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Proc. Nat. Acad. Sci. USA Vol. 71, No. 11, pp. 4341-4345, November 1974

Characterization of Binding Components for Progesterone and 5a--3,20-dione in the Hamster Uterus (3a-hydroxy-5a-pregnan-20-one/cytosol/ receptor/ /) WENDELL W. LEAVITT AND CHARLES J. GROSSMAN Department of Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45219 Communicated by Paul Herget, August 21, 1974

ABSTRACT In the hamster uterus, a specific pro- ing that progestin action is triggered by progesterone itself gesterone (pregn-4-ene-3,20-dione) receptor has been (23, 24). Second, an active progesterone , such as identified in the cytosol fraction. In the present study, we examined hamster uterine cytosol for the possible -exis- dihydroprogesterone (5a-pregnane-3,20-dione), could be the tence of specific macromolecules that bind the proges- intracellular mediator of hormone action (19, 25). Third, the terone metabolite, 5a-pregnane-3,20-dione. When cytosol mechanism of progestin action may vary in the different target was analyzed by density-gradient centrifugation with tissues so that progesterone might regulate certain target cells sucrose-glycerol gradients and by Scatchard plot analysis whereas a progesterone metabolite might control other of [3H]5a-pregnane-3,20-dione binding data, there was no target evidence of specific binding components for this metabo- types. lite. In vivo treatment of proestrous hamsters with un- The experiments reported here were designed to determine labeled progesterone, 5a-pregnane-3,20-dione, or whether or not progestin action in uterine target cells is for 1 hr revealed that only progesterone caused the de- mediated by conversion of progesterone to an "active" me- pletion of progesterone-receptor sites from the uterine cytosol fraction. Incubation of uterine strips which had tabolite such as dihydroprogesterone. Our approach to this been preloaded with two different concentrations of [3HJ- problem was similar to that used previously in the study of progesterone demonstrated that progesterone was meta- target tissues (3, 26). Our working hypothesis was bolized to 5a-pregnane-3,20-dione and to a greater extent to based on the following rationale. If dihydroprogesterone were 3a-hydroxy-5a-pregnan-20-one. The accumulation of 5a- pregnane-3,20-dione during progesterone metabolism the true intracellular mediator of progestin action, uterine appeared to be related to the availability of nonspecifically should exhibit two fundamental properties. First, bound hormone. These studies (i) strongly suggest there uterine cytosol should contain a specific receptor molecule for is no specific receptor system for 5a-pregnane-3,20-dione this metabolite; and second, after progesterone uptake, di- in the uterine cytosol fraction, (ii) confirm the existence hydroprogesterone should accumulate as a result of proges-- of a specific in uterine cytosol, and (iii) provide evidence that progesterone itself mediates the terone metabolism and binding of the metabolite. uterine progestational response via interaction with a MATERIALS AND METHODS specific receptor system. Animals. Adult female golden hamsters (Engles Labora- Considerable evidence has accumulated in support of the tory, Farmersburg, Ind.) were housed in a controlled environ- receptor hypothesis of action. According to ment with a 13:11 photoperiod (lights on 0500-1800 hr). this concept, the binding of steroid hormone by a specific Estrous cvcles were monitored according to the postestrous cytoplasmic receptor protein initiates the intracellular trans- vaginal discharge which occurs on the morning of cycle day 1. location of hormone-receptor complex to responsive sites in Intact females which exhibited at least three regular 4-day the target cell (1). For (-17#), hormone bind- estrous cycles were used at 1000 hr on cycle day 4 (proestrus). ing and transport of the estradiol-receptor complex to active The concentration of progesterone-receptor sites in the uterine sites in the nuclear chromatin takes place without significant cytosol fraction is greatest at the proestrous stage of the cycle metabolism of the steroid molecule (2). In constrast, andro- (15). Adult females were ovariectomized under gens. are actively metabolized within the target cell. After (90 mg/kg) anesthesia. After a 2-week postoperative recovery entry into the target cell, is converted to a potent period, they were primed by subcutaneous administration of metabolite, , which then interacts with a estradiol-17,3 (15 pg/animal per day) for 3 days. Primed ani- specific receptor molecule (3). mals were used 24 hr after the last estradiol . This The mechanism of progesterone (pregn-4-ene-3,20-dione) regimen of estrogen priming is known to produce a maximal action remains to be clarified. Specific progesterone-binding increase in the cellular concentration of cytoplasmic pro- macromolecules have been demonstrated in the cytosol frac- gesterone-receptor sites (15). tion of avian (4-6) and mammalian (7-15) target tissues. However, after progesterone uptake, there is a rapid metabo- . [7a-3H]Progesterone (11.5 Ci/mmol), [1,2-3H]pro- lism of this steroid to a variety of (16-22). Several gesterone (47.8 Ci/mmol), and [1,2-3HJ5a-pregnane-3,20- possibilities must be considered at this time to account for the dione (47.8 Ci/mmol) were obtained from New England mechanism of progestin action. First, there is evidence suggest- Nuclear Corp. The isotopes were checked by means of techniques described elsewhere (27) and were found to be >98% pure. They were stored at 40 in absolute ethanol (100 Trivial names: progesterone, pregn-4-ene-3,20-dione; dihydro- /hCi/ml) until used. Nonradioactive steroids were obtained progesterone, 5a-pregnane-3,20-dione; , 3a-hydroxy- from commercial sources and were recrystallized from aqueous 5a-pregnan-20-one. . 4341 Downloaded by guest on September 23, 2021 4342 Cell Biology: Leavitt and Grossman Proc. Nat. Acad. Sci. USA 71 (1974)

Buffers. Buffer A contained 50 mM Tris HCL (Schwarz/ mated that 30% of the [3H]progesterone had been taken up Mann, ultrapure), 1 mM EDTA, 12mMIthioglycerol (Sigma), by the strips. Then the radioactive medium was decanted, the pH 7.5. Buffer B was 10 mM Tris HCl, 1 mM EDTA, 12 strips were rinsed three times in fresh Hank's solution (40), mM thioglycerol, pH 7.5. Glycerol (reagent, Matheson, Cole- and 3 ml of fresh solution were added per flask. The uterine man & Bell) was added to buffers A and B at a concentration strips were then incubated in a Dubnoff shaker at 370 under of either 10% (v/v) or 30% (v/v) as indicated. 95%02 and 5% CO2 with agitation at 60 strokes/min. At Uterine Cytosol. Uterine tissue was homogenized in 4 vol- various times of incubation, the flask contents were chilled in umes (v/w) of buffer A + 10% glycerol at 2° with a Polytron an ice bath. The medium was collected, and the uterine strips Pt-10 (Brinkmann Instruments), and cytosol was prepared were washed twice with cold Hank's solution. The washes exactly as described before (15). Cellular pellets were ana- were combined with the incubation medium, and radioactive lyzed for DNA; calf DNA was the standard (28). steroids were extracted three times from the medium with 2 Sucrose-Glycerol Gradient Centrifugation. Linear sucrose volumes of ethyl acetate each time. Uterine strips were placed gradients (5-20% in buffer B + 10% glycerol) were prepared in 6 ml of ethanol- (1: 1, v/v) for at least 24 hr. Then in 5-ml polyallomer tubes with a Beckman gradient former. the strips were pulverized with a ground-glass homogenizer, Cytosol, diluted 1/4 (w/v) with buffer A + 10% glycerol, was and the tissue fragments were extracted with ethanol-acetone incubated for 15 min with unlabeled cortisol (1.1 X 10-s M) solution (27). at 40 before labeling with 3H-labeled steroid. This was done to Thirty micrograms each of authentic progesterone, 3a- saturate binding sites on plasma contaminants such as cortico- hydroxy-5a-pregnan-20-one (pregnanolone), and dihydro- steroid-binding globulin. Cytosol was then incubated with 2.1 progesterone were added as carrier steroids to each extract. X 10-8 M of either [1,2. 