Rabbit Uterine Cytosol Research Articles

Evidence that the 90-kDa heat shock protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three other proteins with Mr of 63,000, 56,000, and 50,000.

Monoclonal antibody (mAb) 8D3 and 3G3 are unique antibodies capable of precipitating both free 90-kDa heat shock protein (HSP90) and HSP90-protein complexes. Immunoprecipitation of [35S]methionine-labeled Hepa 1c1c7 cytosolic extracts were performed using mAb 8D3 or 3G3. The resulting immunoprecipitates can be dissociated from the mAb with a 500 mM NaCl wash. These washes were subjected to both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Five major protein spots were detected in addition to HSP90 with the following relative molecular weights: 68,000, 63,000, 56,000, 50,000, and 188,000. On Western blots mAb 3G3 was capable of specifically binding to HSP90. Each of these proteins was localized on two-dimensional gels. Using one-dimensional gel electrophoresis and immunochemical localization on Western blots, the p68 spot was identified as HSP70, and the p56 spot was found to cross-react with polyclonal antibody JP-1 raised against a 59-kDa protein. This 59-kDa protein has been found previously to be associated with several steroid hormone receptors in rabbit uterine cytosol. Immunoprecipitation of [32P]orthophosphate-labeled Hepa 1c1c7 cytosol with mAb 8D3 or 3G3 revealed two major phosphorylated proteins with relative molecular weights of 90,000 and 50,000. The identities of p63 and p188 are currently unknown. This is the first report examining the major proteins that are complexed with HSP90 in mammalian cells.

Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo- p-dioxin: Use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors

The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by “3-methylcholanthrene-type” compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of ≈9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of ≈11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of ≈9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.

Delta 9-[16 alpha-125I]iodo-19-nortestosterone: a gamma-emitting photoaffinity label for the progesterone receptor.

We have synthesized 16 alpha-iodo-4,9-estradien-17 beta-ol-3-one [delta 9-16 alpha-iodo-19-nortestosterone (delta 9-INT)] labeled with 125I (delta 9-[16 alpha-125I]INT) to provide a new gamma-emitting photoaffinity ligand for the progesterone receptor that has many advantages over the currently available [3H]R5020. We have characterized the interaction of delta 9-[16 alpha-125I]INT with the rabbit uterine progesterone receptor and have demonstrated the usefulness of this compound for studies of receptor structure. The binding of 2 nM [3H]progesterone to receptor in rabbit uterine cytosol was specifically competed for by 19-nortestosterone, 16 alpha-iodo-19-nortestosterone, and delta 9-INT. Scatchard analysis demonstrated that delta 9-[16 alpha-125I]INT and [3H]progesterone estimated the same number of binding sites in rabbit uterine cytosol, with a Kd for delta 9-[16 alpha-125I]INT of about 2.7 nM. The binding of delta 9-[16 alpha-125I]INT was inhibited by both progesterone and R5020, whereas testosterone, estradiol, and 5 alpha-dihydrotestosterone were ineffective. In cytosol, delta 9-[16 alpha-125I]INT covalently labeled the same mol wt receptor forms as [3H]R5020. Although the efficiency of cross-linking was similar for [3H]R5020 (3%) and delta 9-[16 alpha-125I]INT (4%), the radioactivity was 10-fold greater due to the higher specific activity of delta 9-[16 alpha-125I]INT and the lack of sample quench. The use of delta 9-[16 alpha-125I]INT greatly increases the sensitivity and efficiency of the photoaffinity labeling technique; it will provide a valuable tool for further studies of the progesterone receptor, allowing the detection of receptor in dilute cytosol after gel electrophoresis under denaturing conditions.

