Cytosolic Progesterone Receptor Research Articles

The effect of RU 38,486 on progesterone and oestrogen receptor concentrations in the decidua and placenta in early pregnancy

Decidual and placental concentrations of progesterone and oestrogen receptors in 15 women having medical terminations of pregnancy with a combination of the anti-progesterone steroid RU 38,486 and prostaglandin E1 analogue have been compared with 10 matched controls undergoing surgical aspiration of pregnancy. In the patients treated with RU 38,486, the mean (SD) decidual cytosolic and total progesterone receptor concentrations of 4.3 (5.6) and 7.6 (7.1) fmol/microgram DNA respectively were significantly lower than the levels of 18.5 (14.4) and 22.3 (17.4) fmol/microgram DNA in the control patients. Progesterone receptor concentrations in the placenta were very low in both groups. No significant change in oestrogen receptor concentrations occurred in the decidua or placenta after treatment with RU 38,486. These data indicate that there may be down-regulation of decidual progesterone receptor concentrations following RU 38,486 treatment.

Synergistic effects of progesterone and oestradiol on rat LH subunit mRNA

The effects of oestradiol and progesterone on LH-subunit mRNA levels were investigated in ovariectomized rats. Four weeks after ovariectomy, rats were implanted with silicone elastomer capsules containing oestradiol and/or injected daily with progesterone in oil (5 mg/rat) for 8 days. The levels of pituitary mRNA encoding alpha and LH-beta were determined using direct hybridization with specific [32P]cDNA probes. After oestradiol implantation in ovariectomized rats, both alpha and LH-beta mRNA decreased with time, with maximum inhibition after 6-8 days of treatment. Progesterone injected alone did not show any effect on alpha and LH-beta mRNA. Cytosolic progesterone receptors, determined using [3H]methyl-17 alpha-progesterone as ligand, were undetectable in control ovariectomized rats. In contrast, 2 days after oestradiol implantation, the number of receptors increased to 287.5 +/- 35.4 (S.E.M.) fmol/pituitary and reached a plateau of 400 +/- 21.8 fmol/pituitary after 4 days. The effects of progesterone were therefore examined by first implanting ovariectomized rats with oestradiol to induce progesterone receptors and then injecting progesterone daily for a further period of 6 days. As a result of this treatment, progesterone induced a decrease in the pituitary gland contents of both alpha and LH-beta mRNAs, and LH release was significantly greater than that observed in the group receiving oestradiol alone. Moreover, the mRNA levels in the animals treated with oestradiol plus progesterone were lower after 8 days of treatment than those observed in ovariectomized rats treated with a tenfold higher dose of oestradiol alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical Detection of Progesterone Receptor in Breast Carcinoma: Comparison of Two Monoclonal Antibodies

Immunocytochemical progesterone receptor (PR) assays employing two different monoclonal antireceptor antibodies (mPRi, JZB39) were compared with each other and with the dextran-coated charcoal assay (DCC) in human breast carcinoma. The immunocytochemical analyses were semiquantitatively scored (histoscore) based on relative staining intensity and the percentage of positively stained carcinoma cell nuclei. The immunocytochemically determined PR status agreed with that obtained by the DCC assay in 76% of the cases when the monoclonal antibody mPRi was used. The agreement was slightly better (82%) with the immunocytochemical assay using the JZB39 antibody. The semiquantitative histoscores correlated significantly with log-transformed cytosol PR concentrations in the DCC assay (mPRi, R = 0.543; JZB39, R = 0.618; P less than 0.0001 for both). The histoscores obtained with the mPRi and JZB39 antibodies were highly correlated with each other in adjacent frozen sections (R = 0.84, P less than 0.0001) the latter antibody giving somewhat higher histoscores. These results further validate the use of immunocytochemical PR assays for routine diagnostic purposes, especially when sufficient material is not available for DCC assays.

