Interaction of Uteroglobin With Progesterone, 5aPregnane-3,20Dione and Estrogens F. FRIDLANSKY AND E. MILGROM Groupe de Recherches sur la Biochimie Endocrinienne et la Reproduction (INSERM U.135), Faculte de Medecine Paris, Sud, 94270 Bicetre, France progesterone (KD = 4.1 x 10~7M) but a threefold higher affinity for 5a-pregnane-3,20-dione (KD = 1.3 x 10~ 7 M). Estradiol was bound but non-specifically with a very low affinity, and its binding was not enhanced by SH-reducing agents. Hormonal specificity of binding to uteroglobin was different from that of binding to rabbit uterine progesterone receptor. Various synthetic progestagens (chlormadinone acetate,* norethisterone, R5020) were bound to the latter but not to the former protein. Diethylstilbestrol had some affinity (15% of that of progesterone) for uteroglobin and no affinity for the progesterone receptor. Uteroglobin incubated in the presence or absence of cofactors (NADH and NADPH) with or without dithiothreitol did not metabolize progesterone. (Endocrinology 99: 1244, 1976)
ABSTRACT. Uteroglobin was obtained from 5 day pregnant rabbits and purified to homogeneity by Sephadex G 75 and DEAE-cellulose chromatographies. Progesterone binding to uteroglobin was decreased by lyophilization and enhanced by SHreducing agents. Dithiothreitol was more effective than dithioerythritol, and /3-mercaptoethanol was only active at 25 to 100 mM concentrations. SHblocking agents (iodoacetate, iodoacetamide, phydroxymercuribenzoate and, dithiobisnitrobenzoic acid) inhibited binding. In the absence of SHreducing agents only one in every 500 uteroglobin
molecules bound the hormone, whereas under optimal conditions (20 mM dithiothreitol) one in every two molecules bound progesterone. There was no significant difference in equilibrium dissociation constants under these two conditions. Uteroglobin had a relatively high affinity for
T TTEROGLOBIN (1) or blastokinin (2) \^J is a protein found in uterine secretions of the pregnant, pseudopregnant or progesterone-treated rabbit. It is also present in the oviducal fluid (3). The physiological role of this protein is not clearly understood although growth stimulatory effects on the blastocyst have been described (4,5). According to some reports uteroglobin binds progesterone (6-9) and also estradiol (6,8). However, these findings have been challenged in other publications (3,10). Our purpose was to see if the reported discrepancies are due to differences in stabililty of uteroglobin under various experimental conditions. The steroid specificity of uteroglobin binding was compared with that of the intracellular progesterone Received April 22, 1976. * Abbreviations used are: chlormadinone acetate: 6chloro-17 - acetoxy - pregna - 4,6 - diene - 3,20 - dione; norethisterone: 17 a-ethinyl-17-hydroxyestra-4-en-3one; R5020: 17,21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione; 5a-pregnanedione: 5a-pregnane-3,20-dione; 17 hydroxyprogesterone: 17 a-hydroxy-pregn-4-ene-3, 20-dione; dihydrotestosterone: 17 j8-hydroxy-5a-androstan-3-one; DTT: dithiothreitol.
receptor of the uterus. The possibility of metabolism of progesterone by uteroglobin was also examined. Materials and Methods Steroids [l,2-3H]Progesterone (Amersham, SA 47.6 Ci/mmole) and [l,2-3H]5a-pregnane-3,20-dione (New England Nuclear, SA 55.7 Ci/mmole) were used. Their purity was checked by silica gel thin layer chromatography (benzene:ethyl acetate, 3:2). [6,7-3H]Estradiol was obtained from Amersham (SA 43 Ci/mmole). Chromatographically pure unlabelled steroids were gifts from RousselUclaf. Buffer Sodium phosphate buffer (73 mM) ph 7.4 (phosphate buffer) was used in most experiments. SH reagents Dithioerythritol, dithiothreitol (Cleland's reagent), p-hydroxymercuribenzoate sodium salt (crystalline) and 5,5'dithiobis-2-nitro-benzoic acid were from Sigma. Iodoacetic acid potassium salt, and iodoacetamide were from Calbiochem.
