Evidence that a non-aromatizable metabolite of norethisterone induces estrogen-dependent pituitary progestin receptors

Neutral reduced metabolites of norethisterone (NET) specifically interact with intracellular estrogen receptors in target organs. To determine if this interaction can effectively initiate estrogen-dependent cellular responses, the effects of an A-ring-reduced NET derivative upon the induction of cytosol-located pituitary progestin receptors (PR) and uterine growth were studied in adult castrated female rats. Different doses of 17 alpha-ethynyl-5 alpha-estran-3 beta, 17 beta-diol (3 beta, 5 alpha-NET) were s.c. administered to ovariectomized animals for 6 days. 17 beta-Estradiol (E2) and oil-treated rats served as experimental controls. Pituitary PR were labeled in vitro by a post-gradient technique using [3H]ORG-2058 as the ligand. PR binding specificity was determined by the use of an excess of radioinert steroids. The results demonstrated that administration of 3 beta, 5 alpha-NET induced specific 8-9S pituitary cytosol PR in a dose-dependent manner. Binding properties of the 3 beta, 5 alpha-NET-induced progestin binding sites (Kd = 1.0 X 10(-9) M; NBS = 1.2 X 10(-9) M) appear indistinguishable from those induced by E2. In addition, 3 beta, 5 alpha-NET administration resulted in a significant increase in uterine weight at the expense of myometrium and endometrium growth in a similar fashion to that observed in the E2-treated group. When 3 alpha, 5 alpha-epimeric alcohol (3 alpha, 5 alpha-NET) was administered, induction of pituitary PR and uterine growth were also observed although to a lesser extent. Inasmuch as the results demonstrate that neutral non-aromatizable NET metabolites induce biochemical and morphological estrogenic responses, they offer an alternative explanation for the mechanism of estrogen-like action of this synthetic contraceptive progestin.     

ORG-2058 as a ligand in the assay of progesterone receptor in breast cancer

Tritiated [(16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione)-6,7-3H] (ORG-2058) and 17,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020) were compared as ligands in the assay of progesterone receptor in human and rat breast tumors. We found that ORG-2058 is a better ligand because of its low nonspecific binding. Most of the nonspecific binding of the other ligand R5020, is to proteins which bind corticosteroids. In cancerous tissue ORG-2058 binds to progesterone receptor linearly in a range of protein concentrations which are normally used in the receptor assay. On the other hand, R5020 exhibits binding linearity over a narrower protein concentration in many tumor biopsies, which may cause severe limitation in the assay procedure or frequent underestimation of receptor content.     

Human mammary cancer cell mutants with altered hormone receptor activity

We have recently isolated retinoic acid-resistant clones U-2 and U-3 from human breast cancer cell line MCF-7 (Ueda et al. (1985) Cancer Res. 45, 3332-3338). Growth of MCF-7 cells was found to be stimulated by estradiol but that of U-2 or U-3 was not. Cytosol from U-2 or U-3 cells contained no detectable estradiol receptor activity, whereas that from the parental MCF-7 cells showed estradiol receptor activity of 32 fmol/mg cytosol protein with a Kd of 2.6 X 10(-10) M by Scatchard analysis. Sucrose gradient centrifugation analysis of the cytosol fraction confirmed the presence of estradiol receptor activity in MCF-7 but not in U-2. Cytosol from MCF-7 and U-2 cells showed progesterone receptor activities of 106 fmol/mg protein with a Kd of 7.4 X 10(-10) M and 13 fmol/mg protein with a Kd of 9.9 X 10(-10) M, respectively. Addition of estradiol to the culture medium of the cells increased the level of progesterone receptor about 2-fold in MCF-7, but not in U-2. U-2 or U-3 cells showed about 5-fold higher resistance to an antiestrogen, tamoxifen, than MCF-7, and they were also 300- to 1,000-fold more resistant to other antiestrogens, epitiostanol and medroxyprogesterone, than MCF-7. The altered cellular sensitivity of U-2 or U-3 to the hormone antagonists is discussed in relation to the absence or presence of hormone receptors.     

