Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

Steroid metabolism by avian ovarian cells during follicular maturation

The profiles of steroid hormones produced by ovarian cells from the domestic hen were examined. Theca cells from the immature, small white follicles (SWFT), the third largest (T3), and largest (T1) preovulatory follicles, and the ruptured, postovulatory follicle (POFT) were incubated for 3 h at 37 degrees with [3H] progesterone (Prog) or [3H] pregnenolone (Preg). Granulosa cells from the largest preovulatory follicle were incubated with [3H] Preg or were coincubated with theca cells and [3H] Preg. The production of specific steroid metabolites was determined on the basis of coelution of radioactivity with known standard compounds, using an isocratic high-pressure liquid chromatography (HPLC) technique. Granulosa cells converted 93% of [3H] Preg substrate to Prog. More Prog was utilized by T3 cells than by T1 and SWFT cells, either when [3H] Prog was the substrate or when coincubated with granulosa cells and [3H] Preg. The major metabolites of Prog were androstenedione, 17-hydroxyprogesterone, and an unidentified compound with an elution time of 53 min. The POFT cells metabolized [3H] Prog to the same extent as T3 cells did, but their profile of steroidogenesis favored production of the unidentified 53 min metabolite. SWFT cells utilized the least amount of [3H] Preg substrate. The results point to marked changes in enzyme activities in theca cells during maturation and following ovulation.     

Binding characteristics of [3H]delta(5)-androstene-3beta,17beta-diol to a nuclear protein in the rabbit vagina

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Estradiol binding by intact cells isolated from DMBA-induced mammary tumors of the rat

Study of hormone binding in intact cells enables one to examine binding under conditions which elicit a biological response. Cells from 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors of the rat were enzymatically dispersed. More than 80% of these cells excluded trypan blue and were used to study binding of [3H] estradiol-17 beta. Specific binding was determined by subtracting the amount of [3H]estradiol bound in the absence and presence of 200-fold excess unlabeled estradiol. Specific binding at 37 degrees was maximal after 15 min. Steroid competition studies indicated that [3H]estradiol binding sites were relatively specific for estrogens, although there was a 9-18% inhibition of binding by androgens and progestins when present at 150-fold molar excess. Scatchard analyses of [3H]estradiol (0.15-5.0 nM) binding by whole cells suggest a single, high-affinity binding site (Kd = 7.5 x 10-10M) of low capacity (6.1 fmol/10(6) cells). More [3H]estradiol was translocated to the nucleus after 1 hr at 37 degrees than at 0 degrees. Preliminary studies indicated that incubations at 37 degrees result in appreciable metabolism of [3H]estradiol to other steroids and/or conjugates when examined by silica gel thin layer chromatography.     

Two sex steroid receptors in SC-115 mammary tumor cells

The growth of the SC-115 mammary carcinoma in mice is androgen dependent. Estrogens antagonize the androgen effect. The high affinity binding of androgens and estrogens has been studied in soluble extracts of the tumor, of primary culture cells and clone MI₁ cells.Results indicate that two distinct specific steroid hormone-binding sites (termed ‘receptors’) are found in all cytosol fractions. The androgen-receptor (A) binds testosterone, androstanolone, cyproterone (an anti-androgen), progesterone and estradiol, but only very weakly non-steroidal diethylstilbestrol. The estrogen-receptor (E) binds estrogenic substances such as estradiol and diethylstilbestrol, but no androgen. The apparent K_{D, eq} for A and E receptors of [³H]androstanolone and [³H]estradiol respectively, is identical (-0.5-1 nM at 4 °C). The affinity of estradiol for the A-receptor, when measured against [³H]androstanolone binding, indicates a K_i = 17.5 nM. The concentration of binding sites is of the order of 0.1 pmole/mg protein (somewhat higher for A than for E receptor) in MI₁ cell cytosol. Studies by ultracentrifugation through glycerol-Tris gradients (low salt medium) reveal the macromolecular nature of the cytosol A and E receptors (7–7.5 S). Evidence is presented of the transfer of the A and the E receptors to nuclei after incubation of tumor slices as well as of clone MI₁ cells with the corresponding hormones.Experiments suggest that the two different binding sites are present on two separated macromolecular moieties. After incubation at 37 °C of tumor slices with 10–20 nM [³H]testosterone, or with 10 nM [³H]estradiol, the corresponding radioactive hormone-receptor complexes are, as expected, found in the nuclear KCl extracts. In parallel experiments, where slices are incubated with non-radioactive hormones at the same concentration and the nuclear KCl extracts subsequently treated by radioactive steroids, no available androgen-binding sites are found in the nuclei after exposure to estradiol, nor estrogen-binding sites after exposure to testosterone.Therefore, in the same cell, two receptors are present which bind androgens and estrogens with high affinity, and one given hormone (estradiol) can be specifically bound (with different affinities) by two different receptors which, however, discriminate a synthetic analog (diethylstilbestrol). The data may give some molecular background for interpreting responses to the same hormone which may differ at various concentrations, for studying effects of analogs, and for analysing the control of tumor growth by antagonistic steroids.     

