Binding of estrogen and progestin in the human fallopian tube

FERTILITY AND STEluLrrY Copyright c 19S1 The American Fertility Society

Vol. 36, No. 5, November 1981 Printed in U.SA.

BINDING OF ESTROGEN AND PROGESTIN IN THE HUMAN FALLOPIAN TUBE

RELJO PUNNONEN, M.D.* ALTTI LUKOLA, M.Sc.t Department of Obstetrics and Gynaecology, Turku University Central Hospital, Turku, Finland, and Department of Biochemistry and Pharmacy, A.bo Akademi, Turku, Finland

The estrogen and progestin receptor concentrations in the human fallopian tube haue been investigated. The binding of estrogen and progestin to their cytoplasmic and nuclear receptor proteins were of high affinity and specificity. The cytoplasmic and nuclear receptor concentrations in the isthmus, ampulla, and fimbria did not differ significantly. The nuclear progestin receptor concentrations in the isthmus and fimbriae were significantly higher (P < 0.05) in the follicular phase than in the luteal phase of the menstrual cycle. In the postmenopausal tubes the receptor concentrations were in general low. In the pregnant tube the estrogen receptor concentration was low, whereas the progestin receptor concentration was quite high. Fertil Steril36:610, 1981

Little attention has been paid to the question of the steroid hormone receptors in the human fallopian tube. Fertilization and the early development of the zygote, however, occurs in this organ. The endosalpinx is influenced by the ovarian hormones and exhibits histologic cyclic changes corresponding to the various phases of the menstrual cycle. 1 Ovarian hormones also play an important role in the regulation of the tubal activity. 2 The purpose of the present study was to investigate the concentrations of the estrogen and progestin receptors in the human fallopian tube during the normal menstrual cycle, after the menopause, and during tubal pregnancy.

had regular menstrual cycles and 4 were postmenopausal. One tube was obtained in connection with a tubal pregnancy. The tubes were put immediately on ice, and all subsequent procedures were performed at 0 to 4o C if not otherwise stated. The tubes were divided along the length into isthmus, ampulla, and fimbria and were cut into small pieces and washed \fith ice-cold physiologic saline. Homogenization was performed in three volumes (v/w) of 40 mM Tris/HCl-1 mM dithiothreitol buffer, pH 7 .4, with an Ultra-Turrax TP 18/10 homogenizer. The homogenates were centrifuged for 1 hour at 100,000 x g in an MSE Superspeed centrifuge. The supernatant fluids were assayed for cytoplasmic receptors. To the pellet was added three volumes MATERIALS AND METHODS (v/w of the original sample) of homogenizing buffer containing 0.6 M KCl and incubated for 1 hour The fallopian tubes were removed in connection at oo C under occasional agitation. The supernaof hysterectomies from 16 women, of whom 12 tant obtained after 1 hour of centrifugation at 100,000 x g was used for assay of nuclear receptors. Received February 12, 1981; revised and accepted May 8, 1981. Aliquots of cytosol (0.1 ml) were incubated *Reprint requests: Reijo Punnonen, M.D., Department of overnight at oo C with various concentrations of Obstetrics and Gynaecology, Turku University Central Hostritiated ligand (0.16 to 10 nM) with or without a pital, 20520 Turku 52, Finland. 200-fold excess of unlabeled ligand (in 0.1 ml hotDepartment of Biochemistry and Pharmacy, Abo Akademi, mogenizing buffer with estradiol or 0.1 ml homogTurku, Finland. 610

Vol. 36, No. 5

BINDING OF ESTROGEN AND PROGESTIN

TABLE 1. Equilibrium Dissociation Constantsa for Estrogen

and Progestin Binding in the Human Fallopian Tube

Cytoplasmic Nuclear

Estradiol

R5020

nM

nM

0.117 ± 0.031 0.093 ± 0.047

0.803 ± 0.230 0.221 ± 0.159

aMean ± standard error of the mean.

