Estrogen target cells

Printed

in Sweden

Experimental

Cell Research 101 (1976) 15-22

ESTROGEN TARGET CELLS Establishment of a Cell Line derived from the Rat Pituitary Tumor MtTIF, C. SONNENSCHEIN, Tufts University

A. M. SOTO, J. COLOFIORE and R. FAROOKHI

School of Medicine,

Cancer Research Center, Boston, MA 0211 I, USA

SUMMARY A ce!l line, designated FPG, derived from the transplantable rat pituitary tumor MtT/F4 has been established in long-term culture. The established cell line carries markers similar to those of the original source of material. Several clones from this line have been isolated. Saturable estrogen binding proteins of high aftinity were characterized both in the cytosol and in the KCI nuclear extract of the MtT/F, tumor growing in castrated animals. Clones C,FPG and CSFPG also showed the presence of estrogen receptors in their cytosol. “Empty” resident nuclear receptors in both cloned cell lines were also characterized. As far as can be determined, these nuclear receptors were not translocated from the cytoplasm in an estrogen, temperature-dependent step. The dissociation constants for the estrogen receptor were essentially the same (lo-r0 M) in the cytoplasmic and nuclear compartments of both the tumor and the cell lines derived from it. The number of binding sites/cell in the two clones did not vary significantly; they have about 7 Oo&lO 0Of1sites/cell. The availability of cell cultures capable of reproducing systems present in whole animals should contribute to an understanding of the mechanism of action of estrogens.

The mechanism of action of sex steroid hormones is being examined by different approaches to explain how these hormones affect their target cells [l]. An alternate approach in the continuing search for uncovering the effect of such hormones in target cells is represented by the use of target cells growing in long-term culture conditions. Long-term cell cultures derived from specialized or highly differentiated tissues or tumors have been useful for the understanding of functions performed in the intact organism. Pituitary cell lines, which can be cloned, represent a good model system for the study of specialized functions, such as prolactin and growth hormone synthesis and secretion [2, 3, 41, ability to act as target cells for estradiol-17/3 (EZ) [5, 61, the presence of thyroid hormone receptors Z-761806

[7], the growth dependency for tri-iodothyronine [7, 81 and the presence of plasma membrane hormone receptors [9]. The establishment and characterization of a rat pituitary cell line derived from the mammosomatotrophic tumor MtT/F, [lo] is described in this report. A novel pituitary factor, the “feminizing factor” or feminotropin, that modifies the sex steroid metabolic pattern in male and female rat liver is secreted by cells from this tumor [ 111. The estrogen target condition of the tumor and two clones of the cell line FPG are defined. EXPERIMENTAL Two adult Fisher rats bearing 2 tumors (one in each thigh) of the transplantable MtT/F, tumor cells were graciously supplied by Dr Robert Bates, Endocrine Research Laboratory, NIAMD, NIH, Bethesda, Md. These tumor cells are known to secrete high levels of at least three pituitary hormones, namely growth hormone, prolactin and adrenocorticotrophin (ACTH) inExp

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to the bloodstream [12]. Indirect evidence for the increased activity of prolactin was the bilateral enlargement of mammary glands which secreted milk spontaneously. Similarly, adrenal glands were larger than normal and histolonicallv the effect of ACTH resulted in a widening of the fascicular layer of cells in the cortex. (For further details on the characteristics of these tumors, see [12-141.) A orimarv culture was initiated from one of the original tumor-bearing animals, sent by Dr Bates. Cells were disuersed bv sterilv mincine the tumor mass with scissors, trying to free’ as many individual cells as possible from the friable tumor. Once cells and small pieces of tumor were obtained, they were kept for IO min in hypotonic saline solution to eliminate red blood cells; this procedure was repeated twice and finally a much reduced red cell mass was obtained with the yellowish tumor cell pellet after low-speed centrifugation. This pellet was seeded on several 250 ml Falcon plastic flasks each containing 10 ml of growth medium. The medium used to initiate the primary culture and for maintenance growth was dulbecco’s modified Eagle medium reconstituted from powder (GIBCo, Grand Islands, N.Y., or Flow Laboratories, Bethesda, Md) plus 15% horse serum and 2.5 % calf serum (Microbiological Ass., Bethesda, Md). Antibiotics (penicillin-streptomycin), Fungizone (Squibb & Sons, Princeton, N.J.) and anti PPLO-agent (Tylomicin, GIBCo) or gentamicin (Schering Co., Port Reading, N.J.) were added to the media. We called this combination medium I. In order to test for the ability of these cells to specifically bind estradiol-l7/3 (E2), they were grown in Dulbecco’s modified Eagle medium plus 10% castrated and adrenalectomized calf serum (medium 2). This serum does not contain estrogen (Dr W. C. Wagner, Iowa State University, Ames, Iowa). The purpose of using medium 2 is to prevent the EZ- and temperaturedependent translocation of the estrogen receptor to the nuclei of target cells [ 151. Procedures for subculturing cell cultures, cloning and freezing have been described in details elsewhere [6]. I

