type II collagen in cartilage-like matrix within the mesenchymal component of heterologous tissue recombinants. Cartilage has never been described during in vivo or in vitro tooth tissue differentiation or associated with the pathology of dental papilla mesenchyme. These results support the hypothesis that epithelial-mesenchymal interactions during embryonic development can selectively induce de novo synthesis of unique gene products.     

Effect of human recombinant mullerian inhibiting substance on isolated epithelial and mesenchymal cells during mullerian duct regression in the rat.

The effect of human recombinant Mullerian Inhibiting Substance (MIS) on the regression of the Mullerian duct (MD) of female rat fetuses was examined in vitro to determine whether MIS acts on MD epithelium and/or mesenchyme at the critical periods of sexual differentiation. Urogenital ridges (URs) of female rat fetuses at 14.5- to 18.5-days of gestation (plug day = 0) were cultured for 3 days with or without recombinant human MIS in CMRL 1066 medium with 10% female fetal calf serum. In URs from 14.5- and 15.5-day-old fetuses, the cranial portion of the MD regressed almost completely during the 3-day culture period in the presence of MIS, whereas the caudal half to third of the MD remained intact but tapered to a fine point cranially. MDs survived in URs from 16.5-day-old fetuses cultured in the presence of MIS except that the cranial portion of the MDs was deformed. MIS did not elicit regression of MDs in URs obtained from 17.5- and 18.5-day-old fetuses, but instead caused the MD epithelium to form bulges projecting into the mesenchyme. MD epithelium at 15.5-days of gestation was separated from the surrounding UR mesenchyme, and both components (MD epithelium and mesenchyme) were cultured separately for 3 days in the presence or absence of MIS. Both epithelial and mesenchymal cells survived in the presence or absence of MIS. MD epithelium formed typical epithelial colonies, whereas UR mesenchyme spread as fibroblastic cells. Analysis of labeling index after incorporation of [3H] thymidine demonstrated that MD epithelial DNA synthesis was not influenced by MIS. In contrast, mesenchymal labeling index was reduced significantly by MIS. This effect of MIS on UR mesenchyme in conjunction with earlier histological observations of mesenchymal condensation during MD regression and an absence of direct effects of MIS on the epithelium suggests that MIS elicits its effect on the MD epithelium via the surrounding mesenchyme.     

Absence of a nuclear androgen receptor in isolated germinal cells of rat testis

Androgen receptors are known to be present in the seminiferous tubules of rat testis and in the present study it has been attempted to compare the binding of [³H] testosterone to androgen receptors in male germinal cells and Sertoli cells. Cell preparations enriched in germinal cells and Sertoli cells were isolated from testicular tissue of 30–35-day-old rats. The cell preparations were either obtained from intact rats and labelled in vitro with [³H] testosterone or were obtained from the testes of hypophysectomized rats which were labelled in vivo with [³H]testosterone prior to the isolation of the cells. The nuclear fractions of the labelled cell preparations were extracted with 0.4 M KCl and the extracts were fractionated by sucrose density gradient centrifugation to estimate specific binding of radioactive steroid.Specific binding of radioactive steroid to nuclear androgen receptors was observed in Sertoli cell preparations but not in preparations of germinal cells (spermatocytes and round spermatids).     

Estrogen responsiveness of normal mouse mammary cells in primary cell culture: association of mammary fibroblasts with estrogenic regulation of progesterone receptors

