Autoradiographische Untersuchungen zur Mitoseaktivität im Hypophysenvorderlappen männlicher Ratten nach Behandlung mit Antiandrogenen und nach Kastration

Zusammenfassung Nach Applikation von ³H-Thymidin wird die Mitoserate im Hypophysenvorderlappen von männlichen Ratten nach 10-bzw. 14tägiger Behandlung mit Antiandrogenen (Cyproteron bzw. Cyproteronacetat) und 10 bzw. 14 Tage nach Kastration autoradiographisch untersucht. Kastrierte oder Cyproteron-behandelte Tiere weisen gegenüber Normaltieren eine 3–4fach erhöhte Zahl markierter HVL-Zellen auf. Nach Behandlung mit Cyproteronacetat ist dagegen die Mitoserate vermindert. — Die Ergebnisse ergänzen die Vorstellung vom Wirkungsmechanismus der genannten Antiandrogene, wonach Cyproteron nur antiandrogen wirkt und damit die FSH-Abgabe im HVL erhöht, wogegen Cyproteronacetat zusätzlich antigonadotrope Wirkung besitzt, die die FSH-Abgabe drosselt.

---

Radioautographic investigations on the rate of mitosis in the anterior pituitary in male rats after treatment with antiandrogenic substances and castration

Summary After application of ³H-thymidine the rate of mitosis in the anterior pituitary of male rats is investigated by means of radioautography after treatment with antiandrogenic substances (cyproterone and cyproterone acetate) for 10 and 14 days respectively and 10 and 14 days after castration. Castrated or cyproterone-treated rats show a 3 to 4 fold increase of the number of labeled anterior pituitary cells as compared to untreated animals. In the anterior pituitary of cyproterone acetate-treated rats the rate of mitosis decreases. — The present results are in accordance with the view, that cyproterone acts only antiandrogenic and stimulates the FSH-release in the anterior pituitary, whereas cyproterone acetate has also an antigonadotrophic effect, which slows down the FSH-release.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Evidence that an extrahypothalamic pituitary corticotropin-releasing hormone (CRH)/adrenocorticotropin (ACTH) system controls adrenal growth and secretion in rats

Within two weeks, hypophysectomy induced in rats a striking decrease in the level of circulating ACTH (the concentration of which was at the limit of sensitivity of our assay system), coupled with a net reduction in the plasma corticosterone concentration and an evident adrenal atrophy. Zona fasciculata, the main producer of glucocorticoids, was decreased in volume, due to a lowering in both the number and average volume of its parenchymal cells. Subcutaneous ACTH infusion (0.1 pmol·min^-1), administered during the last week following hypophysectomy, restored the normal blood level of ACTH and completely reversed all effects of hypophysectomy on the adrenals. Subcutaneous infusion for one week with -helical-CRH or corticotropin-inhibiting peptide (1 nmol·min^-1), which are competitive inhibitors of CRH and ACTH, evoked a further significant lowering of plasma corticosterone concentration and markedly enhanced adrenal atrophy in hypophysectomized rats. These findings strongly suggest that an extrahypothalamic pituitary CRH/ACTH system may be involved in the maintenance of the growth and steroidogenic secretory activity of the rat adrenal cortex.     

Effect of cyproterone acetate on the reproductive system of the female rat. A histological review.

Effects of cyproterone acetate, a synthetic steroidal compound, on the reproductive organs of female rats have been investigated. This agent caused reduction of ovarian weights indicative of suppression of pituitary gonadotrophins. Oestrogenic nature of cyproterone acetate was investigated in intact and ovariectomized rats taking uterine weight and vaginal keratinization as an index of oestrogenicity. Cyproterone acetate in ovariectomized animals induced vaginal keratinization and increased the uterine weights. These effects were parallel to the effect of oestradiol dipropionate in ovariectomized animals, thus indicating oestrogenic activity of cyproterone acetate. We may conclude that the above compound caused antifertility effects due to its oestrogenic nature at the dose level of 2 mg/alternate day in rats when the compound was administered subcutaneously.     