enizing buffer containing 10% glycerol with progestin). The nuclear receptors were quantitated by incubating aliquots of the nuclear extract for 1 hour at 22° C. The concentrations of radioactive ligands and the buffers were the same as used for quantitation of cytoplasmic receptors. Bound and free hormones were separated by the addition of · 0.5 ml dextran-coated charcoal (1% activated charcoal and 0.1% dextran in assay buffer). After 30 minutes at oo C the tubes were centrifuged, and 0.5 ml of the supernatant was used for measurement of radioactivity. The number of binding sites and dissociation constants were determined according to Scatchard. 3 The protein content of the cytosols and nuclear extracts were measured by the method of Lowry et al. 4 Radioactivity was measured in a liquid scintillation counter (LKB Wallac 81000). The scintillation liquid composed 0.6% (w/v) 2,5-diphenyloxazole (PPO) and 0.08% (w/v) 1,4-di(4-methyl-5-phenyloxazolyl)-benzene (POPOP) in toluene. (2,4,6, 7 -3 H)-Estradiol (111.0 Ci/mmol), (17methyl- 3 H)-R5020 (87.0 Ci/mmol) and radioinert R5020 were obtained from New England Nuclear Corporation. Radioinert 17 13-estradiol, estriol, estrone, testosterone, dihydrotestosterone, corticosterone, and progesterone were purchased from Sigma Chemical Company. RESULTS

The equilibrium dissociated constants determined from Scatchard plots for the cytoplasmic and nuclear estrogen and progestin receptors are shown in Table 1. The binding of both estradiol and R5020 showed a higher affinity to the nuclear binding protein than to the cytoplasmic receptor. The specificity of the binding was determined by the ability of different nonradioactive ligands to compete with the 3 H-labeled ligand for binding to their respective receptor proteins. As appears from Table 2, the binding of estradiol and R5020 to their respective receptors was highly specific. Tables 3 and 4 show the estrogen and progestin receptor concentrations in the isthmus, ampulla, and fimbriae during the follicular phase respec-

611

tive to the luteal phase of the menstrual cycle. The corresponding figures for the tubes obtained from postmenopausal women and for the case of tubal pregnancy are shown in Table 5. There were no significant differences (P > 0.05) in the receptor concentrations between the three anatomical sections of the tubes. The differences in the cytoplasmic receptor concentrations between the luteal and follicular phases were also not significant (P > 0.05). The nuclear progestin receptor concentrations in the isthmus and fimbriae were significantly higher (P < 0.05) in the follicular phase than in the luteal phase of the menstrual cycle. In the pregnant tube the estrogen receptor concentration was very low, whereas the cytoplasmic progestin receptor concentration was relatively high in all three sections of the tube. In two cases (patients 8 and 9) the progestin receptor levels were high. In one case (patient 12) no detectable numbers of progestin receptors could be found in the tube. This patient was 51 years of age and had an irregular menstrual cycle. DISCUSSION

The dissociation constant for the estrogen receptor obtained in the present paper is in agreement with the value obtained for the estrogen receptor in rabbit tube,5 but is significantly lower than that reported for the human fallopian tube. 6 The concentration of estrogen receptors has been reported to be higher in the ampullar region than the isthmus. 6 • 7 The anatomic sections revealed no differences in the estradiol binding capacity between the proliferative and secretory phases, but the cytoplasmic estrogen receptor fraction was generally elevated in the follicular phase when compared with the luteal phase. 6 The variations in the estradiol receptor levels in the difTABLE 2. Specificity of the Estrogen and Progestin Binding in the Human Fallopian Tub~ %Binding

Competitor

None Estradiol Estriol Estrone Dihydrotestosterone Progesterone R5020 Corticosterone Testosterone

Estradiol

R5020

100 8 (8)b 13 (12) 10 (11) 70 (75) 92 (95) 98 (96) 99 (99) 98 (99)

100 81 (82) 99 (99) 83 (85) 64 (63) 21 (19) 18 (16) 67 (68) 86 (89)

acompetitor concentration bNuclear binding.