Estrogens enhance proliferation of normal mouse mammary cells in vivo. However, when cultured alone, normal mouse mammary epithelial cells fail to exhibit a proliferative response to estrogen in vitro; the basis for this lack of in vitro responsiveness to estrogen is not known. The purpose of the present study is to determine if cultured normal mouse mammary cells possess estrogen receptors (ER) and/or progesterone receptors (PgR) and if the ER mechanism is functional, as measured by the ability of estrogens to regulate PgR. Recent findings that mammary fibroblasts can influence the behavior of mammary epithelial cells in vitro led us to investigate their effect on epithelial cell responsiveness to estrogen. In these studies, collagenase-dissociated mammary glands of midpregnant BALB/c mice were the source of mixed cultures (containing both epithelial cells and fibroblasts) and epithelial or fibroblast cultures. The purity of epithelial or fibroblast cultures was quantified immunocytochemically using antivimentin antibody as a fibroblast marker. Steroid hormone binding was quantified in intact cultured cells using [3H]R5020 and 17 beta-[3H]estradiol as the ligands. Specific high affinity binding sites for estrogen (Kd = 3.1 +/- 0.8 X 10(-10] and progestins (Kd = 3.3 +/- 1.2 X 10(-9) M) were detected in mixed cultures. To assess the possible role of mammary fibroblasts, we investigated cultures containing only fibroblasts which were derived by differential centrifugation. When 17 beta-estradiol was added to the culture medium, a significant (P less than 0.01) increase in PgR concentration was observed in mixed cultures. While mixed cultures maintain responsiveness to estrogen in vitro, as measured herein, the epithelial cultures, derived by differential centrifugation and Percoll gradient sedimentation, did not. However, estrogenic regulation of PgR appears to be specific to epithelial cells in mixed cultures, since fibroblast cultures neither contained PgR nor displayed estrogen-inducible PgR. The lack of responsiveness of epithelial cultures is not due to a loss or decrease in the ER concentration. Thus, the presence of mammary fibroblasts appears to be associated with epithelial cell responsiveness to estrogen in vitro.     

Analysis of prostatic bud induction by brief androgen treatment in the fetal rat urogenital sinus

The androgen dependency of prostatic bud formation in fetal rat urogenital sinuses was studied using brief treatments with androgen, and the incorporation of androgens by the sinus mesenchyme was followed by steroid autoradiography. Urogenital sinuses from 16.5-day fetuses of both sexes were grown in organ culture and treated with androgens for periods ranging from 4 to 72 h and then transferred to control medium. A minimum treatment of 24 h was required to induce prostatic buds in male sinuses and of 36 h in all female sinuses. This difference in response disappeared after more prolonged treatment. In both sexes the number of prostatic buds increased with the time of exposure to androgens. Prostatic bud formation continued for 24-36 h after transfer to control medium. Steroid autoradiographic analysis showed that the labelled androgen was concentrated in the mesenchymal nuclei. The rate of incorporation rose steeply during the first 12 h and then more slowly. After transfer to control medium the amount of labelled androgen decreased rapidly to half within 12 h and then decreased more slowly. In the competition experiments a 200-fold excess of unlabelled testosterone or dihydrotestosterone in the labelling medium greatly reduced the nuclear labelling with [3H]testosterone.     

Role of androgens in proliferation and differentiation of mouse mammary epithelial cell line HC11

Androgens have been found in mammary epithelium and in milk throughout the cycle of the mammary gland in vivo. The aim of this study was to investigate the possible role of these substances in mammary epithelial growth and differentiation in the mouse HC11 cell line. Cells were stimulated with testosterone, dihydrotestosterone, androstenedione and 5alpha-androstane-3alpha,17beta-diol at concentrations ranging between 0.3 nM and 30 nM. Cyproterone acetate or flutamide, androgen receptor antagonists, (3 microM) were used to block specific androgen effects. Proliferative effects were measured by an MTT (tetrazolium blue) conversion test and [(3)H]thymidine uptake. HC11 cells were transfected with pbetacCAT, a chimeric rat beta-casein gene promoter-chloramphenicol acetyl transferase (CAT) gene construct and CAT ELISA was used to determine gene expression. RT-PCR was performed to detect androgen receptor expression. After 24, 48 and 72 h androgens significantly (P<0.05) increased proliferation. Androgen antagonists significantly (P<0.05) reduced the proliferative effects. Furthermore androgens potentiated the lactogenic effect of prolactin, insulin and dexamethasone (P<0.05). Finally, the androgen receptor gene was expressed in both proliferating and differentiated HC11 cells. These observations lead us to hypothesize an activity of this class of steroids in mammary physiology. In particular, androgens stimulate cell proliferation and beta-casein gene expression; this influence appears to be mediated by androgen receptors.     

