Characterization of ovarian membrane receptor for 17,20beta-dihydroxy-4-pregnen-3-one,a maturation-inducing hormone in yellowtail,Seriola quinqueradiata

In our previous studies, we tentatively identified 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) as a maturation-inducing hormone (MIH) in yellowtail (Seriola quinqueradiata) through in vivo and in vitro experiments. In this study, we investigated the binding sites for radioactive 17,20 beta-P and characterized the receptor binding to the ovarian plasma membrane in yellowtail undergoing first stage of maturation (FSM). Equilibrium binding sites for 17,20 beta-P have been detected within 1h incubation and the binding dissociated completely within 50 min at 4 degrees C and was pH dependent (optimum pH 7.8). Scatchard analyses of specifically bound 17,20 beta-P showed the evidence of a single class of high affinity binding sites (K(D)=22.9 nM), with limited capacity (B(max)=2.1 pmol/g tissue) to the ovarian membrane of yellowtail undergoing FSM. Competition results revealed that ovarian membrane receptor was highly specific for 17,20 beta-P. There was no other steroid competed strongly with the binding sites of [3H]17,20 beta-P, except 17,20 beta-P itself. On the other hand, 17,20 beta-P did not bind to the membrane prepared from maturationally incompetent (MI) and ovulation (OV) stages of oocytes. As the time proceeded after the stimulation of HCG, binding activity increased significantly (0.389+/-0.036 pmol/g tissue) in the ovarian membrane of maturationally competent (MC) oocytes by 12h postinjection. The binding activity was further significant (0.868+/-0.032 pmol/g tissue) at FSM by 24h postinjection and reached its peak (0.920+/-0.115 pmol/g tissue) temporarily at second stage of maturation (SSM) by 36 h postinjection and then sharply declined to the prestimulation levels during OV stage by 48 h postinjection. In addition to our previous findings, the present results indicate that 17,20 beta-P is the MIH in yellowtail.     

Sexually mature male goldfish release large quantities of androstenedione into the water where it functions as a pheromone

Previous studies have demonstrated that ovulatory female goldfish release a variety of sex steroids into the water where they function as a pheromonal blend dominated by C21 steroids that stimulates male hormone release, sperm production and behavior. This study investigated whether male goldfish might also release sex steroids with pheromonal activity. It found that spermiated male goldfish release substantial quantities of androstenedione (AD; about 50 ng/h) together with smaller (10-20 ng/h) quantities of several other related C19 steroids but only very small quantities (<5 ng/h) of C21 steroids. Further, when sexually aroused by females and/or their pheromones, males released even greater quantities of AD (up to 1 microg/h) while C21 steroid release rate changed little. This created a ratio of C19 to C21 steroids of about 50:1 that was dramatically different from that emitted by females (1:7). The male olfactory system was also found to be extremely sensitive to AD, detecting it to near picomolar concentrations. Together with previous studies that have shown water-borne AD to increase male aggressive behavior while suppressing responsiveness to female pheromones, this study establishes AD as a male pheromone in the goldfish. Because ovulating females also release AD but in the presence of C21 steroids, recognition of the male-derived steroid pheromone is presumably mixture dependent.     

Competitive binding studies with glucocorticoid receptors from rat-thymus cells: differential temperature-dependence of steroid binding.

Competitive steroid-binding studies were performed with intact rat thymus cells and with cytosol preparations at different temperatures using [1,2-3H]dexamethasone as the labelled ligand. Steroids lacking a 17 alpha-hydroxyl group, such as corticosterone, were better able to compete with [1,2-3H]dexamethasone for binding to glucocorticoid receptors at 0 degrees C than compounds containing a 17 alpha-hydroxyl substituent, such as cortisol. At 37 degrees C the reverse was true. This temperature-dependent change in relative affinities appeared to be unrelated to steroid metabolism or receptor activation, and to depend only on the thermodynamic parameters of the steroid--receptor interaction. Relative biological activities for different steroids agree more closely with the relative affinities determined at 37 degrees C than with those determined at lower temperatures.     

Some properties of androgen-binding activity in rat testis.