Occurrence of a 6S intermediate form of the progesterone receptor that is sensitive to ribonuclease

Steroid receptors exist in cytosol as 9S, non-DNA-binding species and as 4S (transformed) species that bind to DNA or nuclei. Labeling the progesterone receptor from rabbit uterine cytosol with [3H]progesterone in the presence of 10 mM sodium molybdate revealed a 9S species on sucrose gradient centrifugation. Without molybdate, the receptor sedimented as an intermediate species of 6S, which converted to 4S in 0.3 M NaCl. The 6S species could also be generated from the 4S species by dialysis. Dilution of the same 4S species gave only partial re-aggregation with 50% of the receptor remaining as 4S. Dialysis appeared to retain the association of a macromolecular aggregation factor present in cytosol. Serum did not seem to be the source of the aggregation factor, as perfusion of the uterine vasculature before excision did not affect the S value of the receptor. We tested whether RNA was involved by treating receptor with RNase A (100 micrograms/400 microliters cytosol). While the molybdate-stabilized cytosol receptor (9S) was unaffected, RNase A partially (50%) converted the 6S form of receptor to 4S. RNase A also partially converted the re-aggregated form back to 4S. Protease inhibitors had no effect on this action of RNase. Formation of receptor-ribonucleotide protein particles may play a role in steroid action in the cell.

Characterization of molybdate-stabilized estrogen receptors by hydrophobic interaction HPLC: resolution of two 8S subunits.

This report describes the separation and characterization of estrogen receptors (ER) according to their degree of hydrophobicity and surface charges. Molybdate-stabilized [3H]ER from rabbit uterine cytosol was sequentially purified by passage through a size-exclusion pre-column, an anion-exchange column, and a hydrophobic interaction column. With fresh cytosol, a major radioactive peak was eluted from the DEAE columns; a major peak and a minor, less hydrophobic, peak were eluted from the hydrophobic column. In contrast, ER from frozen cytosol showed one peak in the DEAE-column and exhibited four radioactive peaks in the hydrophobic column. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, [3H] tamoxifen aziridine(TA)-labelled ER showed radioactive bands at 62 and 48 kd. The subunits which were characterized by these radioactive bands were successfully separated by the hydrophobic column; the more hydrophobic subunit corresponded to the 62 kd band. The HPLC-purified [3H]TA-labelled ER subunits sediment at a 7.4-8.5S region in a low-salt sucrose gradient. These results show that differential negative surface charge and hydrophobic areas exist in the holo-receptor and its subunits, and the hydrophobic interaction HPLC column separates the two major 8S steroid binding subunits of ER.

Characterization of rabbit uterine estrogen receptor proteins by radioiodination and partial peptide mapping.

Two estrogen binding proteins (Mr = 50,000 and 65,000) were purified from rabbit uterine cytosol using an improved procedure for affinity chromatography on diethylstilbestrol-agarose. The estrogen receptors were radioiodinated while adsorbed to the resin using the lactoperoxidase or Bolton-Hunter techniques. After elution, the labeled receptors were utilized for peptide mapping studies and investigations of receptor function. Partial peptide mapping revealed strong homology between the Mr 50,000 and 65,000 proteins suggesting common structural features. Estrogen receptors labeled by the lactoperoxidase procedure were rendered unable to bind immobilized heparin or hormone; in contrast, the Bolton-Hunter labeling technique yields proteins that retain both their ability to bind hormone and to absorb on heparin-agarose. The development of these iodination methodologies appears useful for the investigation of both the structure and functional properties of the receptor proteins.

ChemInform Abstract: Synthesis and Evaluation of Potential Radioligands for the Progesterone Receptor.

AbstractGemäß Formelschema werden Iod‐ und Amid‐Steroide (IV), (VI), Und (VIII) dargestellt, die auf Progesteronrezeptor‐Affinität getestet werden.

Effect of ribonuclease on the physico-chemical properties of estrogen receptor

Estrogen receptors (ER) from rat and rabbit uterine cytosol were examined for their sensitivity to ribonuclease (RNase). After RNase treatment, a major part of rabbit uterine ER was converted from the 7S to 3–4S form, and its binding to DNA-cellulose was increased by 40%. Similar treatment on rat uterine ER showed a shift from 7S to 4.5S, and the DNA-cellulose binding was stimulated by 20%. Measurement of endogenous RNase levels showed that lower RNase concentration in rabbit uterine cytosol coincided with larger stimulation of DNA-cellulose binding by exogenous RNase. These results indicate that a major part of 7S ER is susceptible to RNase, and cleavage of bound RNA seems to uncover additional binding sites for DNA. In contrast to the general thinking that 4S to 5S transformation is essential for nuclear binding, we have observed that RNase-treated rat uterine ER did not undergo such a transformation by warming at 25°C, while DNA-cellulose binding of the receptors increased. Thus, temperature activation could occur independent of 4S to 5S transformation.

