Nuclear origin of progesterone receptor of the chick oviduct cytosol. An immunoelectron microscopic study

Progesterone receptor (PR) was studied immunoelectron microscopically from fixed vibratome sections of the chick oviduct and biochemically from the fractionated oviduct homogenate. Immunoelectron microscopically both unoccupied and occupied PR were localized inside the nuclei. Only a few cells showed PR immunoreactivity in the endoplasmic reticulum which probably represents newly synthetized PR. Biochemically unoccupied PR was in the cytosol fraction. The cytosol and nuclear PR as well as the non-transformed 8S-form and the transformed 4S-forms of cytosol PR were recognized by the anti-PR antibody (IgG-RB). The lack of PR immunostaining in the cytoplasm is therefore not due to lack of recognition by IgG-RB. We propose that in the chick oviduct progesterone receptor is a nuclear protein but synthetized in the endoplasmic reticulum.     

Nuclear origin of progesterone receptor of the chick oviduct cytosol

Summary Progesterone receptor (PR) was studied immunoelectron microscopically from fixed vibratome sections of the chick oviduct and biochemically from the fractionated oviduct homogenate. Immunoelectron microscopically both unoccupied and occupied PR were localized inside the nuclei. Only a few cells showed PR immunoreactivity in the endoplasmic reticulum which probably represents newly synthetized PR. Biochemically unoccupied PR was in the cytosol fraction. The cytosol and nuclear PR as well as the non-transformed 8S-form and the transformed 4S-forms of cytosol PR were recognized by the anti-PR antibody (IgG-RB). The lack of PR immunostaining in the cytoplasm is therefore not due to lack of recognition by IgG-RB. We propose that in the chick oviduct progesterone receptor is a nuclear protein but synthetized in the endoplasmic reticulum.     

Antibodies against highly purified B-subunit of the chick oviduct progesterone receptor

A rabbit was immunized with the highly purified B-subunit (110kDa) (20 to 50 micrograms per injection) of the chick oviduct progesterone receptor (PR). Specific antibodies (IgG-RB) were observed 2 weeks after the first booster injection and high antibody titers in the serum were found after the second and third booster injections (with Kdeq of interaction integral of 2 nM). IgG-RB were tested by immunoprecipitation, immunoblotting, density gradient ultracentrifugation and protein A-sepharose assay methods. They recognized not only the B-subunit but also the A-subunit (79K), the nuclear PR, the mero-receptor (proteolytic cleavage product) and the "non-activated" molybdate-stabilized "8S" PR. However, IgG-RB did not interact with the 90K non hormone-binding component of this 8S-PR. IgG-RB did not affect the binding of the hormone to PR, whether incubated with the receptor before or after labelling with tritiated progesterone. They did not cross-react with glucocorticosteroid receptor of the chick oviduct. Weak interaction was observed with estrogen receptor of the chick oviduct and with KC1 activated "4S" forms of the rabbit and human uterus PR.     

Progesterone receptor and hsp90 are not complexed in intact nuclei

In hypotonic cell extract (cytosol), unliganded progesterone receptor (PR) is known to form an oligomeric complex with heat shock protein 90 (hsp90), and this complex does not bind to DNA. Since ligand binding has been shown to render the complex less stable in vitro, it has been proposed that ligand binding regulates DNA binding and receptor activity in vivo by altering the stability of the oligomeric complex. However, there is no direct evidence as to whether this oligomeric complex is present in vivo. The present study addressed this problem. First, we used an immunoelectron-microscopic technique and monoclonal antibodies to ascertain the location of PR and hsp90 in chick oviduct cells. Hsp90 was found in the cytoplasm and PR in the nucleus. To study the relative affinities of the PR and hsp90 antibodies, we then constructed a chimeric protein (PR-hsp90), which was expressed in the HeLa cells. Both hsp90 and PR antigens of the chimera were detected in the nuclei with the same intensity, which indicates that the antibodies have equal sensitivities in detecting their antigens. This suggests that if significant amounts of nuclear hsp90 were present in intact cells, it should have been detected by our method. Our results indicate that the PR does not exist in vivo as an oligomeric, nonDNA-binding form in the cell nuclei and that the oligomeric form found in tissue extracts is possibly formed during tissue processing.     