Plasma concentrations and receptor binding of RU 486 and its metabolites in humans

Using Chromosorb chromatography and HPLC, we measured the plasma concentrations of RU 486, and its monodemethylated (RU 42633), didemethylated (RU 42848) and alcoholic nondemethylated (RU 42698) metabolites up to 72 h following oral ingestion of 100 mg of RU 486 by five female volunteers. The peak plasma level of RU 486 (4.5 mumol/l) occurred within 1 h after ingestion of the compound; at this point significant amounts of the metabolites were also present in the plasma. After the initial redistribution within 6 h the plasma concentrations of RU 486 and three of its metabolites measured remained stable for 24 h. Concentrations of the monodomethylated metabolite exceeded those of the parent steroid during the time period measured, whereas the concentrations of the didemethylated and alcoholic metabolites were lower than those of RU 486, but still notable. At 72 h the concentrations of all the four steroids were still in the micromolar range. The relative binding affinities of these metabolites to human endometrial and myometrial progesterone receptors as well as to human placental glucocorticoid receptors were determined in vitro. The affinity of RU 486 for the human uterine progesterone receptor (Kd = 1.3 X 10(-9) M for RU 486) was higher than that of progesterone but lower than that of ORG-2058, a potent synthetic progestin. The relative binding affinities of the monodemethylated, alcoholic and didemethylated metabolites to the progesterone receptor were 21, 15 and 9%, respectively, compared with the parent compound RU 486; each was lower than that of progesterone (43%). RU 486 had an approx. 4-fold higher relative binding affinity to the glucocorticoid receptor than dexamethasone. Interestingly, the relative binding affinities of the metabolites studied to the human glucocorticoid receptor exceeded those of dexamethasone or cortisol. Compared with the parent compound RU 486, they were 61, 48 and 45% for the monodemethylated, alcoholic and didemethylated metabolites, respectively; each was higher than that of dexamethasone (23%). The affinity of dexamethasone to the human glucocorticoid receptor was 1.6 X 10(-9) M. These data indicate that the pool of certain metabolites of RU 486 may contribute to a significant extent to the antiprogestagenic (23-33%) and even greater extent to the antiglucocorticoid (47-61%) effects of RU 486.     

Estrogen and progesterone receptors in the organs of prenatal cynomolgus monkey and laboratory mouse

The estrogen and progesterone receptors of several organs of the prenatal cynomolgus macaque and the fetal mouse were studied using a combination of the dextran-coated charcoal technique and high-performance liquid chromatography. This procedure permitted the concurrent measurement of both receptors in minute amounts of tissue. Estrogen receptors, but not progesterone receptors, were found in the fetal monkey and mouse uteri. No estrogen or progesterone receptors were detected in the lungs, liver, kidney, heart, brain, adrenal gland, or limbs of mouse or monkey fetuses. The nonspecific binding of radioactive ORG-2058 was not displaced by unlabeled progesterone, 17 alpha-hydroxyprogesterone caproate, or ORG-2058. Because the steroid receptors that are indispensable mediators of steroid hormone action were absent from the nonreproductive tissues, prenatal development of these organs and tissues cannot be adversely influenced by exposure to estradiol, progesterone, or their synthetic analogues.     

Characterization of Leydig cell gonadotropin-releasing hormone binding sites utilizing radiolabeled agonist and antagonist

Gonadotropin-releasing hormone (GnRH) binding sites in intact Leydig cells and in membrane preparations were investigated using 125I-labeled GnRH agonist and antagonist. Binding was saturable and involved a single class of high affinity sites. Intact Leydig cells and a membrane preparation had a higher affinity for GnRH agonist (Kd 3.0 +/- 1.7 X 10(-10) M) than for GnRH antagonist (Kd 10.0 +/- 1.8 X 10(-10) M). With anterior pituitary membranes the Kd was 2.8 +/- 0.7 X 10(-10) M for the agonist and 2.4 +/- 1.4 X 10(-10) M for the antagonist. The Kd for GnRH was similar for Leydig cells and the anterior pituitary. Chymotrypsin and trypsin digestion decreased receptor binding, but neuraminidase increased Leydig cell binding in contrast to the decrease in binding observed with pituitary receptors. The results suggest that the Leydig cell GnRH binding sites may differ from the pituitary receptor which may be related to structural differences in GnRH-like peptides recently described in extracts of rat testis.     

