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Endometrial estradiol-17β-dehydrogenase activity in women wearing Cu-IUD
CONTRACEPTION
ENDOMETRIAL
ESTRADIOL-17B-DEHYDROGENASE L. Tsengl, V. Dramusic2,
ACTIVITY
IN WOMEN WEARING Cu-IUD
and E. Gurpide3*
1 Department of Obstetrics and Gynecology, School of Medicine, State University 11794
of New York of Stony Brook, Stony Brook, New York
2 Division of Pediatric and Adolescent Gynecology, Institute Mother and Child Protection, Beograd, Radiga Dakica 8, Yugoslavia 3 Department of Obstetrics and Gynecology, Medicine, New York, N.Y., 10029
for
Mount Sinai School of
ABSTRACT
In order to determine whether inhibition of the endometrial estradiol-176-dehydrogenase activity by Cu++ could be one of the factors accounting for the enhanced contraceptive efficacy of copper intrauterine devices (CU-IUD), the activity of the enzyme was measured in endometrial biopsies from women wearing this device. The levels of enzymatic activity found in these specimens were within the range of controls (women without IUD), e.g. 0.1-0.8 and 7-27 nmol El formed/mg protein/h in the follicular and luteal phases of the menstrual cycle, respectively. It is concluded that CU-IUD does not modify significantly either the activity of endometrial-17b-dehydrogenase, an enzyme known to regulate the tissular concentration of estradiol, or its response to the inductive effect of progesterone.
Accepted
*
for publication
February
24, 1979
To whom reprints should be addressed.
MARCH
1979 VOL. 19 NO. 3
247
CONTRACEPTION
INTRODUCTION The enhanced contraceptive effectiveness obtained by adding a copper wire to plastic intrauterine devices (1) may be related to For effects of Cut+ on biochemical processes in endometrial cells. instance, Cut+ may influence the production and secretion of proteolytic enzymes, prostaglandins and glycogen (1,2,3). Cupric ions may also interfere with the hormonal action of estradiol (E2) and progesterone (P) on cells by reducing the binding of these hormones to their receptors (4,5). The purpose of the present study was to examine the possibility that Cu++ released from copper intrauterine devices (CU-IUD) may inhibit the activity of the estradiol-178-dehydrogenase (E2DH), an enzyme postulated to regulate the concentration of i2 in the endometrium under physiologic conditions (6,7). Pollow et al. have reported that the - -mitochondrial and microsomal activities of the enzyme in the cytosollc, fractions of human endometrium are inhibited by 10m3 M Cut+ (8-10). The activity of the estradiol dehydrogenase was measured in endometrial biopsies obtained from normally menstruating women wearing CU-IUD for different periods of time. The enzyme activities in endometrium from these subjects were compared with the average activities found in proliferative and secretory endometrium of cycling women who did not use intrauterine devices (11). EXPERIMENTAL Endometrial
biopsies
Endometrial curettings were obtained from 8 healthy, normally menstruating subjects whose ages ranged from 19 to 37 years and who were wearing the Cu-7 intrauterine device (Searle Laboratories) from 8 to 33 months. Most curettings were performed during the period estimated to correspond to the mid-luteal phase of the cycle. All specimens included in this study were dated histologically. Tissue curettings were kept frozen until ready for assay. Previous studies have shown that freezing does not affect estradiol dehydrogenase activity measured in homogenates in the presence of excess substrate and cofactor. Enzyme assay Estradiol dehydrogenase activity in each endometrial specimen was measured as oreviouslv described (6). The assay mixture contained about 1 mg tissue homogenate protein, 1.4 pmol NAD+ and 20 nmol [6,7-3Hl-E2 (specific activitv 2600 dom/ns, New Enoland Nuclear Cot-o..), per ml of 5O'mM Tris buffer; pH 8.0: The amount-of product formed at~various periods of incubation was estimated from the (3H/14C) ratio in estrone (El),separated from E2 by thin layer chromatography after adding to each sample [4-14CI-E1 (specific activity 48 Ci/mmol, New England Nuclear Corp.) to correct for losses incurred during purification. Protein content in the assay mixture was determined by the method of Lowry e_t A. (12). Estradiol dehydrogenase activity was expressed as nmol El formed/mg