Nuclear uptake and retention of androgen by the pituitary gland of the hamster and the rat

Summary Castrated adult male hamsters and castrated adult female rats were injected with either 0.2 g (hamsters) or 0.5 g (rats) of ³H-dihydrotestosterone (107 Ci/mmole)/100 grams body weight and killed 11/2 h later. The pituitary glands were removed and processed for both autoradiography and immunocytochemistry (hamster) or only autoradiography (rats). Localization of the androgen was found in 10–15% of the cells of the pars distalis in both species. Only cells that stained for luteinizing hormone (LH) in the hamster's pars distalis concentrated the androgen. Also cells in both the pars intermedia and pars nervosa (1–5%) concentrated the androgen in both species. Although the number of cells that concentrated the androgen in the pars intermedia and pars nervosa was small, this finding may be related to recent physiologic data that suggest that the gonadal steroids may play a role in regulating water retention and natriuresis.This study was supported by USPHS Research Career Development Award KO4NS0000164 (P.J. Sheridan) and USPHS Grants No. 1 RO1 NS12933, P30 HD10202 and HD 10914     

Immunohistochemical identification of cells in the pars distalis of the pituitary of the lizard anolis carolinensis

Summary Immunocharacteristics of the pars distalis cells of the pituitary of the male lizard A. carolinensis are determined by employing the immunoperoxidase technique with antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, 5 different cell types with characteristic anatomical distribution are recognized. ACTH cells are found in the rostral half of the pars distalis, and PRL cells in the rostral two thirds of the pars distalis. GH and TSH cells are located in the caudal half of the pars distalis. GTH cells are distributed throughout the gland. When consecutive sections are stained with antiserum to ovine FSH or its -subunit and to ovine LH, the same cells show immunoreactivity to all the three antisera. None of the GTH cells show positive immunoreactivity to ovine -LH antiserum. The results suggest the existence of one gonadotropic cell type in the pituitary of this lizard.Supported by U.S. Council for International Exchange of Scholars (to D.R.N.) and PHS Grant NS09914     

Effect of corticosterone on noradrenergic nuclei in the pons-medulla and [3H]NA release from terminals in hippocampal slices

The aim of the present study was to investigate possible membrane and genomic effects of corticosterone on the noradrenergic system of the rat brain. Corticosterone effects were studied in vivo by treating rats s.c. with 10 mg/kg corticosterone for 7 or 14 days. In the first two experiments corticosterone significantly decreased th noradrenaline (NA) and dopamine (DA) levels in the pons-medulla, an area which contains the A1-A7 noradrenergic cell groups, while the NA and DA levels in the dorsal hippocampus remained unchanged. In a third experiment where the locus coeruleus (LC) and the A1 and A2 nuclei (A1,A2) were analysed separately, NA levels were unchanged but total MHPG levels and the total MHPG/NA ratio were decreased in the A1,A2 area. Chronic corticosterone treatment (14 days) did not alter the ₂-adrenoceptor-mediated modulation of [³H]NA release from dorsal hippocampal slices. Neither the spontaneous outflow nor the electrically stimulated release of [³H]NA from dorsal hippocampal slices of untreated rats was affected by exposure of the slices to corticosterone (10^–7 M–10^–4 M) in the superfusion buffer. Thus, chronic corticosterone treatment of rats altered the noradrenergic system of the pons-medulla, but did not change the ₂-adrenoceptor-mediated modulation of NA release in the dorsal hippocampus, a major terminal area of the LC neurons. Corticosterone also did not appear to have a direct membrane effect on the NA terminals in the dorsal hippocampus of the rat.     

Histochemical method for the demonstration of myosin adenosine triphosphatase in muscle tissues

Zusammenfassung In der vorliegenden Arbeit wird die Wirkung der Antiandrogene Cyproteron und Cyproteronacetat auf einige Enzyme in den Leydigzellkomplexen (LZK) des Hodens infantiler und ausgewachsener Ratten untersucht. Neben einer Vergrößerung der LZK — Ausdruck einer vermehrten Gonadotropin-Ausschüttung als Folge eines Kastrations-effektes — ergab sich eine Aktivitätssteigerung der Enzyme des Steroidstoffwechsels (-Hydroxybuttersäuredehydrogenase, Steroid-3-ol-Dehydrogenase), des Energiestoffwech-sels (Laktatdehydrogenase, Succinodehydrogenase) und der Atmungskette (NADH-Cyto-chrom-c-Reduktase). Die Glukose-6-Phosphatdehydrogenase wies dagegen keine Aktivitätsveränderung auf. Unbeeinflußt blieben auch die unspezifischen Esterasen und die sauren Phosphatasen. Der erwartete Unterschied zwischen Cyproteron und Cyproteronacetat blieb aus. Die Gründe hierfür werden diskutiert.