Radioligand purification prior to routine receptor assays

The need to repurify the commercially available radioligands [3H]estradiol and [3H]testosterone before use in routine assays was investigated. Storage of these products for 2 months after delivery led to appreciable degradation of [3H]estradiol compared to [3H]testosterone. Unexpectedly, TLC and even HPLC procedures were ineffective in completely restoring the purity of [3H]-estradiol and the unremoved polar products induced important variations in our estrogen receptor assays. An increase in non-specific binding and a comcomitant decrease in total binding were observed resulting in an underestimation of specific binding sites and of the affinity constant. In some cases Scatchard analysis was not possible. We therefore strongly recommend the repurification of low-stability radioligands and propose an economic time-saving procedure for the purification of [3H]estradiol by solvent differential partition which requires no high-cost investment in apparatus.     

Atypical Androgen Receptor in the Human Melanoma Cell Line IIB-MEL-J

To evaluate the presence of androgen receptors in the human melanoma cell line IIBMEL-J, a Scatchard plot analysis was performed. Cells in culture revealed a single binding component with an apparent dissociation constant (K_D) at 37°C of 11 nM and a binding capacity of 326 fmol/mg protein when measured with [³H]-R1881. Competition analysis revealed an atypical relaxation of specificity, since not only androgen (testosterone, dihydrotestosterone [DHT], R1881) and antiandrogen (hydroxy-flutamide [OH-FLU]) competed for [3H]-R1881 binding, but also estradiol, progesterone, and cortisol at 500-fold excess concentration. Binding of [3H]-estradiol and [3H]-R5020 in the absence of unlabeled DHT were completely suppressed in its presence. Immunohistochemistry of androgen receptor with a monoclonal antibody showed that nuclei were vigorously stained. Different doses of flutamide (FLU) and OH-FLU tested on cultured IIB-MEL-J cells in the presence of serum inhibited significantly cell proliferation in a dose-dependent manner. When cells were incubated with 10 nM DHT and 1%charcoal-adsorbed serum, a significant stimulation of growth that was observed was inhibited by 4 μM OH-FLU. DHT stimulation was completely reversed by the antiestrogen tamoxifen. In addition, male nude mice transplanted with IIB-MEL-J tumor were treated with FLU when tumors were palpable. FLU was effective in diminishing tumor growth and increasing survival rate of the animals. As a conclusion, the presence of functional androgen receptors in these cells has been demonstrated by growth inhibition in vitro and in vivo with antiandrogens, and their atypical nature is suggested by binding cross-reactivity and competition studies.     

Down-regulation of mt1 melatonin receptors in rat ovary following estrogen exposure.

In this study, we have demonstrated that 2-[125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (KD=83 pM; Bmax=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt1 melatonin receptor antibody, we have also detected mt1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d x 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a Bmax=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a Bmax=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt1 melatonin receptors in rat ovary were down-regulated (approximately 95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[125I]-iodomelatonin binding occurred with total 2-[125I]-iodomelatonin binding =3.15 [corrected] fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.     

Steroid synthesis in ovarian homogenates from immature mice treated with diethylstilboestrol in neonatal life