Chick oviduct glucocorticosteroid receptor. Specific binding of the synthetic steroid RU 486 and immunological studies with antibodies to chick oviduct progesterone receptor

The glucocorticosteroid receptor (GR) has been studied in oviduct cytosol prepared from estrogen-primed, 4-week-withdrawn chicken. The equilibrium dissociation constant was 6 nM for dexamethasone, and 18 300 receptor sites/cell were measured assuming that all cells contain identical concentrations of GR. Dexamethasone, used in most studies investigating glucocorticosteroid action, was found not to be the best GR ligand. The affinities of several natural and synthetic glucocorticosteroids for GR increased in the following order: cortisol less than deoxycorticosterone less than dexamethasone less than corticosterone less than triamcinolone acetonide. The synthetic steroid RU 486 was the most specific ligand of GR (its affinity was approximately equal to 10-fold higher than that of triamcinolone acetonide), while it did not bind either to plasma transcortin (which binds dexamethasone nor, surprisingly, to progesterone receptor (PR), contrary to what occurs in mammalian species. The molybdate-stabilized, 8-S form of GR was prepared from withdrawn chick oviduct, whole chick embryo or cultured chick embryo fibroblasts (which do not contain PR), and was labeled with either [3H]dexamethasone or [3H]RU 486. The sedimentation coefficient of radioactive ligand--8-S GR complexes was shifted towards heavier forms after incubation with polyclonal (IgG-G3) or monoclonal (BF4) antibodies generated against the molybdate-stabilized, 8-S form of the chick oviduct PR. Since neither IgG-G3 nor BF4 interacted with the steroid binding 4-S form of GR, it is suggested that these antibodies recognized a non-steroid binding protein common to molybdate-stabilized, 8-S forms of GR and PR.     

Immunoelectron microscopic localization of progesterone receptor in the chick oviduct

A peroxidase-anti-peroxidase (PAP) method using polyclonal anti-PR antibodies was used to localize progesterone receptor (PR) electron microscopically in the chick oviduct. The immunoreaction precipitate indicating PR was localized inside the nuclei of epithelial, glandular and stromal cells. In the estrogen withdrawn oviduct cytoplasmic immunoreaction precipitate was not seen. Inside the nucleus unoccupied PR was localized mainly like the heterochromatin. As visualized by the PAP technique, the localization of PR was not systematically changed after progesterone administration. In conclusion, we suggest that progesterone receptor in the chick oviduct is an intranuclear protein.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Expression of nuclear progesterone receptor and progesterone receptor membrane components 1 and 2 in the oviduct of cyclic and pregnant cows during the post-ovulation period

ABSTRACT: BACKGROUND: Progesterone (P4) may modulate oviductal functions to promote early embryo development in cattle. In addition to its nuclear receptor (PR), P4 may mediate its actions through P4 receptor membrane component 1 (PGRMC1) and its relative, PGRMC2. Two successive experiments were undertaken to characterise the expression of PR, PGRMC1 and PGRMC2 in the bovine oviduct during the post-ovulation period, and to relate their expression to the presence of an embryo, the proximity of the CL and to the region of the oviduct. METHODS: In the first experiment (Exp. I), whole oviduct sections were collected from Holstein cows at Day 1.5, Day 4 and Day 5 post-ovulation (n = 2 cows per stage). The expression of PR, PGRMC1 and PGRMC2 was studied in the ampulla and isthmus by RT-PCR, western-blot and immunohistochemistry. In Exp. II, oviduct epithelial cells were collected from cyclic and pregnant Charolais cows (n = 4 cows per status) at Day 3.5 post-ovulation and mRNA expression of PR, PGRMC1 and PGRMC2 was examined in the ampulla and isthmus by real-time quantitative PCR. RESULTS: In Exp. I, PR, PGRMC1 and PGRMC2 were expressed in all oviduct samples. PGRMC1 was mainly localised in the luminal epithelium whereas PR and PGRMC2 were localised in the epithelium as well as in the muscle and stroma layers of the oviduct. The expression was primarily nuclear for PR, primarily cytoplasmic for PGRMC1 and both nuclear and cytoplasmic for PGRMC2. In Exp. II, mRNA levels for PR, PGRMC1 and PGRMC2 were not affected by either the pregnancy status or the side relative to the CL. However, the expression of PR and PGRMC2 varied significantly with the region of the oviduct: PR was more highly expressed in the isthmus whereas PGRMC2 was more highly expressed in the ampulla. CONCLUSIONS: This is the first evidence of PGRMC2 expression in the bovine oviduct. Our findings suggest that P4 regulates the functions of the bovine oviduct in a region-specific manner and through both classical and non classical pathways during the post-ovulation period.     