β-Adrenergic Receptors in the Rat Mammary Gland during Pregnancy and Lactation: Characterization, Distribution, and Coupling to Adenylate Cyclase

To investigate a possible role of catecholamines in mammary gland growth and differentiation, we have studied the characteristics of a specific beta-adrenergic receptor population during the different reproductive phases of the rat mammary gland, namely pregnancy and lactation. The functional response to mammary beta-adrenergic receptor stimulation was assessed by measurement of adenylate cyclase activity during the same physiological states of the gland [125I]Cyanopindolol (CYP) binds specifically to membranes prepared from lactating mammary glands. Scatchard analysis of the binding data shows the presence of a single class of high affinity sites, with an apparent Kd value of 25.0 +/- 0.4 pM and a binding capacity of 32.5 +/- 1.2 fmol/mg protein in lactating mammary glands at random stages of lactation. The order of potency of a series of agonists to compete for [125I]CYP binding is consistent with the interactions with a beta 2-subtype receptor. The binding of [125I]CYP to mammary glands also shows a marked stereoselectivity; the (-)isomers of isoproterenol and propranolol are more potent than their respective enantiomers. The radioautographic localization of [125I]CYP reveals the presence of specific beta-adrenergic receptors in the epithelial cells, alveoles, ducts, as well as adipocytes. [125I]CYP binding shows a 2- to 3-fold increase during pregnancy. Such a result correlates with parallel increases in stimulation of adenylate cyclase activity, the cytosolic progesterone receptor concentration, as well as plasma 17 beta-estradiol and progesterone levels. At parturition, a sharp decline in beta-adrenergic receptor concentration is observed, a finding concomitant with a drop in progesterone receptor levels as well as plasma estradiol and progesterone concentrations. During midlactation, beta-adrenergic receptors reach their maximal levels. The presence of specific beta-adrenergic receptors functionally coupled to the adenylate cyclase system and the marked changes in receptor capacity and distribution measured during the different physiological states of the mammary gland suggest that the mammary beta-adrenergic receptors are highly sensitive to changes in the hormonal milieu and provide a mechanism for a direct catecholaminergic influence on mammary gland growth and differentiation.

Hormone receptor status in human endometrial adenocarcinoma.

Steroid receptor levels were determined in 196 samples of endometrial adenocarcinoma: cytosol estradiol receptors (ERc) were measured in 171 samples, cytosol progesterone receptors (PRc) in all samples; nuclear estradiol receptors (ERn) and nuclear progesterone receptors (PRn) in 68 samples; total estradiol receptors (ERt = ERc plus ERn) and total progesterone receptors (PRt = PRc plus PRn) were measured in 68 samples. The ERc levels were 88.2 +/- 8.9 (mean +/- SEM) and ERn were 94.4 +/- 15.6 fmol/mg protein; PRc levels were 197.9 +/- 25.9 and PRn 178.3 +/- 55.9 fmol/mg protein. The ERt levels were 162.6 +/- 23.2 and PRt 249.8 +/- 75.7 fmol/mg protein. The presence of PRc was related to the ERc levels according to the cut-off used. Estradiol receptors (ER) and progesterone receptors (PR) were present in the cytoplasmic and nuclear fractions in 60.2% and 36.8% of cases, respectively. The simultaneous presence of both ERt and PRt was observed only in 27.9% of cases. In the normal endometrium ERc and PRc were negatively correlated (r = -0.525, P less than 0.005), whereas in endometrial adenocarcinoma the correlation was positive (r = 0.491, P less than 0.001). In contrast with the normal endometrium the correlation between ERc and ERn was positive (r = 0.582, P less than 0.001) in tumor tissue. In neoplastic tissue Scatchard analysis showed a single class of specific ERc sites with a dissociation constant (Kd) of 1.39 +/- 0.8 X 10(-9) mol/l, one tenth of that found in the normal premenopausal endometrium. Qualitative and quantitative analysis of the receptor status showed that in 30% to 40% of cases studied the behavior of the neoplastic cell was similar to that found in the normal endometrial cell. In a 4-year follow-up of patients affected by endometrial adenocarcinoma there is better survival in the groups of patients with a simultaneous presence of ERt and PRt than in the group with their absence.