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Drugs and isotopes Estrone (E,), estradiol-17g (E,), estriol (E,), progesterone and testosterone were obtained from Sigma and Co. (St Louis, MO). In most cases, steroids were purified by chromatography and were used at a higher than 95% purity. Labelled steroids included [2,4,6,73H]estrone ([3H]E,) (S.A. 106 Ci/mM): [2,4,6,7-3H]estradiol-17P (rSH]E,) (S.A. 106 Ci/mM) 2,4,6,7-r3H]estriol ([3H]E&; [1 ,2-3H]progesterone (S.A. 50.3 Ci/ mM) and [1,2,6,7-aH]testosterone (S.A. 91 CilmM); aII purchased from New England Nuclear Co., Boston, Mass. All the tritiated steroids were chromatographed to determine purity which was more than 95 %.

Tumor transplantation-inoculation into animals When the size of the MtT/F, tumor (l-2 cm in diameter) required so, they were transplanted after mincing to obtain a dispersed population of cells. BeExpCdRes

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tween 1 to 5x l(Yj viable cells were injected in the thigh of inbred Fisher rats (Microbiological Ass.. Bethesda, Md). Experiments were conducted to determine the degree of autonomy of these transplantable tumors. To this effect. an eaual number of cells were iniected into intact and castrated males and females. Shortly after the cell line derived from the MtT/F, tumor was established, an inoculum of several concentrations of cells (from lo6 to 7X IO’ cells) were injetted into immunocompetent adults as well as newborn Fisher rats (within 24 h of birth (5X I@ cells)).

Chromosome studies Chromosome counts of C,FPG and C,FPG cells were performed. Chromosome preparations were carried out by a modification of the method previously published by Hungerford [ 161. Suitable metaphases were photographed and idiograms arranged from prints.

Preparations of subcellular fractions of F, tumors The tumor-bearing animals were castrated 2-3 weeks prior to sacrifice. The rats were killed by decapitation and the tumors removed immediately, placed in icecold 0.9% NaCI, 10 mM Tris-HCl, pH 7.4 (buffer A). After mincine and washing twice with buffer A, the tissue was &suspended in-3 vol of buffer B (10 mM Tris-HCI. 2 mM MeCl. oH 7.4). and disrupted in an allglass homogenizer. The homogenate ‘was filtered through two lavers of cheesecloth and centrifuged at 700 g-for 10 min. The supernatant, “crude cytosol”, was centrifuged at 129000 g for 20 min; this supernatant was called cytosol. The crude nuclear pellet was resuspended with 0.1% Triton X-100, 0.32 M sucrose, 2-mM MgCI,, IO mM Tris-HCI buffer, pH 7.4. After incubation at 4°C for 5 min, nuclei were centrifuged and washed three times with 0.32 M sucrose, 2 mM MgCI,, IO mM Tris-HCI buffer, pH 7.4. Nuclei were extracted with 0.5 M KCI, 10 mM Tris-HCI, 1.5 mM EDTA buffer, pH 7.4 (TKE) by freezing and thawing, followed by centrifuaation at 129000 g for 20 min. Purification of nuclei w&s achieved by Chauveau’s method [ 171when needed. Nuclear recovery is expressed as the ratio of DNA recovered in the nuclear fraction to the DNA present in the total homogenate. DNA was assayed by the Burton method [ 181using calf thymus DNA (Sigma) as standard.

Preparation of subcellular fractions of FPG cells in culture To prepare subcellular fractions from the cultured cells. the cells were collected bv centrifugation, washed twice with Dulbecco’s modiied Eagle medium and resuspended in 3 vol of buffer B, allowed to swell for 10 min at 4”C, and driven through a syringe fitted with a 22 gauge needle. After centrifugation at 700 g for 10 min the crude cytosol and nuclear fractions were prepared following procedures described above

Estrogen target cells in cell culture for tumor fractionation. Lactic dehydrogenase assays and electron microscopy of the nuEle&fraction were performed to confirm the lack of contamination between subcellular fractions [ 191.