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit.

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

An immobilized antiestrogen binds a specific uterine protein in addition to estrogen receptor proteins

An antiestrogen affinity resin was synthesized by conjugating LY117018, a benzothiophene-derived antiestrogen, to epoxy-activated agarose. This affinity resin bound the M r = 50 000 and 65 000 estrogen receptor proteins of rabbit uterine cytosol; in addition, it retained a protein from the cytosols of both rat and rabbit uteri that exhibited an ability to interact specifically with LY117018. The possibility that the LY117018 binding protein, which is distinct from estrogen receptors, may play a role in the antiestrogenic actions of LY117018 is discussed.

Affinity chromatography of estrogen receptors on diethylstilbestrol-agarose

Diethylstilbestrol was coupled to epoxy-activated agarose yielding an affinity resin which is highly efficient for the isolation of estrogen receptors. This resin, diethylstilbestrol-agarose (DES-agarose), bound two proteins ( M r = 50,000 and 65,000) from rabbit uterine cytosol that show a specific interaction with estradiol. A two step procedure—adsorption on DES-agarose followed by a selective elution with p-sec-amylphenol and NaSCN, yielded highly purified estrogen receptors which can be used in the studies of estradiol-receptor interactions with other cell constituents.

Biological activities of 4-fluoro estrogen analogues

As part of a study on the biological activity of γ-emitting estradiol analogues, a series of fluorinated and/or brominated analogues of estradiol were synthesized. 4-Fluoro-1,3,5(10-estratrien-3,17β-diol (4-fluoro-estradiol) and 16α-bromo-1,3,5(10)-estratrien-3,17β-diol (16α-bromo-estradiol) had relative binding affinities (RBA) for the rabbit uterine cytosol receptor which were comparable to those of estradiol. However, while 4-fluoro-estradiol stimulated the increase in uterine weight of immature mice to the same amount as estradiol, 16α-bromo-estradiol was less effective in this assay indicating that its metabolism is probably more rapid than that of estradiol. 16β-Bromo-1,3,5(10)-estratrien-3,17β-diol had a low RBA and was ineffective in stimulating uterine weight at the doses used. 4-Fluoro-16α-bromo-estradiol had a lower RBA than either the 4-fluoro- or 16α-bromo-estradiol and all the 4-fluoro-seleno-estrogens tested possessed low RBA's.

The biologic activity of selenoestrogens.

For differential imaging of mammary tumors with estrogen receptors and without estrogen receptors we required gamma-emitting estrogen analogues. In this paper we report on the binding properties of 7 alpha-, 16 alpha-, and 17 alpha-methylselenoestrogens and 17 alpha-phenylselenoestrogens relative to the binding properties of estradiol. The selenium-containing estrogens retained the ability to displace [3H]estradiol from the estrogen receptor of rabbit uterine cytosol, although in most instances the displacement was small (3-7% compared to estradiol). The most active compounds were 16 alpha-phenylselenoestrone, 16 alpha-methylselenoestradiol, and 17 alpha-methylselenomethyl-estradiol which had relative binding of 23, 27, and 31%, respectively, compared with that of estradiol. 16 alpha-Methylselenoestradiol was able to translocate the estrogen cytosol receptor to the nucleus, in vitro, but was not able to increase the uterine weight when administered to mice in vivo.

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).