Female mice of the NMRI strain were treated with the synthetic oestrogen diethylstilboestrol (DES) for the first 5 days after birth. Pools of ovaries were removed from groups of 6-, 12-, 21-, 28- and 56-day-old females. An homogenate of an ovarian pool was incubated for 1 h in the presence of [3H]pregnenolone. Synthesized steroids were extracted and separated in a two-dimensional thin-layer chromatography system. Homogeneity of tentative steroids was verified with recrystallization to constant specific activity. Synthesis of [3H]progesterone and [3H]testosterone was demonstrated at 6 days, [3H]androstenedione at 12 days, [3H]17 alpha-hydroxyprogesterone at 21 days, and [3H]oestradiol-17 beta at 28 days. Up to 28 days (21 days for progesterone), the synthetic activity was lower in homogenates of DES-exposed ovaries than in control homogenates. After 28 days, values for recovered [3H]progesterone, [3H]androstenedione and [3H]oestradiol-17 beta were higher in DES homogenates than in control homogenates while the reverse was true for [3H]17 alpha-hydroxyprogesterone and [3H]testosterone. The results are compatible with an early and direct DES inhibitory effect on ovarian steroidogenesis and, later in immature life, a DES-induced disruption of the normal FSH-LH stimulation of ovarian development.     

Gonadotropin-releasing hormone analogs inhibit primate granulosa cell steroidogenesis via a mechanism distinct from that in the rat

Gonadotropin-releasing hormone (GnRH) and related peptides are implicated in the local control of rat ovarian function, but evidence to date for direct effects of such peptides on primate ovarian cells is equivocal. In contrast to rat ovaries, where GnRH action is mediated through specific, high-affinity GnRH receptors, no such binding sites have been identified in primate tissue. Using undifferentiated granulosa cells from immature follicles in cyclic (luteal phase) marmoset ovaries, we have observed direct suppression of human (h) FSH-induced steroidogenesis by GnRH analogs in vitro. Granulosa cells from immature (less than 1 mm diameter) follicles were incubated for 4 days in the presence of hFSH and testosterone (aromatase substrate) to stimulate cyclic AMP (cAMP) production and steroidogenesis. The additional presence of GnRH alone (up to 10 microM) had no effect on FSH action. However, the GnRH agonist, [D-Ser(But)6]GnRH 1-9)-ethylamide (Buserelin, 0.1 microM-10 microM), caused time- and dose-dependent inhibition of estradiol (maximum inhibition = 79%; ED50 = 0.55 microM) and progesterone production (maximum inhibition = 93%; ED50 = 0.1 microM). Accumulation of cAMP was also inhibited by up to 54%. Paradoxically, a GnRH antagonist [( N-Ac-D-Nal(2)1,D-pCl-Phe2, D-Trp3, D-hArg(Et2)6, D-Ala10]-GnRH; 10 microM) alone also inhibited hFSH-stimulated cAMP and steroid production by 40% and 70%, respectively. Moreover, the suppressive effects of the GnRH agonist on granulosa cell functions were augmented by the presence of the GnRH antagonist (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged cultivation of rat uterine cells with preserved estrogen responsiveness

A rat uterine cell culture was prepared as an experimental system for investigation of mechanisms of steroid hormone actions. Cells frequently supplemented with fresh medium were successfully cultured for 4 weeks through 2 successive passages. Studies of estrogen responsiveness in the primary culture as well as in it's first subculture were performed by a small scale uptake assay for determination of specific steroid binding. Scatchard analysis of specific ovarian hormone binding confirmed that cultured uterine cells preserve both estradiol and progesterone receptors. Characteristics of specific [3H]estradiol binding detected in cells of the first subculture were comparable to those obtained in the initial primary culture. The number of specific estradiol binding sites was diminished to one third of the initial values only in cells of the second subculture, 22 days after isolation of cells from tissue. In the primary culture and in it's first subculture the cells responded to estradiol with a 2-3-fold increase in progesterone receptor level. The subcellular distribution of steroid receptors was also studied; estradiol receptor complexes were detected predominantly in the nuclei whereas progesterone receptors were nearly equally distributed between nuclei and cytosol.     

Interaction of sex steroids with proteins from the soluble fraction of swine and cattle liver (a preliminary analysis)]

The interactions of [3H]estradiol, [3H]testosterone and [3H]progesterone with soluble proteins from porcine and calf liver were studied. The specific binding of [3H]progesterone and [3H]testosterone in calf liver cytosol seems to be due to serum transcortin or its intracellular precursor (analog). Contrariwise, the specific binding of [3H]progesterone observed in porcine liver cytosol was absent in the serum. This binding was characterized by slow association and dissociation dynamics, moderate affinity for the [3H]-ligand and a high binding capacity. The structural determinants of the ligands were studied by competitive inhibition of the [3H]-ligand binding. The delta 4-3-keto group in the steroid A-ring was found to be the most important determinant. An intensive metabolism of [3H]progesterone was observed during its incubation with cytosol (data from thin-layer chromatography). A 3H-metabolite (presumably, 20 beta-dihydroprogesterone) was predominant in the bound ligand fraction. The data obtained suggest that proteins of a steromodulin type are widely distributed in the mammalian liver.     