Metabolism of testosterone by the epithelium and mesenchyme of the rat urogenital sinus

Testosterone metabolism was measured in separated epithelium and mesenchyme from the urogenital sinuses of 17- and 19-day-old male and female rat embryos and compared with testosterone metabolism in the intact sinus. Both the epithelium and the mesenchyme converted testosterone to 5 alpha-dihydrotestosterone. The epithelium produced much more androstanedione and androsterone but less 3 alpha, 17 beta-androstanediol than did the mesenchyme. The whole sinus synthesized all four metabolites, but in different proportions, producing relatively more androsterone than either of its two component tissues. These data suggest that androsterone is formed by the joint action of epithelium and mesenchyme. Metabolism of testosterone did not differ with sex or foetal age in either of the separated tissues or in the intact sinus, implying that the failure of urogenital mesenchyme from 19-day-old female foetuses to induce prostatic morphogenesis is not due to the loss of 5 alpha-reductase. It is suggested that this lack of inductive capacity may be attributable to a decline in androgen levels with age in female mesenchyme.     

Microautoradiographic studies on distribution of 5 alpha-dihydrotestosterone, cyproterone acetate and oestradiol-17 beta in human prostatic hyperplasia tissue transplanted to juvenile rats

While maintaining the actual conditions prevailing in benign prostatic hyperplasia (BPH) in man, transplantation of BPH tissue to new born rats proved a suitable model for examining the distribution of sexual hormones in different tissue compartments of BPH. In combination with the microautoradiographic method, it was possible to demonstrate the residence of the radio-active androgen 5 alpha-dihydrotestosterone (5 alpha-DHT), the antiandrogen cyproterone acetate (CA) and the oestrogen oestradiol-17 beta (E2) in the epithelium and/or stroma of human BPH tissue. Quantitative evaluation in the form of a point per area count on photographic pictures yielded a silver grain distribution ratio in epithelium and stroma of 1.3: 1 and 1.5: 1 for epithelium to stroma after administration of [3H] 5 alpha-DHT and [3H]CA administration respectively and 0.5: 1 after [3H]E2. The high tracer recovery rate throughout the stroma following E2 administration supports the current view that the stromal proliferation is attributable mainly to oestrogen influences. The relatively high silver particle proportion throughout the stroma following 5 alpha-DHT administration corroborates recent findings which suggest that the exclusive androgen dependency of the glandular epithelium can only be considered in conjunction with an active metabolization of androgens in the stroma. The correspondence in the distribution of the radioactive tracer after [3H] 5 alpha-DHT and [3H]CA administration both in the epithelium and stroma suggest that an antagonism may also exist in the stroma.     

Follicle-stimulating hormone regulates androgen receptor mRNA in Sertoli cells

Follicle-stimulating hormone (FSH) and testosterone stimulate the production of a variety of proteins by immature Sertoli cells. A highly purified Sertoli cell preparation was incubated for 3 days with FSH and testosterone. Both androgen receptor protein and mRNA concentrations were markedly increased by FSH. Testosterone also increased the androgen receptor protein concentration, but did not increase the expression of the androgen receptor mRNA. It is concluded that FSH plays a role in the responsiveness of Sertoli cells to testosterone.     

Estrogen stimulation of deoxyribonucleic acid synthesis in uterine epithelial cells which lack estrogen receptors

Autoradiographic studies from this laboratory have previously indicated that the uterine epithelium of the neonatal mouse is devoid of estrogen receptors. The neonatal murine uterus is composed of an undifferentiated mesenchyme and a simple columnar epithelium lining the lumen. In the present study, a method for measuring whole cell uptake of 16 alpha-[125I]iodoestradiol ([125I]iodo-E2) was developed and applied to enzymatically separated, isolated epithelial and mesenchymal cells of neonatal (4-5 days postnatal) uteri. Epithelial cells and fibromuscular cells (stromal and myometrial cells) from uteri of 21-day-old animals were used to validate the assay method. A component of the uptake of [125I]iodo-E2 by cells from 21-day-old uteri was shown to be specific, saturable, and of high affinity. Kd values for specific uptake by uterine mesenchymal and epithelial cells were 1.2-1.3 nM. Maximal specific uptake was 9.3 and 4.2 fmol/micrograms DNA for epithelial and mesenchymal cells, respectively. The uterine epithelial cells of 4- and 5-day-old mice showed no measurable specific uptake of [125I]iodo-E2, while the mesenchymal cells from these animals had a maximal specific uptake of 7.9 fmol/micrograms DNA, with a Kd of 1.3 nM. DNA synthesis increased within the uterine epithelium of neonatal mice after estrogen treatment. The thymidine labeling index was doubled 10 h after a single dose of diethylstilbestrol (DES) and returned to pretreatment values by 18 h. The epithelial mitotic index was also 2-fold higher than control values 16-18 h after DES treatment. The increase in the thymidine labeling index was specific to estrogen treatment. DES did not induce the production of estrogen receptors in neonatal uterine epithelium. Epithelial cells of 5-day-old mice that were treated with DES showed no specific [125I]iodo-E2 uptake, while whole cell uptake by mesenchymal cells from these animals exhibited a specific, high affinity component, with a maximal binding of 8.4 fmol/micrograms DNA. Autoradiographic analysis of [3H]estradiol uptake by uterine tissues from 5-day-old mice 12 h after DES treatment did not show nuclear concentration of the steroid in the epithelial cells. These results indicate that the uterine epithelium of the neonatal mouse is indeed devoid of estrogen receptors, and yet the rate of DNA synthesis within this tissue is responsive to estrogen stimulation. The epithelial cells remain devoid of estrogen receptors after DES stimulation, indicating that intraepithelial estrogen receptors are not required for induction of DNA synthesis in these cells in situ. Possible mechanisms by which this phenomenon may occur are discussed.     

