The Cytoplasmic Estradiol Receptors of Bovine Uterus

The interconversions and properties of uterine cytoplasmic receptor proteins have been studied. In general the properties analyzed are those pertaining to the binding of the cytosol receptors to membranes or nucleohistone. Specific cytoplasmic estradiol receptors are found in both the endometrial and myometrial layers of both mature and immature heifers. The level of receptors is highest in immature tissues. The 4 S and 9 S receptors have similar binding properties and probably represent different levels of molecular complexity of the same receptor. The 4 S and 5 S receptors have very different binding properties in that the 4 S estradiol-receptor binds membranes in a temperature dependent process requiring the presence of divalent cations, whereas the 5 S receptor binds nucleohistone in a very rapid reaction even at 4’, in the absence of divalent cations. A small amount of 4 S receptor also binds to nucleohistone but only at elevated temperatures (25”) and in the absence of divalent cations. A small measure of specificity for target organ material is seen in the binding of 4 S estradiol-receptor to membranes. No specificity for target tissue was noted for 5 S estradiol-receptor binding to nuclear material. The 4 S form of the estradiol-receptor complex is converted into the 5 S estradiol-receptor in a reaction which does not require the preseyce of chromatin, but is dependent upon an elevated temperature and the presence of estradiol. Immature uterine tissue does not contain the 5 S receptor if assayed directly, but if the tissue is preincubated with estradiol, the formation of the 5 S receptor can be demonstrated, arguing that the 4 S-5 S conversion occurs within the cell in a reaction requiring the presence of estradiol. Since after incubation the 5 S estradiol-receptor is present in the cytoplasm in viuo and can bind nuclear material instantaneously at 4”, we have raised the possibility that the 5 S estradiol-receptor found associated with nuclear material could have been translocated and bound during the homogenization of the tissue.

The interconversions and properties of uterine cytoplasmic receptor proteins have been studied. In general the properties analyzed are those pertaining to the binding of the cytosol receptors to membranes or nucleohistone. Specific cytoplasmic estradiol receptors are found in both the endometrial and myometrial layers of both mature and immature heifers. The level of receptors is highest in immature tissues. The 4 S and 9 S receptors have similar binding properties and probably represent different levels of molecular complexity of the same receptor. The 4 S and 5 S receptors have very different binding properties in that the 4 S estradiol-receptor binds membranes in a temperature dependent process requiring the presence of divalent cations, whereas the 5 S receptor binds nucleohistone in a very rapid reaction even at 4', in the absence of divalent cations. A small amount of 4 S receptor also binds to nucleohistone but only at elevated temperatures (25") and in the absence of divalent cations. A small measure of specificity for target organ material is seen in the binding of 4 S estradiol-receptor to membranes. No specificity for target tissue was noted for 5 S estradiol-receptor binding to nuclear material. The 4 S form of the estradiol-receptor complex is converted into the 5 S estradiol-receptor in a reaction which does not require the preseyce of chromatin, but is dependent upon an elevated temperature and the presence of estradiol. Immature uterine tissue does not contain the 5 S receptor if assayed directly, but if the tissue is preincubated with estradiol, the formation of the 5 S receptor can be demonstrated, arguing that the 4 S-5 S conversion occurs within the cell in a reaction requiring the presence of estradiol. Since after incubation the 5 S estradiol-receptor is present in the cytoplasm in viuo and can bind nuclear material instantaneously at 4", we have raised the possibility that the 5 S estradiol-receptor found associated with nuclear material could have been translocated and bound during the homogenization of the tissue. It has been proposed that an early event in the mechanism of estradiol action in the uterus involves transport of the hormone to the cell nucleus in order to modify nuclear activity. It has been argued that transport occurs by way of a hormone receptor which first binds t.he hormone in the cytoplasm and subsequently migrates and binds to nuclear material.
Similar mechanisms have been suggested for the action of progesterone in the chick oviduct (1) autl dihydrostestosterolie in the ventral prostate (2). The transport hypothesis has been based upon three lines of evidence.