---

The effect of the antiandrogens cyproterone and its acetate on the interstitial cells of juvenile and adult rat testes was studied histochemically

Summary Under both steroids, the interstitial cell complex enlarged which is considered evidence of increased gonadotropin secretion mediated by an antiandrogen blockade of hypothalamic androgen receptors. There was also an increase in the activity of enzymes involved in steroid metabolism (-hydroxy-butyrate and 3-hydroxy-steroid-dehydrogenase) and energy metabolism (lactic and succinic dehydrogenases) as well as an increase in NADH-cytochrome-c-reductase activity. On the other hand, no changes in the activity of glucose-6-phosphate dehydrogenase, non-specific esterases and acid phosphatases were found. Unexpectedly, there was no difference between the effects of cyproterone and cyproterone acetate. The reasons for this are discussed.

     

Immunocytochemical localization of ACTH-like immunoreactivity in rat adrenal glomerulosa and fasciculata cells

Summary The role of ACTH in the synthesis of the adrenocortical hormones has been largely described. In order to investigate the localization of this peptide at the subcellular level of the adrenal glomerulosa and fasciculata cells, an immunocytological method was used. Rat adrenals were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-(1–24) ACTH or anti (17–39) ACTH sera. The antigen-antibody reaction was detected by PAP complexes (revealed by 4-chloro-1-naphthol) or with protein A-colloidal gold or IgG-colloidal gold.The results obtained were the same whatever the antisera of the technique employed. All the cells of the adrenal zona glomerulosa and zona fasciculata were labelled. ACTH-like immunoreactivity in zona glomerulosa and zona fasciculata cells was observed at the plasma membrane level, in cytoplasmic matrix, mitochondria and nucleus (in the euchromatin close to the heterochromatin aggregations and, occasionally, associated with the nucleolus). No immunoreactivity was observed when non-immune serum or anti-ACTH serum preincubated with ACTH were used, nor there was any modification of the immunocytochemical reaction when anti-ACTH serum incubated with heterologous antigens was employed. These data, demonstrate the presence of endogenous ACTH in both adrenal glomerulosa and fasciculata cells, and suggest that the peptide is internalized after binding to the plasma membrane.T. Garcia-Caballero has a Postdoctoral Fellowship from Xunta de Galicia.     

Localization of corticotropin- and endorphin-related peptides in the intermediate lobe of the rat pituitary

Summary The question is examined whether -melanocyte stimulating hormone (-MSH), adrenocorticotropic hormone (ACTH), met-enkephalin and -endorphin are detectable by enzyme immunocytochemistry in the cells of the intermediate lobe (PI) of the rat pituitary. By applying antibodies against MSH, ACTH and -endorphin on light microscopic sections, intense immunostaining was found in all PI-cells. At the ultrastructural level, after treatment of consecutive serial sections with these three antibodies the immunoreactivity was localized in the same secretory granules. No specific metenkephalin immunoreactivity could be detected in the cells of the intermediate lobe.Supported by Deutsche Forschungsgemeinschaft SFB 87/B2     

FMRF -amide-like immunoreactivity in brain and pituitary of the hagfish Eptatretus burgeri (Cyclostomata)

Summary Paraffin sections of brain and pituitary of the hagfish Eptatretus burgeri were immunostained with an antiserum to FMRF-amide. Immunoreactivity was visible in a large number of neurons in the posterior part of the ventromedial hypothalamus and in long neuronal processes extending cranially from the hypothalamus to the olfactory system and caudally to the medulla oblongata. FMRF-amide-like immunoreactivity was also found in cells of the adenohypophysis. These observations suggest that the hagfish possesses a brain FMRF-amide-like transmitter system and pituitary cells containing FMRF-amide-like material.Antisera to ACTH, -MSH and pancreatic polypeptide gave no immunoreaction in hagfish brain or pituitary. D aspartic acid - F phenylalanine - L leucine - M methionine - R arginine; - W tryptophan - Y tyrosine     

DNA fragmentation, DNA repair and apoptosis induced in primary rat hepatocytes by dienogest, dydrogesterone and 1,4,6-androstatriene-17beta-ol-3-one acetate