3H ]progesterone or [1 ,2-3H Idihydro- The extracts were taken to dryness, dissolved in 1 ml of progesterone for 2 hr at 4°. A 0.2-ml fraction was layered on methanol, and two 10-,l aliquots were removed for the de- each gradient. Gradients were centrifuged at 2° at 202,000 termination of the initial specific activity (dpm/Mug of carrier X g for 18 hr in a model L2-65B preparative ultracentrifuge steroid) (27). Each extract was purified by thin-layer chro- (Beckman). Gradients were fractioned from the bottom (22 matography with narrow plates (5 X 20 cm) coated with fractions/gradient), and the radioactivity content of each silica gel HF-254+366 (Brinkmann) in -acetonie fraction was counted as before (15). Sedimentation coefficients (9:1, v/v) (17, 27). The distribution of radioactivity on the were estimated by the method of Martin and Ames (29) with plates was determined with a Nuclear Chicago model 1002 bovine serum as the standard. A 4.2S sedimentation radiochromatogram scanner. Carrier steroid spots were located coefficient was assumed for bovine serum albumin. by the RF values of standards and by direct visualization Scatchard Plot Analysis. Uterine cytosol was diluted '/,6 under UV light. Zones corresponding to the migration of (w/v) with buffer A + 30% glycerol. Measurement of the dihydroprogesterone, progesterone, and pregnanolone were specific progesterone-binding capacity of uterine cytosol frac- eluted individually with ethyl acetate-methanol (1:1, v/v). tions was carried out exactly as described previously (15). Each eluate was reduced to dryness, and the residue was Specific binding of [3H]progesterone was the differencebetween acetylated overnight (31). Each steroid fraction was rechro- total binding and nonspecific binding. Specific binding data matographed on thin-layer plates in chloroform-ethyl were analyzed according to Scatchard (30). The relationship acetate (13:1, v/v) (27). Plates were scanned for the distribu- between the bound to free ratio and the concentration bound tion of radioactivity as before, and the zone corresponding to (nM) was subjected to linear regression analysis (15) provid- the migration of authentic steroid standard (either dihydro- ing a correlation coefficient (r), slope (m = KA) and the con- progesterone, progesterone, or pregnanolone acetate) was and taken to centration of progesterone receptor sites (pmol/ml of cytosol). eluted, washed three times with distilled wvater, was tested for dryness. Each sample was dissolved in 100 ,1 of ethyl acetate, In addition, proestrous uterine cytosol specific of [3H]dihydroprogesterone binding with the same assay proce- and two 10-,41 aliquots were removed for the measurement the radioactivity recovered (27). The remainder of the sample dure but modified as follows: (1) [3H ]dihydroprogesterone was steroid substituted for [3H]progesterone, and (2) nonspecific binding was used for determining the recovery of carrier by or columns means of a series of tubes an gas-liquid chromatography; 1% QF1 1% OV17 was determined by containing of the was excess of unlabeled- (4 X 10 IM) in addition to 3H-labeled were used (27, 32). Radiochemical purity samples of the labeled steroid established by fraction collection of the column effluent during dihydroprogesterone. The concentration A radio- was varied between 0.053 X 10-8 M and 1.03 X 10-8 M. chromatography as described elsewhere (32). single with the carrier steroid in all Statistical treatment