Effect of molybdate on the molecular organization of the estrogen receptor system of porcine uterus

Molybdate was shown to have complex effects in modulating the molecular organization of the constituents of the estrogen receptor (ER) system of porcine uterus. We showed previously the presence of one basic ER molecule (vero-ER) (sedimentation coefficient, 4.5S; Stokes radius, 44 A) and ER-binding factors (ERBFs) ["8S" ER-forming factor ("8S" ER-FF), (component A) X (component B)6; "6S" ER-FF, (component B)6; "5S" ER-FF, component A] in the porcine uterus [Fukai, F. & Murayama, A. (1981) J. Biochem. 95, 1697-1704]. Molybdate regulates the specific interaction of vero-ER with ERBFs in a complex way. The apparent Kd value (6.7 X 10(-10) M) of vero-ER with "8S" ER-FF in the presence of molybdate (30 mM) was decreased remarkably as compared with that (2.7 X 10(-9) M) in the absence of molybdate. In contrast, the apparent Kd value (3.7 X 10(-9) M) of vero-ER with "5S" ER-FF observed in the presence of molybdate (30 mM) was increased over ten-fold as compared with that in the absence of molybdate. Meanwhile, the affinity (Kd, 5 X 10(-9) M) of vero-ER for "6S" ER-FF was scarcely influenced by molybdate. These results reveal the mechanism by which molybdate selectively stabilizes "8S" ER. Molybdate further affected the molecular constitution of ERBFs. The dissociation of "8S" ER-FF into component A and component B, which takes place under hypertonic (0.4 M KCl) conditions at higher temperature (25 degrees C), was suppressed almost completely by molybdate (30 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of the steroid-binding core of porcine estrogen receptor

The steroid-binding core of estradiol receptor was purified from pig uterus cytosol by a protocol consisting of (1) adsorption to heparin-sepharose, (2) enzymatic release of the receptor core, (3) DEAE-chromatography, (4) Sephadex G-150 filtration and (5) chromatography on heparin-sepharose. The final product was approximately 18000-fold enriched over the starting material. It consisted of at least 18% core protein resembling dimeric microsomal receptor with a molecular mass of 75 kDa and an isoelectric point of 5.8 (microheterogeneity). A goat antiserum raised against the preparation contains immunoglobulins G precipitating estradiol-receptor complexes, and antibodies releasing the steroid from its binding site.     

The steroid binding domain of porcine estrogen receptor

For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.     

Aprotinin inhibits the hormone binding of the estrogen receptor from calf uterus

Micromolar concentrations of the proteinase inhibitor, aprotinin, produced a dose-dependent inhibition in the binding capacity of the estrogen receptor from calf uterus. Aprotinin inhibition was greater at 28 degrees C than at 4 degrees C and only occurred when conditions allowed the receptor transformation. When aprotinin was tested in the presence of transformation inhibitors, its effect was no longer seen. The binding capacity of the highly purified estrogen-binding subunit was similarly inhibited.     

Purification of the receptor for platelet-derived growth factor from porcine uterus

The receptor for platelet-derived growth factor has been purified to homogeneity on a large scale from porcine uterus. The purification procedure utilizes solubilization of uterus membranes by Triton X-100, followed by sequential chromatographies on wheat germ agglutinin-Sepharose, fast protein liquid chromatography Mono-Q, and anti-phosphotyrosine-Sepharose. About 160 micrograms of homogeneous and functionally active 170-kDa receptor could be purified from 5 kg of uterus tissue. The pure receptor responded to platelet-derived growth factor stimulation by autophosphorylation, indicating that the receptor has a kinase domain as an integral part of the molecule. A rabbit antiserum was produced against the pure receptor, which specifically recognizes the intact 170-kDa receptor.     

Role of porcine endometrial estrogen sulfotransferase in progesterone mediated downregulation of estrogen receptor