Interaction of [3H]nomegestrol acetate with cytosolic progesterone receptors from the rat uterus

The binding characteristics of the progestin 17 alpha-acetoxy-6-methyl-19-[3H]norpregna-4,6-diene-3,20-dione, nomegestrol acetate ([3H]NOM-Ac) to progesterone receptors (PgRs) of uterus were determined in the rat. Scatchard plot analysis of the equilibrium binding data showed that [3H]NOM-Ac binds to uterine PgR with a Kd of 5.44 +/- 1.27 nM and a Bmax of 1.51 +/- 0.11 pmol/mg protein. Analysis of dissociation kinetics showed that [3H]NOM-Ac dissociates slowly from the PgR, k - 1 = 4.9 +/- 0.5 10(-5) s-1. Competition experiments against [3H]NOM-Ac showed the specificity of the binding with a sequence in relative affinity as follows: ORG 2058 greater than P greater than NOM-Ac greater than medroxyprogesterone acetate greater than megestrol acetate greater than cyproterone acetone greater than NOM.     

Hormonal regulation of oestrogen and progesterone receptors in cultured bovine endometrial cells.

Changes in the number of progesterone and oestradiol receptors in the endometrium are thought to play a role in the induction of luteolysis. The effect of oestradiol and progesterone on the regulation of their receptors in cultured bovine uterine epithelial and stromal cells was examined to determine the mechanisms involved in this process. Cells were obtained from cows at days 1-3 of the oestrous cycle and were cultured for 4 or 8 days in medium alone (RPMI medium + 5% (v/v) charcoal-dextran stripped newborn calf serum) or with oestradiol, progesterone or oestradiol and progesterone. At the end of culture, receptor binding was measured by saturation analysis. Specific binding of both [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor (6,7-3H) pregn-4-ene-3,20-dione) and [3H]oestradiol to epithelial and stromal cells showed high affinities (Kd = 1.1 x 10(-9) and 6 x 10(-10) mol l-1, respectively, for progesterone receptors; Kd = 5.5 x 10(-9) and 7 x 10(-10) mol l-1, respectively, for oestradiol receptors). In the stromal cells, oestradiol (0.1-10 nmol l-1) increased the number of oestradiol receptors from 0.21 +/- 0.06 to 0.70 +/- 0.058 fmol microgram-1 DNA and the number of progesterone receptors from 1.4 +/- 0.83 to 6.6 +/- 0.70 fmol microgram-1 DNA in a dose-dependent manner after 4 days of culture (P < 0.01). After culture for 8 days, the stimulatory effect of oestradiol increased. Progesterone (50 nmol l-1) had no effect on the number of oestradiol or progesterone receptors (P > 0.05). However, progesterone inhibited the stimulatory effect of oestradiol. In epithelial cells, the lower concentrations of oestradiol (0.1 and 1 nmol l-1) stimulated the number of progesterone receptors (P = 0.05) after 4 days culture, whereas the highest concentration of oestradiol (10 nmol l-1), progesterone (50 nmol l-1) and progesterone (50 nmol l-1) plus oestradiol (1 nmol l-1) had no effect. After culture for 8 days, the stimulatory effect of oestradiol decreased. In contrast to progesterone receptors, the number of oestradiol receptors increased with oestradiol concentration (P < 0.01). These data show that the number of progesterone receptors was higher in the stromal cells than in epithelial cells, whereas the number of oestradiol receptors was higher in the epithelial cells than in stromal cells. Oestradiol upregulates its own receptor and increases the number of progesterone receptors in both cell types in vitro, whereas progesterone has little effect, but inhibits the effects of oestradiol on progesterone receptors.     