of the data was by Student's t-test. active peak was associated peak Results were considered significant at P < 0.05. cases. The percentage of pregnanolone or dihydroprogesterone Uterine Uteri from produced was calculated from the final specific activity (dpm Metabolism of [31]Progesterone by Strips. of carrier steroid recovered) divided by the hamsters were placed in cold recovered perutg estrogen-primed-ovariectomized X 100. The of progesterone Hank's balanced salt solution, pH 7.4 (BSS). Each uterus was initial specific activity percentage utilized equalled 100-(final specific activity . initial slit longitudinally into two strips per horn. After blotting and utilized 25-ml flask each specific activity X 100). The amount of progesterone weighing, four uterine strips were used per calculated from this ml Hank's solution. 6 of or metabolite produced was percentage containing 3 of Approximately 4Ci initial tissue concentration of or was added and from the [3H]progesterone either [1 ,2-3H ]progesterone [7a-3H ]progesterone of to each flask in 10 ,ul of ethanol. Flasks were placed in a plat- (ng/g tissue). form shaker at 40 and incubated with gentle agitation for 90 RESULTS at 40 was monitored Properties of binding components for [3Hjprogesterone min. Uptake of radioactivity by taking in uterine of medium at 15, 30, 45, 60, and 90 min and and [(Hldihydroprogesterone cytosol 10-MI aliquots 0, with the measuring the disappearance of radioactivity from the me- A specific progesterone-binding macromolecule dium. Thus, after incubation at 40 for 90 min, it was esti- properties of a hormone receptor was demonstrated previously Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Uterine Progesterone Receptor 4343

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0 1 2 3 4 05 1 2 3 4 0 0I BOUND (nM) 2 610b 1'4 1'822 2 6 1014 1822 4 8 12 16 20 4 8 12 16 20 FIG. 1. Scatchard plots of [3H]progesterone- and [3H]dihy- TOP BOTTOM TOP BOTTOM droprogesterone-binding data in uterine cytosol obtained from FRACTION proestrous hamsters. (A) [3H]Progesterone binding (O-0) demonstrates the presence of a limited quantity of high affinity FIG. 2. Sucrose-glycerol gradient centrifugation. Distri- (KA = 1 X 109 M-1) binding sites. [3H]Dihydroprogesterone bution of radioactivity after centrifugation (18 hr at 202,000 X binding (---- -) is nonsaturable and nonspecific. (B) Specific g, 20) of 3H-labeled uterine cytosol fractions on 5-20% sucrose [3H]progesterone binding in proestrous uterine cytosol after pre- gradients containing 10% glycerol. The sedimentation peak of treatment in vivo with unlabeled cortisol (Ox-), dihydro- bovine serum albumin (arrows) run under identical conditions is progesterone (,-- -A), or progesterone (O - ). The number indicated above (4.2 S). Proestrous uterine cytosol was used of binding sites relative to tissue weight was obtained from the after in vivo pretreatment for 1 hr with unlabeled cortisol (O0 .*0) intercept on the abscissa. Each point represents the mean of dihydroprogesterone (A- - -A), or progesterone (0 0). (A) duplicate determinations. [3H]Progesterone-labeled cytosol; (B) [3H]dihydroprogesterone- labeled cytosol. in the hamster uterine cytosol fraction (15). This substance exhibited a 6-7S sedimentation coefficient upon density gesterone could not be demonstrated in proestrous cytosol by gradient centrifugation in 5-20% sucrose gradients containing Scatchard plot analysis of its binding data (Fig. 1A). Although 10% glycerol. It possessed a high affinity for progesterone it did bind to uterine this was = cytosol components, binding (equilibrium association constant, KA 1.7 X 109 M-l) as nonspecific in nature as indicated by the failure of unlabeled measured by Scatchard plot analysis of specific [3H ]progester- dihydroprogesterone (4 X 10-6 M) to competitively inhibit one-binding data. The receptor showed