The Mechanism of Action of the Copper Intrauterine Device*

FERTILITY AND STERILITY Copyright < 1976 The American Fertility Society

Vol. 27, No.7, July 1976 Printed in U.S A.

THE MECHANISM OF ACTION OF THE COPPER INTRAUTERINE DEVICE* TERUHIKO TAMAYA, M.D., YOSHINORI NAKATA, M.D., YOUSUKE OHNO, M.D., SUMINORI NIOKA, M.D., NORIO FURUTA, M.D., AND HIROJI OKADA, M.D. Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan

The effects of copper ions on the binding of steroids to receptors revealed that the inhibitory effect of Cu++ was apparent at 10-6 M, and the binding capacities decreased to 10% at 10-2 M Cu++. The kinetic study demonstrated that Cu++ was a competitive inhibitor of steroid hormone-receptor binding (K; ~ 2.7 x 10-·' M to estrogen receptor; K; ~ 5.1 x 10--<; M to progesterone receptor). These results indicate that copper ions interfere at the steroid-binding site of receptor and that progesterone receptor is more affected by copper ions than is estrogen receptor. The sedimentation pattern showed the dissociation and aggregation of receptor macromolecules by copper. These phenomena may indicate the biologic inactivation of receptor. In fact, morphologically, progestational proliferation was severely inhibited and estrogenic action seemed to be inhibited. The Timm stain showed copper uptake by endometrial epithelium and superficial stromata. The copper content apparently increased in the cytoplasm of uteri bearing a copper intrauterine device, compared with controls. In vivo, the concentration of cytoplasmic copper was approximately 1.4 x 10-6 M, which was obviously inhibitory to steroid hormone-receptor interaction. However, complete morphologic suppression of the progestational effect by copper cannot exclude the coexistence of some other mechanism in these phenomena.

Copper is known to enhance the contraceptive effect of an intrauterine device (IUD). 1 In clinical trials the increased contraceptive efficacy, the decreased expulsion rate, and the occurrence of fewer complications (e.g., hemorrhage) have been proved. 2 ' 5 Accumulated evidence indicates that steroid hormone receptors mediate their known biologic effects in target organs. The presence of estrogen and progesterone receptors has been demonstrated m Accepted February 5, 1976. *Presented at the Twentieth Memorial and General Meeting of The Japanese Society of Fertility and Sterility, October 2 to 4, 1975, Sendai, Japan.

the rabbit uterus. 6 In this study the mechanism of action of the copper IUD (CuIUD) was investigated in rabbit uteri. The effects of copper on steroid hormonereceptor interaction and morphologic effects of copper were studied; additionally, the uterine copper content was measured experimentally. The in VIVO and in vitro results are compared.

MATERIALS AND METHODS

Preparation of Uterine Cytosol. Immature female rabbits (2 months old and weighing 1 kg) were primed by the subcutaneous injection of 50 fLg of 17{3-

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estradiol in sesame oil for 8 days. They were decapitated 2 days later and the uteri were excised, minced, and homogenized (three strokes) with a Teflon homogenizer using 3 volumes (v/w) of buffer A (0.01 M Tris. 1.5 mM ethylenediaminetetraacetic acid, pH 7.4). The suspension was centrifuged at 800 x g for 10 minutes, and the supernatant was again centrifuged at 27 4,200 x g (maximum) for 60 minutes at 4° C in a Hitachi RPS-55-T rotor. This supernatant was used as the uterine cytosol. Radioactive Steroid and Counting of Radioactivity. 1,2-3 H-Progesterone (4 7.8 Ci/mmole) and 6,7- 3 H-17,B-estradiol (48.0 Ci/mmole) were purchased from New England Nuclear Corporation, Boston, Mass. Radioactivity was counted with a Packard 3390 liquid scintillation spectrometer. Usually, a 0.2-ml aliquot of the sample was mixed with 3.0 ml of methanol and 10 ml of toluene-scintillation fluid (2,5-diphenyloxazole [PPO], 4 gm/liter; 1,4-bis[2-(5-phenyloxazolyl)]benzene [POPOP], 0.1 gm/liter in toluene). Determination of Progesterone Binding to Uterine Cytosol. Aliquots of uterine cytosol (0.5 ml) were incubated at 4o C for 2 hours with 3H-progesterone or estradiol plus buffer A (0.5 ml) containing various concentrations of copper ions. At the termination of the incubation, 0.5 ml of a charcoal suspension (0.1% Norite A and 0.001% dextran in buffer A plus 0.25 M sucrose) was added to separate unbound and weakly bound progesterone. Unbound steroid denotes steroid removed by charcoal absorption. The kinetic analysis was done by double-reciprocal plot. 7 The K; (inhibitor constant) was calculated by the following equation: K;