Binding of estrogen receptors The parameters studied were the dissociation constant for hormone-receptor interaction (Kd) and the concentration of receptors. Briefly, the subcellular extracts were incubated at 4°C with a fixed concentration of [3HJEz and an increasing concentration of unlabelled hormone ranging from 0 to lOA7M. After equilibrium was attained, the bound and free hormones were separated by hydroxylapatite adsorption [19], and the data obtained was analysed according to Scatchard

WI. Competition experiments 0.1 ml aliquots of cvtosol or nuclear extracts were incubated ai 4°C witd saturating amounts of [3H]E2 and increasing amounts of steroids (E,, E,, E,, T and P) ranging f;om 0 to 10e5 M. A& equilibrium was achieved, bound and free hormones were separated by hydroxylapatite adsorption and bound fractions were analysed for radioactivity.

Sucrose tritiated gradients Extracts from subcellular fractions were analysed through sucrose tritiated gradients as described by Harrison & Toft [21] with some minor modifications. Briefly, linear sucrose gradients between 5-20% were prepared from stock solutions containing 2X IO+’ M [3H]E, in 10 mM Tris, 1.5 mM EDTA, pH 7.4 or TKE buffers. The cytoplasmic and nuclear extracts were layered on the 4.8 ml gradient and centrifuged at 40000 rpm for 16 h in a Spinco SW50.1 Rotor. Between 30-38 fractions were collected by puncturing the bottom of the tube. Bound and free E, were separated by hydroxylapatite adsorption. The adsorbed fractions were analysed for radioactivity. Sedimentation values were calculated by comparison with BSA standards [22]. Linearity was checked by adding phenol red to the low density stock solution; the concentration of phenol red was determined by adsorbance at 400 nm.

Protein determinations Estimation of proiein concentrations were performed by the method of Lowry et al. [23] utilizing crystalline bovine serum albumin as a standard.

RESULTS Establishment of the cell lines Several attempts were made to establish a cell line from the MtT/F, tumor in our laboratory. In all of these experiments cell

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death followed the transulantation of the animal tumor to the plastic flask. Between the 2nd and 4th day after initiation of the culture, most cells appeared dead according to the dye exclusion test. In one attempt, viable cells were seen after 5 days of cultivation. Most of what were considered viable cells were growing in clumps floating in the growth media. This culture was named FPG. An aliquot of the viable cell clumps was injected back into the thigh of an adult Fisher female rat after 3 days of culture. The animal was injected with a long acting estradiol compound (estradiol valerate, 2 mg, Delestrogen, Squibb & Sons) assuming that this hormone would help the injected cells to grow, and after 25 days, a tumor about 1.5~ 1x 1 cm in size was obtained. Eventually, this transplantable tumor kills the animal as the original MtT/F, does. No significant difference in growth rate was recorded between the MtT/F, tumors transplanted from animal to animal and the one obtained from cells grown in culture conditions for 3 days. Out of this tumor, developed from cells kept f or 3 days in culture, a new cell line was established. The new primary culture was designated FPG-B. After 7 days of incubation, the primary culture clumps of FPG-B cells were reinjected into a Fisher female rat and again another tumor developed after 30 days, but no cell line could be established from the latter source of material. No obvious explanation could be given to this failure. Once the cell lines FPG and FPG-B became established in culture, single cell clones were isolated from wells of Microtest II plates (Bioquest, Cockeyville, Md) where single cells were seeded. Fifteen clones were grown in medium A and several ampules containing 1-2x 106cells were kept frozen in a liquid nitrogen tank. To deterE.vp Cdl /?c,.\ /o/ (1976)

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mine the specific binding of labeled estrogens to subcellular fractions of these clonal cell lines, cells from clones CFPG and C,FPG were studied (see below). After the establishment of cell lines FPG and FPG-B, new attempts to grow pituitary tumor cells from MtT/F, tumors did not succeed. We continue trying to establish new cell lines from the MtT/F, tumor.