Progesterone receptor concentration differences in the chick oviduct cells and apparent down-regulation by ligand. A semiquantitative immunohistochemical study

A semiquantitative immunohistochemical technique was developed for identification of chick progesterone receptor (PR). The mouse monoclonal antibody PR6 was used. The nuclear PR concentration was analyzed with Leitz Orthoplan MPV-3 light microscope. The target tissue was chick oviduct, with epithelial, glandular, mesenchymal, smooth muscle and peritoneal cells analyzed separately. PR concentration varied between different cell types and also from cell to cell within a single cell type. A significant decrease of PR concentration, as noted by decrease in staining, was also observed in all studied cell types, 6 h after a single injection of progesterone. This technique allows for histological identification of biochemical events that should help lead to the understanding of the role of PR changes in a variety of experimental situations.     

Changes in quantities of high-mobility-group protein 1 in oviduct cellular fractions after oestrogen stimulation.

Antiserum against chick oviduct high-mobility-group protein 1 (HMG 1) has been induced in the rabbit. With this antiserum, immunobiochemical techniques have been used to probe the quantitative change of HMG 1 in the cellular fractions of chick oviduct before or after oestrogen stimulation. HMG 1 is detectable in the cytosol, microsomal and nuclear fraction of the chick oviduct cell. After administration of oestrogen to young chicks in vivo for 5 days, the quantity of HMG 1 is increased 4-fold in the cytosol, 3.5-fold in the microsomal fraction and 1.6-fold in the nuclear fraction. The finding of large amounts of HMG 1 in cytoplasm of oviduct cell is not likely due to its leakage from the nucleus. We anticipate that HMG 1 is synthesized in the cytoplasm and then transported into the nucleus. The synthesis and transportation of HMG proteins is probably regulated by oestrogen.     

Sex-steroid-sensitive stromal cells and oviduct differentiation

The chick oviduct differentiates during sexual maturation before the age of 20 weeks. In the present work we used immunohistochemistry to study sexual maturation associated progesterone receptor (PR) expression in the chick oviduct as an indication of progesterone sensitivity. Since the PR is estrogen inducible protein, its expression also reflects the effects of endogenous estrogens. Thus PR expression can be used as a marker for action and sensitivity of cells to these sex steroids. In the luminal epithelium and mesothelium (peritoneal epithelium) the PR was expressed in high concentrations from the time before hatching (the constitutive PR). The PR was not detectable in stromal cells of immature chicks. At the age of 7-10 weeks the PR was detected in submucosal but not in mucosal stromal cells (the inductive PR). The appearance of these PR-expressing cells was associated with an increase in luminal epithelial cell proliferation. At the age of 14-16 weeks the mucosal plicae increased in height and the PR-expressing stromal cells were seen in the center of these mucosal plicae. There were also areas in the mucosal plicae where a large number of stromal cells expressing the PR were seen in the mucosal layer. Thereafter the size of the oviduct increased rapidly and the gland formation commenced. In the fully matured oviduct (over 18 weeks of age) virtually all stromal cells both in mucosa and submucosa expressed the PR. It is concluded that the PR expression in the luminal epithelium and mesothelium was constitutive (independent of sexual maturation). In stromal cells this was expressed during sexual maturation (probably induced by endogenous estrogen) and was associated with histological changes in the oviduct. We propose that direct effects of estrogen and progesterone in the oviduct growth and glandular formation are mediated through these stromal cells.     