These are: (a) If immature uterine tissue is incubated with &radio1 and the cells subsequently disrupted and fractionated, 5 S estradiol-receptor complexes of cytoplasmic origin can be recovered from the nuclear material (3). (b) The amount of 5 S cstradiol-receptor complex bound to nuclear material increases as a function of hormone concentration (4) and time of incubation (5). The increase in nuclear bound 5 S estradiol-receptor is paralleled by a concomitant decrease in the amount of cytoplasmic receptor.
(c) A number of studies have argued that cytoplasmic receptors can bind specifically to target organ chromatin in in vitro assays (6). This paper is concerned with a re-examination of the interaction between cytoplasmic estradiol-receptors and chromatin in terms of the nature and location of the binding.
We have a.lso examined the na.ture of the interconversion between the two major species of cytoplasmic receptors and the relative capacities of these two receptor molecules to bind chromosomal material.
The results argue that cytoplasmic estradiol-receptors (particularly the 5 S receptor) could bind to chromosomal material as an artifact of isolation and that there is doubt as to the phgsiological significance of cytoplasmic estradiol-receptor complexes found in association with nuclear material.

Preparation of Estradiol-Receptor
Complexes--Bovine uterine tissue (40 g) was blended in 50 ml of either Buffer B (10 mM 'L-is-Cl, 1.5 m&f EDTA, p1-I 7.5) or Buffer A (100 rnM NaCl, 5.0 mM KCl, 3.0 m&f MgC12, 1.5 mM CaC12, 10 mM Tris-Cl, pH 7.0) as described in the text. After centrifugation at 27,000 x g for 20 min the supernatant was added to the required amounts of [3H]estradiol-17/3 and centrifuged at 130,000 x g for 60 min. The supernatant (containing the cytoplasmic receptor) was treated with freshly prepared charcoal-dextran G-60 (7) to a final concentration of 0.257, charcoal-0.0025% dextran. After incubation for 20 min at 4", the charcoal was removed by centrifugation at 10,000 x g for 5 min. In cases where a number of 1627 different concentrations of estradiol were needed for Scatchard plots, the [3H]estradiol-17P was added to the cytosol solution after the centrifugation at 130,000 x g. Preparation of Nuclei and Microsomes-Nuclei and microsomes from thymus, lung and immature uteri (4 to 8 week old heifers) were prepared by the procedure previously outlined (8). For experiments involving incubations in Buffer B, the nuclear pellets from the centrifugation in 2.4 M sucrose-Buffer A were washed twice with Buffer B prior to use.
In order to avoid the presence of free estradiol during incubation with nuclear material, such incubations were conducted in the presence of fresh charcoal-dextran which was added immediately before mixing cytoplasmic receptors with nuclei or membrane material.
The final concentration of nuclei or microsomes was such that incubation systems contained 3 to 5 mg of DNA per ml or 3 to 5 mg of protein per ml (microsomes), respectively. After incubation, 0.2-ml aliquots were collected both before and after centrifugation at 27,000 x g for 5 min, and the samples were assayed for radioactivity.
A correction for charcoal adsorption was made by a control incubation in the absence of nuclear material.
The nature of such a correction is described in the legend to Fig. 2.
Dissociation of Cytoplasmic Receptor-Nuclear Complexes-After incubation with estradiol-receptor complex, the nuclei (or microsomes) were collected by centrifugation at 10,000 X g for 1 min (nuclei) or at 27,000 x g for 5 min (microsomes).
The pellets were washed once in the incubation buffer and then in Buffer B. The pellets were extracted in 0.4 M KCI-3% sucrose-10 mM Tris-Cl-l.5 mM EDTA, pH 8.5 (Buffer C) for 1 hour at 4" and layered on 5 to 20% sucrose gradient in Buffer C. Centrifugation was in a Beckman SW 56 rotor at 300,000 x g for 19 hours at 4'. Bovine serum albumin, (.szo,~ = 4.5) was used as a marker in a separate tube (9). After centrifugation, fractions (10 drops) were collected and assayed for [3H]estradiol by counting in Bray's solution (10).