=

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The incubate (0.4 ml) was layered on 4.5-ml gradients containing 5 to 20% sucrose and centrifuged at 40,000 rpm in a Hitachi RPS-55-T rotor at 4° C for 16 hours. Cow fibrinogen and bovine serum albumin were used as sedimentation coefficient markers to standardize the gradients. Experimental Rabbits Bearing CuIUDs. Immature female white rabbits were primed by the subcutaneous injection of 50 11-g of 17,B-estradiol in sesame oil for 7 days. On the 8th day, a Cu-IUD was inserted into the right uterine horn as illustrated in Figure 1. Progesterone (1.0 mg) or 17,8-estradiol (50 JJ-g) in sesame oil was injected subcutaneously for another 7 days. On the next day these rabbits were decapitated and the uteri were excised. Sections of the uterine horns were stained by the Timm 8-hematoxylin combined method. Each uterine horn (1 gm) was minced and homogenized with a Teflon homogenizer (three strokes). The 1500 x g supernatant of this homogenate was used as cytoplasm. The cytoplasmic copper content of each uterine horn was measured by atomic absorption spectroscopy.

(iK,1)/(K" -K,1 )

where i is the concentration of inhibitor and K 1, is the pretending dissociation constant in the presence of inhibitor. Sucrose Gradient Centrifugation.

FIG. 1. Intraluminal insertion of the CuIUD into the rabbit uterus. The Cu-IUD was constructed of 6 em of polyethylene tubing (Fs. 4) coiled with copper wire (0.15 mm in diameter and 60 em long). I, Cu-IUD contacted; II, Cu-IUD not contacted; Ill, inert IUD.

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ly less affected by copper than that of the progesterone receptor. The kinetic study depicted in Figure 3, determined by dextran-coated charcoal, \ 75% showed that copper was a competitive \ inhibitor of steroid hormone-receptor . \ ~X lQQ ( Control binding: Ki =i = 2.7 x 10-s M to estro\ 50% gen receptor (K d =i= 1.3 x 10-9 M), and \ K; =i= 5.1 x 10- 6 M to progesterone I receptor (Kd 8.1 x 10-to M). These I 25% results indicate that copper acts through direct interference at the steroid-binding site of the receptor, resulting in the enhanced contraceptive effect of the IUD. Copper concentration Because the progesterone receptor was more affected by copper ions than was the FIG. 2. The effect of copper on steroid horestrogen receptor, it is suggested that the mone-receptor binding in rabbit uterine cytosol. . , - · - • - , 3 H-Estradiol-receptor binding; e, - - , estrogen receptor is more stable than the 3H-progesterone-receptor binding. progesterone receptor; thus the biologic effect of copper seems to be somewhat estrogenic. RESULTS It was demonstrated by 5 to 20% Effect of Copper on Steroid Hormone- sucrose linear gradient centrifugation Receptor Binding. Copper ions be- (Fig. 4) that estrogen and progesterone gan to interfere with steroid hormone- receptors, which both sedimented at 8 S, receptor binding at a concentration of were changed to more sedimenting forms 10- 6 M Cu++, and 10% binding of both in the presence of 10- 4 M Cu++, and progesterone and estradiol was ob- were dissociated to a 6.5 S form with served at 1 o-z M Cu++, as is shown moderate loss of steroid hormone binding in Figure 2. The binding capacity of the affinity in 10-z M Cu++. This dissociation estrogen receptor appeared to be slight- and aggregation of the receptor macroI