1244
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STEROID BINDING BY UTEROGLOBIN
1245
Collection of uterine fluid
and cut into small pieces. All further operations were performed at 0 C. Pregnant New Zealand rabbits, 4 months old, The tissue was homogenized in 4 volumes of were killed on the 5th day of pregnancy. Each buffer containing Tris 0.01M, EDTA 1.5 mM, uterine horn was perfused with 3 ml of saline. dithiothreitol 2 mM and HCl added to pH 7.4. After centrifugation at 5000 x g for 10 min the After centrifugation at 105,000 x g for 90 min the uterine fluid was either frozen at — 20 C or cytosol was obtained and diluted with one lyophilized. In some cases it was concentrated volume of buffer containing 60% glycerol. by ultrafiltration through a dialysis membrane before freezing or lyophilization. Radioactivity Purification of uteroglobin (11) Six ml of concentrated uterine fluid containing 90 mg of protein were chromatographed on a column of Sephadex G 75 (2.5 x 85 cm). Flow was 10 ml/h and 2 ml fractions were collected. Optical density at 230 nm was recorded. The fractions corresponding to the uteroglobin peak were pooled. At this stage the protein is over 90% pure as judged by polyacrylamide gel profiles. The purification was continued on DEAEcellulose (Whatman DE52). The uteroglobin solution was dialyzed against Tris-0.05M HCl pH 8.0 buffer for 20 h at 0 C. Twenty-five ml containing 10 mg of protein was then chromatographed on a 1.5 x 37 cm column. Flow was 30 ml/hour and 1.8 ml fractions were collected. Elution was performed with a 0 to 0.5M NaCl gradient in the Tris-HCl buffer. Uterglobin was eluted at 0.05M NaCl. The protein was then pure when analyzed by polyacrylamide gel electrophoresis (11). The preparation was dialyzed against phosphate buffer and kept frozen at - 2 0 C until use. Equilibrium
dialysis
Radioactive and unlabelled steroids were dried in a Packard counting vial and redissolved in 15 ml of phosphate buffer. The protein solution (1 ml) was dialyzed (dialysis tubing, 8/100 feet, Union Carbide Corporation, Chicago) against steroid solution at 4 C with stirring (Tefloncoated magnetic disc) for 40 hours. Preliminary experiments had indicated that this time was necessary to reach equilibrium. Preparation of uterine cytosol for receptor studies Immature female rabbits three months old, were injected on three successive days with 50 /ug estradiol/0.25 ml sesame oil. On the fourth day the animals were killed, the uteri excised
Small volumes (
ABSTRACT. Uteroglobin was obtained from 5 day pregnant rabbits and purified to homogeneity by Sephadex G 75 and DEAE-cellulose chromatographies. Progesterone binding to uteroglobin was decreased by lyophilization and enhanced by SHreducing agents. Dithiothreitol was more effective than dithioerythritol, and /3-mercaptoethanol was only active at 25 to 100 mM concentrations. SHblocking agents (iodoacetate, iodoacetamide, phydroxymercuribenzoate and, dithiobisnitrobenzoic acid) inhibited binding. In the absence of SHreducing agents only one in every 500 uteroglobin
molecules bound the hormone, whereas under optimal conditions (20 mM dithiothreitol) one in every two molecules bound progesterone. There was no significant difference in equilibrium dissociation constants under these two conditions. Uteroglobin had a relatively high affinity for
T TTEROGLOBIN (1) or blastokinin (2) \^J is a protein found in uterine secretions of the pregnant, pseudopregnant or progesterone-treated rabbit. It is also present in the oviducal fluid (3). The physiological role of this protein is not clearly understood although growth stimulatory effects on the blastocyst have been described (4,5). According to some reports uteroglobin binds progesterone (6-9) and also estradiol (6,8). However, these findings have been challenged in other publications (3,10). Our purpose was to see if the reported discrepancies are due to differences in stabililty of uteroglobin under various experimental conditions. The steroid specificity of uteroglobin binding was compared with that of the intracellular progesterone Received April 22, 1976. * Abbreviations used are: chlormadinone acetate: 6chloro-17 - acetoxy - pregna - 4,6 - diene - 3,20 - dione; norethisterone: 17 a-ethinyl-17-hydroxyestra-4-en-3one; R5020: 17,21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione; 5a-pregnanedione: 5a-pregnane-3,20-dione; 17 hydroxyprogesterone: 17 a-hydroxy-pregn-4-ene-3, 20-dione; dihydrotestosterone: 17 j8-hydroxy-5a-androstan-3-one; DTT: dithiothreitol.
