The nuclear conversion of pregnenolone to progesterone and subsequent binding to the nuclear progesterone-binding protein in the guinea pig adrenal cortex: a possible regulatory role for the pregnenolone-binding protein.

Progesterone, which is normally produced in the endoplasmic reticulum, was found to be rapidly degraded in the cytosolic fraction of the guinea pig adrenal cortex in vitro. Assuming this finding reflects what happens in vivo raises a question as to the source of progesterone for interacting with a nuclear progesterone-binding protein (P4-BP) that exists in this model system. It was subsequently found that pregnenolone, which in contrast to progesterone is relatively stable in the cytosol, was converted to progesterone by endogenous nuclear 3 beta-ol dehydrogenase. It was also determined that the nuclear-derived progesterone specifically bound to the nuclear P4-BP which is distinct from the classical progesterone receptor. The guinea pig adrenocortical cytosol contains a specific pregnenolone-binding protein (P5-BP) that could be virtue of its pregnenolone binding activity regulate the conversion of pregnenolone to progesterone in the nuclear compartment and thereby reduce the binding of progesterone to the nuclear P4-BP. A partially purified P5-BP preparation markedly inhibited the nuclear conversion of pregnenolone to progesterone and reduced the binding of progesterone to the nuclear P4-BP (P5-BP did not directly inhibit binding of progesterone to the nuclear P4-BP). The ability of P5-BP to inhibit the conversion of pregnenolone to progesterone was destroyed by heat and alkaline phosphatase treatment. The binding of pregnenolone to the P5-BP, as previously reported, is regulated by phosphorylation/dephosphorylation, and alkaline phosphatase-treated P5-BP loses the ability to bind pregnenolone; this process can be reversed by a cytosolic kinase. This provides a mechanism for controlled release of bound steroid. These results suggest that P5-BP regulates the nuclear conversion of pregnenolone to progesterone and thus the binding of progesterone to the nuclear P4-BP.     

Medroxyprogesterone acetate: a steroid with potent progestational activity but low receptor affinity in the guinea pig uterus.

The guinea pig progestin receptor appears to be unique among mammalian receptors studied to date in that it displays a low binding affinity for some biologically potent 17 alpha-substituted progestinss. [3H]Medroxyprogesterone acetate (MPA) was synthesized and used to investigate this dichotomy between binding affinity and biological activity. The comparison of cytoplasmic and nuclear receptor binding characteristics suggested that progesterone and MPA were bound to the same receptor but with different affinities. Following an intravenous injection of 3H-steroids, the plasma level of progesterone was lower than that of MPA at all time points. Correspondingly, for up to 6 hours following steroid administration, progesterone levels were lower in uterine cytoplasm and higher in nuclei than those of MPA. However, by 24 hours, MPA nuclear levels were higher than those of progesterone, in accordance with plasma levels. We conclude that the potent biological activity of MPA relative to progesterone is due in part to its slower rate of metabolism and longer nuclear retention.     

Antibodies to rabbit progesterone receptor: crossreaction with human receptor.

Progesterone receptor from rabbit uterine cytosol was purified to a specific activity of approximately 2 nmol of bound hormone per mg of protein. A goat was immunized with this preparation and, after two injections of 0.7-0.8 nmol, yielded antireceptor antibodies. The antiserum reacted with both cytosolic and nuclear rabbit progesterone receptor and also with progesterone receptor from other rabbit tissues (vagina and pituitary). A crossreaction was observed with progesterone receptors from other mammalian, especially human, tissues (cytosolic receptor from rat and guinea pig uterus, cytosolic receptor from human breast cancer, and nuclear receptor from human endometrium). On the contrary, there was no interaction with a nonmammalian receptor (chicken oviduct progesterone receptor). The antibodies did not crossreact with other rabbit steroid receptors (uterine estradiol receptor and liver glucocorticoid receptor) or with nonreceptor progesterone-binding proteins (transcortin from plasma and uteroglobin from uterine fluid).     

Progesterone-binding proteins: in vitro binding and biological activity of different steroidal ligands.