considerable hormonal binding of [HIdihydroprogesterone. specificity in that estrogen, , and existence of a failed To further test for the specific dihydropro- to compete effectively for progesterone-binding sites. gesterone binding system in the uterus, we pretreated pro- In addition, the receptor showed a greater binding affinity for estrous hamsters with either unlabeled progesterone, dihy- progesterone than for progesterone metabolites. in 0.2 ml of oil In droprogesterone, or cortisol (1 mg of steroid per the present study, we measured the specific progesterone- animal). After 1 hr, the uterus was removed, and the cytosol binding capacity of hamster uterine cytosol prepared at the The concentration of proestrous stage the (Fig. In agreement fraction was prepared for study. pro- of 1A). gesterone receptor sites was measured by Scatchard plot with previous results (15), proestrous cytosol contained a analysis (Fig. 1B), and the sedimentation properties of [3H]- significant quantity of specific progesterone-receptor sites material (Table 1). However, specific binding of [3H]dihydropro- progesterone- and [3H]dihydroprogesterone-binding was determined by density gradient centrifugation of labeled TABLE 1. Specific progesterone-binding capacity of cytosol fractions (Fig. 2). Pretreatment of proestrous hamsters uterine cytosol fractions with cortisol or dihydroprogesterone did not alter significantly the progesterone-binding capacity of proestrous uterine Progesterone binding sites cytosol (Fig. 1, Table 1). In comparison, pretreatment with Steroid pmol/ml of pmol/g of pmol/mg of unlabeled progesterone depleted receptor sites from the treatment cytosol tissue DNA cytosol fraction as evidenced by a 50% reduction in the progesterone binding capacity (Fig. 1B, Table 1). None The sedimentation profile of [3H]progesterone-binding com- (control) 2.83 4 0.12 56.6 i 5.2 30.1 ±- 4.8 ponents in uterine cytosol from the three steroid-treated Cortisol 3.16 ± 0.14 63.2 ± 5.4 30.7 ± 3.2 in 2A. from Dihydropro- groups is depicted Fig. Uterine cytosol obtained gesterone 2.84 4± 0.15 56.7 ±t 6.3 33.2 ± 3.6 cortisol- or dihydroprogesterone-pretreated animals contained Progesterone 1.35 ± 0.08* 26.9 ± 2.3* 16.6 ± 2.1* a [3H]progesterone-binding peak in fractions 10-11 (6-7S sedimentation coefficient). After pretreatment with pro- * P < 0.01, steroid treatment compared to control. Proestrous gesterone, [3H]progesterone binding was reduced in the 6-7S hamsters were pretreated for 1 hr with 1 mg of steroid per animal. region of the gradient, and a peak was observed in fraction 8 Each value represents the mean i SEM for four determinations. (about 4S sedimentation coefficient). Previous studies demon- Downloaded by guest on September 23, 2021 4344 Cell Biology: Leavitt and Grossman Proc. Nat. Acad. Sci. USA 71' (1974)

strated the material sedimenting in the 6-7S region of the gradient was progesterone receptor, but the identity of com- ponents sedimenting in the 4-5S region has not been estab- 2500-09.. lished (15). Thus, the present results are consistent with a 6-7S component that is a receptor responsive to progesterone action but not to dihydroprogesterone or cortisol action at the 200- dosage used. The loss of [3H]progesterone binding from the -0.6 6-7S region of the sucrose-glycerol gradient after in vivo progesterone administration suggested that receptor material -0.5 150 - had been removed from the cytosol fraction. This interpreta- tion is supported by the finding of a significant reduction in /4 // ~~~~~~-0.4U)D) the concentration of progesterone receptor sites after pro- LU * - gesterone treatment (Table 1). 