Four steroids that share the 17-hydroxy-3-oxopregna-4,6-diene structure - cyproterone acetate, chlormadinone acetate, megestrol acetate, and potassium canrenoate - have been shown previously to behave with different potency as liver-specific genotoxic agents, the response being markedly higher in female than in male rats, but similar in humans of both genders. In this study, performed to better define the relationship between chemical structure and genotoxicity, dydrogesterone (DGT) with double bonds C4=C5 and C6=C7, dienogest (DNG) with double bonds C4=C5 and C9=C10, and 1,4,6-androstatriene-17beta-ol-3-one acetate (ADT) with double bonds C1=C2, C4=C5 and C6=C7, were compared with cyproterone acetate (CPA) for their ability to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes from three rats of each sex. At subtoxic concentrations, ranging from 10 to 90 microM, all four steroids consistently induced a dose-dependent increase of DNA fragmentation, which in all cases was higher in females than in males; their DNA damaging potency decreased in the order CPA > DNG > ADT > DGT. Under the same experimental conditions, the responses provided by the DNA repair-synthesis assay were positive or inconclusive in hepatocytes from female rats and consistently negative in hepatocytes from male rats. In the induction of apoptotic cells, examined in primary hepatocytes from female rats, CPA was more active than ADT and DGT, and DNG was inactive. Considered as a whole these findings suggest that a liver-specific genotoxic effect more marked in female than in male rats might be a common property of steroids with two or three double bonds.     

Comparison of the biopotency of corticosterone and dexamethasone acetate in glucocorticoid receptor down regulation in rat liver

The effects of long treatment with dexamethasone 21-acetate and corticosterone on the glucocorticoid receptor in rat liver cytosol were compared. Dexamethasone acetate (5 micrograms/ml or 10 micrograms/ml water) or corticosterone (100 micrograms/ml water) was given to adrenalectomized animals as drinking solution for 6 days, and glucocorticoid receptor concentration was determined at 0, 12, 24, 48 and 72 h after steroid withdrawal. Dexamethasone acetate caused a dose dependent depletion of cytosol receptor. There was no measurable binding at time 0; the values of Bmax for the glucocorticoid receptor with decreased at 12, 24 and 48 h after the steroid withdrawal. Increased dissociation constant (Kd) were calculated for 12 and 24 h samples. The effect of corticosterone on receptor depletion was less pronounced. Bmax for the receptor was decreased at 0, 12, 24 h after steroid withdrawal with no change in Kd. The extent of steroids-induced receptor depletion showed good correlation with the induction of tyrosine aminotransferase (TAT), however, maximum TAT activity measured immediately after withdrawal of dexamethasone acetate was lower than that found after a single injection of dexamethasone acetate. We conclude that both steroids cause down regulation of the glucocorticoid receptor in rat liver cytosol, with both the extent and the duration of depletion being dependent on the biopotency of the glucocorticoid.     

Sex difference in triglyceride/fatty acid substrate cycling of rat adipose tissue: indirect regulation by androgens.

Male Sprague-Dawley rats displayed significantly higher rates of triglyceride/fatty acid (TG/FFA) substrate cycling in subcutaneous, perigenital, and mesenteric white adipose tissue, compared to females. To investigate possible regulation via androgens and estrogens, male rats were treated with the androgen antagonist, cyproterone acetate (10 mg daily in subcutaneous injections), or estradiol polyphosphate (0.3 mg intramuscularly, given as a single dose). Estradiol treatment did not affect TG/FFA cycling. Treatment with cyproterone acetate significantly decreased TG/FFA cycling in perigenital (epididymal) tissue. This effect could however largely be ascribed to concomitant inhibition of food intake by cyproterone acetate. The effects of cyproterone acetate on the two axes of TG/FFA cycling (lipolysis and re-esterification) were further studied in vitro. Norepinephrine-stimulated glycerol release from perigenital adipocytes was inhibited, whereas activities of esterification enzymes (GPAT and PPH) was essentially unaffected. We conclude that androgens seem to affect TG/FFA cycling indirectly via the lipolytic axis.     