Progesterone-binding proteins from human, rabbit, sheep and guinea pig myometrial cytosol, all induced with oestradiol, as well as from pregnant guinea pig myometrium and plasma were investigated. The physico-chemical properties of the oestradiol-induced binding proteins were very similar in all the species studied. In all, 63 steroids were tested for their ability to compete with tritiated progesterone for the binding sites on these six proteins and their relative affinities were determined. The studies reveal that the ligand specificities of oestrogen-induced myometrial binding proteins from human, rabbit and sheep are rather similar, whereas that from guinea pig myometrium has different binding characteristics. The properties of the binding proteins from pregnant guinea pig uterus and plasma differ substantially from all of the induced proteins. It is clear from the different physico-chemical characteristics and binding specificities that the oestrogen-induced myometrial protein of the guinea pig is not the same as that which appears in the myometrium and plasma during pregnancy. The binding energies of the well bound progestational compounds were of the order of -12 Kcal/mole, half of which stems from the interaction of the steroid nucleus with the protein. The specific interaction of the protein with the two functional groups, the 3-keto-4-ene system and the acetyl sid chain each contributed-3 Kcal/mole. In the case of the rabbit, sheep and human proteins a 17alpha-ethynyl-17beta-hydroxyl function could replace the acetyl side chain. For a large number of steroids reasonable agreement existed between the degree of binding to the rabbit myometrial protein and in vivo biological activity (Clauberg-McPhail test) in the same species. The data suggest that as far asthe binding aspect is concerned, the rabbit is an appropriate model for assessing the biological activity of compounds under development for human application. The in vitro binding system is also a useful tool to assess whether steroids need to be bio-activated before eliciting a biological response.     

Oestrogen-inducible uterine progesterone receptors. characteristics in the ovariectomized immature and adult hamster.

In order to characterize the uterine progesterone-binding proteins of oestrogen-primed and unprimed, ovariectomized immature and adult golden hamsters, cytosols were incubated with [3H]progesterone and/or other steroids and analyzed by sucrose-glycerol density gradient ultracentrifugation. Progesterone-binding components with sedimentation coefficients of 7S and 4.5S were found in the uterine cytosols, but not in the cytosols from the hypothalamus, pituitary, diaphragm, or small intestine. Oestradiol-17beta markedly elevated the level of 7S uterine receptor and this increase appeared to be due to new receptor synthesis, since actinomycin D and cycloheximide blocked this response. Fifty to 100 mug of oestradiol-17beta per kg body weight was found to promote a maximum increase in the 7S macromolecular component. The 7S receptor showed a tendency to saturate at 1 X 10(-7) M [1,2-3H]progesterone, indicating limited capacity. At a molar ratio of 100:1, unlabeled progesterone competed effectively for 7S and 4,5S [3H]progesterone binding, whereas 5alpha-pregnane-3,20-dione, oestradiol-17beta and testosterone did not. Moreover, [1,2-3H]cortisol and [1,2-3H]corticosterone did not bind to the 7S receptor, implying steroid specificity. CI-628, a non-steroid oestrogen antagonist, completely prevented [6,7-3H]oestradiol-17beta binding to its 8.5S uterine cytosol receptor, but was entirely without effect on 7S and 4.5S [3H]progesterone binding. Pronase, but not DNase or RNase, abolished 7S and 4.5S progesterone binding, suggesting that the binders are at least in part protein. Protamine sulphate and p-hydroxymercuribenzoate also obliterated 7S binding, implying that this receptor may be an acidic protein which contains sulfhydryl groups that are necessary for progesterone binding.     

Progesterone-binding globulin and testosterone-binding activity in guinea pig serum during pregnancy: relationship to progesterone and oestrogens.

Progesterone levels in serum have been determined throughout pregnancy in guinea-pigs by a competitive protein-binding technique, using pregnant guinea pig plasma protein as binding agent. The concentrations of this protein (progesterone-binding globulin, PBG) as well as the testosterone-binding activity (TBA) have been quantitated by means of equilibrium dialysis. In order to correlate these parameters to the endogenous oestrogen production, the urinary oestrogen excretion was recorded. The concentration of progesterone, PBG and TBA showed a sharp rise on days 14-18 of gestation, reaching a maximal level on days 30-44. The progesterone level thereafter declined significantly towards parturition, followed by a sharp drop post partum. PBG and TBA followed a similar course, the decline towards parturition, however, being non significant. Neither progesterone, PBG nor TBA were significantly correlated to the urinary oestrogen excretion. The concentration of PGB was remarkably high, amounting to 1-2 x 10(-5) M during most of the guinea pig pregnancy. This binding capacity generally exceeded the endogenous progesterone concentration by a factor of 20, as calculated on a molar basis. A mean of 64% of the binding sites was available for testosterone binding, corresponding to a free binding capacity of 400 mug testosterone per 100 ml.     