Fractionation of Nuclear Material into Membrane and Nucleohistone-After incubating nuclei and chromatin with [%]estradiol-receptor the material was washed twice with Buffer B and twice with distilled water to form a viscous gel which was sheared in a VirTis 45 homogenizer at 70 volts in 3 bursts of 15 s each over a lo-min span as described previously (8). After shearing, the solution was centrifuged at 150,000 x g for 90 min, which sediments -95% of the membrane and leaves >90% of the DNA (as nucleohistone) in the supernatant. The ionic environment was adjusted to a final 10 mM Tris-Cl, 1.5 mM EDTA, pH 7.2 and the solution was centrifuged at 150,000 x g for 90 min to pellet the nucleohistone.
Aliquots (0.2 ml) were .taken both before and after centrifugation to determine the amount of estradiol sedimented and for DNA analysis (11).
Receplor Binding to Chromatin, Microsomes, and Nucleohistone in Absence of Esfradiol--,k solution containing cytoplasmic receptor (estrogen not added) in Buffer B (5 ml) was incubated at 25" for 30 min with a dispersed nucleohistone pellet (10 mg of DNA).
The nucleohistone was removed by centrifugation at 27,000 x g for 5 min at 4". Subsequently a fresh pellet of nucleohistone (10 mg of DNA) was added for a second incubation. After removal of the nucleohistone, the supernatant (0.6 ml) was added to increasing concentrations of [3H]estradiol-178 and incubated at 4" for 4 hours to obtain a Scatchard plot for an analysis of the number of cytoplasmic receptor binding sites remaining in solution.
An identical procedure was followed using chromat.in (8 mg of DNA per pellet) or microsomes (20 mg of membrane protein per pellet).
[3H]Estradiol-l 70 Binding in Tissue Incubations-The tissue (40 g) was incubated at 37" for 45 min in Eagle's medium (100 ml) (12)  After incubation the tissue was washed three times in water (4") and then incubated at 4" for 5 hours in Eagle's medium (50: I, v/v) containing a 5000.fold excess of unlabeled cstradiol.
After two such incubations the uteri were washed twice in distilled water and frozen or homogenized directly in the appropriate buffer.

RIC3ULTS
Cytoplasmic Estradiol-Receptors of Bovine Uterus-Uterine tissue was collected from immature aud mature heifers and dissected into the endometrial and myometrial layers. The sedimentation coefficients of the cytoplasmic receptors from these tissues were determined by sucrose density gradient analysis after binding [3H]estradiol at 4" in vitro with the results shown in Fig. 1. In agreement with many workers in other systems (13, 14) we find both 4 S and 9 S receptor proteins in these systems. These gradients contain no magnesium and it is possible that at least part of the 9 S material comes from aggregation of the 4 S protein, as the 4 S receptor is the primary component of adult uterine tissue when the cytoplasmic extract is prepared in the presence of magnesium (see below).
Binding of Estradiol-Receptors to Nuclei in Presence of Divalent Cations-The following experiments were performed in the pres-CIICC of magnesium and calcium ions which inhibit the conversion of the 4 S and 9 S receptors to the 5 S form a.nd its subsequent binding to nuclei (vide infm).
The cytoplasmic binding proteins were incubated with [311]cstradiol and unbound hormone removed at 4" using charcoal as described under "Materials and Methods." The hormone-receptor complexes were incubated with either nuclei or microsomal membranes from immature uterine tissue and from thymus at either 25' or 4" for various time intervals, as shown in Fig. 2. The binding of the estradiolreceptor complexes is dependent upon temperature and at 4" the extent of binding amounts to less than 10% of the input radioactivity.
At 25" we see considerable binding though t,here is little specificity of binding and the uterine estradiol-receptor complexes can bind efficiently to both nuclei and microsomal membrane.
J<oth nuclei and microsomes from immature utcriue tissue show a small adtlitioJla1 binding capacity, relative to nuclei or microsomes from thymus.
The data of Fig. 2 concern, at least ill part, a ternary complex of cytoplasmic receptor-estradiol-nucleus (or microsomes) rather than a shift of [3H]cstracliol from the cytoplasmic receptor to a nuclear receptor.
This was demonstrated by isolating uterine nuclei from such an incubation and extracting t.he bound hormone in 0.4 M KCl. hs shown in Fig. 3A the estradiol is released as the 4 S cstradiol-receptor.
Not all of the nuclearassociated hormone is &ractable in KC1 and this may reflect a low affinity binary estradiol-nuclear complex of t.he type discussed previously (8).