Effects of the β-isomer of hexachlorocyclohexane on estrogen-sensitive human mammary tumor cells

The effects of the β-isomer of hexachlorocyclohexane (β-HCH) on the induction of the cytosolic progesterone receptor (PgRc), on the redistribution of the estrogen receptor (ER), and its affinity for ER were investigated in the estrogen-sensitive human mammary tumor cell line MCF-7. The effects of β-HCH were compared to those of estradiol-17β (E 2) and 2,4,6-trichlorophenol (TCP), a major urinary metabolite of β-HCH in rats. β-HCH in concentrations higher than 1 μ m caused induction of PgRc whereas TCP was essentially without effect up to a cytotoxic concentration of 100 μ m. Furthermore, β-HCH (10 μ m) caused redistribution of ER, i.e., decrease of cytosolic ER concentration and increase of nuclear ER concentration, in a way similar to E 2. In contrast to this, β-HCH up to a molar excess of 6 × 10 4 (concentration 30 μ m) caused no significant displacement of [ 3H]E 2 from ER indicating that β-HCH has no substantial affinity for ER. It is concluded that β-HCH has estrogenic properties which are at variance with the failure to demonstrate binding of β-HCH to ER.

Tamoxifen decreases the estradiol induced progesterone receptors by interfering with nuclear estrogen receptor accumulation

The retention time of the estrogen receptor in the nucleus of target cells after antiestrogen treatment has been shown to be longer than after estradiol. This paper describes the accumulation of nuclear estrogen receptors and the obtention of estrogenic responses (i.e. synthesis of cytosolic progesterone receptors and DNA) in the rat uterus after tamoxifen treatment in the presence or absence of estradiol. One-week ovariectomized adult rats were implanted with a silicone elastomer capsule containing corn oil or 25 μg estradiol/capsule (0 h). 48 h after implantation rats were injected with corn oil or 2 mg tamoxifen/kg and decapitated at 72,96 or 120 h after implantation. In parallel experiments the implants were removed just before the injections of tamoxifen or oil. Tamoxifen injected into rats implanted with oil increased both the occupied nuclear receptors and the progesterone receptors at 96 h. In rats implanted with estradiol, tamoxifen did not increase the occupied nuclear receptors and decreased the levels of progesterone receptor and DNA at 96 h. In rats whose estradiol implants were removed at 48 h tamoxifen did not change the level of occupied nuclear receptors at 72 h but it increased them abruptly at 96 and 120 h. In these rats progesterone receptors decreased at 72 h but they increased at 96 and 120 h, and DNA decreased at 120 h to a lower level than before implantation. The results suggest that when estradiol is acting, tamoxifen is not able to increase the level of occupied estrogen receptor and it acts as an antiestrogen by decreasing the high level of progesterone receptors previously induced by estradiol. When estradiol is not acting tamoxifen behaves as a partial estrogen agonist by inducing progesterone receptors. However, the antiestrogenic action of tamoxifen on the rat uterus DNA does not seem to be affected by estradiol.

Cytoplasmic steroid receptors in tumoral and “normal” endometrial samples from symmetrical uterine zones

Cytosol progesterone (PgR) and estradiol (E 2R) receptors were quantified simultaneously in “normal” and tumoral endometrium samples, located symmetrically on the longitudinal axis of the uterine cavity. With this experimental model two different groups of patients were detected. In the first group (7 of 10 women), the endometrial carcinoma had a greater cytosolic concentration of PgR than the corresponding “normal” endometrium, both kinds of tissue being affected by the same circulating hormonal “environment” peculiar to each patient. The opposite occurs in the other group (3 of 10 women), since the “normal” endometrium was found to be “richer” in receptors than the tumoral endometrium. It is suggested that this difference in the capacity of the tumor for synthesizing PgR and even E 2R as compared to the “normal” endometrium may be a marker which improves selection of patients who will be more likely to respond favorably to endocrine therapy.