High-affinity (Ka approximately equal to 5 X 10(8) M-1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant (t1/2 = 3 min, 0 degrees C, 30% glycerol buffer) using dextran-coated charcoal to separate bound from free hormone. Because of this fact, exchange of endogenous and labeled hormone was complete in the assay incubation time (16 h, 0 degrees C) and Scatchard plots of the high-affinity binding data were shown to measure total as contrasted to available sites. The binding was highly specific for androgens. Polyacrylamide gel electrophoresis separated high-affinity androgen-binding protein (Rf 0.54) from albumin (Rf 0.62). Binding site estimates under saturating conditions or by Scatchard analysis of electrophoresis data utilizing [3H]dihydrotestosterone agreed reasonably well with estimates made by the charcoal technique using [3H]testosterone.     

Androgen metabolism in rat anterior pituitary cells in culture

The metabolism in vitro of 4 androgens, namely testosterone, androstenedione, 5α-dihydrotestosterone and 3α-androstanediol has been studied in male and female rat anterior pituitary cells in primary culture. When testosterone was used as precursor, androstenedione, 5a-dihydrotestosterone and 3α-androstanediol were the main metabolites whereas androstenedione was mainly converted into testosterone, 5α-androstanedione, 5α-dihydrotestosterone and androsterone. Studies on the metabolism of 5α-dihydrotestosterone and 3α-androstanediol showed that these compounds were easily interconverted and were also significantly metabolized to 5α-androstanedione and androsterone. No aromatized compounds could be detected suggesting that androgen action in the pituitary cell occurs directly via the androgen receptor rather than through prior conversion into estrogens.     

Glucocorticoid receptor in human embryo fibroblasts. I. Kinetic and physicochemical properties.

The kinetics of dexamethasone binding to L 809 E cell line cytosol have been investigated by means of the protamine sulfate precipitation assay. The KDeq for dexamethasone was 1.1--3.3 nM. Binding was specific for glucocorticoids. The mean association rate constant (k+1) was 8.5 x 10(5) M-1 x min-1 and the dissociation rate constant was 4.6 x 10(-5) min-1 at 0 degrees C. The concentration of binding sites was 0.3 pmol/mg of cytosol protein. Binding kinetics were compatible with a model of positive cooperativity. The receptor sedimented at 7.5--9 S in glycerol gradients. By a combination of calibrated ultracentrifugation and polyacrylamide gel electrophoresis, a Stokes radius of 8.5 nm, a molecular weight of 268 000 daltons and a frictional ratio of 1.8 were determined in low ionic strength conditions. When the cells were incubated with 10 nM [3H]dexamethasone for 1 h, a more than 90% depletion of cytosol receptor and an equivalent accumulation of nuclear dexamethasone--receptor complexes was observed.     

Cytochrome P-450 and the aromatization of 16alpha-hydroxytestosterone and androstenedione by human placental microsomes.

When measured in vitro using human placental microsomal preparations, the aromatization of 16alpha-hydroxytestosterone to estriol and androstenedione to estrone and estradiol proceeds at almost identical initial rates. Important differences between 16alpha-hydroxytestosterone and adrostenedione aromatization are evident, however. While substantial findings have implicated cytochrome P-450 in placental aromatization, the aromatizaiton of androstenedione is insensitive to CO although it is competitively inhibited by metyrapone. 16alpha-Hydroxytestosterone aromatization, in contrast, is inhibited 50-60% by CO and is strongly inhibited by metyrapone. 16alpha-hydroxytestosterone aromatization is strongly inhibited in a competitive manner by androstenedione, while 16alpha-hydroxytestosterone has essentially no effect on androstenedione aromatization, althogh at very high 16alpha-hydroxytestosterone concentrations (65 muM) and subsaturating androstenedione concentrations, 16alpha-hydroxytestosterone appears to noncomptitively inhibit androstenedione aromatization. The apparent Km for the aromatization of androstenedione is 95 nM and for 16alpha-hydroxytestosterone, 7 muM. Both androstenedione and 16alpha-hydroxytestosterone cause type I spectral perturbations associated with binding to cytochrome P-450 when added to placental microsomes; however, the deltaA390-420 is twice as great in response to saturating amounts of androstenedione than in response to 16alpha-hydroxytestosterone. If androstenedione is added to 16alpha-hydroxytestosterone, the same spectral change as that caused by androstenedione alone is expressed. The apparent spectral dissociation constant for androstenedione is 93 nM while for 16alpha-hydroxytestosterone it is 11muM; both essentially the same as the comparable apparent Kms for aromatization. The evidence suggests the presence of two aromatase P-450's in human placenta.     

Interaction of glucocorticoid hormones with rat skeletal muscle: catabolic effects and hormone binding.