248
MARCH
1979 VOL. 19 NO. 3
CONTRACEPTION
protein/h. The correlation coefficients of the regression lines used to estimate rates of reaction were, in all cases, greater than 0.99. Measurement
of enzymatic
activities
in the presence of Cu++
In order to verify the influence of Cu++ on the activity of the estradiol-17e-dehydrogenase activity, solutions of copper sulfate in 50 mM Tris buffer were added to the enzymatic assay mixture to obtain Cu++ concentrations ranging from 10e7 to 10m3 M. The possibility that an inhibitory effect of Cut+ on the enzymatic activity -in vivo could be overlooked because of dissociation of CIA++ from the enzyme during preparation of the tissue homogenate and dilution in the assay mixture, was evaluated as follows. In one experiment, the activity of the enzyme was measured in the homogenate and in the particulate fraction obtained by centrifugation of the homogenate at 100,000 x g for 30 min. In the other two experiments, a similar fraction, which contained nuclei, mitochondria and microsomes, was resuspended in 1O-3 M Cu++ buffered solution (50 mM Tris) at 4C, the suspension was centrifuged at 100,000 x g, and the pellet was washed once with Tris buffer. Half of the washed pellet was assayed for estradiol dehydrogenase activity, to determine whether the inhibition by Cu++ was detectable in spite of washing and dilution. The other half was assayed in the presence of 10m3 M Cut+ to estimate the maximal inhibition obtained with cupric ion. RESULTS Figure 1 shows the activity of the enzyme at various concentrations of Cut+ in the assay mixture. No significant inhibition was noted when the Cut+ level was below 10s5 M; 90% inhibition was obtained at 1O-3 M.
Figure 1.
MARCH
Inhibition of estradiol-17H-dehydroqenase (E$H) activity at various concentrations of Cu++ in the assay mixture.
1979 VOL. 19 NO. 3
249
CONTRACEPTION
Figure 2 presents results from the tests of reversibility of inhibition of the enzymatic activity by Cu++ in 2 endometrial specimens. Ninety to 95% of total estradiol-17B-dehydrogenase activity in the tissue was found in the 100,000 x 9 pellet, with a specific activity 1.4 times higher than that measured in the whole homogenate. The activity of the enzyme was lower in the washed pellet previously treated with 10S3 M Cut+ than in the untreated pellet. This finding indicates that the inhibition is only partially removed by dilution. Consequently, it can be concluded that if Cu++ were associated with the enzyme and inhibited its action -in vivo, the reduction in activity would be detectable in the tissue homogenate.
Tissue homogenate (E2DH activity % 73%)
. Resuspended
in 10S3 M Cut+ at 4C
. Centrifuged at 100,000 x 9
u'. Washed once with 50 mM Tris Pellet /$ / ” Assayed in 50 mM Tris / E2DH activity 'L 3%
Figure 2.
\
Assayed in 10S3 M Cut+, 50 mM Tris E2DH activity 2r 3%
Test to demonstrate irreversibility of the inhibition by Cut+ of the estradiol-l7g-dehydrogenase activity by dilution.
The Table lists the enzymatic activity found in endometrial curettings of Cu-IUD wearers, together with information on age of patients, length of time elapsed between insertion of the device and the biopsy, and histologic dating of the specimen.