Temporal effects of progesterone inhibition of occupied nuclear oestrogen receptor retention in the rat uterus.

Previous studies have shown that progesterone rapidly inhibits retention of uterine nuclear oestrogen receptor in several mammalian species. This effect of progesterone may constitute a general mechanism by which progesterone modulates oestrogen action. The objective of the present study was to examine the temporal pattern of progesterone inhibition of retention of occupied nuclear oestrogen receptors in the rat uterus at various sustained serum concentrations of progesterone. Silicone elastomer implants (1 cm) packed with crystalline oestrogen were placed s.c. in the flank region of ovariectomized adult rats. Twenty-four hours after placement of the implants, animals were either injected s.c. with 5 mg progesterone in corn oil every 24 h, treated with 2 x 5 cm implants of progesterone, or treated with 1 x 5 cm silicone elastomer implants of progesterone. Serum concentrations of progesterone at the time of necropsy were 0.47 +/- 0.02, 0.18 +/- 0.02 and 0.10 +/- 0.01 mumol/l respectively. Control animals were given oestrogen implants alone and had a serum progesterone level of 0.03 +/- 0.01 mumol/l. Occupied nuclear oestrogen receptor and cytosolic oestrogen and progesterone receptor levels (pmol/uterus) were measured between 0 and 48 h following progesterone treatment. Cytosolic progesterone receptor levels were suppressed similarly in all progesterone-treated groups compared with controls given oestrogen alone throughout the 48-h test period. Cytosolic oestrogen receptor levels were significantly suppressed at 12 h following progesterone treatment in all groups. Except for the highest (pharmacological) serum progesterone concentration, cytosolic oestrogen receptor exhibited a replenishment phase between 12 and 48 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of progestin-specific response in the chicken oviduct.

Avidin is a host acute defense protein induced by progestins and by inflammation caused by injurious factors such as microbes, viruses, toxic factors or tissue trauma. In the reproductive tract of egg-laying vertebrates avidin has evolved into a progestin-dependent secretory protein involved in anti-microbial action through its biotin avidity. For "progestin-dependent avidin" production, cellular differentiation by estrogen is necessary. In contrast, the expression of "progestin-independent or inflammation-induced avidin" does not require differentiation. Many cell types such as macrophages, heterophils and fibroblasts can produce avidin after non-specific cellular injuries. The wide distribution of avidin in avian, reptilian and amphibian species could be explained on the basis of its vital functions such as antimicrobial or antifungal, metabolic and immunomodulatory actions. The ontogeny of the progestin-dependent avidin synthesis is a complex event involving oviductal differentiation by steroid hormones leading to a specific gene expression. The first phase in oviductal differentiation by estrogens is characterized by a new chromatin organization and by an infiltration of progesterone receptor (PR)-containing mesenchymal cells into the subepithelial mucosa leading to epithelial cell differentiation ("mesenchymal and epithelial cell interaction"). The second phase in the differentiation of progestin-induced response is dependent on the presence of PR in the secretory cells. Two kinds of PR expression occur in the oviduct. The first is a "constitutive PR" and is found in the epithelial, submucosal and peritoneal cells of the immature chick oviduct without steroid treatment, and the second is an "inducible PR" found especially in the mucosal mesenchymal and smooth muscle cells. Avidin production requires PR in the target cells, but not all PR-containing cells can produce avidin. Therefore, in addition to PR, other transcription factors are needed to define the target cell specificity of the response to progestins. Earlier biochemical studies suggested that cytosolic and/or nuclear unoccupied PR was complexed as an 8 S form with the heat shock protein 90 (hsp90). Our immunohistochemical results, however, indicate that PR in vivo is not bound to hsp90, which is located entirely in the cytoplasm, whereas PR is an entirely nuclear protein in both ligand-occupied and unoccupied forms. Therefore, we assume that PR is a monomeric (4S) or homodimeric (5S) (chromatin?) protein associated to DNA. Ligand binding to PR appears to lead to a conformational change, dimer formation, tighter binding to PRE (progesterone responsive element) and to transcription factors, phosphorylation and proteolysis of PR as well as a chromatin change.(ABSTRACT TRUNCATED AT 400 WORDS)