100%

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•

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+

5. 0

5.0

11 I .

12 I .

4. 0 (!/Bound) xlO"M

1

3. 0

10-"M Cu

Kii:2. 7xlo- 5 M

10-"M Cu

3. 0

(Kp~4 .4,10- 9 MI

Ki~S.lxl0- 6 M (Kpofl. 7x!Q- 6 M)

2. 0

1.0

1.0

2.0

(!/Free) xlO~M~ 1

FIG. 3. Double reciprocal plot. (1 ), Effect of copper ion on estrogenreceptor binding; (2), effect of copper ion on progesterone-receptor binding.

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~strogen

(2) Progesterone receptor

receptor

10,000

dpm

c

~- .

.· 5,000

A

Bottom

10

20

Fraction number

30 Top

20 Bottom 10 Fraction number

30 Top

FIG. 4. The effect of copper ion on the sedimentation pattern in rabbit uterine cytosol. A, No ion; B, 10- 4 M Cu++; C, 10- 2 M Cu++. BSA, Bovine serum albumin.

FIG. 5. Timm-hematoxylin stain of histologic sections from rabbit uteri. Each rabbit was primed with estrogen and a Cu-IUD was inserted as illustrated in Figure 1, followed by progesterone (A and B) or estrogen (C and D) treatment (see "Materials and Methods"). A and C, section from III; B and D, section from I, where copper is stained black and is seen in the endometrium and superficial stromata.

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ppm/mg(protein)

2xlo-' 2.4xlQ- 2 pprn/mg(protein) 2.2xlQ- 2 pprn/rng(protein) 0.67Xl0- 2 ppm/rng(protein)

FIG. 6. Concentration of copper in rabbit uterine cytoplasm. The protein concentrations were determined by Mehl's method." I, Cu-IUD contacted; II, Cu-IUD not contacted; III, inert IUD.

molecules may result in biologic inactivation of receptors. Morphologic Effects of Copper. In the uteri of rabbits bearing a Cu-IUD, the Timm-Hematoxylin combined stain showed that progestational proliferation was markedly inhibited (Fig. 5, A and B) and that estrogenic activity was also inhibited (Fig. 5, C and D). Copper was demonstrated in the epithelium and superficial stromata of both experimental uteri bearing a Cu-IUD (Fig. 5, B and D). Copper Content. The copper content of the cytoplasm is shown in Figure 6. It is obvious that the copper content of uteri bearing a Cu-IUD was increased. DISCUSSION

The increased contraceptive efficacy of copper has been considered to result from biochemical and morphologic reactions of uterine and oviductal mucosa, 9 direct influence of the blastocyst, 10 inhibited implantation, 11 and spermatocidal effect. 12 Oster 15 reported that the chemical action of copper was the degradation of S--8 contained protein, resulting in precipitation of albumin, inactivation of enzyme, and decreased elasticity of uterine mucus. It is known that the steroid hormone receptor has an S--8 binding capacity16· 17 ; therefore, the receptor might be inactivated by copper. In our study, copper inhibited steroid hormone-receptor binding, with Ki 2.7 x 10- 5 M to

+

estrogen receptor and with Ki ~ 5.1 x I0- 6 M to progesterone receptor (Fig. 3). Copper was more sensitive to progesterone receptor than to estrogen receptor. The competitive inhibition by copper of ste:r:oid hormone-receptor interaction suggests that copper affects the steroid hormone binding site of the receptor. The sedimentation pattern demonstrated that copper aggregated or dissociated steroid hormone receptor macromolecules. These changes make receptors biologically inactive. Hernandez et al. 18 have reported that the dissociation of polysomes by copper is one effect of a Cu-IUD. Morphologically, progestational proliferation was severely inhibited (Fig. 5B) and estrogenicity seemed also to be inhibited (Fig. 5D). This may result in dominancy of the estrogenic effect. The greater stability of the estrogen receptor in the presence of copper suggests an increased estradiol uptake in rat uteri bearing a Cu-IUD. 19 · 20 Stromal hypertrophy has been noted in Cu-IUD-bearing rabbits, 1 and deciduoma formation was inhibited by copper in the rat. These biologic effects are considered estrogenic instead of the result of inactivated progestational activity. The Timm stain demonstrated copper intake by endometrial epithelium and superficial stromata (Fig. 5, B and D). The copper content of the cytoplasm (2.4 x 10- 2 pprn!mg of protein), as measured by atomic absorption spectros-