Progesterone receptor in the chick bursa of fabricius: characterization and immunohistochemical localization

A high affinity progesterone-binding site was studied in the chick bursa of Fabricius. The dissociation constant for progesterone was 1.4 nM, and the concentration of progesterone-binding sites increased with estradiol treatment. In estradiol-treated bursas, the receptor concentration was about 240 fmol/mg protein. The binding site was specific for progestins, with the following order of affinities: ORG 2058 greater than progesterone greater than promegestone. Androgens, dexamethasone, and estradiol were weak competitors for progesterone binding in the bursa cytosols from estradiol-treated chicks. Immunoglobulin G fraction of antiserum (immunoglobulin G-RB) raised in rabbit against the B-subunit of chick oviduct progesterone receptor (PR) was used for an immunohistochemical study. The PR was found only in the interfollicular cells, which were most probably nonlymphoid cells. Staining was localized exclusively in the elongated nuclei of these cells. No staining was seen in the bursal epithelium or inside the lymphoid follicles. The results indicate that the interfollicular cells of the bursa contain specific PRs which are under estrogen regulation as in the oviduct. Thus, these cells might be under direct progesterone regulation.     

Cytoplasmic and nuclear progesterone receptors in the guinea pig uterus.

Although progesterone receptors have been studied in the uterine cytoplasm of many species, relatively little was known about the nuclear content of these binding proteins. In the present study, a nuclear progesterone receptor was deteced in the guinea pig uterus. The binding of progesterone to the nuclear receptor was hormone and tissue specific. Furthermore, the nuclear localization of the progesterone receptor complex in the uterus was both time and temperature dependent. Since the nuclear receptor was extracted in high salt buffer, the effects of KCl on several physical properties of the cytoplasmic and nuclear binders were studied. In the presence of high salt, cytosol and nuclear receptors were virtually indistinguishable. These proteins were clearly distinguished upon removal of the KCl by rapid dialysis: the nuclear receptor had a slower sedimentation rate, a faster rate of dissociation and a higher binding affinity than did the cytosol receptor for progesterone. We conclude that the cytosol and nuclear receptors for progesterone in the guinea pig uterus are distinct macromolecules. These observations are consistent with the postulate that the cytoplasmic receptor is a precursor of that in the nucleus.     

Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures.

Oviduct cells from estradiol-treated chicks were grown in primary culture. After 3-5 days of culture in medium containing estradiol, 90% of the cellular progesterone binding sites were detected in the cytosol. After exposure to [3H]progesterone at 37 degrees C, 80% of the progesterone binding sites were found in nuclear fractions. Progesterone receptor phosphorylation was assessed after incubating the cells with [32P]orthophosphate. Receptor components were immunoprecipitated with a specific polyclonal antibody (IgG-G3) and analyzed by NaDodSO4/PAGE and autoradiography. In the cytosol, constant amounts of 32P-labeled 110-kDa subunit (the B subunit, one of the progesterone-binding components of the receptor) and of the non-steroid-binding heat shock protein hsp90 were found, whether cells had been exposed to progesterone or not. No 32P-labeled 79-kDa subunit (the A subunit, another progesterone-binding subunit) was detected. Various procedures were used to solubilize nuclear progesterone receptor (0.5 M KCl, micrococcal nuclease, NaDodSO4), and in no case was 32P-labeled B subunit detected in the extracts. However, nonradioactive B subunit was detected by immunoblot in a nuclear KCl extract of progesterone-treated cells. These results suggest that the fraction of the B subunit that becomes strongly attached to nuclear structures is not phosphorylated upon exposure of cells to progesterone.     

Cytoplasmic progesterone receptors in uterine tissue of the snapping turtle (Chelydra serpentina)