Assessing the early luteal phase in in vitro fertilization cycles: relationships between plasma steroids, endometrial receptors, and endometrial histology

The corpus luteum-endometrial unit was investigated in in vitro fertilization (IVF) cycles using endocrine, morphologic, and biochemical measurements on the day normally scheduled for embryo transfer (day 16), in 12 stimulated and 4 natural cycles. Advanced endometrial histologic maturity was recorded in 9 of the 12 stimulated cycles. No in-phase endometria were seen when the preovulatory plasma estradiol (E2) was greater than 500 pg/ml or the day 16 plasma progesterone (P) greater than 10 ng/ml in natural or stimulated cycles. Significant negative correlations were noted between both preovulatory E2 and day 16 P and the concentration of cytosolic progesterone receptor (PRc). Advanced endometrial maturity tended to be associated with low concentrations of PRc. Regardless of endometrial maturity, the natural cycle was characterized by low cytosolic E2 receptors (ERc) and high PRc, whereas the concentration of both receptors was usually greatly reduced in stimulated cycles. It is concluded that the advanced endometrial maturation observed in stimulated IVF cycles is a consequence of the production of supraphysiologic levels of sex steroids by the corpus luteum that cause profound modifications of endometrial receptor dynamics.

Comparative effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and progesterone on estrogenic responses in rats

The relative activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and progesterone were determined in the 25 day old female Long Evans rats. Both 2,3,7,8-TCDD and progesterone treatment result in significant decreases in nuclear and cytosolic progesterone and estrogen receptor (PR and ER). The major difference between the two compounds is associated with the relative persistence of the effects of 2,3,7,8-TCDD. Using an in vitro uterine strip assay, similar effects on ER and PR levels were observed following 2,3,7,8-TCDD treatment on the uteri. Protein and RNA synthesis inhibitors, namely puromycin, cycloheximide and actinomycin D were preincubated with the uteri prior to progesterone or 2,3,7,8-TCDD treatment. Previous studies by Leavitt and coworkers suggested that progesterone-induced decreases in nuclear ER levels were dependent on both RNA and protein synthesis. With 2,3,7,8-TCDD, the protein synthesis inhibitors did not prevent the observed decreases in ER and PR levels, whereas the RNA synthesis inhibitor actinomycin D inhibited the effects of 2,3,7,8-TCDD on uterine ER and PR levels. These results suggest that the mechanism of 2,3,7,8-TCDD-mediated decreases in ER and PR levels is different from that of progesterone.

Tissue specific effects of progesterone on progesterone and estrogen receptors in the female urogenital tract

The effect of progesterone administration on progesterone and estrogen receptors in the uterus, vagina and urethra of rabbits was studied. After 24 h of progesterone treatment the concentration of cytosolic progesterone receptors decreased to about 25% of the control value in the uterus, whereas no significant change in receptor concentration was observed in the vagina or the urethra. The concentration of the nuclear progesterone receptor did not change in any of the three tissues studied. The apparent dissociation constant ( K d) of nuclear progesterone receptor increased after progesterone treatment in all three tissues. Although the K d of the cytosolic progesterone receptor also increased in all tissues, the difference was significant for only the vagina and urethra. The concentration of cytosolic estrogen receptors in the uterus decreased significantly ( P < 0.001) after progesterone treatment whereas the K d value increased slightly ( P < 0.05). In vagina or the urethra, there was no change in either estrogen receptor concentration or K d values after progesterone treatment. These data clearly showed that the reduction by progesterone of progesterone and estrogen receptor concentrations occurs only in the uterus and not in the vagina or the urethra.