Site of 4 S Esfradiol-Receptor
The nuclear and microsomal binding of [sH]estradiol-receptor involves a correction due to the absorption of free 1%. estradiol on to charcoal (particularly at 25") which is necessary to prevent any dissociated estradiol from binding to membranes.
The amount of label bound to nuclei and charcoal which cosediments is compared to that bound and sedimented on charcoal alone in the absence of nuclei. Subsequently the hormone bound to nuclei is obtained by simple subtraction.
::I Microsomes were washed once and nuclei twice with Buffer B prior to KC1 extraction unless otherwise stated.
T.IBI.E I Eflect of divalent calions on estradiol-receptor binding to nuclei in in vitro incubations Nuclei were isolated from immature uterine tissue as described under "Materials and Methods" and incubated in Buffer A at 25" for GO min. bation and fractionated into the membrane and nuclcohistot~c components.
The data shown in Table I demonstrate that the bulk of the la.bel wa.s associated with the membrane fract.ion. The various subnuclcar fractions were then extracted with 0.4 M KC1 and analyzed on sucrose gradients as shown in Fig. 3.
An &radio1 binding component of sedimentation coefficient 4 S was extracted both from whole nuclear material and from the nuclear membrane fraction, but was not obt.ained from the nucleohistone fraction, again confirming (a) that the 4 S estradiol-receptor is unchanged after binding nuclei in the presence of divalent cations; and (b) that it binds primarily to the membrane fraction.
The data of Fig. 3B also serve to document that the estradiol-receptor complex binds to microsomal membranes in a form which is also extracted as a 4 S protein.
Conversion of 4 S Estradiol-Receptor to 6 S Estradiol-Receptor-The cytoplasmic extract containing the 4 S binding protein was incubated in Buffer U at 25" for 30 min in the presence of estradiol. Subsequently two binding complexes of sedimentation coefficients 4 S and 5 S were observed upon sucrose gradient analysis as shown in Fig. 48. In a control experiment we incubated cgtoplasmic receptor under identical conditions except that the hormone was omitted.
The incubation mixture was cooled to 4" and [3H]cstradiol added to label the receptor proteins. Upon analysis of the sucrose gradient ( Fig. 4) we can dctcct only a single 4 S peak under these circumstances.
The ability of magnesium and calcium ions to inhibit the conversion of the 4 S to the 5 S receptor is documented in Fig. 4B. That the divalent cations inhibit the forward reaction rather than activating the reverse 5 S to 4 S conversion was shown by incubating a mixture of 4 S and 5 S receptor (prepared in Buffer 13) with magnesium and calcium ions and showing that the 5 S receptor did not revert to the 4 S form (Fig. 4B). Finally, the cytosol solution from the incubation at 25" for 30 min which contains both 4 S and 5 S receptors was incubated with chromatin at 4" in Buffer B for 5 min; after cent.rifugation the suprrnatant was collected and analyzed on a sucrose gradient as shown in Fig. 4A. The 5 S estradiol-receptor has been effectively removed by binding to the chromatin in 5 min at 4', an incubation condition under which binding of the 4 S rcccptor to chromatin is not observed, as described above.
Thus, the 4 S receptor protein can be converted in part to the 5 S receptor in a reaction which requires elevated temperature (25') and the presence of estradiol, but is independent of the presence of chromatin.
Similar observations have been reported by Jensen (15) for the rat uterus. to Nuclear Material--The following expcriment.s were conducted in a divalent cation-free medium as this facilitates the conversion of the 4 S to the 5 S form. Cytoplasmic estradiol-receptor complex in Buffer B was prepared in the usual manner.
It was then incubated for 30 min at either 4" or 25" so that we expect the 4" incubation to contain only the 4 S estradiol-receptor and the 25" incubation to contain a mixture of 4 S and 5 S estradiol-receptors.