animals at basically the same time (20 days) regardless of the sex and treatment. This result is in accordance with published reports [24]. The cell line FPG and FPG-B and some clones derived from the former were tested at different times after their establishment, for their ability to develop into tumors at the site of inoculation when injected into Fisher rats. Cells were injected into (a) intact males and females; (6) estradiol valerate (Delestrogen) monthly injected males and females, to establish whether estradiol may influence the growth of these cells; (c) into newly-born Fisher rats in order to avoid the possibility that the cells would be rejected by the host for their antigenicity (cells were injected within 24 h of birth); (d) into Wistar/Furth female rats in order to discard the possibility that these FPG and FPG-B cells which are phenotypically indistinguishable from GH, cells [25], and/or the RAP series of rat pituitary cells [6] could be a contamination by these cells which are also growing in our laboratory. In each of these groups of animals, FPG cells and FPG-B cells growing in culture were injected in doses starting at lo6 cells and going up to 7x IO7 cells. Results in all these groups of animals under all the variables described were consistently negative, i.e., no tumor developed at the site of inoculation in any of the animals used to test malignancy.

Tumor transplantation The original MtT/F4 tumor obtained from Dr Bates was tested for its ability for growing in inbred animals with different hormonal backgrounds. Equal numbers of cells (106) were injected into normal and castrated male and female rats. Tumors developed at the site of inoculation in all the

Chromosomal studies Chromosome counts were performed on 120 C,FPG cells between passages 9 and 11 after cloning. The modal chromosome number of these cells is 61. An approximation of the morphological distribution of chromosomes is as follows: 14-18 small metacentrics and submetacentrics and 40-50

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Fig. 1. Abscissa: Ez bound (X 10m9M); ordinate: bound/free Ez. (Lef) cytosol; (right) nuclear. Scatchard plot representation of the binding of estradiol to A, cytosol (Kd=0.3x lo+’ M); and 0, nuclear (Kd= 0.4~ lo-r0 M, 1.5fmoleslmg protein) extracts of MtT/F* tumor. Portions (0.1 ml) of cytosol or nuclear extract were incubated with 10m9M [3H]E2 in Tris buffer (0.1 ml) and increasing concentrations of unlabelled E, (0-3x lo-* M) to give a total volume of 0.3 ml and incubated overnight at 4°C to ectuilibrium. Bound and free E, were separated by hydroxylapatite adsorption; after equilibration the slurrv was filtered through Whatman no. 1 paper and washed with 10 ml of 5% mM Tris, 5 mM KH,PO, buffer. nH 7.4. The dried resin containing the-bound hormone was counted. The results were corrected for the non-specific binding that remained adsorbed to hydroxylapatite. For details, see Materials and Methods.

Exp

Cell

RPS 101 (1976,

Estrogen target cells in cell culture

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Fig. 2. Abscissa: E2 bound (X IO-$ M); ordinate: bound/free E,. Scatchard plot representation of the binding of E, to A, cytosol (Kd=0.5X10-10 M, 126 fmoles/mg protein) and to 0, nuclear (Kd=O.8x lo-lo M, 64 fmoles/mg protein) extracts of C,FPG cells. See fig. 1 and Materials and Methods for details.

acrocentrics and telocentrics. It is worth mentioning the presence of two big metacentric chromosomes of dissimilar size in 90% of the cells of this clone. At least one of these two “marker” chromosomes is always present. Cells of clone C,FPG were also studied to determine their chromosome complement. The modal number is 63 and the two big metacentric chromosomes are also present in these clonal cells. In about 10% of the cells a medium-sized submetacentric chromosome has been observed. Estrogen binding in MtTIF, tumor extracts Fig. 1 represents a Scatchard plot of E, specific binding in cytosol and nuclear extracts. The Kd is 0.3 X lo-lo M in cytosol and 0.4~ lo-lo M in nuclear extracts. The concentration of E,-binding sites is 115 fmoles ( lo-l5 mole)/mg of protein in cytosol and 15 fmolesimg of soluble protein in the KC1 extractable nuclear preparation. The KC1 extractable nuclear receptor represents lO20% of the total tumor receptor present in the whole preparation (cytosol+nuclear).

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Estrogen binding in FPG cells Both cell lines C, and C,FPG have cytoplasmic and nuclear receptors. The Kd for Ez is similar to those described for E, target organs in general, 0.2 to 1x lo-lo M. About 30 % of the total cell specific binding was in the nuclear compartment. Fig. 2 represents a Scatchard plot of data obtained with C,FPG cells in culture. The Kd values were 0.5 and 0.8~ lo-i0 M in cytosol and nuclear extracts, respectively. The concentration of binding sites in cytoplasmic extract was 126 fmolesfmg of protein which corresponds to about 4 000 sites/ cell; and 64 fmoles/mg of soluble protein, corresponding to 2500 sites in the nuclear extract. The low protein concentration (approx. 75 kg/lo” cells) reflected by these figures is probably due to the unavoidable loss of protein in obtaining the clean nuclei preparations. Studies conducted with C,FPG indicate the presence of about 10000 sites/ cell.