The antiprogesterone RU486 stabilizes the heterooligomeric, non-DNA-binding, 8S-form of the rabbit uterus cytosol progesterone receptor

The salt-induced (0.3 M KCl) transformation of the non-transformed, heterooligomeric 8S-form of the rabbit uterus cytosol progesterone receptor (PR) was analyzed by density gradient ultracentrifugation (8S → 4S conversion) and DNA-cellulose chromatography (non-binding → binding forms). After 1 h treatment at 2 C, > 90% of agonist (R5020 or Org2058)-PR complexes were transformed, contrary to antagonist (RU486)-PR complexes, which did not undergo any transformation. Thus, there is stabilization of the non-transformed receptor form by RU486 as compared to the effect of agonist binding. The hydrodynamic parameters of both agonist-and antagonist-bound non-transformed receptors were similar and the calculated Mr were ≈ 283,000 and ≈ 293,000, respectively. In both cases, purification indicated the presence of a 90-kD non-hormone-binding protein associated with the hormone binding unit(s). Transformation of RU486-PR complexes occurred after exposure to high salt at increased temperature and was correlated to the dissociation of the 90-kD protein from the receptor. Both agonist- and antagonist-bound transformed forms of PR had apparent similar affinities for DNA-cellulose. Molybdate-stabilized and KCl-treated RU486-PR complexes were more stable, as assessed by steroid binding, than the corresponding R5020-PR complexes, arguing in favor of a stabilizing effect of both the 90-kD protein and RU486 against inactivation. These cell-free experiments support the concept that RU486 in the rabbit uterus system stabilizes the 8S non-DNA binding, non-transformed form of the receptor at low temperature. The possibility that impaired dissociation of the heterooligomeric receptor form is involved in the antiprogesterone activity of RU486 is discussed.

In vitro binding of progesterone, cronolone and medroxyprogesterone acetate to uterine progesterone receptors of sheep, rabbit and mouse

Various aspects of the binding of the synthetic progesteongens, cronolone (9 alpha-fluoro-11 beta-hydroxy-17 alpha-acetoxypregn-4-ene-3,20-dione) and medroxyprogesterone acetate (6 alpha-methyl-17 alpha-acetoxypregn-4-ene-3,20-dione, MAP) to uterine cytosol progesterone receptors of the sheep, rabbit and mouse were studied, in an attempt to explain interesting species differences in the biological activity of these steroids. For the sheep, data for binding-site concentration, relative binding affinity (RBA), dissociation constant (Kd) and rates of association and dissociation indicate specific binding of cronolone to the progesterone receptor and these would seem to explain in part the high progestational activity of cronolone in this species. By contrast, with the mouse, there was only a low level of specific binding of cronolone and this appears to explain its inability to maintain pregnancy in this species. Results for the binding activity of cronolone in rabbit uterus were similar to those for the sheep and thus inability of cronolone to maintain pregnancy in the rabbit is not explained by a failure to bind the progesterone receptor. Species differences in binding to the progesterone receptor were also seen with MAP where the RBA, with respect to progesterone, was high in the sheep and rabbit and lower in the mouse. The results, however, do not relate directly to the progestational activity of MAP in these species. Overall, the data indicate that species differences in the binding activity of steroid receptors constitute one factor that causes species-dependent variation in biological responses to progestogens.

Transformation of human progesterone receptor in the presence of the progestin (R5020) and the antiprogestin (RU486)

