Further characterization of estrogen receptors in the human oviduct

FERTILITY AND STERILITY ~gh~,"'J983, The Alp.erjcan Fertility, Society

Vol. 39, No.6, June 1983 Printed in U.8A.

Further characterization of estrogen receptors in the human oviduct

Eberhard K. Muechler, M.D.* Donna Cary, B.A. Department of Obstetrics and Gynecology, University of Rochester School of Medicine and Dentistry, Rochester, New York

Estrogen binding in human oviducts was studied in vitro by the dextran-coated charcoal assay and sucrose density ultracentrifugation. Estrogen binds with high affinity and limited capacity to cytosol of the human oviduct. The concentration of competitive inhibitors to produce 50% reduction in estrogen binding was 8 x 10- 8 M for the antiestrogen CI-628, 8 x 10- 7 M for the progestogen norethynodrel, and 3 x 10 - 6 M for the testosterone derivative danazol at the ligand concentration of 1 nM estradiol. Nuclear estrogen binding was not inhibited by a 100-fold excess of progesterone or by a 10-fold excess of norethynodrel. Estrogen-binding protein with a sedimentation coefficient of 48 was seen in oviductal cytosol of all three anatomic segments. The nuclear 48 peak of estrogen binding was demonstrated in the ampullary tubal segment. Fertil 8teril39:819, 1983

Recent studies by us and others l - 6 have demonstrated the presence of estrogen receptors in the human oviduct. There is no agreement about the segmental distribution of estrogen receptors in the fallopian tube. Several reports l -3 , 6 indicate higher concentrations of estrogen receptors in the ampulla than in the isthmus or infundibulum. In contrast, one study 5 reports uniform distributions of estrogen receptors in the anatomic segments of the fallopian tube for both the proliferative and secretory phases of the menstrual cycle. These studies utilized mostly the dextran-coated charcoal (DCC) assay for separation of free and bound estradiol (E 2). We have used in this study the

Received October 14, 1982; revised and accepted January 19, 1983. ' *Reprint requests: Eberhard K. Muechler, M.D., Department of Obstetrics and Gynecology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642. Vol. 39, No.6, June 1983

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technique of sucrose density ultracentrifugation to establish the concept that previously measured estrogen binding in the oviductal segments is indeed conforming to biochemical qualities ofreceptor binding. Furthermore, we have investigated the interaction of progesterone (P), norethynodrel, and danazol with the estrogen receptor of the human oviduct by sucrose density ultracentrifugation and DCC assay. MATERIALS AND METHODS CHEMICALS AND BUFFERS

E2 (2,4,6,7 _3H) with a specific activity of 93 Cil mmol was purchased from New England Nuclear Corporation, Boston, MA. The following radioinert compounds were purchased: P and norethynodrel (17cx-ethynyl-17~-hydroxy-5 (10) estrene-3-one) were purchased from Sigma Chemical Company, St. Louis, MO. CI-628 (cx-[4-pyrrolidinoethoxy] pheny1-4-methoxy -cx- ni trostil-

Muechler and Cary Estrogen receptors in human oviducts

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bene) was donated by Parke-Davis, Ann Arbor, MI. Danazol (17a-ethinyl-17~-hydroxy-4 androsteno [2,3-dJ isoxazoD was a gift from SterlingWinthrop, Rensselaer, NY. The buffer used for cytosol experiments consisted of 50 mM Tris-2 mM ethylenediaminetetraacetic acid (EDTA)-l mM dithiothreitol (DTT), pH 7..4, at 4° C. The preparation of nuclear extracts for sucrose density ultracentrifugation was performed with 50 mM Tris-2 mM EDTA-1 mM DTT-O.4 M KCL buffer. Sucrose and Norit A were purchased from Fisher Scientific Company, Waltham, MA, and dextran T-70 from Pharmacia Company, Piscataway, NJ. Krebs-Ringer-Henseleit (KRH) buffer was prepared fresh and used for incubation of oviductal tissue. Aqueous counting scintillant from Amersham, Arlington Heights, IL, was used as liquid scintillation fluid. CYTOSOL PREPARATION

Human fallopian tubes were collected from seven women undergoing hysterectomy and salpingectomy. Tissue specimens were examined by the pathologist and immediately placed in icecold buffer solution. All oviducts were processed the same day for experimental assays. The serosal and connective tissue were separated from the oviduct proper. The oviductal tissue was homogenized in 5 volumes of Tris-EDTA-DTT buffer solution with a Tekmar instrument (Tekmar Company, Cincinnati, OH). Homogenization was performed with three pulses of 10 seconds' duration at the maximal speed setting. The homogenate was centrifuged at 160,900 x g for 60 minutes in a Beckman L5-65 ultracentrifuge (Beckman Instruments, Palo Alto, CA). The supernatant was incubated the same day with ligand.