100 /, Sucrose-glycerol gradient centrifugation of [3H ]dihydro- *) - L0 progesterone-labeled uterine cytosol revealed a major binding an approximate sedimentation coefficient of 4 S 50 z peak with -1~0.,X,// _~~~~~~~~~0.21z (fractions 7 + 8, Fig. 2B). This region of the gradient is occupied by steroid-binding proteins derived from plasma -01 X t ...... x ...... Xi such as -binding globulin and albumin as well as -. other unidentified steroid-binding components (15). There- 0 10 30 60 90 120 180 240 100 -7 1 T- fore, the tissue specificity of binding material in this region can -@ -03 be questioned, and the steroid specificity of 4-5S components is uncertain. Inasmuch as none of the steroid pretreatments 02 affected the amount of [3H Idihydroprogesterone binding in 50 the 4S region of the gradient, we conclude that the 4S peak of this binding represents nonspecific binding material(s). Thus, these results coupled with those from the Scatchard plot 0 ....--.. 0.. 010 30 60 90 120 180 240 analysis demonstrate that hamster uterine cytosol does not INCUBATION TIME (min) contain a specific receptor system for dihydroprogesterone. FIG. 3. Progesterone metabolism by uterine strips from [3HJProgesterone metabolism by uterine strips in vitro ovariectomized-estrogen-primed hamsters. Uterine strips were Uterine strips containing two different concentrations of [3Ii I- preincubated with [3H]progesterone for 90 min at 4°. Then the progesterone were incubated for different times at 370 (Fig. tissue was removed from the radioactive medium, placed in 3). From the results, it appeared that enough [3H ]progesterone fresh medium, and incubated at 370. (A) Experiment 1: initial = was present to saturate the receptor system in both cases. tissue concentration (shaded area) of [7a-3H] progesterone 258 4t 9.4 ng/g of tissue. (B) Experiment 2: initial tissue con- However, the amount of nonspecific [3H ]progesterone binding i in the first experiment (236 ng/g centration (shaded area) of [1,2-3H]progesterone = 62 2.2 was estimated to be greater the specific progesterone- second ng/g of tissue). ng/g of tissue. In both experiments, of tissue) than in the (40 binding capacity was 22 i 1.2 ng/g of tissue as determined by There was a rapid metabolism of [3H]progesterone during Scatchard plot analysis. Progesterone utilization (e- *), 3a- the first 30 min of incubation, and this was followed by a hydroxy-5a-pregnan-20-one production (O- - -0), and 5a- slower [jH]progesterone utilization from 30 to 240 min (Fig. pregnane-3,20-dione production (X * X) were measured by 3). Progesterone metabolism (ng/g of tissue) was greater in reverse isotope dilution analysis as described in Materials and those strips with the higher initial concentration of [3H]- Methods. Each point represents the combined results of separate progesterone (Fig. 3A) than in those containing the lower con- analyses done on tissue and medium. centration (Fig. 3B). Two radioactive metabolites of [3H]- progesterone were identified on the basis of (1) chromato- progesterone receptor titer was the same in both experiments, graphic mobility in two different thin-layer chromatography it seems pertinent that more dihydroprogesterone was pro- systems, and (2) a retention time identical to that of authentic duced in tissue containing the higher level of nonspecifically steroid standard determined by gas-liquid chromatography bound [3H]progesterone (compare dihydroprogesterone pro- with 1% QF1 and 1% OV17 columns. The major metabolite duction in Fig. 3A and B). formed during incubation was 3a-hydroxy-5a-pregnan-20-one DISCUSSION (pregnanolone), and smaller amounts of 5a-pregnane-3,20- from studies of the action of , dione were produced (Fig. 3). In addition, a small quantity of Information derived shown that in all cases the target polar metabolite(s) was observed to accumulate in the estrogen, and androgen have tissues contain a limited quantity of soluble receptor protein medium, but this material was not identified. The The time-course of pregnanolone production followed the which selectively binds hormone with high affinity (1). pattern