Progesterone receptor is constitutively expressed in chicken intestinal mesothelium and smooth muscle

We have previously shown that progesterone receptor (PR) is expressed in the mesothelium of the chick oviduct and ovary and in the smooth muscle cells of the oviduct and the bursa of Fabricius. Here, we investigated the presence of PR in different parts of the peritoneum and abdominal organs using an immunohistochemical staining based on monoclonal antibodies against chicken PR. In 4-week-old sexually immature chicks, PR expression was located in the mesothelial cells of different parts of the peritoneum, in a thin layer of muscle cells of the ileum and throughout the muscle tissue of the colon and cloaca. In chicks of the same age treated with estrogen, PR was demonstrated similarly in the peritoneum and in the smooth muscle cells of the ileum, colon and cloaca. Using 25-week-old mature chickens, PR was also detected in identical tissues. Immunoblotting of the cloacal cytosol revealed the B form, but no A form of PR, both of which were found in the oviduct samples. Muscle cells of the duodenum and jejunum were not found to contain PR. Estrogen treatment was not needed to stimulate the production of PR in any of the tissues examined. We therefore conclude that the B form of PR is constitutively expressed in the mesothelial cells in different parts of the peritoneum and also in the smooth muscle cells of the ileum, colon and cloaca.     

Protein kinase activity of purified components of the chicken oviduct progesterone receptor

Preparations of the 90K and 110K components of the chick oviduct progesterone receptor (PR) purified to near homogeneity were tested for protein kinase activity. The 90K component was shown to incorporate radioactive phosphate from [γ-³²P]-ATP in the presence of Ca²⁺ but not of Mg²⁺ ions, while the 110K component was phosphorylated in the presence of Mg²⁺, but not of Ca²⁺. The enzymatic activity of the 90K polypeptide appeared selective, since added proteins (histones) did not become phosphorylated. However, all proteins present in the 110K preparations were phosphorylated in the presence of Mg²⁺. These data suggest that components of the chick oviduct PR display protein kinase activity.     

Different immunoelectron microscopic locations of progesterone receptor and HSP90 in chick oviduct epithelial cells.

The intracellular locations of the components of the heterooligomeric progesterone receptor (PR), heat-shock protein (hsp90), and the ligand-binding component were studied by immunoelectron microscopy in the chick oviduct, using immunogold double labeling and peroxidase techniques with monoclonal antibodies (MAb) against hsp90 (7D alpha and 4F3) and against PR (PR6 and PR13). PR was located in the nuclei of epithelial cells independently of the presence or absence of ligand. Cells with apically located nuclei were often PR negative. Ten minutes after progesterone administration no apparent change was seen in PR immunoreactivity, but chromatin underwent extensive rearrangement and PR was seen at the margins of the hetero- and euchromatin. The nucleoli did not contain PR. Hsp90 was located in the cytoplasm as aggregates, often inside small vesicles. In the apical part of the cell, hsp90 was located at the Golgi complex. The nuclei contained no detectable amounts of hsp90 except for that in the nucleoli. Ten minutes after progesterone administration the location or immunoreactivity of hsp90 did not alter. Thus, there seems to be a clear difference in the intracellular distribution of PR and hsp90. The epithelium also exhibited some cells with high levels of hsp90 and no or low levels of PR. These results raise the question of whether PR is associated with hsp90 in intact cells.     

Hormone-dependent processing of the avian progesterone receptor

Avian progesterone receptor exists as two forms, A and B, with molecular weights of 79,000 and 110,000 daltons, respectively. The origin and significance of these two forms is an area of active investigation and debate. Monoclonal antibodies produced against these two forms were used to examine receptor stability in cytosol and changes in the receptor forms induced by hormone binding. The lability of hormone binding at elevated temperatures is well documented. Analysis by Western blotting showed the receptor was stable in freshly prepared oviduct cytosol for 2 hr at 37°C, while hormone binding was lost within 30 min. However, loss of receptor through degradation was seen when cytosol was prepared from frozen tissue or when homogenization was excessive. Progesterone was injected into diethylstilbestrol-stimulated chicks to examine, in vivo, effects of hormone treatment on receptor forms in the cytosol and nuclear fractions. Progesterone treatment caused a time- and dose-dependent conversion of the A receptor to a form (A′) with a slower electrophoretic mobility. The cytosolic progesterone receptor was divided equally between the B and A forms, while the nuclear receptor was predominantly A′. The amount of nuclear receptor was consistently less than cytosolic receptor. Receptor phosphorylation was analyzed by incubating tissue minces with [³²P]orthophosphate with or without progesterone followed by immune isolation of receptor forms. Progesterone treatment caused a time-dependent increase in cytosol receptor phosphorylation which was evident after 5 min of treatment. This phosphorylation was observed with both the A and B receptor forms. The results indicate that receptor phosphorylation is a very early event during progesterone action.     