A high affinity progesterone-binding component was detected in the cytosol of the uterus of the snapping turtle, Chelydra serpentina. Density gradient centrifugation indicated that binding of [3H]progesterone and [3H]promegestone (R5020) was to a fraction with a heavier sedimentation coefficient than bovine serum albumin (BSA) appearing as a broader peak in the 6-7 S region; it was not affected by excess cortisol. Another binding peak, lighter than BSA and appearing with [3H]R5020 and [3H]progesterone near the 4 S region, was affected by excess cortisol. Excess progesterone decreased both the heavier and lighter peaks. Analysis of steroid specificity revealed that, of the natural steroids, progesterone had the highest affinity for the uterine cytosol. This was followed by deoxycorticosterone, 5 alpha-pregnanedione, testosterone, oestradiol-17 beta, corticosterone, 5 alpha-dihydrotestosterone and cortisol. Non-linear regression analysis of saturation data indicated the presence of two classes of high affinity binding sites: progesterone-binding sites (R-sites) with equilibrium association constants (Ka) of 2.9 +/- 0.28 litres/nmol (mean +/- 95% confidence limit) for [3H]R5020 and 0.34 +/- 0.20 litres/nmol for [3H]progesterone, and corticosteroid-binding globulin-like sites (G-sites) with Ka of 4.5 +/- 1.6 litres/nmol for progesterone. The concentration of R-sites was between 0.66 +/- 0.10 and 2.6 +/- 0.55 pmol/mg protein while that of G-sites was between 0.73 +/- 0.05 and 5.0 +/- 0.27 pmol/mg protein. DEAE-cellulose filtration assay also confirmed the presence of R-sites and G-sites in the cytosol. R-sites were detectable without oestrogen priming during the preovulatory and vitellogenic phases (low progesterone, high oestrogen concentrations) when the ovarian follicles are mature (18-22 mm diameter).(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors.

A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgG1 and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.     

Immunochemical studies of guinea pig progesterone-binding plasma protein.

An antiserum specific for guinea pig progesterone-binding plasma protein (PBP) has been prepared. Using a very sensitive immunoenzymatic assay, PBP could be detected not only in pregnant guinea pig plasma (970 microgram/ml of plasma at day 40--60 of pregnancy), but also in the plasma of fetuses (2.77 micrograms/ml), umbilical arteries (1.79 micrograms/ml), umbilical vein (2.90 micrograms/ml), and in amniotic fluid (0.47 microgram/ml). The protein was also found in low concentration in the plasma of nonpregnant females (2.10 micrograms/ml) and males (1.56 micrograms/ml). The antiserum was used to search for immunological similarities between various steroid-binding proteins. No cross reaction was found with cavian or human corticosteroid-binding globulin and human sex steroid-binding plasma protein. There was no cross reaction between guinea pig PBP and PBP of other pregnant hystricomorphs (viscacha, degu, and coypu). Moreover, no evidence was found of an interaction between guinea pig uterine progesterone receptor and the anti-PBP antiserum.     

Search for a progesterone-binding protein in secretory granules of the ovine corpus luteum

The hypothesis that electron-dense granules present in the corpus luteum contain progesterone in a protein-bound form was examined using differential and sucrose gradient centrifugation. Ovine luteal tissue was fractionated by differential centrifugation at 1,000 X g (P1 pellet), 10,000 X g (P2), and 82,000 X g (P3; supernatant S3). Samples of P2, P3, and S3 were further fractionated o 20-40% (P2 and P3) or 5-25% (S3) sucrose gradients and examined for progesterone-binding activity by measuring the progesterone content and/or the specific binding of [3H]progesterone of sucrose gradient samples. In addition, saturation binding assays were performed with steroid-free samples of P2 and S3. Saturable binding of progesterone was not found in P2, the fraction containing electron-dense granules. In S3, two progesterone-binding proteins with sedimentation rates of 3.2S and 8.6S and an affinity of 7.1 X 10(5) M-1 for progesterone were detected. The sedimentation behavior of these proteins was distinct from that of ovine plasma transcortin, a 4S protein. The view that a binding protein is released into the interstitial fluid during the exocytosis of granules was examined by measuring the progesterone-binding activity of protein released by slices of corpus luteum in vitro. No binding activity was found. The results of this investigation do not support the hypothesis that putative progesterone-secreting granules observed in luteal tissue contain a binding protein     

Determination of human blood and urine progestins by the method of competitive protein binding on the basis of guinea pig transprogestin

The use as a testing system of transprogestin or of progesterone-binding protein (obtained from a pregnant guinea pig) with a high specificity to progesterone and a number of its affiliated metabolites increased the sensitivity of the method considerably, permitting to determine progestins in the blood and urine of nonpregnant women (at various phases of the sexual cycle) and in men. Dynamics of the given progestins in the urine correlates with the changes of their blood level and permits complete and objective assessment of the ovarian and placental functions in women with various physiological conditions.     