sured by the method of Lowry et al. B Unlabeled danazol, norethynodrel, and CI-628 were added to cytosol in increasing concentrations (10- 9 to 10- 5 M) with the 3H-E 2 concentration held constant (1 nM). SUCROSE DENSITY GRADIENTS OF CYTOSOL AND NUCLEAR EXTRACT

Oviducts were separated into anatomic segments of fimbria, ampulla, and isthmus. Aliquots of oviductal tissue minces (250 mg) were incubated in 5 ml KRH buffer at 25° C with 1 nM 3H-E 2 • The incubation was carried out for 1 hour under an O2 atmosphere in a Dubnoff shaking water bath, Precision Scientific, Chicago, IL. The reaction was stopped by chilling the incubate on ice. After centrifugation at 700 x g for 5 minutes, the pellet was washed with 10 ml KRH buffer and recentrifuged three times. The pellet was then suspended in Tris-EDTA-DTT buffer and homogenized in the presence of 10- 6 M unlabeled E 2 • The homogenate was centrifuged at 700 x g for 15 minutes. The supernatant was recentrifuged at 160,900 x g for 45 minutes and then incubated with ligand. Aliquots were layered on sucrose gradients. The nuclear pellet was then homogenized in 1 ml Tris-EDTA-DTT-O.4 M KCL buffer.

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Aliquots of 0.2 ml oviductal cytosol were incubated with 0.7 to 3.5 nM 3H-E 2 at 4° C overnight. The reaction was terminated by the addition of 1 ml ofDCC suspension (0.05% dextran-0.25% Norit A in Tris-EDTA-DTT buffer). After 10 minutes of incubation at 4° C and centrifugation at 700 x g, the supernatant was decanted into scintillation vials. The bound radioactivity was counted in a liquid scintillation spectrophotometer. Blank tubes were prepared and subtracted. The apparent dissociation constant (K.!) and E 2 -binding capacity were determined by Scatchard analysis. 7 The protein concentration in cytosol was mea820

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Muechler and Cary Estrogen receptors in human oviducts

Fertility and Sterility

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10-9 10-8 10-7 10-6 10-5 INHIBITOR [M] Figure 2 Inhibition of 3 H-E 2 binding in human oviductal cytosol by the antiestrogen CI-628, the progestin norethynodrel, and danazol at a ligand concentration of 1 nM. Oviducts were obtained from a 49-year-old climacteric woman.

The nuclear extract was layered onto 5% to 20% sucrose gradients prepared in Tris-EDTA-DTT0.4 M KCL buffer. Centrifugation was performed with a SW 60 rotor at 208,000 x g for 16 hours in a Beckman L5-65 ultracentrifuge. Fractions of ten drops were collected from the bottom of the tube with a Buchler piercing unit (Searle Analytic Inc., Fort Lee, NJ). The radioactivity of each fraction was counted in a Mark I liquid scintillation counter (Nuclear Chicago, Chicago, IL). 14C_ labeled bovine serum albumin was prepared and used as an external marker for determination of the sedimentation coefficient. 9 In other experiments, unlabeled P (10- 7 M) or norethynodrel (10- 8 M) were added to the incubation of tissue minces to determine competition of nuclear E2 uptake.

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RESULTS Estrogen binding in cytosol of the human oviduct is characterized by high affinity (Kd = 0.9 x 10 -10 M). The available estrogen-binding capacity is estimated to be 38.9 fmol/mg protein (Fig. Vol. 39, No.6, June 1983

1). These results define estrogen binding in the human oviduct to be representative of receptor binding. The inhibitory action of several unlabeled compounds on estrogen receptors of the human fallopian tube can be seen in Figure 2. In comparison with the antiestrogen CI-628, there is considerably weaker inhibition of estrogen binding by the progestin norethynodrel and the testosterone derivative danazol. Concentrations of norethynodrel and danazol have to be lO-fold and 38-fold higher, respectively, to achieve competitive inhibition of estrogen binding in the human oviduct similar to CI-628. The interaction of P and norethynodrel with the nuclear estrogen receptor can be seen in Figure 3. The nuclear estrogen receptor in the human oviduct sediments in sucrose gradients with a sedimentation coefficient of 4S. P and norethynodrel in 100-fold and 10-fold excess of 3 H-E 2, respectively, lack competitive action on nuclear estrogen binding in the human oviduct. All three anatomic segments of the human oviduct demonstrate estrogen binding with a sedimentation value of 4S. The sedimentation coefficient of the nuclear estrogen receptor of the human oviduct can be seen in Figure 4. Nuclear estrogen receptor with a sedimentation coefficient of 4S is present predominantly in the ampullary segment of the human fallopian tube.