Immature uterine chromatin was added to each incubation system and the incubation continued according to the following protocol: (a) 4" preincubatioll-4" second incubation; (b) 4" preincubation-25" second incubation; (c) 25" preincubation-4" second incubation; and (d) 25" preincubation-25" second incubation. The rate of [W]est.radiol binding t.o chromosomal material as a function of time is shown in Fig. 5. Material which had not been incubated at 25" at either stage in the experiment (and therefore contains only 4 S estradiol-receptor) fails to bind, as expected, since the 4 S receptor dots not bind chromosomal material at 4". Preincubation at 25' followed by binding at 25" generates masimal interaction and sucrose gradient analysis of the 0.4 M KC1 extract demonstrates that both 5 S and 4 S receptors are bound undrr these conditions (Fig. 6). Estradiolreceptor prcincubatcd at 25" and subsequently treated with chromosomal material at 4" shows an immediate binding which amounts to 5Or/, of the masimum, 1~ow~'vcr, the extent of binding dots not incrrahc during the second incubation at, 4". We surmise t.hat this might bc due t.0 an initial rapid binding of the 5 S cstradiol-receptor produced during the preincubation, since t.he remaining 4 S receptor is convcrtctl neither to 5 S nor is it itself bound at. the temperat.ure (4") of the second incubation. This interpretation n-as confirmed by extracting the chromosomal material with 0.4 M KC1 after a 25"/4" incubation.
The distribution of [3H]estradiol is primarily in the 5 S form as shown in Fig. 6.
Binding of Receptor to Nucleohistone, Microsomes, and Chromatin in Absence of Estradiol--iZ cyt)osol solution in Buffer n was incubated at 25" for 30 min with the appropriate substrate (chromatin, nucleohistonc, or microsomes) as described under "hlnterials and Methods." After removal of these organelles (together with associated receptor proteins) by centrifugation, the supernatants were assayed for the number of receptor binding sites remaining in the supernatant using the Scatchard procedure. The results for the binding determinations are shown in Table II. To ensure that the receptor is actually binding to the various organelles and not being destroyed in some way (e.g. by proteolysis), the chromatin pellets from the incubation were incubated with [3H]estradiol in Buffer B at 4" for 2 hours. The nuclei were then extracted in 0.4 M KC1 and sedimented on a sucrose gradient. Fig. 7 shows that the receptor is indeed extracted and exists as the 4 S receptor.
A control experiment, utilizing chromatin which had Ilot beer) treated with receptor, does riot show the presence of cytoplasmic binding proteins upon KC1 extraction.

EJect of Divalent Cations on Binding of Eslradiol-Receptor to
Nudei from Immature Uterus-The estradiol-receptor complex was prepared at 25" in Buffer B (the solution will then contain both 4 S and 5 S receptors) and subsequently magnesium and  The procedure for assay of hormone binding is as described in Table I  calcium were added to a final concentration of 4.5 mM. This solution was then incubated with nuclei at 4". A control iucubation was performed in the absence of divalciit cations. The data of Fig. 8 show that much more of the cytoplasmic hormone rcccptor binds to chromosomal material in the presence of I%uffcr B t,han when divalent cations arc present. This is solely due to the binding of the 5 8 estradiol-receptor complex (the 4 S form will not bind at 4") to the chromosomal material as was shown in Fig. 5.
Site oj 5 S Estradiol-Receptor Binding to Chromosomal dlaterial-The cytoplasmic estradiol-receptor comples was prepared aiid incubated with chromatic in Buffer U for 60 mill at 25". The chromatin was then collected and fractionated into membrane and uuclcohistone components and each was assayed for associated [aH]estradiol.
The data of Table 111 show that in co11trast to an incubation in the presence of magnesium and calcium (Table I) a substantial part of the label is uow associated with the nucleohistone moiety, although a somewhat smaller portion remains associated wit.h the mrmbrane fraction.
Tot.al chromatic as well as the membrane and nuclcohistonc subfractions, were extracted with 0.4 III KC1 and examined by sucrose gradient techniques.
As seen in Fig. 9, chromatic contains both 4 S and 5 S receptor following such au incubation.
However, the 5 S-receptor is bound primarily to nuclcohistone, whereas the membrane fraction is much enriched in t.he 4 S receptor, confirming the notion that different estradiol-receptor complexes bind to different nuclear sites. There is also relatively little binding of the 5 S receptor to microsomal mcmbrancs (see Fig. 3) as had been predicted from the binding data above.