Steroid hormone-binding proteins in blood plasma

Two types of proteins form dissociable complexes with the circulating steroid hormones in blood serum: albumin, the most abundant plasma protein, and the highly specific glycoproteins that occur in low concentrations: corticosteroid-binding globulin (CBG or transcortin), sex steroid-binding protein (SBP) and progesterone-binding globulin (PBG). Albumin interacts with low affinity, predominantly by hydrophobic bonding; the affinity constants of the steroid complexes with the specific globulins are higher by several orders of magnitude. CBG and SBP have molecular weights similar to that of albumin; PBG appears to be larger. A fairly general characteristic of these steroid-binding glycoproteins is their tendency to polymerize. Rat CBG forms dimers, tetramers and octamers upon removal of steroid from the complex; stoichiometric recombination with corticosterone completely reverses the polymerization. Estrogenic hormones increase the level of CBG and of SBP; androgenic hormones have the opposite effect. A CBG-depressing influence of corticosteroid hormones has been observed in the rat; adrenalectomized rats have increased CBG activity. The thyroid-stimulating hormone of the pituitary is required for the estrogen-induced rise of CBG synthesis in the rat; the effect is mediated by the thyroid gland. In accordance, thyroidectomy reduces the CBG activity whereas administration of thyroxin results in an increase. In contrast to all other species investigated, guinea pig serum contains separate proteins specific for binding of corticosteroid (CBG) and of progesterone (PBG). PBG levels increase about hundred-fold in pregnancy. PBG is a glycoprotein of χ₂- or β-globulin mobility with an approximate molecular weight of 100,000 and an association constant for progesterone-binding higher than any of the other steroid-serum protein complexes. Association with serum proteins suppresses the biological activity of steroid hormones. Bound hormone in blood constitutes an inactive pool which protects the hormone from metabolic and chemical alterations and provides, by reversible dissociation, a buffer against sudden changes in active hormone concentrations.     

Primary structure and functional expression of a guinea pig kappa opioid (dynorphin) receptor.

A full-length cDNA encoding the guinea pig kappa opioid (dynorphin) receptor has been isolated. The deduced protein contains 380 aa and seven hydrophobic alpha-helices characteristic of the G protein-coupled receptors. This receptor is 90% identical to the mouse and rat kappa receptors, with the greatest level of divergence in the N-terminal region. When expressed in COS-7 cells, the receptor displays high affinity and stereospecificity toward dynorphin peptides and other kappa-selective opioid ligands such as U50, 488. It does not bind the mu- and delta-selective opioid ligands. The expressed receptor is functionally coupled to G protein(s) to inhibit adenylyl cyclase and Ca2+ channels. The guinea pig kappa receptor mRNA is expressed in many brain areas, including the cerebellum, a pattern that agrees well with autoradiographic maps of classical guinea pig kappa binding sites. Species differences in the pharmacology and mRNA distribution between the cloned guinea pig and rat kappa receptors may be worthy of further examination.     

Close examination of steroidogenesis disorders in a DOC- and progesterone-producing adrenocortical carcinoma

We report a case of hypertension, hypokalemia, and amenorrhea accompanying an adrenocortical carcinoma. A 27-year-old woman was admitted to our hospital because of a left adrenal incidentaloma. She presented with hypertension, hypokalemia, and amenorrhea; her plasma renin activity was low, but her plasma aldosterone concentration was normal, as were cortisol and androgens. By contrast, her serum concentrations of deoxycorticosterone (DOC), 18-hydroxydeoxycorticosterone, and progesterone were high, and her urinary steroid profile showed elevated secretion of 17-deoxysteroids and 11-deoxysteroids (progesterone, DOC, 11-dehydrocorticosterone, and 11-deoxycortisol), and 3β-hydroxy 5-en steroids (pregnenolone, 17-hydroxypregnenolone, and DHEA). Decreased ratios of metabolites of (1) 17-OHpregnenolone to pregnenolone and 17-OHprogesterone to progesterone, (2) corticosterone to DOC and cortisol to 11-deoxycortisol, and (3) progesterone to pregnenolone, 17-OHprogesterone to 17-OHpregnenolone and androstenedione to DHEA suggested the impairment of 17α-hydroxylase, 11β-hydroxylase, and 3β-HSD activities, respectively. After the tumor was removed, levels of all adrenal steroids were normalized. Based on the Weiss criteria, the tumor was diagnosed as an adrenocortical carcinoma, and immunohistochemical analysis of steroidogenic enzymes revealed disorganized steroidogenesis in the tumor tissue. With adrenocortical carcinomas, heterogeneity of individual steroid producing enzymes within tumor cells can lead to hypersecretion of various steroid intermediates, even when steroid end products are within the normal range.