Cellular and subcellular 3H-estradiol localization in the pituitary by autoradiography

Summary The cellular and subcellular localization of 6.7-³H-estradiol-17 in the pituitaries of five immature and two mature castrated female rats was studied by autoradiography. The autoradiographic technique used minimizes or eliminates translocation. It is based on low temperature tissue preparation of freeze-dried sections which are dry-mounted on dried photographic emulsion, excluding known sources of translocation artifacts such as liquid fixatives, dehydrating and clearing fluids, embedding media and thawing. The animals were given from 0.093 to 0.63 g of ³H-estradiol and were sacrificed at 15 min, 1, 2, or 6 hours after the injection. A large portion of the anterior pituitary cells was found to be labeled; the extent of this labeling varied with dose, time of sacrifice after the injection, and photographic exposure time, but apparently not with the endocrine status of the animal. The portions labeled were 76 and 86 per cent for the immature and mature rats respectively, exceeding single tinctorial light-microscopic groups. Gomori trichrome chromophiles and chromophobes, cells with intense and weak pyronin basophilia, as well as morphologically defined castration cells, were all partially labeled and unlabeled. Acidophiles appeared to be labeled in a somewhat higher proportion. Cells of the intermediate and posterior lobe were generally unlabeled except for occasionally interspersed cell groups or single cells, especially at the border between intermediate and posterior lobe, probably identical with basophilic invaginations in man and other mammals. The subcellular concentration of radioactivity was always nuclear. The findings are interpreted as suggesting a) feedback control on the pituitary level, in addition to the diencephalic level, b) pluripotentiality of anterior pituitary cells, and c) possible positive feedback mechanism of estradiol with secondary negative effect. Dry-mount autoradiography with labeled hormones, as applied in this study, provides a new methodological approach to the elucidation of pituitary physiology and pharmacology.Supported by USPHS Grants 1-ROl-AM-12, 649-01, GRS Grant FR-5367, ACS Grant IN-41-H, and Otho Sprague Memorial Institutional Grant. — The author thanks Dr. N. S. HALMI for consultation.     

Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas

Summary Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelanocortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and -neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B-and A-cells, and -endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1–32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary.     

ACTH radioimmunocytochemistry (RICH) on rat anterior pituitary cells

Summary Radioimmunocytochemistry (RICH) was applied to detect corticotrophs in adult rat pituitaries and 8-day-old anterior pituitary monolayers by incubating sections and cultures with ¹²⁵I-ACTH-anti ACTH immune complexes. After incubations autoradiography was made. In comparison, conventional immunostaining was carried out on adjacent sections and parallel cultures. It has been esteblished that RICH is suitable for detection of corticotrophs.     

An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique

Summary A simple procedure is described for removing Epon resin from semi-thin 1 m sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4–7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.     

Regulation of calmodulin content in synaptic plasma membranes by glucocorticoids

Synaptic plasma membranes (SPM) from the brain are known to have specific binding sites for several steroid hormones, but the mechanisms of membrane transduction of steroid signals is not understood. In this study, corticosterone was found to prevent temperature-dependent dissociation of endogenous calmodlin (CaM) from highly purified SPM from rat cerebral cortex. The steroid stabilizes Ca²⁺-dependent membrane binding of endogenous CaM (78% of total CaM), whereas Ca²⁺-independent binding of CaM (the other 22%) is not affected. The stabilization of membrane binding of endogenous CaM by corticosterone is concentration-dependent, with the maximal effect occurring at steroid concentration of 1 M. The EC₅₀ is estimated as 130 nM, which is almost identical to the K_d of specific binding of the steroid to SPM (120 nM) reported previously. The effect in stabilizing membrane binding of CaM is specific to corticosterone and other glucocorticoids (cortisol, dexamethasone and triamcinolone); gonadal steroids (17-estradiol, progesterone and testosterone) are ineffective. Furthermore, corticosterone administration in vivo (2 mg/kg, i.p.) produced a rapid increase of CaM content in SPM, occurring within 5 min after steroid injection and persisting for at least 20 min. Since CaM mediates a variety of biochemical processes in synaptic membranes, we hypothesize that the effect of glucocorticoids in promoting membrane binding of CaM may lead to a cascade of consequences in synaptic membrane function.Special issue dedicated to Dr. Sidney Ochs.     

Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone,or 3H-estradiol to male rhesus monkeys

Summary Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with ³H-testosterone, ³H-dihydrotestosterone, or ³H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either ³H-testosterone (4% of pars distalis cells and 5 dpm/g DNA) or ³H-dihydrotestosterone (5% of cells and 13 dpm/g DNA) than following the administration of ³H-estradiol (43% of cells and 214 dpm/g DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78–94%). In prostate, seminal vesicles, and penis, ³H-dihydrotestosterone was the predominant form of nuclear radioactivity following both ³H-testosterone (67–90%) and ³H-dihydrostestosterone (94–97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, ³H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.