250
MARCH 1979 VOL. 19 NO. 3
CONTRACEPTION
E2DH Activity Subject
Age
in Endometrial
TABLE Specimens
Time elapsed since insertion of Cu-IUD (months)
from Women Wearing Cu-XUD
Histologic dating
E2DH activity nmol El
(
LI
37
8
RS
37
10
Early secretory Mid-secretory
:: BB ZP
41 37 33 35
9 6 8 33
Mid-secretory Late secretory Late secretory Secretory Mean + SD Controls
MS
MI
35 36
(n=21) Mean + SD
Proliferative Late proliferative
9 24
Mean Controls
(n=20) Mean + SD
mg prot x h
1
19 7 27 8 5 13 13 _ + 8 15 +6 0.1 0.8 0.4 1.4 + 0.5
It is apparent from these data that the enzymatic activities in secretory endometrium exposed to Cu-IUD fell within the range of the controls. The . ._ activity in 1 of the 2 specimens of proliferative endometria analyzed was Low considerqbly lower than the mean value for the corresponding controls. levels of E2DH activity, however, are also occasionally found in proliferative endomeiria of subjects not wearing intrauterine devices. DISCUSSION The results from this study show that it is unlikely that interference of Cu++ with the activity of estradiol dehydrogenase is a factor in the enHagenfeldt& & (13) reported hanced contraceptive efficacy of the CU-IUD. that the concentration of copper in endometrial specimens from women wearing CU-IUD for 1 year was about 1-1.7 Pg/g (3.7-6.2 x low6 M), a concentration apparently insufficient to exert inhibitory effects. It can be concluded from the results obtained that Cu-IUD does not prevent the increase of endometrial E2DH activity which occurs during the luteal phase of the menstrual cycle under the influence of progesterone In contrast, intrauterine devices releasing progesterone (PrOgeStaSertR) appear to influence the level of enzymatic activity, which was found to be elevated in endometrial specimens obtained during the follicular phase from women wearing these devices (7). ACKNOWLEDGEMENTS Mr. J. Mazella provided skillful technical assistance. This wnrk was supported by Grants HC 07197 and HD 10933 of the National Institutes of Health.
MARCH 1979 VOL. 19 NO. 3
251
CONTRACEPTION
REFERENCES 1. 2. 3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
Hasson, H.M. Copper IUDs. J. Reprod. Med. 20:139-154 (1978). Oster, G. and Salgo, M.P. The copper intrauterine device and its N. Engl. J. Med. 293:432-438 (1975). mode of action. Lopez de la Osa, E., Hagenfeldt, K. and Diczfalusy, F. Effect of Cu-T device on the glycogen content of the human endometrium. Contraception 6: 449-457 (1972). Janne, O., Kontula, K., Luukkainen, T. and Vihko, R. Oestrogeninduced progesterone receptor in human uterus. J. Steroid Biochem. 6:501 -509 (1975). Young, P.C.M., Cleary, R.E. and Ragan, W.D. Effect of metal ions on the binding of 17B-estradiol to human endometrial cytosol. Fertil. Steril. 28: 459-463 (1977). Tseng, L. and Gurpide, E. Estradiol and 20a-dihydroprogesterone dehydrogenase activities in human endometrium during the menEndocrinology 94: 419-423 (1974). strual cycle. Tseng, L. and Gurpide, E. Induction of human endometrial estradiol Endocrinology 97: 825-833 (1975) dehydrogenase by progestins. Pollow, K., Lijbbert, H., Jeske, R. and Pollow, B. Studies on 178hydroxysteroid dehydrogenase in human endometrium and endometrial Acta Endocrinol. 79: 146-156 (1975). carcinoma. Pollow, K., Lubbert, H. and Pollow, B. Studies of 17B-hydroxysteroid dehydroqenase in human endometrium and endometrial carcinoma. III-. Partial purification and characterization of the microsomal enzvme. Acta Endocrinol. 80: 355-364 (1975). Pollow; K., Lijbbert, H. and Pollow, B. On the'mitochondrial 17Bhydroxysteroid dehydrogenase from human endometrium and endometrial carcinoma: characterization and intramitochondrial distribution. J. Steroid Biochem. 7: 45-50 (1976). Tseng, L., Gusberg, S.B. and Gurpide, E. Estradiol receptor and 175dehydrogenase in normal and abnormal human endometrium. Ann. N.Y. Acad. Sci. 286: 190-198 (1977). Lowrv. O.H.. Rosebrouah, N.J.. Farr. A.L. and Randall. R.J. Protein measurement with the-Foliniphenol reagent. J. Bioi. Chem. 193: 265-275 (1961). Hagenfeldt, K. Intrauterine contraception with the Copper-T device. Contraception 6: 37-54 (1972).