receptor of the uterus. The possibility of metabolism of progesterone by uteroglobin was also examined. Materials and Methods Steroids [l,2-3H]Progesterone (Amersham, SA 47.6 Ci/mmole) and [l,2-3H]5a-pregnane-3,20-dione (New England Nuclear, SA 55.7 Ci/mmole) were used. Their purity was checked by silica gel thin layer chromatography (benzene:ethyl acetate, 3:2). [6,7-3H]Estradiol was obtained from Amersham (SA 43 Ci/mmole). Chromatographically pure unlabelled steroids were gifts from RousselUclaf. Buffer Sodium phosphate buffer (73 mM) ph 7.4 (phosphate buffer) was used in most experiments. SH reagents Dithioerythritol, dithiothreitol (Cleland's reagent), p-hydroxymercuribenzoate sodium salt (crystalline) and 5,5'dithiobis-2-nitro-benzoic acid were from Sigma. Iodoacetic acid potassium salt, and iodoacetamide were from Calbiochem.
1244
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 00:46 For personal use only. No other uses without permission. . All rights reserved.
STEROID BINDING BY UTEROGLOBIN
1245
Collection of uterine fluid
and cut into small pieces. All further operations were performed at 0 C. Pregnant New Zealand rabbits, 4 months old, The tissue was homogenized in 4 volumes of were killed on the 5th day of pregnancy. Each buffer containing Tris 0.01M, EDTA 1.5 mM, uterine horn was perfused with 3 ml of saline. dithiothreitol 2 mM and HCl added to pH 7.4. After centrifugation at 5000 x g for 10 min the After centrifugation at 105,000 x g for 90 min the uterine fluid was either frozen at — 20 C or cytosol was obtained and diluted with one lyophilized. In some cases it was concentrated volume of buffer containing 60% glycerol. by ultrafiltration through a dialysis membrane before freezing or lyophilization. Radioactivity Purification of uteroglobin (11) Six ml of concentrated uterine fluid containing 90 mg of protein were chromatographed on a column of Sephadex G 75 (2.5 x 85 cm). Flow was 10 ml/h and 2 ml fractions were collected. Optical density at 230 nm was recorded. The fractions corresponding to the uteroglobin peak were pooled. At this stage the protein is over 90% pure as judged by polyacrylamide gel profiles. The purification was continued on DEAEcellulose (Whatman DE52). The uteroglobin solution was dialyzed against Tris-0.05M HCl pH 8.0 buffer for 20 h at 0 C. Twenty-five ml containing 10 mg of protein was then chromatographed on a 1.5 x 37 cm column. Flow was 30 ml/hour and 1.8 ml fractions were collected. Elution was performed with a 0 to 0.5M NaCl gradient in the Tris-HCl buffer. Uterglobin was eluted at 0.05M NaCl. The protein was then pure when analyzed by polyacrylamide gel electrophoresis (11). The preparation was dialyzed against phosphate buffer and kept frozen at - 2 0 C until use. Equilibrium
dialysis
Radioactive and unlabelled steroids were dried in a Packard counting vial and redissolved in 15 ml of phosphate buffer. The protein solution (1 ml) was dialyzed (dialysis tubing, 8/100 feet, Union Carbide Corporation, Chicago) against steroid solution at 4 C with stirring (Tefloncoated magnetic disc) for 40 hours. Preliminary experiments had indicated that this time was necessary to reach equilibrium. Preparation of uterine cytosol for receptor studies Immature female rabbits three months old, were injected on three successive days with 50 /ug estradiol/0.25 ml sesame oil. On the fourth day the animals were killed, the uteri excised
Small volumes (