Specificity of 5 S Estradiol-Receptor Binding to Chromosomal diaterial-The cytoplasmic cstradiol-receptor complex was prepared in Buffer I3 and incubated in the presence (Buffer -4) or absence (13uffcr 13) of magnesium ions with subcellular fractions from uterus, thymus, or lung. The last two tissues do not respond specifically to estradiol.
The binding data are presented in Fig. 10. There is a small degree of specificity seen in the binding to microsomal mcmbrancs particularly in the divaleut cation-containing medium (Huffcr A) as shown in Fig. IOA, however, nuclear material from all three tissue binds the estratliol-receptor complcs in an itlcntical fashion iutlcpcndent of the ionic euvironmcrit (Fig. IOB), The association rate for the biuding is also t.hc same for all three in the prcscucc or abscucc of divalcnt cations (Fig. 1OC compared to Fig. 2). The binding also appears to be nonsaturable. Estractiou (0.4 M KCl) of lung aud thymus chromatin after binding gives rise to 4 8 aud 5 8 cstradiol-receptor complexes (data not shown) in esseutially the same proportions and quantity to that fouud for uterine chromatin (Fig. 9).
Presence of 5 S Estradiol-Receptor within Immature Uterine Tissue-We have demonstrated that the conversion of 4 S to 5 S receptor occurs if the cytosol solution is incubated at 25" in the presence of estradiol.
We now wanted to determine whether this same conversion could occur in the cytoplasm of the immature uterine cell. Immature uteri were incubated at 37" in the presence of [311]estradiol for 45 min. The uteri were rinsed and suspended at 4" in a 5000.fold excess of unlabeled cstradiol (two changes over 10 hours) so that the radioisotope could be depleted iu the interstices of the tissue and thus avoid the artifact of binding of [3H]estradiol to cytoplasmic receptors upou tissue disruption.
Any cstradiol-receptor binding which had occurred nithiu t.hc ccl1 is essentially irreversible at 4" for 10 hours (17). The tissue was disrupted in lsuffcr 1% slut1 tlic cytosol solution analyzed on a 5 to 20% sucrose gradient with the results shown in Fig. 11. Hoth 4 8 and 5 K cstradiol-receptors wcrc prcscilt iu the cytosol. In a control cspcrimcut, immature utcrinc tissue was incubated at 4" with cstradiol The sucrose gradient analysis of the cytosol (Fig. 11) shows that only the 4 8 receptor is present.
These results indicate that 5 8 receptor proteins are produced iu the cytoplasm, only when cstradiol is present at elevated temperatures.
If the cytosol solut.ion from t,he incubation at 37" is treated with chromatin at 4" for 5 min, the 5 S estradiol-receptor is almost tot,ally removed from solution (Fig. 11). Thus, we conclude that the conversion of 4 S receptor to the 5 S form t,akes place within the cytoplasm of the uterine cell at 37" in t.he presence of estradiol and that subsequently the 5 S estradiol-receptor is available for a rapid binding to nuclear material at 4" in the absence of divalent cations.
Availability of Receptor within Alature Endomeirial Cell-Cytoplasmic receptors can bind estradiol rapidly at 4" in vitro [3H]EstradioLreceptor (from 37" tissue though the rate of dissociation at this temperature is low indeed (17). Thus, the level of estradiol-receptor complex obtained after tissue disruptiou (at 4") will reflect the actual level occurring within the cell, together with any additional bincling to e&radio1 located within the interstices of the tissue and made available to the receptor upon tissue homogenization.
That the interstitial labeled estradiol can make a sizable contribution to the level of the estradiol complex is shown in Table IV. In this experiment endometrial tissue which had been incubated at 37" with [3H]estradiol was divided into equal fractions, one-half was washed and disrupted immediately and the other half was washed and incubated at 4" for 10 hours in the presence of fresh medium before tissue disruption.
The level of free hormone falls from 14,000 dpm per ml in the tissue which was not postincubated to 3400 dpm per ml in the tissue which was. Similarly, the level of receptor-estradiol complex falls by a comparable amount indicating that as much as 60% of the hormone receptor complex can be generated during the homogenization procedures. That such a postiucubation is sufhcieut for removal of interstitial hormone is documented iii I'ig. 12 which shows the timcdcpcndcut rclcasc of [311]estradiol from tissue previously ificubated at 37" (which permits hormone entry into the cell) alid at 4" in which case horrnonc does not cntcr the cell and the subscqucnt hormoric release is simply that rcmovcd from tlie ilitersticcs of the tissue (18). This last point is confirmed by the lack of receptor binding after homogenization of the latter tissue in the presence of charcoal.