observed for progesterone utilization throughout the present results confirm the existence of a specific progesterone 3). In contrast, receptor in the hamster uterine cytosol fraction (15). Pro- incubation period in both experiments (Fig. frac- dihydroprogesterone production increased during the first 30 gesterone receptor sites were depleted from the cytosol vivo but no a was observed for the remainder of the tion in response to in progesterone action, response min and then plateau amount of di- incubation period. The maximal amount (ng/g of tissue) of was elicited by treatment with an equivalent accumulated was related to the hydroprogesterone or cortisol. The competitive binding dihydroprogesterone which for initial tissue concentration of [3H]progesterone. Since the activity of dihydroprogesterone and cortisol progesterone Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Uterine Progesterone Receptor 4345

receptor sites is 20% and <1%, respectively, as compared-to We are indebted to Dr. Bert O'Malley for helpful suggestions the activity of progesterone (15). From this, it would appear and Ms. Arline Moore for excellent technical assistance. This that the has a work was supported by Population Council Grant M73.149 and receptor considerably greater binding affinity USPHS Grant HD06982-01. for progesterone than for dihydroprogesterone. Thus, the present findings are consistent with a relationship between the of the 1. O'Malley, B. W. & Means, A. R. (1974) Science 183, 610-620. binding affinity progesterone receptor for a steroid and 2. Jensen, E. V. & De Sombre, E. R. (1973) Science 182, 126- the biological activity of the steroid. Such a relationship has 134. been observed in the case of estrogen and androgen receptor 3. Fang, S. & Liao, S. (1971) J. Biol. Chem. 246, 16-24. systems (33, 34). Furthermore, there is evidence indicating 4. O'Malley, B. W., Toft, D. 0. & Sherman, M. R. (1971) J. that the translocation of from Biol. Chem. 246, 1117-1122. hormone-receptor complex the 5. Sherman, M. R., Corvol, P. L. & O'Malley, B. W. (1970) J. cytosol fraction to active intranuclear sites is a primary event Biol. Chem. 245, 608.5-6096. in the mechanism of hormone action (2). The progesterone- 6. Toft, D. 0. & O'Malley, B. W. (1972) Endocrinology 90, induced depletion of receptor sites from the uterine cytosol 1041-1045. fraction may represent an early step in the mechanism of 7. Wiest, W. G. & Rao, B. IR. (1971) in Advances in Bio- sciences, ed. Raspe, G. (Pergamon Press, Oxford), Vol. 7, progestin action, i.e., transport of progesterone-receptor com- pp. 2.51-266. plex to acceptor sites in a particulate fraction. 8. Reel, J. R., Van D)emark, S. 1)., Shih, Y. & Callantine, Although dihydroprogesterone has been suggested as a M. It. (1971) Sieroids 18, 441-461. 9. M. & C. W. possible intracellular mediator of progestin action (19, 25), we Corvol, P., Falk, R., Freifeld, Bardin, (1972) were unable to Endocrinology 90, 1464-1469. demonstrate a specific binding macromolecule 10. Faber, L. E., Sandmann, M. L. & Stavelv, H. E. (1972) J. for this steroid in the hamster uterine cytosol fraction. The Biol. Chem. 247, 8000-8004. apparent lack of a specific receptor would imply that the 11. Feil, P. 1., Glasser, S. It!., O'Malley, B. W. & Toft, 1). 0. mechanism of progestin action in the uterus does not involve (1972) Endocrinology 91, 738-746. conversion of to an active 12. McGuire, J. L. & Bariso, C. (1972) Endocrinology 90, 496- progesterone metabolite as appears 506. to be so for androgen target tissues (26). However, hamster 13. Milgrom, E., Atger, M., Perrot, AM. & Baulieu, E. E. (1972) uterine tissue metabolized progesterone to both dihydro- Endocrinology 90, 1071-1078. progesterone and pregnanolone during incubation of uterine 14. Rao, B. It., Wiest, W. G. & Allen, W. M. (1973) Endo- strips which had been with crinology 92, 1229-1240. preloaded [3H]progesterone. 