Stereospecificity of the intracellular binding of norethisterone and its A-ring reduced metabolites

The interaction of norethisterone (NET) and four A-ring reduced metabolites of NET with cytosol receptors for progesterone (PR), androgen (AR), and estrogen (ER) was investigated. Cytosol preparations from: uteri of adult estrogen-primed castrated rats, ventral prostates of adult castrated rats and uteri of immature rats were used as the source of PR, AR, and ER respectively. 3H-Labeled ORG-2058, R-1881, and 17 beta-estradiol were used as the radioligands. The results of competitive studies disclosed that: the most efficient competitor for PR binding sites was NET (Ki = 1.1 X 10(-7) M) followed by 5 alpha-dihydro NET (5 alpha-NET), whereas the 3 alpha,5 alpha; 3 beta,5 alpha and 3 alpha,5 beta-tetrahydro NET derivatives were ineffective the most efficient competitor for AR binding sites was 5 alpha-NET (Ki = 1 X 10(-8), immediately followed by NET, while the three tetrahydro NET derivatives were not competitors and remarkable competition for ER binding sites was only exhibited by the 3 beta,5 alpha-tetrahydro NET derivative (Ki = 4.6 X 10(-8) M) and to a lesser extent by its 3 alpha,5 alpha-epimeric alcohol, while NET and 5 alpha-NET were completely ineffective. These findings demonstrate the stereospecificity of the intracellular binding of NET and its reduced metabolites with cytosol steroid putative receptors, and provide biochemical support to the understanding of the variety of hormone-like effects observed after the in vivo administration of NET.     

Effects of interaction between estradiol-17 beta and progesterone on the proliferation of cloned breast tumor cells (MCF-7 and T47D)

We have previously reported that the proliferation of cloned MCF-7 and T47D human mammary tumor cells can be inhibited by increasing concentrations of charcoal-dextran stripped female human serum (CDFHS). The maximal proliferation rate was restored by the addition of 3 X 10(-11) M estradiol-17 beta to the culture media. These observations suggest that the proliferation of T47D and MCF-7 cells is regulated by a blood-borne inhibitor whose effects are neutralized by estrogens. In the present report we explore the possibility that progesterone alters the estrogenic response. MCF-7 cells were grown in DME containing 2-40% CDFHS. Progesterone, at 3 X 10(-7) M to 3 X 10(-12) M, had no effect on the yield of MCF-7 or T47D cells that were cultured in the presence or absence of estradiol-17 beta.     

Assessment of progestin receptor polymorphism by various synthetic ligands using HPLC

Tritiated R5020 and [3H]ORG-2058 were utilized to investigate apparent polymorphism of progestin receptors by vertical-tube gradient centrifugation and HPLC in size exclusion (HPSEC), ion-exchange (HPIEC) and chromatofocusing (HPCF) modes. Rapid centrifugation (3 h) following molybdate stabilization (1 h) showed mainly 8-9S receptor species with 90-96% recovery. [3H]R5020 appeared to associate with a receptor isoform sedimenting faster than that bound to [3H]ORG-2058. Excess unlabeled R5020 did not eliminate all of the [3H]R5020 binding by the 8-9S component suggesting some nonspecific association while excess unlabeled ORG-2058 suppressed this binding by either ligand. Separate labeling of cytosol with each ligand and mixing prior to gradient separation showed at least two receptor species isoforms sedimenting in the 8-9S region with mol. wt of 190,000 and 173,000 D. Sephacryl S-300 chromatography revealed two radioactive peaks with either ligand but with slight molecular weight differences. HPSEC confirmed the presence of isoforms with different molecular size and shape as a function of the radioactive ligand employed. HPIEC showed the presence of two labeled receptor species irrespective of the ligand used. The first peak appeared at the void volume of the column (10 mM), co-eluted with free ligand, indicating the possibility of ligand stripping by the column. The second peak bound both steroids specifically and eluted with 100 mM phosphate. HPCF identified a single specific receptor eluting at a pH of 5.6-6.1, but with free steroid in the void volume irrespective of the ligand used. [3H]ORG-2058 appeared to be less prone to the stripping phenomenon than was [3H]R5020. These data suggest these ligands either bind to different progestin receptor species or they modify receptor characteristics in a fashion that allows separation based upon size and shape.     