=

2

X

w-s M.

------~---------....._._........_.__;_

_

___;

__

___,~

November 1981

PUNNONEN AND LUKOLA

TABLE 3. Number of Estradiol and Progestin Binding Sitesa in Different Segments of the Human Fallopian Tube during the Follicular Phase of the Menstrual Cycle R5020

Estradiol Patient

Age n2

n3

nl

n2

n3

46.1 27.4 79.0 75.0 2.5 18.4 2.5 8.1 113.2 6.6 22.7 11.4 40.0 20.0

44.9 52.2 123.0 43.0 2.7 10.8 4.2 6.9 64.5 3.9 28.5 24.7 22.0 16.4

305.2 70.8 548.0 86.6 101.4 106.0 176.8 72.5 37.6 8.8 19.7 9.6 42.9 10.3

327.4 48.4 963.0 220.0 222.0 32.7 124.6 35.7 55.7 7.8 11.0 2.6 55.0 13.8

214.0 55.4 1440.0 106.0 131.3 62.8 209.6 68.7 56.1 10.4 21.0 47.8 22.9 8.1

43.7 ± 15.3 23.8 ± 8.9

41.4 ± 15.8 22.5 ± 6.9

175.9 ± 72.6 52.0 ± 19.6

251.2 ± 125.7 51.5 ± 28.7

299.2 ± 192.5 51.3 ± 12.8

nl

1

42

2

39

3

55

4

51

5

53

6

47

7

34

c 20.4 N 26.6 c 58.0 N 45.8 c 3.5 N9.7 c 3.7 N 6.2 c 61.0 N 4.0 c 14.7 N 7.0 c 26.4 N 12.5

c 26.8 ± 9.0 N 15.9 ± 5.7

Mean± SEMb

i

t;

I

j

~ = fmol/mg protein. C = cytoplasmic; N = nuclear; n1 = isthmus; n2 = ampulla; n3 = fimbriae.

bMean ± standard error of the mean.

I_

despite continued treatment with estradiol. 9 In our study, too, the lowest estradiol receptor concentrations were found in the isthmus, and the concentrations were reduced during the luteal phase in all- parts of the tube. The depression of the binding sites during the luteal phase, however, was not so dramatic as has been reported for rhesus monkey. 9 The mechanism of the depressive action of progestins on estrogen receptor levels is obscure. It is, however, evident that this effect is due to the inhibition of estrogen receptor replenishment. 10• 11 In the myometrium, as well as in the fallopian tube, progestin appears to act as an estrogen antagonist- by reducing the estrogen-induced increase in the receptor concentrations. At the endometrial level, however, proges-

ferent portions of the tube has been suggested to be related to the variation inthe cellular components-of the individual segment. Thus, for example, the ampulla has more abundant endosalpinx than the isthmus and therefore contains more receptors. 6 Radioactive labeling in autoradiography, however, has been reported to be more intense in the muscle and connective tissue than in the endosalpinx. 8 During an artificial menstrual cycle in the rhesus monkey, the highest estrogen receptor concentrations were found during the follicular phase.9 In the isthmus the estrogen receptor concentrations were lower than in the ampullar region. Progesterone treatment provoked a severe depression in the estrogen receptor concentration,

TABLE 4. Number of Estradiol and Progestin Binding Sitesa in Different Segments of the Human Fallopian Tube· during the Luteal Phase of the Menstrual Cycle R5020

·Estradiol Patient

Age n2

n3

nl

n2

n3

6.6 5.6 17.5 9.2 16.4 3.9 8.4 2.5 None 2.4

6.7 5.7 24.8 8.7 18.6 10.9 8.0 4.3 None 3.1

125.6 None 26.4 37.5 9.3 21.3 27.9 None None

126.3 None 239.0 28.2 55.4 10.3 10.8 12.1 None None

227.3 None 329.7 21.8 57.6 5.2 9.3 10.0 None None

9.7 ± 3.2 4.7 ± 2.8

11.6 ± 4.4 6.5 ± 1.4

46.1 ± 25.0 12.7 ± 6.1

86.3 ± 44.1 10.1 ± 5.1

-124.7 ± 65.5 7.4 ± 4.0

nl

8

50

9

24

10

26

11

50

12

51

Mean± SEMb

C4.9 N 4.0 CNone N3.3 c 13.5 N8.7 c 5.9 N4.5 c 3.2 N 5.0

c 5.5 ± 2.2 N 5.1 ± o-.9

~n = fmollmg protein; C = cytoplasmic; N bMean ± standard error of the mean.