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copy (Fig. 6), was equivalent to 1.4 x 10- 6 M Cu++, if calibrated under the cytosol preparation (6.6 X 10- 7 M cu++ in control). The concentration 1.4 x 10- 6 M Cu++ appears to be effective in the steroid hormone-receptor interaction.

REFERENCES 1. Zipper J, Medel M, Prager R: Suppression of fertility by intrauterine copper and zinc in rabbits. Am J Obstet Gynecol 105:529, 1969 2. Roy S, Cooper D, Mishell DR Jr: Experience with three different models of the copper T intrauterine contraceptive device in nulliparous women. Am J Obstet Gynecol 119:414, 1974 3. Shaila NG, Lane ME, Sobrero AJ: A comparative randomized double-blind study of the copper-T200 and copper-7 intrauterine contraceptive devices with modified insertion techniques. Am J Obstet Gynecol 120:110, 1974 4. Timonen H, Toivonen J, Luukkainen T: Useeffectiveness of the copper-T300 during the first year. Am J Obstet Gynecol120:466, 1974 5. Zipper JA, Tatum HJ, Pastene L, Medel M, Rivera M: Metallic copper as an intrauterine contraceptive adjunct to the "T" device. Am J Obstet Gynecol 105:1274, 1969 6. Tamaya T, Nakata Y, Nioka S, Furuta N, Ishihara S, Okada H: The effect of metallic ions on steroid hormone receptor binding. Acta Endocrinol Jap 51:586, 1975 7. Lineweaver H, Burk K: The determination of enzyme dissociation constants. JAm Chern Soc 56:658, 1934 8. Timm F: Zur Histochemie der Schwermetalle das Sulfid-Silberverfahren. Dtsch Z Ges Gerichtl Med 46:706, 1958

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9. Chang CC, Tatum HJ: A study of the antifertility effect of intrauterine copper. Contraception 1:265, 1970 10. Naeslund G: Blastocystotoxic effect of copper in vitro. Contraception 6:281, 1972 11. Chang CC, Tatum HJ, Kind FA: The effect of intrauterine copper and other metals on implantation in rats and hamsters. Fertil Steril 21:274, 1970 12. MacLeod J: Sulfhydryl groups in relation to the metabolism and motility of human spermatozoa. J Gen Physiol 34:705, 1951 13. Ullmann G, Hammerstein J: Inhibition of sperm motility in vitro by copper wire. Contraception 6:71, 1972 14. White IG: The interaction between metals and chelating agents in mammalian spermatozoa. J Exp Biol 33:422, 1956 15. Oster GK: Chemical reactions of the copper intrauterine device. Fertil Steril 23:18, 1972 16. Jensen EV, Hurst DJ, DeSombre ER, Jungblut PW: Sulfhydryl groups and estradiol-receptor interaction. Science 158:385, 1967 17. Kontula K, Jimne 0, Luukkainen T, Vihko R: Progesterone-binding protein in human myometrium. Influence of metal ions on binding. J Clin Endocrinol Metab 38:500, 1974 18. Hernandez 0, Ballesteros LM, Mendez JD, Rosado A: Copper as a dissociating agent of liver and endometrial polysomes. Fertil Steril 25:108, 1974 19. Adadevoh BK, Dada OA: Effect of intrauterine copper on the uptake of estradiol-C 14 by rat tissues. Fertil Steril 24:54, 1973 20. Aedo AR, Zipper J: Effect of copper intrauterine devices (IUD's) on estrogen and progesterone uptake by the rat uterus. Fertil Steril 24:345, 1973 21. Mehl JW: The buiret reaction of proteins in the presence of ethylene glycol. J Biol Chern 157: 173,1944