The molar rat.io of bound cstradiol to free estrndiol (B :F ratio) in 27 ml of homogenate at, 4" is obt.ained directly from Table 1V (Column 11) and has a value of 1.26. Since t,he mass arid the density (1.03 g per ml) of this tissue are known, u-e can compute the volume of the tissue (10.3 ml) used for the incubations.
Upon a 2.7-fold dilution (for homogenization) the 12 :F ratio decreases by a factor of 1.48, from which we estimate that in the cell the actual molar "bound to free" ratio is 1.48 X 1.26 = 1.87 at 4". In addition, since the cytosol receptor has AH0 = -18 Cal per from the centrifugation at 130,000 X g was treated with charcoal three times to insure removal of all free est.radiol. The other half of the tissue was incubated at 4' for 10 hours and Centrifugation then homogenized in the presence (Column D) or absence (Column was at 480 X g/10 min (nuclei); 7,700 X g/10 min (fragmented B) of excess unlabeled estradiol (5,000 times).
chromosomal material); 27,000 X g/20 min (mitochondria and For tissue that has heavy microsomes); and 130,000 X g/60 min (lighf microsomes).  (19), it is possible to compute that the dissociation constant for hormone-receptor binding shifts from 1.35 X 10e9 M at 4" (the temperature of homogenization) to 1.08 X lo-lo M at 37". The attendant shift in L3:F ratio is directly computed from the Scatchard equation and we conclude that the molar l3:F ratio within the cell at 37" should be about 2.3.
The observed molar ratio of bound estradiol relative to that which is free within the cells after extensive postincubation treatment need not necessarily reflect the ratio existing within the cell prior to tissue disruption.
The molar LI:F ratio would be equal to that observed posthomogenization only if the hormone and the receptor are both as free to move within the cell as they are in the homogenizat.ion medium.
Certainly, t,he observations reported previously concerning binding of free estradiol to the nuclear membrane (8) would argue that the internal domain open to the hormone is quite extensive.
However, if the receptor were compartmentalized within the cell in such a way so that it could not bind estradiol, then the B : F ratio within the cell would be appreciably less than that observed in the fully dispersed homogenization medium, particularly if the blending process exposes, or releases, receptor binding capacity.
If the 13:F ratio within the cell prior to homogenization could be estimated, then a simple calculation would give a direct indication of the extent of compartmentalization of the receptor molecules. Fortunately, it is relatively simple to obtain an estimate of the amount of free and bound estradiol within the cell prior to homogenization by first removing interstitial esfradiol as described above and then blending the t.issue in the presence of a large excess of unlabeled estradiol.
This will compete with the bulk of the free labeled intracellular hormone in binding any released receptors.
The amount of free and bouud hormone will then be a fairly accurate reflection of the amounts in the tissue before cooling and homogenizing.
If such an experiment is performed, then as seen from the data of Table IV, the I%:F ratio is 0.7. This value is some 30$& of that obtained in t,he absence of unlabeled estradiol and as such argues that in the intact cell a substantial fraction of the receptors is unavailable for interact.ion with cstradiol, though this is not the case after tissue rupture.
It is possible to calculate the amount of receptor unavailable from the follo!ving relationship: When we refer to the presence of free estradiol in the endometrial cell, we are measuring that estradiol which is not bound to the cytoplasmic receptor.
This measurement with charcoal does not preclude the existence of lower affinity sites which can bind estradiol.
Charcoal binds estradiol very strongly and can therefore destroy low affinity binding, providing t.here is a significant dissociation rate for this binding. Therefore, free estradiol may be bound to sites of lower affinity in the cell such as membranes or enzyme systems which might be controlled by this hormone and we would not detect these using this approach.