Studies on the indicators for the responsiveness of human breast cancer to sex steroids (author's transl)

This study was designed to obtain indicators for endocrine responsiveness in human breast cancer. In this study steroid receptors in breast cancer tissue were first characterized as follows : estradiol-17β (E2) -estrogen receptor (ER) had a dissociation constant (Kd) of 5.50 ± 0.39 × 10-10M in premenopause or 5.60 ± 0.43 × 10-10M in postmenopause, R5020progesterone receptor (PR) had a Kd of 4.80 ± 0.63 × 10-10M in premenopause or 4.20 ± 0.40 × 10-10M in postmenopause, 5α-dihydrotestosterone (DHT) -androgen receptor (AR) had a Kd of 5.04 ± 0.55 × 10-10M in premenopause or 4.12 ± 0.47 × 10-10M in postmenopause, and R1881-AR had a Kd of 2.90 ± 0.50 × 10-10M, determined by charcoal adsorption at 4°C. Such steroid receptors were sedimented to 7-6S and 5-4S regions, and the 7-6S binding was easily dissociated during 5-20 percent sucrose gradient centrifugation. R1881-cytosol bindings contained AR and PR. Cytosolic steroid receptor levels were determined in each case. Maximal binding sites (Bm; fmol/mg cytosol protein) analyzed by Scatchard plots analysis were as follows : 65.6 ± 11.4 (premenopause) and 81.0 ± 17.1 (postmenopause) in ER (NS), 19.2 ± 4.4 (premenopause) and 44.4 ± 10.3 (postmenopause) in PR (p<0.05), 71.6 ± 17.6 (premenopause) and 77.9 ± 12.1 (postmenopause) in AR (DHT) (NS), and 25.3 ± 7.5 (premenopause) and 14.4 ± 9.5 (postmenopause) in AR (R1881) (NS). The nuclear receptor was determined by exchange assay. Nuclear PR and AR were detected in few cases. Nuclear ER was detected where in some cases cytosolic ER was not detected. The necessity of determining nuclear ER whenever ER is present or absent in the tumor cell will be discussed.In examining the nuclear binding of a steroid receptor complex, the ER and PR of rabbit uterine cytosol were substituted for those of breast cancer tissues. The nuclear binding was dependent upon the dose and incubation time of the rabbit-steroid uterine cytosol complex. The dissociation constant of the nuclear binding of the E2-ER complex was 6.1 × 10-11M, and that of the R5020-PR complex was 1.5 × 10-10M in a case. In determined cases, even when the ER or PR was present in either the cytoplasm or nucleus of the tumor cell, the steroid-receptor complex formed in the rabbit uterine cytosol did not bind to the tumor nuclei in some cases. These results suggest that part or all of the nuclear receptor-binding sites of breast cancer tissues are defective in some tumor nuclei.When the E2 -ER complex bound to the tumor nuclei, the nuclear RNA synthesis was stimulated in the time-dependent process of the E2-ER complex. Through the determined cases, even when the E2-ER complex bound to the tumor nuclei, the nuclear RNA synthesis was not stimulated in some cases.The above results suggest that the presence of a steroid receptor, the nuclear binding of a steroid-receptor complex, or the stimulation of nuclear RNA synthesis by steroid receptors occurring in breast cancer cells are more precise indicators of endocrine responsiveness in human breast cancer.

Progesterone receptor binding characteristics following freezer storage of uterine cytosol

Uterine cytosol prepared from estrogen treated rabbits was stored for various lengths of time at −60°C. The avidity for [ 3H]-progesterone, the number of progesterone binding sites, and the relative binding affinities of five steroids were determined for fresh cytosol, and for cytosol subjected to freezer storage. None of the binding parameters differed from those determined for fresh cytosol after 1, 2, 4, 5 or 8 weeks of storage. However, after 16 weeks of storage the relative binding affinities of three steroids had increased significantly. Rabbit uterine cytosol may be stored at −60°C for 2 months without any perceptible change in the binding properties of the progesterone receptor.