Identification and initial characterization of an autocrine pheromone receptor in the protozoan ciliate Euplotes raikovi

The polypeptide pheromone Er-1, purified from the ciliate Euplotes raikovi of mating type I and genotype mat-1/mat-1, was iodinated with 125I-Bolton-Hunter reagent to a sp act of 0.45-0.73 mu Ci/microgram of protein. This preparation of 125I-Er-1 bound specifically to high affinity binding sites on the same cells of mating type I. Binding of 125I-Er-1 occurred with an apparent Kd of 4.63 +/- 0.12 X 10(-9) M in cells in early stationary phase. It was estimated that these cells carry a total number of approximately 5 X 10(7) sites/cell, with a site density that falls in the range of 1,600-1,700/microns 2 of cell surface. Unlabeled Er-1, other homologous pheromones such as Er-2 and Er-10, antibodies specific for Er-1, and human IL-2 were shown to act as effective inhibitors of specific binding of 125I-Er-1 to mating type I cells. The "autocrine" nature of the identified specific high affinity binding sites for Er-1 was further substantiated by cross-linking experiments. These experiments revealed that mating type-I cell membranes contain one protein entity of Mr = 28,000 that is capable of reacting specifically with the homodimeric native form of Er-1.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male

The in vitro binding of a synthetic androgen, methyltrienolone ([3H]-R1881), to brain and pituitary (PIT) cytosol and nuclear extracts was determined in male and female rats. Purified cytosol was prepared from PIT or hypothalamic-preoptic area-amygdala (HPA) and incubated in the presence of 0.1 to 10 nM [3H]-R1881. Scatchard analysis revealed the presence of a single, saturable, high-affinity binding site in PIT cytosol with a dissociation constant (Kd) of 0.42 X 10(-10) M in females and 0.95 X 10(-10) M in intact males. The Kd of HPA cytosol was much less in castrated males [0.47 +/- 0.05 (SEM) X 10(-10)M, n = 7] and females (0.63 +/- 0.1 X 10(-10) M, n = 4) than in intact males (5.8 +/- 1.1 X 10(-10) M, n = 8). Treatment of castrated males with dihydrotestosterone (DHT) for 24 h (250 micrograms/100 g of body weight) increased the Kd of HPA cytosol only slightly (1.6 X 10(-10) M, mean of two replicates). Scatchard analysis of salt-extracted nuclear androgen receptor (ARn) showed a single, high-affinity binding site with similar Kd values in PIT and HPA of intact and castrated, DHT-treated male rats (PIT Kd = 7.3 X 10(-10) M, 9.3 X 10(-10) M; HPA Kd = 1.5 X 10(-9) M, 1.3 X 10(-9) M, respectively). Competition studies involving a range of several radioinert steroids revealed that the binding of [3H]-R1881 to cytosol (ARc) and nuclear extract was specific for androgen receptor when triamcinolone acetonide (10 microM) was added. The ARc and ARn levels were quantified in PIT, preoptic area (POA), hypothalamus (HT), amygdala, hippocampus, and cortex by single point estimation. Significantly (p less than 0.01) greater amounts of ARc were detected in PIT of ovariectomized females (32.7 +/- 2.9 fmol/mg of protein) than in that of orchidectomized males (22.33 +/- 1.6 fmol/mg of protein). The highest levels in the brain were seen in HT and POA. Pituitary ARc in females varied throughout the estrous cycle. Significantly (p less than 0.01) greater amounts were detected on estrus (45.8 +/- 2.2 fmol/mg of protein) and proestrus (39.0 +/- 1.9 fmol/mg of protein) than on diestrus (29.2 +/- 1.5 fmol/mg of protein). These data confirm the existence of specific receptors for androgen in male and female brain and PIT, and suggest an important role for androgen in the control of PIT hormone secretion in the female.     