Fig. 3. Abscissa: unlabelled steroid/[3H]E,; ordinate: % [3HJE2bound. Competition experiments with cytosd preparations of MtT/F, tumor. Unlabelled EQ(0-O); E, (A-A); and E, (X-X) inhibited 50% of the [3H]Ep binding at different concentrations (E2>E3>EI). Testosterone (Cm) and progesterone (O-O) were unable to compete with [3H]EZ at all the tested concentrations. For details, see Results. Exp Cell Res 101 (1976)

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Sonnenschein et ~1. A

BSA

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fraction no.; ordinate: cpmx IO-“. Sucrose density gradient sedimentation of cytosol and nuclear fractions of clonal cell line C,FPG. (A) Pattern of [3H]E2 binding in cytosol at low ionic strength; (B) sedimentation pattern of the same cytosol extracted with 0.5 M KCI; (C) sedimentation pattern of the nuclear fraction extracted with 0.5 M KCI. BSA (4.45 S) was used as standard. For details, see text.

Fig. 4. Abscissa:

phenomenon was also observed by Harrison & Toft in chick oviduct cytosol [ZI]. Fig. 4 shows the sedimentation profiles of cytoplasmic and nuclear receptors of cells from C,FPG clonal line obtained through sucrose “tritiated” gradients. The cytoplasmic receptor has a sedimentation coefficient of 7.5s when analyzed through S20% sucrose tritiated gradients in low ionic strength buffer and 3.9s when the KC1 concentration is 500 mM. The KC1 nuclear extracts analysed through high salt gradients show a 5.355: receptor. The different S values obtained for cytoplasmic and nuclear receptors analyzed in high salt conditions eliminates the possibility of contamination of the nuclei by cytoplasmic receptor. DISCUSSION

The evidence accumulated indicates that a cell line derived from the transplantable rat Competition experiments pituitary tumor MtT/F, has been esFig. 3 shows the ability of different steroid tablished in a long term culture condition. hormones to compete with [3H]Ez in cytosol As in many other instances, the established preparations of F4 tumor. Unlabelled Ez cell line carries markers similar to those was able to reduce the binding of [“H]E, to present in the original source of material. 50% of maximum at lower concentrations The data presented show that MtT/F, than E3 and E, (0.73, 4.7 and 8.0~ lo+’ M, tumor, and clones C,FPG and C,FPG have respectively). Testosterone and progester- specific high affinity, saturable, estrogen one were ineffective even at relative con- receptors. The values obtained for the discentrations of 10000. These results are in sociation constants (Kd’s) are within the agreement with the Kd values obtained range reported for E, receptors in the pituifrom Scatchard analysis using E2, E, and tary [26] and other target organs, such as El. The degree of affinity was E2>E3>E1 uterus [27]. The degree of affinity for steroid hormone by those receptors is E,> (not shown). E,>E,, and no affinity for P and T as indiSucrose gradient analysis cated by competition studies. The presence of a specific, E2 nuclear When subcellular extracts were analysed receptor in the tumor and in the clonal cell through conventional sucrose gradients, partial dissociation of the E, receptor- lines derived from it is, however, a decomplex was observed. In addition, the free parture from the occurrence in E, target orhormone present in the lower density region gans in castrated animals. The possibility sometimes masked the 4s receptor. This that what we call nuclear receptor may be Exp

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Estrogen target cells in cell culture cytoplasmic contamination carried by the nuclear preparation seems unlikely, because (1) experiments carried out in our laboratory for nuclear receptor in the uteri of the same strain of rats (castrated) were negative; (2) the sedimentation coefficient of E,R present in nuclear preparations was different from that obtained in cytosol when both extracts were analyzed in high ionic strength sucrose gradients, which indicates a conformational and/or structural difference between the receptor in those cell compartments; (3) nuclear preparations obtained as described in Methods have shown, in systematic studies designed to check for cytoplasmic contamination, that the latter could not account for the high level of nuclear receptor found [ 191; and (4) the presence of a nuclear receptor in cell lines growing in estrogen-depleted media makes it unlikely that the cytoplasmic receptor was translocated into the nucleus. Furthermore, if this were the case, it would be difficult to explain why it is unoccupied under the experimental conditions used to characterize the presence of receptors. The presence of a “resident” nuclear receptor in GHB cells [5] has also been reported by another group of investigators [28]. Additionally, preliminary evidence for a translocation process similar to that described in target organs of animals is not apparent in this system. The amount of E, entering the nuclei of cells that already have a sizeable amount of “empty resident” nuclear receptor is small and does not follow the pattern seen in normal uterus (Soto & Sonnenschein, unpublished data). The mechanism by which this receptor is present in nuclei needs further investigation. One can speculate on a possible relationship between this “empty” receptor and the translocated 5s receptor occurring in target tissues exposed to Ee. In vitro trans-