Estrogen sulfotransferase (EST) is a progesterone (Pg) induced secretory endometrial enzyme which may effect estrogen receptor levels by esterifying estradiol-17 beta (E2) to an inactive, sulfate form. The effects of this enzyme were studied using specific inhibitors of EST that do not bind to estrogen receptor (ER): 4-nitroestrone 3-methyl ether and 4-fluoroestrone 3-methyl ether. A 1 h pulse with 4 nM E2 caused ERn (i.e. E2-bound, chromatin-bound receptor) to increase 40% in incubations of proliferative gilt endometrium (no EST activity), while the same E2 treatment of secretory endometrium (high EST activity) caused no increase in ERn. ERn accumulation was completely restored in these experiments by preincubating secretory endometrium with 4 microM 4-fluoroestrone 3-methyl ether. Gilt endometrial explants cultured 7 days with 1 nM E2 plus 1 microM Pg (which induced EST activity) possessed half the ERn as explants devoid of EST activity which were cultured in E2 alone. The addition of 10 microM 4-nitroestrone 3-methyl ester to the cultures of secretory endometrium restored ERn to the levels seen in minces cultured with E2 alone. Furthermore, ovariectomized gilts injected daily with 250 micrograms E2 plus 25 mg Pg had much lower ERn (0.06 fmol/micrograms DNA) than gilts injected with E2 only (0.21 fmol/microgram DNA). ERn was restored completely by supplementing the E2 plus Pg injections with 0.5 g 4-nitroestrone 3-methyl ether administered by vaginal suppositories.(ABSTRACT TRUNCATED AT 250 WORDS)     

A biphasic action of estradiol on estrogen and progesterone receptor expression in the lamb uterus

Regulation of the uterine expression of estrogen and progesterone receptors was studied in 20 three-month-old lambs that were not treated or treated with estradiol- 17beta. Determinations of receptors were performed by binding assays in the nuclear and cytosolic fractions, receptor mRNAs by solution hybridization, and estrogen receptor protein by an enzyme-immunoassay. Estradiol treatment decreased the receptor binding capacity of both receptors and the levels of immunoreactive estrogen receptor 12 h after injection in the absence of decreased receptor mRNAs, suggesting that the initial decrease is due to degradation of the proteins or that mRNAs are translated into new receptor proteins at a reduced rate. The mRNA levels increased after estradiol treatment suggesting that the replenishment phase consists of synthesis of new receptors rather than recycling of inactivated receptors.     

Enhanced inhibition of estrogen receptor nuclear binding in the uterus of aged mice

We have studied why uteri from aging mice show a decrease nuclear concentration of estrogen receptors (UER). While 50-60% of available cytosolic UER from ovariectomized (OVX) mice aged 4-8 months, upon physiochemical activation, are able to bind either to DNA-cellulose or nuclear suspensions from young animals, only 20-30% of comparable concentrations of cytosolic UER from mice aged 15-18 months did so under identical experimental conditions. Nuclear dilutions with uterine cytosolic fractions from estrogen treated OVX mice prior to determination of [3H] UER binding sites in nuclear suspensions decreased the number of nuclear ER sites in both age groups. However, we observed that cytosols from aged animals showed a greater ability to prevent [3H]E2 binding to nuclear sites when compared to young ones (inhibition index: 0.286 +/- 0.013 (SE) vs. 0.137 +/- 0.025, P less than 0.05). These changes occur independently of protein concentration and result from dilution of a specific endogenous inhibitor of [3H]E2 binding to nuclear sites. The significance of these observed differences is discussed.     

Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal

We previously showed that progesterone rapidly down regulates nuclear estrogen receptor (Re) in the estrogen-primed rodent uterus. We have now extended these studies to test the response of the Re system in sheep uterus to progesterone withdrawal. Since the estrogen-Re complex is believed to regulate hormone-dependent gene expression, it was of interest to determine whether withdrawal of progesterone under constant estrogen stimulation would lead to the recovery of nuclear Re levels and estrogen action, i.e. oxytocin receptor (ROT) synthesis. Ovariectomized ewes were primed with estradiol-17 beta and serum steroid levels were maintained by constant infusion of estradiol (0.5 microgram/h) and progesterone (500 micrograms/h) for 5 days. The animals were anesthetized with fluothane/O2, and uterine samples were excised 1 h before and 3, 6 and 12 h after progesterone withdrawal. Estradiol infusion was continued during the experiment in order to maintain estrogen levels at a steady state (14 pg/ml plasma). Re, ROT and progesterone receptor (Rp) were measured in endometrium and myometrium using standard 3H-hormone binding assays. Following progesterone withdrawal, the nuclear Re concentration increased in both uterine compartments, and the nuclear Re level was correlated significantly with the ROT concentration in the membrane fraction of both uterine tissues (endometrium, r = 0.79; myometrium, r = 0.86). Although cytosol Re rose between 6 and 12 h in the endometrium, cytosol Re levels remained unchanged in myometrium. Cytosol Rp appeared to increase in endometrium but not in myometrium. Uterine tissue sampled from a control animal before stopping the progesterone infusion revealed that the observed changes in receptor concentration following progesterone withdrawal were not due to regional differences in receptor levels. These results demonstrate that the recovery of nuclear Re in the ovine endometrium and myometrium following progesterone withdrawal represents a selective effect on Re retention in the nucleus rather than on cytosol Re availability or Re activation which was controlled by constant estrogen infusion. Thus, these results are consistent with the hypothesis that progesterone induces an Re regulatory factor which acts to down regulate nuclear Re, and that the activity of this factor diminishes rapidly after progesterone withdrawal.     

Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus

Populations of macrophages and neutrophils in the uterus are under the control of the female sex steroids estrogen and progesterone (P4). Their influx is induced by estrogen, while P4 can both stimulate and inhibit leukocyte influx depending on the timing of P4 with respect to estrogen. Regulation of leukocytes has been implicated in changes in uterine immune responses during the estrous cycle, pregnancy, and implantation. This work demonstrates that P4 given concurrently with estrogen to ovariectomized mice for 4 days antagonizes the ability of estrogen to recruit macrophages and neutrophils into the mouse uterus. Using progesterone receptor knockout (PRKO) mice, we show that this effect is dependent on progesterone receptors (PR). In the absence of PR, neutrophils recruited by estrogen were found to be degranulated, partially explaining the edema that is observed with long-term treatment of PRKO mice with estrogen and P4. Populations of B lymphocyte cells were shown to be unchanged by estrogen and P4 treatment in both wild-type and PRKO mice. The neutrophil chemotactic chemokine MIP-2 was examined for down-regulation by P4 but was found to be unaffected by hormonal treatment. Together, these observations demonstrate that PR has a strong anti-inflammatory role in the mouse uterus when estrogen and P4 are present together.     

Identification of calpain II in porcine sperm

The role that proteolytic enzymes may play in membrane-associated phenomena of sperm has been the subject of extensive investigation. In the present study, we have examined the possibility that a Ca2+-activated, neutral protease, calpain II, may be associated with sperm membranes. Using indirect immunofluorescence with primary antibodies, which are polyclonal and monoclonal antibodies directed against the 80 kDa subunit of calpain II, we have established the presence of this antigen in porcine sperm. Staining by anticalpain II (80 kDa subunit) of the apical segment of the acrosomal cap and basal body (centriolar) region was seen consistently. Variable staining of the sperm tail was also observed. These observations, combined with our positive identification of a 80 kDa protein in acrosomal membranes (via immunoblot), document the association of this protease with sperm membranes. The proximity of calpain II to the acrosome suggests a potential role for the protease in the Ca2+-mediation of the acrosome reaction.     

Effect of the antiprogestin RU-486 on progesterone inhibition of occupied nuclear estrogen receptor in the uterus

The ability of the antiprogestin, RU-486, to reverse progesterone (P) antagonism of occupied nuclear E receptor retention was studied in the rat and hamster uterus. RU-486 was shown to effectively displace [3H]P binding from rat uterine cytosolic P receptor in in vitro competition assay. In contrast, no competition by RU-486 for [3H]P binding was observed for uterine cytosolic P receptor from the hamster uterus. In the presence of sustained serum levels (silastic implants) of P and estradiol (E), occupied nuclear E receptor was significantly inhibited in the rat uterus. At 6, 12 and 24h after RU-486 treatment (5 mg/animal, s.c.) uterine receptors for E and P were determined. No significant differences in cytosolic E and P receptors were observed between treated (E + P, + RU-486) and control (E + P alone) animals. However, by 6 h following RU-486 treatment, occupied nuclear E receptor retention increased significantly (0.30 +/- 0.05 vs 0.60 +/- 0.09, pmol/uterus) and reached a peak between 12 h (1.32 +/- 0.09) and 24 h (0.83 +/- 0.09). The increase in nuclear E receptor approached the level observed in animals with an E implant alone (1.55 +/- 0.15). Measurement of uterine fluid accumulation following RU-486 treatment showed an increase which paralleled that observed for occupied nuclear E receptor retention. A similar in vivo experiment in the hamster showed no reversal of P inhibition of occupied nuclear E receptor. These results show that: 1. RU-486 is an effective competitor for rat uterine P receptor but not hamster P receptor; 2. RU-486 can rapidly reverse P inhibition of uterine occupied nuclear E receptor in the presence of sustained serum levels of E and P; 3. The recovery of occupied nuclear E receptor is coincident with a resumption of E action (uterine fluid accumulation). The studies also provide a novel means by which antiprogestin activity can be assessed in vivo in the presence of sustained E and P serum levels, e.g. the reversal of P inhibition of uterine nuclear E receptor retention.