Cyproterone acetate prevents translocation of the androgen receptor in the rat prostate

The translocation of the androgen receptor in prostatic tissue has been studied under the influence of different ligands (testosterone, methyltrienolone and cyproterone acetate) in vivo and in vitro. Nuclear and cytoplasmic androgen receptors were estimated using an exchange assay with [3H]methyltrienolone ( [3H]R1881) 1 h and 16 h after injection in castrated rats of either 100 micrograms testosterone (T), 10 mg cyproterone acetate (CA) or the combination of T and CA. Within 1 h after T administration, nuclear receptor levels increased with a concomitant depletion of cytosol receptors. In the CA-treated rats nuclear receptor levels were not different from those of control castrated animals and there was no depletion of cytosol receptors. The combined treatment of T and CA resulted in a partial depletion of cytosol receptors and a simultaneous increase of nuclear receptors. The absence of an increase in nuclear androgen receptors in CA-treated animals cannot be explained by a delay in translocation, because even 16 h after CA injection, only a very small number of nuclear receptors were detectable. Incubation of minced prostatic tissue with [3H]CA or [3H]R 1881 resulted in receptor translocation only in the R1881 incubations and confirmed the in vivo results. Competition studies with different steroids and cytosol receptor (non-activated, 8S form in low salt gradient) or nuclear receptor (activated 3.6S form in high salt gradient) of prostatic tissue show that CA can compete with R1881 for specific androgen-binding sites with a similar relative binding affinity for both receptor preparations. The present results provide evidence that CA prevents translocation of the androgen receptor to the nucleus, although CA can be bound with similar affinities to the nuclear receptor and the cytoplasmic receptor. We propose that the anti-androgenic action of CA involves an inhibition of receptor translocation.     

Effect of LH injections on testicular steroidogenesis, cholesterol side-chain cleavage P450 mRNA content and Leydig cell morphology in hypogonadal mice

Hypogonadal (hpg) mice have a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH) and the gonads consequently lack exposure to gonadotrophins during development. We injected male hpg mice with LH for 10 days to investigate whether LH alone can stimulate normal steroidogenesis in these animals. Control animals had an inactive interstitium and very few germ cells. Testicular content of androgens was undetectable by radioimmunoassay in control animals unless a single injection of LH was given 1 h before death, when androgens were just detectable. Control testes incubated in vitro with [3H]pregnenolone demonstrated that without gonadotrophin stimulation pregnenolone was metabolized only to progesterone in significant amounts. Assay for cholesterol side-chain cleavage cytochrome P450 (P450scc) mRNA showed basal expression in saline-treated hpg mouse testis. LH treatment induced hypertrophy and hyperplasia of Leydig cells and division of germ cells. Testicular androgen content increased significantly, with testosterone and androstenedione as the major androgens. LH-treated testes incubated with [3H]pregnenolone in vitro had a greater synthetic capacity for testosterone, suggesting an increase in 17 alpha-hydroxylase/C17-20-lyase activity. Basal and human chorionic gonadotrophin-stimulated androgen production in vitro increased markedly following LH treatment to levels previously described in the normal adult animal. LH treatment caused a rapid and transient increase in the hybridization of P450scc mRNA which was sevenfold greater than that of saline-treated controls when the animals were killed 1 h after the last injection but fell to control levels within 24 h of cessation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)