We have examined and compared the binding characteristics and transformation in vitro of human uterine cytosolic progesterone receptor (PR) bound to either the progestin agonist, R5020 or the antiprogestin, RU486. Incubation of cytosol with 5–20 nM [ 3H]R5020 and [ 3H]RU486 yielded macromolecular complexes that sedimented in the 4S and 8S regions in 10–35% glycerol gradients. The 8S peaks of radioactivity due to macromolecular-bound [ 3H]R5020 or [ 3H]RU486 could be eliminated by a preincubation of the uterine cytosol with 1–2μ M progesterone or RU486. The [ 3H]R5020 binding in the 4S peak was not competable with either steroid. In contrast, the binding of [ 3H]RU486 in the 4S peak could be abolished by a pretreatment of uterine cytosol with excess RU486, but not progesterone. Selective fractionation of the cytosol with ammonium sulfate, in the presence of sodium molybdate, eliminated the non-specific 4S [ 3H]R5020 binder. The thermal (23°C) transformation of the [ 3H]RU486-receptor complex, as a function of the loss of the area under the 8S peak, appeared to be comparable to that achieved with [ 3H]R5020-receptor complex. The 8S [ 3H]RU486 peak was reduced by only 46% compared to the [ 3H]R5020 peak, which was reduced by 60%. These results demonstrate that in human uterine cytosol, R5020 and RU486 bind in a specific and saturable manner to an 8S PR, which is susceptible to thermal 8S to 4S transformation. In addition, [ 3H]R5020 also interacts with a nonsaturable 4S macromolecule, whereas the 4S [ 3H]RU486 binder is saturable and specific for RU486. The above observations indicate the heterogeneity of the steroid binding components present in the human uterine cytosol, and suggest that caution should be taken when interpreting data which shows the presence of different molecular forms of the steroid receptors.

Binding and biologic activity of diethylstilbestrol in the hamster: Influence of a serum component on estrogen receptor binding and estrogenic activity

The purpose of this study was to compare the biological activity and estrogen receptor (Re) binding affinity of diethylstilbestrol (DES) and estradiol (E 2). Uterine weight response and cytosolic progesterone receptor (Rp) induction were equivalent following daily (3 days) injections of DES or E 2 to ovariectomized animals. The biological equivalence of DES and E 2 was not reflected by competition assays done with either uterine cytosolic or nuclear Re: the relative binding affinity (RBA) of DES to cytosolic Re was 46 ± 5.3 and to nuclear Re was 380 ± 42 compared to E 2 (100). The RBAs of estrone, estriol and enclomiphene with cytosolic or nuclear Re were not significantly different. Further studies showed that this discrepancy in RBA of DES between cytosolic and nuclear Re could not be attributed to salt concentration but could be mimicked by addition of serum to nuclear Re preparations. The RBA of DES done with ammonium sulfate precipitated cytosolic Re approached that observed for nuclear Re. Gel filtration chromatography (Sephacryl S-300) of serum bound tritiated DES was shown to coelute with bovine serum albumin. These results suggest that a serum component (tentatively identified as albumin) can bind DES and cause a decrease in in vitro binding affinity and a reduction in biological activity in vivo.

The Ontogeny of Sex Differences in Estrogen-Induced Progesterone Receptors in Rat Brain*

The sex and age-related changes in the concentration of cytosol progesterone receptors (CPR) induced by estrogen (E) treatment in rat brain and pituitary were investigated by a modification of the Palkovits punch procedure using fresh tissue. Young male and female gonadectomized rats, 15, 21, 30, and 42 days of age, were treated for 44 h by a single sc injection of synthetic E. [Moxestrol (Ru2858)]. Adult gonadectomized animals were treated for 72 h by three injections of estradiol benzoate. Cytosol from pituitary and from punches of ventromedial nuclei (VMN), medial preoptic area (mPOA), arcuate nucleus (ARC), and cerebral cortex was labeled with 0.4 nM [3H]promegestrone (Ru5020) to maximize signal to noise and specificity of labeling of CPR. The developmental patterns of CPR differed across brain regions and between males and females. In VMN, females showed higher CPR levels after estrogen priming at 15, 21, and 42 days of age and in the adult; in ARC, females showed higher CPR levels after E priming at 15, 21, and 30 days of age, but not at 42 days or in the adult. In mPOA and pituitary, no consistent sex differences in CPR induction were found. Cortex showed no induction of CPR by E priming. Radioautography of [3H]Ru5020 uptake in VMN of E-primed 15-day-old male and female rats revealed significantly higher labeling in females, thus showing that the CPR levels in vitro reflect a difference in hormone retention in vivo. Female VMN contained more neurons with a higher labeling index than male VMN. Before puberty (approximately = 30 days), there was a decline in CPR levels induced by E priming in both sexes in pituitary, ARC, mPOA, and VMN. At 30 days, the female greater than male sex difference disappeared and tended to reverse in mPOA and VMN, only to be restored again by 42 days of age. Thus, the CPR induction by E priming may reflect underlying changes in E-sensitive brain regions associated with the preparation for puberty, as well as underlying sex differences in response to estrogen programmed by perinatal exposure to testosterone.