Estrogen-inducible progesterone receptor in primary cultures of rat glial cells

The presence of an estrogen-inducible progesterone receptor was demonstrated in primary cultures of newborn rat glial cells by biochemical and immunohistochemical techniques. The progesterone receptor (PR) was measured 3-4 weeks after primary culture in estradiol-containing or control medium. Cells were labeled with the synthetic progestin [3H]R5020 followed by ultracentrifugation analysis of the cellular extracts. A "9 S" PR was observed in the cytosol and a "4-5 S" PR was found in the nuclear high salt, tungstate ions containing extract of estradiol-treated cells. When the antiprogestin [3H]RU486 was used instead of [3H]R5020 as a ligand, a 9 S PR was also found in the cytosol, but a nonactivated "8.5 S" receptor complex was identified in the high salt nuclear fraction in presence of tungstate ions. The levels of PR, as measured by whole cell assay, were significantly increased when glial cells were cultured in the presence of 50 nM estradiol, as compared to nonestradiol-treated controls. The estrogen induction of PR was suppressed by the antiestrogen tamoxifen, but tamoxifen by itself had no effect on PR concentration. When the glucocorticosteroid receptor and PR were measured in parallel after estradiol treatment of the same primary culture, only the levels of PR were increased. The PR was visualized inside glial cells by immunohistochemical studies with a monoclonal antibody specific for the B-form of PR (KC 146), which was recognized by fluorescein-linked or biotinylated secondary antibodies. Strong staining was observed in estradiol-treated cultures, when compared to a weaker staining in control cultures. This is the first demonstration of PR in rat glial cells, and we present evidence of its induction by estradiol in primary cultures.     

The effects of various steroids on testosterone metabolism by the sexually mature rabbit epididymis

We examined the influences of steroids present in the epididymis on androgen metabolism by epididymal tissue and on the binding of androgen metabolites to the epididymal androgen receptor in castrated adult rabbit epididymides under in vitro conditions. The conversion of [3H]testosterone to [3H]17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT) and to [3H]5 alpha-androstane-3 alpha (beta), 17 beta-diol was inhibited by unlabeled steroids in the following manner progesterone greater than testosterone greater than estradiol. Unlabeled 5 alpha-DHT did not inhibit [3H]testosterone metabolism indicating that product inhibition is not an important regulatory event. The antiandrogen cyproterone acetate did not inhibit the formation of 5 alpha-reduced metabolites of [3H]testosterone. All of the compounds used inhibited androgen binding to the classically defined cytoplasmic and nuclear androgen receptor.     

Aromatase and testosterone fatty acid esters: the search for a cryptic biosynthetic pathway to estradiol esters.

The estradiol fatty acid esters (lipoidal derivatives, LE2) are extremely potent estrogens that accumulate in fat, including fat of menopausal women. These steroidal esters are protected from metabolism and are converted to the free, biologically active steroid through the action of esterases. Previous studies have shown that biosynthetic pathways in the adrenal gland exist in which steroid fatty acid esters are substrates. This led us to determine whether a cryptic aromatase pathway exists in which testosterone esters could be converted directly into LE2. We tested a representative fatty acid ester, testosterone stearate, both as an inhibitor and as a substrate for the aromatase enzyme from human placental microsomes. This ester had neither activity. In addition, we tested [1 beta-3H]testosterone acetate as a substrate for this enzyme complex, measuring the production of 3H2O as evidence of aromatization. Although the rate of reaction was considerably slower than that of testosterone, 3H2O was produced. However, when [2, 4, 6, 7-3H]testosterone acetate was incubated and the steroidal products isolated, we found that hydrolysis of the substrate had occurred. Both [3H]-labeled testosterone and estradiol were found, and very little if any [3H]estradiol acetate was formed. Thus, we conclude that an aromatase pathway involving testosterone esters does not exist and that the sole source of LE2 is through direct esterification of estradiol.     