The mechanism of action of glucocorticoid hormones on rat skeletal muscle was studied by following their effect on muscle weight, free amino acid content, activity of amino acid-metabolizing enzymes, and binding to cytoplasmic receptor proteins. A significant reduction of gastrocnemius muscle and body weight occurred following administration of cortisol, triamcinolone diacetate, and triamcinolone acetonide to adrenalectomized rats. Treatment with triamcinolone diacetate also reduced the level of several free amino acids and enhanced the activity of a myofibrillar protease in skeletal muscle. The hormone had, however, no effect on the activity of various enzymes involved in amino acid catabolism in muscle. In nephrosis, another condition of muscle wasting, the level of several muscle amino acids were also reduced to a lesser extent. Cortisol and triamcinolone acetonide, both of which induce muscle wasting, were found to bind to two distinct cytoplasmic proteins in muscle. Binding of the labeled hormones was followed at 0 C and could be observed in presence of a 1000-fold excess of the catabolically inactive steroid epicortisol. Binding of 3H-triamcinolone acetonide. In vitro competition experiments further suggest a correlation between steroid binding to the 3H-dexamethasone or 3H-triamcinolone acetonide site and their potency to induce muscle catabolism. It is concluded that skeletal muscle is a direct target organ for glucocorticoids, and that muscle responsiveness involves binding of the active hormones to cytoplasmic receptor sites.     

Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

Glucocorticoid-induced cell-size changes and nuclear fragility in rat thymocytes

The events preceding glucocorticoid-induced lymphocytolysis have been studied in isolated rat thymocytes. Incubation of thymocytes at 37°C in the presence of 1 μM dexamethasone resulted in the progressive appearance of pyknotic cells of modal diameter 4.6 μm, distinct from normal cells of diameter 5.2 μm. The rate of appearance of the pyknotic cells was determined by selective electronic cell counting, and was shown to be accompanied by increased nuclear fragility. The production of pyknotic cells was glucocorticoid-specific, dose-dependent, blocked by cycloheximide, and preceded the loss of cell viability as determined by dye exclusion. The pyknotic cells were separated from the non-pyknotic cells by density-gradient centrifugation and shown to be solely responsible for the observed nuclear fragility.     

Differentiation between steroid hormone receptors CBG and shbg in human target organ extracts by a single-step assay

The steroid complexes of (plasma) corticosteroid-binding globulin can be distinguished from intracellular steroid-receptor complexes by agar electrophoresis at low temperature in neuraminidase-treated tissue extracts. With this method, the presence of progesterone receptor has been demonstrated in heavily plasma-protein-contaminated human uterus “cytosol”, but not in human mammary carcinoma extracts. SHBG and “basic” receptors for estradiol and dihydrotestosterone in human uterus cytosol could also be assayed simultaneously.     

Androgen-binding proteins in rat epididymis: properties of a cytoplasmic receptor for androgen similar to the androgen receptor in ventral prostate and different from androgen-binding protein (ABP)

The cytoplasmic receptor (CR) in rat epididymal 105,000 $\text{g}$ supernatant was separated from the androgen-binding protein (ABP) by gel electrophoresis following labeling with [1,2,6,7-³H]-testosterone in vivo. ABP disappeared from epididymal supernatants after castration 01 hypophysectomy, while CR remained unchanged. CR was evenly distributed between caput and cauda, while much more ABP was present in caput. Properties of CR in epididymis and prostate were similar and distinctly different from ABP. Binding to CR was destroyed by charcoal treatment (1 mg/mg protein) of supernatant at 0 °C for 6 h, heating at 50 °C for 30 min, or exposure to the sulfhydryl blocking reagent, $\text{p}$-chloromercuriphenylsulfonate (1 mM) at 25 °C for 30 min, while binding to ABP was unaffected. The isoelectric pH of CR (5.8) was higher than that of ABP (4.6). Dissociation of radioactive 5α-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 °C >2 days), while dissociation from ABP was rapid (half-time at 0 °C ~ 6 min). Cyproterone acetate (250 mg/100g body weight) inhibited binding to CR both in epididymis and ventral prostate but did not affect binding to ABP. Nuclear uptake was inhibited by cyproterone to the same extent as binding to CR, indicating that nuclear uptake and binding are dependent on CR and independent of ABP. The time-course of uptake and binding in epididymal supernatant and nuclear fractions was essentially the same 1 day after bilateral castration when both CR and ABP were present or 8 days after castration when CR alone was present. It is concluded that the cytoplasmic receptor for androgen in rat epididymis has properties very similar to the androgen receptor in ventral prostate but different from ABP.