Properties and compartmentalization of the testicular receptor for 1,25-dihydroxyvitamin D3

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) with properties similar to 1,25-(OH)2D3 receptors in other tissues. The receptor sediments at 3.5 +/- 0.2 S20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH)2D3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH)2D3 (Kd = 3 X 10(-11) M), but low capacity (Nmax = 8 fmol/mg protein) and is specific for 1,25-(OH)2D3 (Affinity: 1,25-(OH)2D3 greater than 1,24(R),25-(OH)3D3 greater than 25-OH-D3 greater than 1 alpha-OH-D3 greater than 24(R),25-(OH)2D3 much greater than 17 beta-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [3H]1,25-(OH)2D3 and at 0 degrees C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature (t1/2 (0 degrees C) much greater than 48 h). At 0 degrees C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 X 10(7) M-1 min-1 and 2 X 10(-5) min-1, respectively. Receptors for 1,25-(OH)2D3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [3H]1,25-(OH)2D3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.     

Role of high-density lipoprotein in transport of circulating bilirubin in rats

The analbuminemic rat strain established by Nagase et al. (Nagase, S., Shimamune, K., and Shumiya, S. (1979) Science 205, 590-591) exhibits hereditary deficiency in albumin biosynthesis. Serum bilirubin concentration is rather lower in homozygous (aa) rats (0.009 +/- 0.002 mg/dl) as compared with heterozygous (Aa) rats (0.047 +/- 0.009 mg/dl) or wild-type Sprague-Dawley (AA) rats (0.034 +/- 0.006 mg/dl) as evidenced by high pressure liquid chromatography analysis of bilirubin. After intravenous administration of various amounts of [heme-3H]hemoglobin in rats, [3H]bilirubin derived from [3H]heme of hemoglobin in vivo is more efficiently excreted into bile in aa rats than in Aa or AA rats. [3H]Bilirubin is exclusively bound with high-density lipoprotein (HDL) in aa rats, and a significant amount of [3H]bilirubin is shown to bind with HDL in Aa or AA rats in vivo. Scatchard plots revealed that [3H]bilirubin is bound with HDL in three binding modes depending on the molar ratio of [3H]bilirubin to HDL: Kd = 0.8 X 10(-7) M (molar ratio, 0.02-0.06), Kd = 1.6 X 10(-6) M (molar ratio, 0.06-0.41), and Kd = 1.2 X 10(-4) M (molar ratio, 0.79-9.02). Even under extreme conditions of excess hemoglobin administration, the molar ratio remains under 0.041; and thus, expected the Kd value would remain around 0.8 X 10(-7) M. Binding of [3H]bilirubin to rat serum albumin revealed two distinct binding modes depending on the molar ratio of [3H]bilirubin to rat serum albumin: Kd = 3.6 X 10(-7) M (molar ratio, 0.03-0.21), and Kd = 5.0 X 10(-6) M (molar ratio, 0.21-2.46). Under physiological conditions in Aa or AA rats, the former mode would be more reliable than the latter. Thus, HDL could bind with approximately 4.5 times higher affinity than rat serum albumin in Aa or AA rats under physiological conditions in vivo.     

BanAI a new isoschizomer of the type II restriction endonuclease HaeIII discovered in a Bacillus anthracis isolate from Amazon Basin

Steroid binding sites with high affinity for progesterone (Kd=40+/-14 nM determined by binding, and Kd=71+/-22 nM determined by displacement studies) and lower affinity for 21-hydroxyprogesterone and for testosterone, but no affinity for estradiol-17beta, onapristone and alpha-naphthoflavone were detected in the enriched plasma membrane fraction of the fungus Rhizopus nigricans. The amount of steroid binding sites is in accordance with the value of B(max)=744+/-151 fmol (mg protein)(-1). In the membrane fraction, progesterone induced about 30% activation of G proteins over basal level, as determined by GTPase activity (EC50=32+/-8 nM) and by the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding rate (EC50=61+/-21 nM). The affinity of receptors for progesterone was substantially decreased in the presence of GTPgammaS and of cholera toxin. Our results suggest the existence of progesterone receptors in the membrane of Rhizopus nigricans and their coupling to G proteins.     