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formation of 4s to 5s receptor in the cytosol of these cells has been explored, and our results showed an estradiol-temperature dependency. In the absence of Ez, the receptor remained as a 4s subunit (not shown). Since several groups [29, 321 reported the E,-independent translocation of unoccupied receptor from cytoplasm to nucleus in immature rat uterus exposed to testosterone, the existence of a pool of cytoplasmic receptor able to be translocated into the nucleus in the absence of E2 may be postulated. Whether this translocation mechanism is dependent on the transformation of the 4s cytoplasmic receptor to the 5s “activated” receptor is not clear. If this were the case, once the receptor is transformed it is localized in the nuclear compartment; the pool of transformable receptor would be transient and therefore would not be apparent by in vitro heating. Another possibility is that a 4s subunit may exist, able to move between the nuclear and cytoplasmic compartments without restraint. Once in the nucleus it is attached to a chromatin fraction forming a KC1 extractable 5s complex. Also, the 5s “empty” receptor may be the conventional nuclear receptor after dissociation from the Ez, although this seems unlikely, since cells growing in E,-depleted medium for more than 40 generations still have 30% of their receptor located in nuclei. Finally, the possibility that this 5s unoccupied nuclear receptor could be a different molecule, unrelated to the cytoplasmic one, cannot be ruled out. A totally independent or endogenous nuclear receptor has already been described in chick liver [30] and mature heifer uterine nuclear membrane [3 11. We thank Gary Murphy, Heather Duram, Dan Kiracafe and Mary Fong for their technical assistance. This research was supported in part by grants CA Exp Cc// Res IO1 (1976)

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12338, CA 13410and CA 12924from the USPHS and by the ACS (Massachusetts Division). Dr C. Sonnenschein is a Research Career Development Awardee from the USPHS (CA 47410).

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15. Jensen, E V. Numata, M, Brecher. P I & de Sombre, E R, The biochemistry of steroid hormone action (ed R M S Smelie) p. 133. Academic Press, New York and London (1971). 16. Hungerford, D A, Stain technol40 (1965) 333. 17. Chauveau, J, Mot&, Y & Rouiller. C H. Exp cell res 11 (1956) 317. 18. Burton, K, Biochem j 62 (1956) 3 15. 19. Soto, A M, Rosner, A L. Farookhi, R & Sonnenschein, C, Methods in cell biol 13(1976) 175. Scatchard, G, Ann NY acad sci 5 I (1949) 660. Harrison, R W & Toft, D, Endocrinology -_ % (1975) 199. 22. Martin, R G & Ames, B N. J biol them 236 (l%l) 1372. 23. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193(195I) 265. 24. Nickerson, P A, Molteni, A & Nakayama, I, Cancer res 33 (1973) 3135. 25. Tashjian, A H, Yasumura, Y, Levine, L, Sato, G & Parker. M L. Endocrinoloev 82 ( 1%8) 34 I. 26. Kahwanago, L’Heinrichs, W-L & Herrman, W L, Endocrinology 86 (1970) 1319. 27. Toft, D, Shyamala, G & Gorski, J, Proc natl acad sci US 57 (l%7) 1740. 28. Haut, E, Noess, 0 & Gautvik, K M, Acta endocrinol, suppl. 199(1975) 201. 29. Ruh, T S, Wassilak, S G & Ruh, M F, Steroids 25 (1974) 257. 30. Lebeau, M C, Massol, N & Baulieu, E E, Eur j biochem 36 (1973) 294. 31. Jackson, V & Chalkley, R, J biol them 249 (1974) 1627. 32. Rochefort, H, Lignon, F & Capony, F, Biochem biophys res commun 47 (1972) 662. Received March 3, 1976 Accepted March 16, 1976