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Figure 3 Sedimentation of the nuclear estrogen receptor in the presence of P and norethynodrel during tissue incubation with 3H-E 2 • Bovine serum albumin sediments at 4.6S. Oviducts from the proliferative phase were used.

Muechler and Cary Estrogen receptors in human oviducts

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Human Fallopian Tube Cytosol

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Figure 4 Sucrose density gradient centrifugation of estrogen binding in the anatomic segments of the human oviduct from a climacteric 52-year-old woman. The bound radioactivity was corrected for variable protein concentration. (B), Sedimentation of the nuclear estrogen receptor in the different anatomic segments of the human oviduct. Protein concentrations of nuclear extracts were: ampulla 14 mg/ml, isthmus 10 mg/ml, and fimbria 10 mg/ml from a climacteric 52-year-old woman.

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Only small shoulders of nuclear estrogen binding are present in the isthmus and fimbria_ DISCUSSION

Our investigation of available estrogen receptors in the human oviduct complements our previous observations of steroid hormone binding in the oviduct. 1, 3, 4, 10, 11 The apparent equilibrium Kd of 10 - 10 M and estrogen binding capacity of 38.9 fmollmg protein in oviductal cytosol is con822

sistent with earlier reports. 4 , 5 Other identified properties of estrogen binding in cytosol from the oviduct include sedimentation at 88 or 48 in sucrose density gradients, 1, 4, 10 steroid specificity,4, 10 thermolability,lO and sensitivity to proteolytic enzymes. 4 All these characteristics fulfill the criteria for the definition of a receptor. We have attempted to clarify further the interaction of therapeutic agents that are used for contraception, abnormal genital bleeding, and endometriosis with the estrogen receptor of the human oviduct. The antiestrogen CI-628 was selected for study because of its known interaction with the estrogen receptor in the animal uterus 12 and the rabbit fallopian tube. 13 The concentration of CI-628 necessary to produce a 50% reduction in oviductal cytosol binding was 8 x 10 - 8 M. This result is almost identical to observations by Ruh and Ruh 14 in the rat uterus. The progestin norethynodrel was selected because of its previously determined interaction with estrogen binding in the rabbit uterus. 15 The concentration of norethynodrel for reducing estrogen binding in human oviductal cytosol by 50% is 8 x 10 -7 M. This indicates a weak affinity of norethynodrel for the estrogen receptor in human oviductal cytosol. The binding affinity of 19-norprogestogens for the estrogen receptor depends on the molecular structure of the steroid molecule. Experiments with the estrogen receptor of the rabbit uterus have shown that acetate or hydroxyl groups at the 313 and 1713 positions result in higher affinity.16 The lack of a phenolic A ring is probably responsible for the overall weak affinity of 19-norprogestogens. These observations on animal tissue explain the low affinity of norethynodrel for the estrogen receptor in the human oviduct in our study. Our results with danazol as competitor for the estrogen receptor in the human oviduct indicate that danazol has a low affinity for the estrogen receptor of the human oviduct (3 x 10- 6 M). This is in agreement with findings by Barbieri et al. 17 in the rat uterus. In contrast, results by Chamness et al. 18 failed to show any effect by danazol on estrogen binding in the rat uterus. The presence of estrogen, P, and androgen binding has been demonstrated in cytosol of human endometriosis tissue. 19 , 20 Danazol appears to be an effective inhibitor of androgen and P binding in animal tissue. 17 , 18 It remains uncertain whether competitive inhibition of estrogen binding by pharmacologic doses of danazol may exert biologic effects in endometriosis.