252
MARCH 1979 VOL. 19 NO. 3
ENDOMETRIAL
ESTRADIOL-17B-DEHYDROGENASE L. Tsengl, V. Dramusic2,
ACTIVITY
IN WOMEN WEARING Cu-IUD
and E. Gurpide3*
1 Department of Obstetrics and Gynecology, School of Medicine, State University 11794
of New York of Stony Brook, Stony Brook, New York
2 Division of Pediatric and Adolescent Gynecology, Institute Mother and Child Protection, Beograd, Radiga Dakica 8, Yugoslavia 3 Department of Obstetrics and Gynecology, Medicine, New York, N.Y., 10029
for
Mount Sinai School of
ABSTRACT
In order to determine whether inhibition of the endometrial estradiol-176-dehydrogenase activity by Cu++ could be one of the factors accounting for the enhanced contraceptive efficacy of copper intrauterine devices (CU-IUD), the activity of the enzyme was measured in endometrial biopsies from women wearing this device. The levels of enzymatic activity found in these specimens were within the range of controls (women without IUD), e.g. 0.1-0.8 and 7-27 nmol El formed/mg protein/h in the follicular and luteal phases of the menstrual cycle, respectively. It is concluded that CU-IUD does not modify significantly either the activity of endometrial-17b-dehydrogenase, an enzyme known to regulate the tissular concentration of estradiol, or its response to the inductive effect of progesterone.
Accepted
*
for publication
February
24, 1979
To whom reprints should be addressed.
MARCH
1979 VOL. 19 NO. 3
247
CONTRACEPTION
INTRODUCTION The enhanced contraceptive effectiveness obtained by adding a copper wire to plastic intrauterine devices (1) may be related to For effects of Cut+ on biochemical processes in endometrial cells. instance, Cut+ may influence the production and secretion of proteolytic enzymes, prostaglandins and glycogen (1,2,3). Cupric ions may also interfere with the hormonal action of estradiol (E2) and progesterone (P) on cells by reducing the binding of these hormones to their receptors (4,5). The purpose of the present study was to examine the possibility that Cu++ released from copper intrauterine devices (CU-IUD) may inhibit the activity of the estradiol-178-dehydrogenase (E2DH), an enzyme postulated to regulate the concentration of i2 in the endometrium under physiologic conditions (6,7). Pollow et al. have reported that the - -mitochondrial and microsomal activities of the enzyme in the cytosollc, fractions of human endometrium are inhibited by 10m3 M Cut+ (8-10). The activity of the estradiol dehydrogenase was measured in endometrial biopsies obtained from normally menstruating women wearing CU-IUD for different periods of time. The enzyme activities in endometrium from these subjects were compared with the average activities found in proliferative and secretory endometrium of cycling women who did not use intrauterine devices (11). EXPERIMENTAL Endometrial
biopsies
Endometrial curettings were obtained from 8 healthy, normally menstruating subjects whose ages ranged from 19 to 37 years and who were wearing the Cu-7 intrauterine device (Searle Laboratories) from 8 to 33 months. Most curettings were performed during the period estimated to correspond to the mid-luteal phase of the cycle. All specimens included in this study were dated histologically. Tissue curettings were kept frozen until ready for assay. Previous studies have shown that freezing does not affect estradiol dehydrogenase activity measured in homogenates in the presence of excess substrate and cofactor. Enzyme assay Estradiol dehydrogenase activity in each endometrial specimen was measured as oreviouslv described (6). The assay mixture contained about 1 mg tissue homogenate protein, 1.4 pmol NAD+ and 20 nmol [6,7-3Hl-E2 (specific activitv 2600 dom/ns, New Enoland Nuclear Cot-o..), per ml of 5O'mM Tris buffer; pH 8.0: The amount-of product formed at~various periods of incubation was estimated from the (3H/14C) ratio in estrone (El),separated from E2 by thin layer chromatography after adding to each sample [4-14CI-E1 (specific activity 48 Ci/mmol, New England Nuclear Corp.) to correct for losses incurred during purification. Protein content in the assay mixture was determined by the method of Lowry e_t A. (12). Estradiol dehydrogenase activity was expressed as nmol El formed/mg