High affinity progesterone binding sites of human uterine microsomal membranes

Microsomal membranes sedimented at 40 000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [3H]progesterone at 4 degrees C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable (Kd approximately 4 X 10(-9) M, concentration of binding sites 400-900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17 alpha-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0-20.0% (R.A. of progesterone 100%). 5 alpha-Dihydroprogesterone and 5 alpha-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.     

Comparisons of gonadotropin binding sites and progesterone concentrations among the corpora lutea of individual pigs

In polyovular species, it is unclear whether the characteristics of each individual corpus luteum (CL), such as mass, progesterone concentration and receptors for luteinizing hormone (LH), are representative of those of its cohorts during the ovarian cycle. The current study was performed 1) to characterize the conditions for estimation of binding parameters for LH receptors in porcine CL, and 2) to compare LH binding sites, luteal progesterone concentrations and luteal masses among CL of ovaries within individual pigs. Gonadotropin binding sites in porcine CL were characterized via specific binding of 125I-human (h) LH to 20,000 X g particulate fractions of luteal tissue. Specific binding was directly proportional to tissue content and was detectable at the lowest content tested (0.5 mg tissue equivalents/tube). Specific uptake of 0.25 ng LH by 5.0 mg tissue equivalents was time- and temperature-dependent; steady-state binding was achieved within 20 h at 37 and 25 degrees C. Binding of LH after 20 h incubation at 37 degrees C (4718 +/- 192 cpm, means +/- SEM) and 25 degrees C (4112 +/- 340 cpm) was greater than that at 4 degrees C (1930 +/- 5 cpm, P less than 0.01). Luteal particulates from individual CL of ovaries collected from four mature nonpregnant pigs (13-23 CL/pig) were incubated with eight concentrations of 125I-hLH. Steady-state binding depended upon hormone concentration until reaching saturation at 2.5 ng 125I-hLH/tube. Scatchard analyses yielded linear plots. Binding capacities for LH ranged among pigs from 0.71 +/- 0.03 to 3.69 +/- 0.13 fmol/mg CL equivalents and receptor affinities (Kd) ranged from 0.92 +/- 0.05 to 4.89 +/- 0.41 X 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone and prostanoid production by bovine binucleate trophoblastic cells

A procedure for preparing highly enriched suspensions of bovine binucleate trophoblastic cells was developed and data showing that these cells produce progesterone, prostacyclin (PGI2), and prostaglandin E2 (PGE2) were obtained. Approximately 200 X 10(6) enzymatically dissociated cells from bovine cotyledons were applied to the surface of a density gradient of 2% to 4% Ficoll-400 using the Wescor CELSEP sedimentation chamber. After 90-120 min of sedimentation at unit gravity, fractions containing binucleate trophoblastic cells were obtained and washed in HEPES-buffered Medium 199. Preparations of 90% to 100% binucleate trophoblastic cells were obtained routinely; viability was 50% to 80%. After incubation at 37 degrees C, concentrations (ng/10(5) cells) of progesterone were greater in those fractions containing binucleate cells than in those containing primarily smaller, mononucleate cells. Total progesterone secreted (mean +/- SEM) after 4 h by 1 X 10(5), 2 X 10(5), 4 X 10(5), 8 X 10(5), and 1.6 X 10(6) binucleate cells was 0.27 +/- 0.03, 1.01 +/- 0.09, 4.02 +/- 0.37, 10.31 +/- 0.92, and 20.96 +/- 2.23 ng, respectively (r = 0.997). Addition of 10% fetal bovine serum (FBS) or normal anestrous cow serum increased (P less than 0.05) production of progesterone by binucleate trophoblastic cells. Luteinizing hormone, follicle-stimulating hormone, prolactin, thyrotropin, and 8-bromo-adenosine 3',5'-cyclic monophosphate had no effect. Binucleate trophoblastic cells also produced PGI2 in relation to number of cells incubated (r = 0.996). Time courses for production of PGI2, PGE2, and progesterone were similar. Aspirin inhibited production of PGI2 and PGE2 by about 50% at a dose of 100 microM; FBS stimulated production of both prostanoids.