Sexual maturation-associated and estrogen-induced progesterone receptor expression in the bursa of Fabricius

The expression of the progesterone receptor (PR) was studied in the chicken bursa of Fabricius (BF) in both sexes from the time of hatching until the bursal involution. Steroid binding studies, immunohistochemistry, and autoradiography were used to characterize and localize the receptor. Three different polyclonal antibodies (IgG-RB, IgG-G3, and IgG-RB2) directed against the chick oviduct progesterone receptor were used for the studies. With immunohistochemistry, no receptor-positive cells were detected in the bursae of young chicks. The first receptor-positive cells were occasionally seen at the age of 10 wk in the frozen sections, not in the paraffin sections. In older female chicks, the staining became more abundant. In males, the PR was expressed only after estradiol treatment. The staining was located in the nuclei of the subepithelial and the interfollicular cells, which were probably mesenchymal in origin. The bursal epithelium and the lymphocytes were not stained. By using a combined technique of autoradiography and immunohistochemistry, we were able to demonstrate that the same cells also concentrated tritiated ORG 2058 (a specific synthetic progestin) in their nuclei. In steroid binding studies with tritiated ORG 2058, the receptor concentration after the age of 10 wk was 50 to 120 fmol/mg protein. Low-level ORG 2058 binding was also detected in young chicks of both sexes before the age of 10 wk. The progestin-binding molecule resembled the progesterone receptor of the chick oviduct in molecular size (studied with HPLC) and binding properties. The PR expression in the BF was preceded by the expression of PR in the oviduct stromal cells and by an increase in oviduct epithelial proliferation, indicating the BF is affected by factors associated with sexual maturation. It is concluded that the subepithelial and the interfollicular stromal cells in the BF, but not the epithelial or follicular cells, are estradiol-sensitive in both sexes immediately after hatching. The endogenous estrogens, however, are not able to induce PR until after the onset of sexual maturation, and only in females. This implies that estrogen and progesterone may affect the structural organization of the BF through the stromal cells, but probably not before the onset of puberty.     

Biochemical characterization of the progesterone receptor in the chick bursa of Fabricius

In a previous work we demonstrated estrogen-inducible progesterone binding sites in the bursa of Fabricius. In the present study these were characterized and compared to the progesterone receptor (PR) in the chick oviduct. When the size of the binding sites was analyzed with sucrose gradient centrifugation, 2 peaks of bound progesterone were obtained. The sedimentation coefficients of the peaks were 8-9 S and 3-4 S. In size exclusion HPLC only 1 peak was seen with a size corresponding to the 8-9 S in the sucrose gradient. The Stokes radius was 7.7 nm. When the ionic strength was elevated or CaCl2 was added, smaller steroid binding forms were detected. The sizes of these progesterone binding molecules at low and high ionic strength and in the presence of CaCl2 were equal in bursa and oviduct when analyzed with HPLC. The Stokes radii of these forms were 5.6 nm in high salt and 2.1 nm with CaCl2. The steroid binding components in the bursa cytosol eluated as 2 peaks from the DEAE column with KCl gradient. The peaks corresponded to the so-called A and B components in the chick oviduct. In the presence of molybdate, bound progesterone eluated as one peak from DEAE in both oviduct and bursa. The progesterone binding capacity was shown to be heat labile with equal half-lives in the bursa and the oviduct. Progesterone and ORG 2058 had a high affinity for the binding site and their binding was specific for progestins. It is concluded that the estrogen-inducible progesterone binding site in the bursa of Fabricius resembles the oviductal progesterone receptor in structural and binding properties.