Receptor binding of fluorescein labeled steroids

Fluorescent derivatives of estradiol-17β (E2), desoxycorticosterone (DOC) and prednisolone (Pr) were synthesized by coupling E2-17β-hemisuccinate (E2-HS), DOC-21-HS and Pr-21-HS respectively to N-fluoreseeinyl-5,N'-(6-amino)-hexyl-thiourea. The long chain of C and N atoms between the steroid and fluorescein was introduced to avoid steric hindrance of the steroid-receptor interaction. The binding affinity of E2-fluorescein-conjugate (E2-F1) was determined by competitive inhibition of the specific [ 3H]-E2 binding to rabbit uterine cytosol receptors. Dissociation constants were found to be 0.8 nM for E2 and 1.5 nM for E2-F1. In the same way, affinity of the DOC-fluorescein-conjugate (DOC-'F1) binding to the progesterone receptor of rabbit uterus was measured using [ 3H]-R 5020 as radioactive ligand. K D was 9.7 mM for DOC-F1 compared to 2.3 nM for progesterone. Pr-fluorescein-conjugate (Pr-F1) bound to the glucocorticoid receptor with a K D of 7.3 nM compared to 3.4 nM for dexamethasone.

Effect of buffers and electrolytes on the stability of uterine progesterone receptors

We have investigated the effects of buffers and electrolytes on the stability of the 7S progesterone-receptor complex of guinea pig and rabbit uterine cytosol. Receptor half-life was greater in 5 m m phosphate than in the cationic or zwitterionic buffers tested. The best medium contained 5 m m phosphate, 10% glycerol, and 10 m m monothioglycerol, and was degassed under vacuum. Exposure to 100 m m sodium phosphate, like exposure to 300 m m KCl, resulted in the disappearance of the 7S progesterone binding component and a concurrent increase in binding to a 4 to 5S component. However, Scatchard analyses suggest that 300 m m KCl led to a reduction in the apparent association constant, whereas the “ K a” observed in 5m m phosphate was maintained in 100 m m phosphate.

Properties of the cytoplasmic progestin-binding protein in the rabbit uterus.

An exchange assay for the measurement of total cytoplasmic progestin binding sites has been developed on rabbit uterine cytosol using the highly potent progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) labelled to a high specific activity. This compound has several advantages over progesterone: it is not bound by plasma corticosteroid binding globulin; it has high affinity for the progestin receptor; it binds virtually as fast as progesterone to the receptor, but the complex formed dissociated 8 times slower; its binding is not displaced by more than 2% by compounds devoid of progestational activity (estrogens, testosterone, dexamethasone, aldosterone). Bound endogenous progesterone was exchanged by tritiated R 5020 in a time compatible with receptor stability. At 0 C, total exchange of filled sites occurred in less than 4 h; at this temperature the R 5020-receptor complex was stable for at least 28 h. The conformation of the R 5020-receptor complex was investigated in sucrose density gradients under various experimental conditions. Unlike progesterone, it was possible to detect a 7S peak in uterine cytosol obtained from rabbits injected with a tracer dose of [3H]R 5020 1 h prior to sacrifice.

Contribution of functional groups of 19-nor-progestogens to binding to progesterone and estradiol-17beta receptors in rabbit uterus.

The structural elements of 19-norprogestogens which may be essential for binding to progesterone and estradiol-17beta(E2) receptors were investigated in the rabbit uterine cytosol. The kinetic study showed that 19-nor-progestogens are competitive inhibitors of progesterone-receptor (8S) binding and E2-receptor binding. The affinities of steroids for the progesterone receptor were as follows: norethindrone (Ki of 2.3 X 10(-9)M) greater than 5alpha-dihydronorethindrone greater than norethindrone acetate greater than lynestrenol greater than 17alpha-ethynyl-estra-4-ene-3beta, 17beta-diol greater than ethynodiol diacetate (Ki of 1.3 X 10(-7) M). The affinities of steroids for the E2 receptor were as follows: ethynodiol diacetate (Ki of 1.3 X 10(-7)M) greater than 17alpha-ethynyl-estra-4-ene-3beta, 17beta-diol greater than norethindrone acetate greater than norethindrone greater than 5alpha-dihydronorethindrone greater than lynestrenol (Ki of 8.4 X 10(-7)M). The results indicate that 3-ketone and 17beta-hydroxyl groups, and the plane of ring A/B of 19-norprogestogen are important for binding to the progesterone receptor. The affinities of 19-nor-progestogens for the E2 receptor were very weak. Their affinities for the E2 receptor increased with addition of acetate or hydroxyl groups at the 3beta and 17beta positions, and were decreased by the elimination of a 3 oxygen function or the reduction of ring A.