DISCUSSION
Cytoplasmic estradiol-receptors are found in the uterine tissue of both mature and immature heifers. These recept.ors are found in good yield in both the endomct.rial and the mgometrial layers which compose the bulk of the uterine Gssue. The endometrium and the mgomctrium respond differently to &radio1 with only the entlometrium of adult cows showing a proliferative response. The uterus of very young animals shows a small hyperemic response to estrogen.
As the animal grows the uterine response increases until at a stage somewhat before puberty it demonstrat.es the full hyperemic and hyperplastic response to hormone administrat.ion (20). The high level of cytoplasmic receptors in very immature uteri was surprising as these receptors were thought to be involved in the proliferative response (21).
It has been argued that the cytoplasmic receptors are involved in transporting hormone through the cytoplasm to the nucleus where it was thought to be involved in differential gene activation, Since we had previously shown that estradiol binds nuclear membrane with high specificity during whole tissue incuba-EXTRACELLULAR q q + I INTRACELLULAR tion, we wondered if the cytoplasmic receptors would show a high and specific affinity for membranes.
We have assayed the binding of the various receptor proteins to microsomal membranes and to chromosomal material (ill-&ding the nuclear membrane). The 4 8 estradiol-receptor protein binds primarily t.o membranes in a temperature-depe1ldent process (very slow at 4") and shows a small specificity for uterine microsomal material over that of thymus microsomes. Furthermore, this binding occurs even if the 4 S receptor protein is not, complexed with estradiol.
The interaction between the 4 S receptor and membranes is disrupted at moderately elevated ionic strength (0.4 M KCl).
The 4 S receptor in media containing divalent cations (Buffer A) has no capacity to bind to norimembraneous chromosomal material (i.e. nucleohistone). In contrast, the 5 S estradiol-receptor complex in Buffer 13 binds predominately to uucleohistone in a rapid reaction even at 4". The conversion of the 4 S estradiol-receptor (and presumably the 9 S form) to the 5 S receptor requires elevated temperature (25"), and the presence of estradiol.
It is inhibited by hyperphysiological concentrations of divalent cations. Chromatin or other nuclear material is not required for this reaction.
Thus, in uterine tissue (in the presence of cstradiol) it is entirely feasible that the 5 S form of the receptor might be formed in the cytoplasm, and indeed this has been shown in the present work. Since the 5 S receptor can bind chromatin instantly even at 4", this raises the possibility that immediately up011 tissue disruption at 4", the 5 8 cstradiol-receptor complex could rapidly bind to nuclei to give an appearance of having been bound t.o the chromosomal material before isolation and thus generate a disturbing artifact.
If no compartmeutalizat,ion of receptors occurs in the cell, then conditions of elevated temperature (37") should facilitat,c the biuding of 4 8 cstradiol-receptor to nuclear material, a subsequent KC1 extract of isolated nuclei would bc similar to that seen in Fig. 6 for chromatin incubated at 25" with estradiolreceptor.
Yet as has been shown previously (5,22), practically all of the estradiol bound to immature uterine chromatic isolated from tissue previously incubated with the hormone, is bound to the 5 S estradiol-receptor.
These observations argue that the 4 S receptor is sequestered in a discrete part of the cell separate from the nucleus (for instance associated with the plasma mcmbrane).
We have demonstrated in this paper that 700/, of the 4 S receptor is restricted even from binding estradiol wit.hin t.he (f) 5 S receptor is reconverted to 4 S receptor.
(g) 4 S receptor reassociates with the plasma membrane.
(h) Estradiol IIOW in t.he free form is fret to associate to the high affinit,y site on t,he nuclear membrane in mat.ure endometrium or t.o bind allosterically t.o other proteins in the cytoplasm of bot.h mat.ure and immature tissues. The model has the merit that it fit.s all the available experimental dat.a (except poitlt f which is an assumption) and also that it provides an explanation for the ability of the hydrophobic est,radiol to pcnctratc rapidly into the cell. It is clearly important to know more about the 4 S and 5 S conversion such as whether it is purely a conformational shift (albeit large) or if the 4 S estradiol-receptor complex associates with another molecule to give a complex with increased mass. In this model we have ncglcctcd to consider the 5 S cstradiol-rcccptor protein as a protcin involved in transport of estxadiol to the nucleus. From the observations reported in this paper wc have severe rescrvations about such a function.