=

nuclear; n1

=

isthmus; n2

·=

ampulla; n3

=

fimbriae.

BINDING OF.ESTROGENAND PROGESTIN

Vol. 36, No.5

613

TABLE 5. Number of Estradiol and Progestin Binding Sitesa in Different Segments of the Human Fallopian Tube Obtained from Postmenopausal Patients and in One Case of Tubal Pregnancy Estradiol Patient

n2

n3

nl

n2

n3

6.5 7.7 14.9 5.3 11.6 3.6 16.1 2.8 4.3 1.8

6.6 7.8 13.8

217.5 31.4 96.2 17.0 1.8 1.0 28.5 None 68,8 8.9

218.5 31.5 102.2

12.0 1.0 16.7 5.2 2.0 1.5

177.8 10.0 48.0 8.1 None None 34.8 None 94.0 8.0

nl

13

52

14

52

15

76

16

/53

17 (pregnant)

R5020

Age

34

c 8.4 N 3.7 c 7.4 N 2.5 c 2.1 N 1.9 c 23.1 N 1.5 c 8.4 N 1.0

2.1 None 25.8 None 105.8 17.1

Mean± SEMb

c 10.2

± 4.5

12.2 ± 2.1

12.2 ± 2.2

65.1 ± 38.8

86.0 ± 48.1

87.0 ± 48.7

Patients 13-16

N 2.4 ± 0.4

4.8 ± 1.0

4.6 ± 1.9

5.2 ± 2.4

12.3 ± 7.4

10.5 ± 10.4

an = fmoVmg protein. C = cytoplasmic; N bMean ± standard error of the mean.

=

nuclear; n1 = isthmus; n2 = ampulla; n3 = fimbriae.

tins seem to act synergistically with estrogens by increasing the estrogen receptor concentration.12, 13 The dissociation constant for the progestin receptors in the present paper is in good agreement with the value previously reported for the human tube. 14 A considerably lower affinity for the human tubal progestin receptor has, however, been reported. 15 The dissociation constants for the progestin receptor in cat16 and beagle17 tubes are also higherthan the values presented in this paper. The KCl-extractable fraction had a higher affinity for the ligand than the cytoplasmic receptor protein, a finding that is in agreement with previous reports. 15· 17 The progestin receptor concentrations in the human myometrium and endometrium are somewhat higher during the follicular than the luteal phase of the menstrual cycle. 18· 19 The situation seems to be similar in the fallopian tube as well. The lowest progestin concentrations were found in the isthmus and at the end of the luteal phase. In some cases the cytoplasmic progestin receptors may remain at quite a high level after the menopause (patient 13). The reason for this phenomenon is not fully understood. Estrogens are capable of inducing new progestin receptors. 11 • 20 The augmentation of cytoplasmic receptors by estrogen action involves the stimulation of RNA and protein synthesis, and the mechanism of estrogen-induced cytoplasmic progesterone receptors appears to be regulated by the cytoplasmic estrogen receptor system. 21 The mechanism for inactivation of progesterone receptors by progestins is obscure but does not seem to require protein synthesis.