15. W. 1). was the Leavitt, W., Toft, O., Strott, C. A. & O'Malley, Pregnanolone major metabolite produced by hamster B. W. (1974) Endocrinology 94, 1041-1053. uterus, and this product accumulated during incubation in 16. Wiest, W. G. (1963) J. Biol. Chem. 238, 94-99. proportion to the amount of progesterone utilized. In contrast, 17. Wichmann, K. (1967) Acta Endocrinol. (Copenhagen) the amount of dihydroprogesterone which accumulated during Suppl. 116 55, 1-98. incubation increased the first 30 18. Sweat, M. L. & Bryson, M. J. (1970) Amer. J. Obstet. only during min. Also, the Gynecol. 106, 193-201. production and accumulation of this metabolite appeared to 19. Armstrong, D. T. & King, E. R. (1971) Endocrinology 89, be related to the amount of nonspecifically-bound progester- 191-197. one which was available for metabolism rather than to the 20. Howard, P. D. & Wiest, W. G. (1972) Steroids 19, 33-45. amount which was bound. 21. Morgan, M. 1). & Wilson, J. D. (1970) J. Biol. Chem. 245, specifically 3781-3789. In the rat, dihydroprogesterone and pregnanolone were 22. O'Malley, B. W. & Strott, C. A. (1973) in Handbook of identified as the principal progesterone metabolites formed in Physiology, eds. Greep, R. 0. & Astwood, E. B. (Williams the uterus (8, 16, 19). When progestational activity of these & Wilkins Co.. Baltimore, Md.), Sect. 7, Vol. II, Part I, two metabolites was tested in the rat, dihydroprogesterone pp. 591-602. and 23. Csapo, A. I. & Wiest, W. G. (1969) Endocrinology 85, 735- pregnanolone possessed little biological activity in the 746. uterus (35). The uterotrophic activity of dihydroprogesterone 24. Wiest, W. G. (1970) Endocrinology 87, 43-48. has been tested in the hamster, and a similar lack of effect was 25. Armstrong, D. T. & King, E. It. (1970) Fed. Proc. 29, 250 observed (36). Our failure to detect a specific receptor system abstr. for this metabolite in the hamster uterus 26. Wilson, J. D. & Gloyna. R. E. (1970) Recent Progr. Horm. coupled with bio- Res. 26, 309-336. results that is an assay indicating it inactive progesterone 27. Leavitt, W. W. & Blaha, G. C. (1972) Steroids 19, 263-274. metabolite lead us to the conclusion that progesterone is the 28. Burton, K. (1956) Biochem. J. 62, 315-323. primary regulator of the progestational response in the uterus. 29. Martin, R. G. & Ames, B. N. (1961) J. Biol. Chem. 236, The present results with the hamster are in agreement with a 1372-1379. previous study with the rat (20) which indicated that uterine 30. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. progesterone metabolism probably represents a mechanism 31. Leavitt, W. W., Carlson, I. H. & Meyer, R. K. (1971) for clearing active hormone (progesterone) from the target Endocrinology 88, 16-25. cell. This conclusion does not exclude the 32. Leavitt, W. W., Lowenhaupt, R. W., Blaha, G. C., Kennan, possibility that A. L. & Meyer; B. K. (1972) Endocrinzology 90, 362-370. progesterone metabolites may play an active role in the 33. Korenman, S. G. (1969) Steroids 13, 163-177. mechanism of hormone action in nonuterine target tissues. 34. Liao, S., Liang, T. & Tymoczko, J. L. (1972) J. Steroid Dihydroprogesterone stimulates release Biochem. 3, 401-408. in both the rat (37) and hamster (38), and it is capable of 35. Sanyal, M. K. & Villee, C. A. (1973) Endocrinology 92, stimulating avidin synthesis in the estrogen-primed chick 83-93. oviduct (22). Therefore, dihydroprogesterone may be involved 36. Blaha, G. C. (1974) Anat. Rec. 178, 311 abstr. in the mechanism of 37. Sanyal, M. K. & Todd, 1t. B. (1972) Proc. Soc. Exp. Biol. progestin action in neural and oviduct Med. 141, 622-624. tissue, but the present results indicate that this is not so in the 38. Boslev, C. G. & Leavitt, W. W. (1972) Fed. Proc. 31, 257 uterus. abstr. 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