248
MARCH
1979 VOL. 19 NO. 3
CONTRACEPTION
protein/h. The correlation coefficients of the regression lines used to estimate rates of reaction were, in all cases, greater than 0.99. Measurement
of enzymatic
activities
in the presence of Cu++
In order to verify the influence of Cu++ on the activity of the estradiol-17e-dehydrogenase activity, solutions of copper sulfate in 50 mM Tris buffer were added to the enzymatic assay mixture to obtain Cu++ concentrations ranging from 10e7 to 10m3 M. The possibility that an inhibitory effect of Cut+ on the enzymatic activity -in vivo could be overlooked because of dissociation of CIA++ from the enzyme during preparation of the tissue homogenate and dilution in the assay mixture, was evaluated as follows. In one experiment, the activity of the enzyme was measured in the homogenate and in the particulate fraction obtained by centrifugation of the homogenate at 100,000 x g for 30 min. In the other two experiments, a similar fraction, which contained nuclei, mitochondria and microsomes, was resuspended in 1O-3 M Cu++ buffered solution (50 mM Tris) at 4C, the suspension was centrifuged at 100,000 x g, and the pellet was washed once with Tris buffer. Half of the washed pellet was assayed for estradiol dehydrogenase activity, to determine whether the inhibition by Cu++ was detectable in spite of washing and dilution. The other half was assayed in the presence of 10m3 M Cut+ to estimate the maximal inhibition obtained with cupric ion. RESULTS Figure 1 shows the activity of the enzyme at various concentrations of Cut+ in the assay mixture. No significant inhibition was noted when the Cut+ level was below 10s5 M; 90% inhibition was obtained at 1O-3 M.
Figure 1.
MARCH
Inhibition of estradiol-17H-dehydroqenase (E$H) activity at various concentrations of Cu++ in the assay mixture.
1979 VOL. 19 NO. 3
249
CONTRACEPTION
Figure 2 presents results from the tests of reversibility of inhibition of the enzymatic activity by Cu++ in 2 endometrial specimens. Ninety to 95% of total estradiol-17B-dehydrogenase activity in the tissue was found in the 100,000 x 9 pellet, with a specific activity 1.4 times higher than that measured in the whole homogenate. The activity of the enzyme was lower in the washed pellet previously treated with 10S3 M Cut+ than in the untreated pellet. This finding indicates that the inhibition is only partially removed by dilution. Consequently, it can be concluded that if Cu++ were associated with the enzyme and inhibited its action -in vivo, the reduction in activity would be detectable in the tissue homogenate.
Tissue homogenate (E2DH activity % 73%)
. Resuspended
in 10S3 M Cut+ at 4C
. Centrifuged at 100,000 x 9
u'. Washed once with 50 mM Tris Pellet /$ / ” Assayed in 50 mM Tris / E2DH activity 'L 3%
Figure 2.
\
Assayed in 10S3 M Cut+, 50 mM Tris E2DH activity 2r 3%
Test to demonstrate irreversibility of the inhibition by Cut+ of the estradiol-l7g-dehydrogenase activity by dilution.
The Table lists the enzymatic activity found in endometrial curettings of Cu-IUD wearers, together with information on age of patients, length of time elapsed between insertion of the device and the biopsy, and histologic dating of the specimen.