Translocatable estrogen receptors in rat splenocytes

Estradiol has previously been shown to suppress the response of the cellular immune system of the rat while enhancing the production of IgM antibodies. Analysis of the cytosol from rat splenocytes showed saturation of specific binding sites at concentrations of between 80 and 160 nM [3H]-estradiol with an approximate Kd of 12 nM. Competitive binding studies showed a dose-dependent decrease in the binding of [3H]-estradiol to the receptor in the presence of increasing concentrations of unlabeled estradiol. Dexamethasone, progesterone and R1881 (synthetic androgen) had no effect on the binding of [3H]-estradiol. The in vivo administration of estradiol resulted in increased nuclear binding of [3H]-estradiol as compared to vehicle treated controls. These results indicate that rat splenocytes possess specific, translocatable estrogen receptors which may be responsible for the observed modulation of the immune system.     

Nuclear estradiol binding in rat adipocytes. Regional variations and regulatory influences of hormones.

The nuclear estrogen receptor was characterised in isolated rat adipocytes. The binding reaction with [3H]estradiol was performed with intact isolated rat adipocytes and the radioactivity associated with the nucleus was subsequently determined after cell lysis. The nuclear uptake of [3H]estrogen in rat adipocytes was temperature dependent and steroid specific. The steady-state binding was achieved after 30 min at 37 degrees C and was constant for several hours. Estradiol was found to bind to a homogeneous class of nuclear receptors in epididymal adipocytes with an apparent Kd of 3.1 +/- 0.76 nM and a Bmax of 7.98 +/- 1.11 fmol/10(6) cells corresponding to about 4800 receptors per nucleus. The estradiol binding exhibited regional variations in isolated adipocytes. In lean rats the highest receptor number was found in epididymal adipocytes, whereas there was a significantly lower number of nuclear binding sites in perirenal and subcutaneous adipocytes (P less than 0.05), unlike in older and more obese rats where the nuclear estradiol binding was greatest in adipocytes from the perirenal fat depot. Incubations with isoproterenol (10 microM) and dibutyryl-cAMP (2.5 mM) both reduced estradiol binding by 56% (P less than 0.005), while insulin (1 nM) enhanced the estradiol binding by 37% (P less than 0.01). In conclusion, a specific and high affinity nuclear estradiol receptor was demonstrated in rat adipocytes and regional differences in nuclear estradiol binding were detected. Furthermore, it was demonstrated that nuclear estradiol binding could be modulated by other agents known to affect adipocyte metabolism.     

Estradiol esters can replace 17 beta-estradiol in the stimulation of DNA and esterase synthesis by MCF-7 cells: a possible role for the estrogen-sensitive MCF-7 cell esterase

In this communication we extend our earlier observations on estrogen-sensitive carboxyl esterases in MCF-7 human breast cancer cells able to hydrolyze esters of estradiol. Using either estradiol acetate or p-nitrophenyl hexanoate as substrates, esterase activity was found to increase 2-3-fold in MCF-7 cells maintained in the presence of 10(-8) M estradiol. Following sucrose density centrifugation, over 85% of total esterase activity was found in the cytoplasmic fraction. No esterase activity was found in spent media from growing cells. By size exclusion chromatography, estradiol acetate esterase activity exhibited a mol. wt of 45-50 kDa. Attempts to demonstrate incorporation of [3H]estradiol into estradiol fatty acid esters by the above MCF-7 cell line (203P) were unsuccessful, although, such incorporation could be demonstrated in two other MCF-7 cell sublines. Incubation of the 203P cells with 10 nM [3H]estradiol in the presence of 0.5 mM radioinert estradiol acetate resulted in the incorporation of 35 +/- 12% of the label into the estradiol acetate in 10 min. In the absence of radioinert estradiol acetate, no incorporation was observed. When MCF-7 cells were incubated with [3H]estradiol in the presence of a large excess of radioinert estradiol valerate, label was found only in estradiol valerate. Similarly, when the incubation was carried out in the presence of a mixture of radioinert estradiol acetate and valerate, label was incorporated into both esters. We conclude that the apparent formation of radiolabeled estradiol esters by MCF-7 cells incubated under the above conditions, results at least in part, from an esterase-catalyzed exchange reaction. Under conditions where no ester hydrolysis could be detected in the absence of cells, valerate and stearate esters of estradiol were found to be as effective as unesterified estradiol in stimulating esterase synthesis and the incorporation of [3H]thymidine into DNA. These results are consistent with a model in which an intracellular esterase in MCF-7 cells can generate estradiol from an exogenous lipoidal steroid and elicit an estrogen response.     

Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.