Muechler and Cary Estrogen receptors in human oviducts

Fertility and Sterility

Our experiments on the nuclear binding of E2 in the human oviduct demonstrate that the nuclear 48 estrogen receptor is not changed qualitatively or quantitatively by 10- 7 M P and 10- 8 M norethynodrel. These in vitro studies suggest that activation of the cytoplasmic receptor complex and nuclear estrogen binding are not affected by P or norethynodrel under our in vitro conditions of tissue incubation. Other experimental conditions have provided information about two possible mechanisms of interaction for progestogens with estrogen receptor. Hsueh et al. 21 have proposed that progestogens inhibit the replenishment of cytoplasmic estrogen receptor, whereas Okulicz et al. 22 have favored the reduction of occupied nuclear estrogen receptors by progestogens. The sedimentation value of estrogen binding has been studied for the entire human oviduct but not for the separate anatomic segments. Our present observations have demonstrated estrogen binding protein in the cytosol and nuclear extract of isthmus, ampulla, and fimbria with the same sedimentation coefficient as for the entire oviduct. No attempt was made to determine quantitative differences of estrogen binding in all three anatomic segments of the oviduct. These findings confirm earlier observations of estrogen binding in the separate segments of the human oviduct, which were made predominantly by DCC assays.1-3, 5, 6 Experimental evidence for correlation of estrogen binding with the biologic response of tubal contractility was demonstrated convincingly by the in vitro studies of Clemens et al. 13 More studies are needed for investigation of the relationships between estrogen binding, anatomic characteristics, ciliogenesis, contractility, and gamete transport in the different segments of the human oviduct. In conclusion, we have described specific properties of the estrogen receptor of the human fallopian tube that may lead to a better understanding of tubal physiology and aid in explaining pharmacologic effects of widely used drugs on the pathophysiology of the oviduct. REFERENCES

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female genital tract of humans and monkeys. Ann NY Acad Sci 180:197, 1977 Muechler EK, Kohler D: Properties of the estrogen receptor in the human oviduct and its interaction with ethinylestradiol and mestranol in vitro. J Clin Endocrinol Metab 51:962, 1980 Punnonen R, Lukola A: Binding of estrogen and progestin in the human fallopian tube. Fertil Steril 36:610, 1981 Pollow K, Inthraphuvasak J, Manz B, Grill H-J, Poll ow B: A comparison of cytoplasmic and nuclear estradiol and progesterone receptors in human fallopian tube and endometrial tissue. Fertil Steril 36:615, 1981 Scatchard G: The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660, 1949 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurements with the folin phenol reagent. J Bioi Chern 193:265, 1951 Rice RH, Means GE: Radioactive labeling of proteins in vitro. J Bioi Chern 246:831, 1971 Muechler EK, Flickinger GL, Mikhail G: Estradiol receptors in the oviduct and uterus of the rabbit. Fertil Steril 25:893, 1974 Muechler EK, Flickinger GL, Mastroianni L Jr, Mikhail G: Progesterone binding in rabbit oviduct and uterus. Proc Soc Exp Bioi Med 150:275, 1976 Katzenellenbogen BS, Bhakoo HS, Ferguson ER, Lan NC, Tatee T, Tsai T-LS, Katzenellenbogen JA: Estrogen and antiestrogen action in reproductive tissue and tumors. Recent Prog Horm Res 35:259, 1979 Clemens LE, Shih Y-H, Brink HO, Callantine MR: Physiologic significance of 1713-estradiol binding in the rabbit fallopian tube. Am J Obstet Gynecol 122:113, 1975 Ruh TS, Ruh M: The effect of antiestrogens on the nuclear binding of the estrogen receptor. Steroids 24:209, 1974 Terenius L: Affinities of progestogen and estrogen receptors in rabbit uterus for synthetic progestogens. Steroids 23:909, 1973 Tamaya T, Nioka S, Furuta N, Shimura T, Takano N, Okada H: Contribution offunctional groups of 19-norprogestogens to binding to progesterone and estradiol-1713 receptors in rabbit uterus. Endocrinology 100:1597, 1977 Barbieri RL, Lee H, Ryan KJ: Danazol binding to rat androgen, glucocorticoid, progesterone, and estrogen receptors: correlation with biologic activity. Fertil Steril 31:182, 1979 Chamness GC, Asch RH, Pauerstein CJ: Danazol binding and translocation of steroid receptors. Am J Obstet Gynecol 136:426, 1980 Janne 0, Kauppila A, Kokko E, Lantto T, Ronnberg L, Vihko R: Estrogen and progestin receptors in endometriosis lesions: comparison with endometrial tissue. Am J Obstet Gynecol 141:562, 1981 Tamaya T, Motoyama T, Ohono Y, Ide N, Tsurusaki T, Okada H: Steroid receptor levels and histology of endometriosis and adenomyosis. Fertil Steril 31:396, 1979 Hsueh AJW, Peck EJ Jr, Clark JH: Control of uterine estrogen receptor levels by progesterone. Endocrinology 98:438, 1976 Okulicz WC, Evans RW, Leavitt WW: Progesterone regulation of the occupied form of nuclear estrogen receptor. Science 213:1503, 1981

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