250
MARCH 1979 VOL. 19 NO. 3
CONTRACEPTION
E2DH Activity Subject
Age
in Endometrial
TABLE Specimens
Time elapsed since insertion of Cu-IUD (months)
from Women Wearing Cu-XUD
Histologic dating
E2DH activity nmol El
(
LI
37
8
RS
37
10
Early secretory Mid-secretory
:: BB ZP
41 37 33 35
9 6 8 33
Mid-secretory Late secretory Late secretory Secretory Mean + SD Controls
MS
MI
35 36
(n=21) Mean + SD
Proliferative Late proliferative
9 24
Mean Controls
(n=20) Mean + SD
mg prot x h
1
19 7 27 8 5 13 13 _ + 8 15 +6 0.1 0.8 0.4 1.4 + 0.5
It is apparent from these data that the enzymatic activities in secretory endometrium exposed to Cu-IUD fell within the range of the controls. The . ._ activity in 1 of the 2 specimens of proliferative endometria analyzed was Low considerqbly lower than the mean value for the corresponding controls. levels of E2DH activity, however, are also occasionally found in proliferative endomeiria of subjects not wearing intrauterine devices. DISCUSSION The results from this study show that it is unlikely that interference of Cu++ with the activity of estradiol dehydrogenase is a factor in the enHagenfeldt& & (13) reported hanced contraceptive efficacy of the CU-IUD. that the concentration of copper in endometrial specimens from women wearing CU-IUD for 1 year was about 1-1.7 Pg/g (3.7-6.2 x low6 M), a concentration apparently insufficient to exert inhibitory effects. It can be concluded from the results obtained that Cu-IUD does not prevent the increase of endometrial E2DH activity which occurs during the luteal phase of the menstrual cycle under the influence of progesterone In contrast, intrauterine devices releasing progesterone (PrOgeStaSertR) appear to influence the level of enzymatic activity, which was found to be elevated in endometrial specimens obtained during the follicular phase from women wearing these devices (7). ACKNOWLEDGEMENTS Mr. J. Mazella provided skillful technical assistance. This wnrk was supported by Grants HC 07197 and HD 10933 of the National Institutes of Health.
MARCH 1979 VOL. 19 NO. 3
251
CONTRACEPTION
REFERENCES 1. 2. 3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
Hasson, H.M. Copper IUDs. J. Reprod. Med. 20:139-154 (1978). Oster, G. and Salgo, M.P. The copper intrauterine device and its N. Engl. J. Med. 293:432-438 (1975). mode of action. Lopez de la Osa, E., Hagenfeldt, K. and Diczfalusy, F. Effect of Cu-T device on the glycogen content of the human endometrium. Contraception 6: 449-457 (1972). Janne, O., Kontula, K., Luukkainen, T. and Vihko, R. Oestrogeninduced progesterone receptor in human uterus. J. Steroid Biochem. 6:501 -509 (1975). Young, P.C.M., Cleary, R.E. and Ragan, W.D. Effect of metal ions on the binding of 17B-estradiol to human endometrial cytosol. Fertil. Steril. 28: 459-463 (1977). Tseng, L. and Gurpide, E. Estradiol and 20a-dihydroprogesterone dehydrogenase activities in human endometrium during the menEndocrinology 94: 419-423 (1974). strual cycle. Tseng, L. and Gurpide, E. Induction of human endometrial estradiol Endocrinology 97: 825-833 (1975) dehydrogenase by progestins. Pollow, K., Lijbbert, H., Jeske, R. and Pollow, B. Studies on 178hydroxysteroid dehydrogenase in human endometrium and endometrial Acta Endocrinol. 79: 146-156 (1975). carcinoma. Pollow, K., Lubbert, H. and Pollow, B. Studies of 17B-hydroxysteroid dehydroqenase in human endometrium and endometrial carcinoma. III-. Partial purification and characterization of the microsomal enzvme. Acta Endocrinol. 80: 355-364 (1975). Pollow; K., Lijbbert, H. and Pollow, B. On the'mitochondrial 17Bhydroxysteroid dehydrogenase from human endometrium and endometrial carcinoma: characterization and intramitochondrial distribution. J. Steroid Biochem. 7: 45-50 (1976). Tseng, L., Gusberg, S.B. and Gurpide, E. Estradiol receptor and 175dehydrogenase in normal and abnormal human endometrium. Ann. N.Y. Acad. Sci. 286: 190-198 (1977). Lowrv. O.H.. Rosebrouah, N.J.. Farr. A.L. and Randall. R.J. Protein measurement with the-Foliniphenol reagent. J. Bioi. Chem. 193: 265-275 (1961). Hagenfeldt, K. Intrauterine contraception with the Copper-T device. Contraception 6: 37-54 (1972).
252
MARCH 1979 VOL. 19 NO. 3