In connection with ovarian follicle cysts (patients 1 and 2), the serum estrogen level is generally elevated and the progestin concentration low. The high cytoplasmic progestin receptor levels observed in these patients is most likely a reflection of the circulating estrogen level. The relatively high receptor levels found in the KCl-extract is, however, difficult to explain. The most likely explanation is the contamination of the high-speed pellet by cytoplasmic receptor molecules. Since we did not find any clear correlation between the cytoplasmic and nuclear receptor concentrations, we conclude that the values obtained do not express the true nuclear levels. In the case of tubal pregnancy (patient 17), we found quite high cytoplasmic progestin receptor levels, in spite of the fact that the tube was exposed to high levels ofprogesterone. The explanation may be the concomitant high circulatory estrogen level. Further studies are, however, required to clarify the mechanism of hormone action in the pregnant tube. Acknowledgments. This work was supported by the Paulo Foundation and the Tor, Pentti and Joe Borgs Foundation.

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4. Lowry OH, Rosebrough NJ, FarrA, Randall RJ: Protein measurement with folin phenol reagent. J Biol Chern 193: 265, 1951 5. Muechler EK, Flickinger GL, Mikhail G: Estradiol receptors in the oviduct and uterus of the rabbit. Fertil Steril 25:893, 1974 6. Flickinger GL, Muechler EK, Mikhail G: Estradiol receptor in the human fallopian tube. Fertil Steril25:900, 1974 7. Robertson DM, Landgren B-M: Oestradiol receptor levels in the human fallopian tube during the menstrual cycle and after menopause. J Steroid Biochem 6:511, 1975 8. Stumpf WE, Baerwaldt C, Sar M: Autoradiographic cellular and subcellular localization of sexual steroids. In Basic Actions of Sex Steroids on Target Organs, Edited by PO Hubinont, F Leroy, P Galand. Basel, S. Karger, 1970, p3 9. Brenner RM, Resko JA, West NB: Cyclic changes in oviductal morphology and residual cytoplasmic estradiol binding capacity induced by sequential estradiol-progesterone treatment of spayed rhesus monkeys. Endocrinology 95:1094, 1974 10. Bhakoo HS, Katzenellenbogen BS: Progesterone modulation of estrogen-stimulated uterine biosynthetic events and estrogen receptor levels. Mol Cell Endocrinol 8:121, 1977 11. Hsueh AJW, Peck EJ Jr, Clark JH: Control of uterine estrogen receptor levels by progesterone. Endocrinology 98:438, 1976 12. Martel D, Psychoyos A: Progesterone-induced oestrogen receptors in the rat uterus. J Endocrinol 76:145, 1978

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13. West NB, Verhage HG, Brenner RB: Suppression of the estradiol receptor system by progesterone in the oviduct and uterus of the cat. Endocrinology 99:1010, 1976 14. Kumra R, Sen KK, Hingorani V, Talwar GP: Binding of progesterone in the human fallopian tube. Am J Obstet Gynecol119:762, 1974 15. Verhage HG, Akbar M, Jaffe C: Cyclic changes in cytosol progesterone receptor of human fallopian tube. J Clin Endocrinol Metab 51:776, 1980 16. Verhage HG, Akbar M, Jaffe RC: Cytosol and nuclear progesterone receptor in cat uterus and oviduct. J Steroid Biochem 11:1121, 1979 17. Lessey BA, Gorell TA: Analysis of the progesterone receptor in the beagle uterus and oviduct. J Steroid Biochem 13:1137, 1980 18. Bayard F, Damilano S, Robel P, Baulieu E-E: R.ecepteurs del'oestradiol et de la progesterone dans l'endometre humain au cours du cycle menstrual. CR Acad Sci Paris 282:1341, 1975 19. Baulieu E-E, Atger M, Best-Belpomme M, Corvol P, Courvalin J-C, Mester J, Milgram E, Robel P, Rochefort H, DeCatalogne D: Steroid hormone receptors. Vitam Horm 33:649, 1975 20. Coulson PB, Pavlik EJ: Effects of estrogen and progesterone on cytoplasmic estrogen receptors and rates of protein synthesis. J Steroid Biochem 8:205, 1977 21. Leavitt WW, Chen TJ, Allen TC: Regulation of progesterone receptor formation by estrogen action. Ann NY Acad Sci 286:210, 1977