Relationship between radioligand binding assay, immunoenzyme assay and immunohistochemical assay for estrogen receptors in human breast cancer and association with tumor differentiation

We have studied the merit of a new enzyme immunoassay (EIA) in relation to the results obtained with a conventional dextran-coated charcoal assay (DCC) of estrogen receptors (ER) in cytosols and nuclear extracts of human breast cancer tissue. The results of the two assays were related to cytosolic progesterone receptor content (PgR), semiquantified ER content in formalin-fixed paraffin embedded tissue specimens and tumor differentiation. The EIA was found stable at low cytosol protein concentrations ( 0.5 mg/ml). The EIA and DCC assays were highly correlated both in cytosols ( r = 0.92, n = 57) and nuclear extracts ( r = 0.82, n = 25), but the EIA slightly overestimated the ER values in both ER fractions. A significant correlation between ER in nuclear (ER(N)) and cytosolic (ER(C)) fractions was established with both assays (DCC: r = 0.90, n = 56; EIA: r = 0.83, n = 24). A qualitative relationship was established between PgR and ER fractions as determined with both assays, the best quantitative association was between PgR and ER(N(DCC)) ( r = 0.58, n = 34, P < 0.001). A significant qualitative and quantitative relationship was found between semiquantified ER content in formalin-fixed, paraffin-embedded tissue and ER(C(DCC)) ( r = 0.88), ER(N(DCC)) ( r = 0.86)), ER(C(EIA)) ( r = 0.60), ER(N(CIA)) ( r = 0.64) and PgR ( r = 0.65). Finally, we found tumor differentiation to be significantly associated with ER content as determined with all assays except for ER(N(EIA)). We recommend the use of the DCC assay for routine analysis of ER until the clinical correlation of EIA results has been established.

Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking.

The nontransformed forms of the chick oviduct cytosol progesterone receptor of sedimentation coefficient approximately 8 S (8S-PR) are heterooligomers including one hormone binding molecule, either B, approximately 110,000, or A, approximately 79,000, and two non-hormone binding subunits recently identified as heat-shock protein Mr approximately 90,000 (hsp 90) [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., & Baulieu, E. E. (1984) Biochemistry 23, 6016-6023]. In the crude cytosol, bisimidates reacted under mild conditions and gave rise to complexes, binding progesterone and reacting with BF4, an anti-hsp 90 monoclonal antibody. These complexes have a sedimentation coefficient of 8.4 S and Rs of 8.1 nm in the presence of 0.4 M KCl and in the absence of molybdate ions, i.e., in conditions that would transform non-cross-linked 8S-PR to Rs approximately 5 nm forms of approximately 4-S sedimentation coefficient. All bisimidates tested, of an effective reagent length between 0.73 and 1.09 nm, gave comparable results in the cytosol prepared with or without molybdate ions, confirming that the latter were not responsible for the formation of the cross-linked 8S complexes. It was found that the dimethyl pimelimidate cross-linked 8S-PR was more resistant to inactivating conditions, urea, or heat treatment than the non-cross-linked 8S-PR. The 8S-PR cross-linked in the cytosol was purified by affinity chromatography in the absence of molybdate ions. After purification, it also reacted with the monoclonal antibody BF4 and had the same Rs (8.0 nm), sedimentation coefficient (approximately 8.5 S), and thus Mr (approximately 290,000) as the original cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)