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Estradiol Receptor Activity in Uteri of Pregnant Mice Given Lead Before Implantation
F'ERTIUTY AND STERIUTY Copyright < 1980 The American Fertility Society
Vol. 34, No.5, November 1980 Printed in V.SA.
ESTRADIOL RECEPTOR ACTIVITY IN UTERI OF PREGNANT MICE GIVEN LEAD BEFORE IMPLANTATION
MARIANN WIDE, PH.D,* LEIF WIDE, M,D.
Institute of Zoology and Department of Anatomy, University of Uppsala, and Department of Clinical Chemistry, University Hospital, Uppsala, Sweden
Lead chloride given intravenously to mice on day 4 of pregnancy has been shown previously to inhibit implantation that normally occurs on the following day. The effect of such lead treatment on the estradiol receptor activity in uterine cytosol from day 5 of pregnancy was investigated. The quantity of estradiol receptors, expressed per uterus, per wet weight of uterine tissue, and per amount of protein, was significantly (P < 0.001) larger in lead-treated animals than in control animals. The estradiol receptor binding affinities were similar for both groups. A possible relationship between the increased estradiol receptor activity and implantation failure in the lead-treated mice is discussed. In vitro addition of 1 and 5 mmoles of lead chloride/liter of cytosol resulted in reduced estradiol binding, which is in accord with previous reports. However, our study indicates that this reduction is due to co-precipitation of lead and receptor proteins. Fertil Steril 34:503, 1980
cytosol was reported by Emanuel and Oakey3 in cows and by Young et a1. 4 in humans. No such decrease was observed for the binding ofprogesterone. 5 In the present investigation the effect of the in vivo addition of lead (intravenously injected on day 4 of pregnancy) on the capacity of uterine cytosol to bind 17[3-estradiol was studied in mice on day 5 of pregnancy. An increase in estradiol receptor activity was found. Since this finding seemed to contradict the results of the abovementioned in vitro addition experiments, our study was extended to include the in vitro addition of different amounts oflead to mouse uterine cytosol.
Implantation of mouse blastocysts has been shown to be inhibited by 1 mg of inorganic lead given intravenously to mothers on the 4th day of pregnancy, the day before implantation. 1 This interference of implantation could be overcome by the administration of 17[3-estradiol and progesterone to the lead-treated mice. 2 These results suggested that the lead had interfered with the synthesis and/or action of ovarian steroid hormones. Since the serum levels of 17[3-estradiol were found to be almost identical in lead-treated and control animals, 2 the investigation of a possible effect of lead on the uterine estradiol receptors seemed ofimportance. In fact, it is well known that heavy metals may affect the binding of estradiol and progesterone to their receptors in uterine cytosol after either in vivo or in vitro. addition of the metals. The effect of lead has been studied only in experiments with in vitro addition of a lead salt. A decrease in the binding of estradiol in endometrial
MATERIALS AND METHODS
Animals and Preparation of Cytosol Albino mice approximately 8 weeks old and weighing 25 to 28 gmreceived intravenous injections of lead chloride (analytic grade, Mallinckrodt, St. Louis, Mo), 75 f.J.g/gm of body weight, in
Received February 12, 1980; revised June 26, 1980; accepted July 7,1980. *Reprint requests: Mariann Wide, Ph.D., Institute of Zoology, Box 561, S-751 22 Uppsala, Sweden.
503
504
November 1980
WIDE AND WIDE
0.15 ml of an aquaeous solution on the morning of day 4 of pregnancy (day 1 = day of vaginal plug). The dose of lead was the same as that which had been found in previous studies to inhibit implantation. 2 , 6 The mice were killed by cervical dislocation 24 hours after the lead injection, and the uteri were excised between the oviducts and the cervix. Uteri from three animals in each experimental and each control group were pooled, weighed, and cut into 1- to 2-mm pieces. These were homogenized, and 1 ml of cold Tris-HCI buffer was added per 80 mg of uterine tissue. The homogenate was centrifuged at 1000 x g for 10 minutes, after which the supernatant was centrifuged again at about 105,000 x g for 60 minutes to yield the cytosol. The entire preparation procedure was performed at temperatures below 4° C. The average (± standard deviation) wet weights of uteri from control mice and lead-treated mice were 95.7 ± 17.2 mg and 87.7 ± 10.6 mg, respectively. The average (± standard deviation) protein concentrations in the cytosol, expressed per gram uterine wet weight, were 46.7 ± 7.4 mg and 53.9 ± 10.2 mg/gm for the two groups of mice, respectively. These differences between control and leadtreated mice were not significantly different.
Reagents Tris-HCI buffer (0.01 M, pH 7.4) was used as a diluent in the assays. 2,4,6,7 -3H-Labeled 1713estradiol (96.0 Ci/mmole) was obtained from New England Nuclear Corporation, Boston, Mass. An "antiestrogen" compound, Cl-628, was supplied by Parke-Davis, Morris Plains, N. J. Dilutions of PbCl2 (analytic grade, Mallinckrodt) were made in two times glass-distilled water. A charcoal suspension of 0.125% Norit A (Serva, Fein Biochemica, Heidelberg, West Germany) and 0.0125% dextran T70 (Pharmacia Fine Chemicals, Piscataway, N. J.) in the buffer was used in the separation of bound from unbound steroid after the incubation procedure. Aqualuma (Lumac) was used in the scintillation counting; 1 pmole of 3H-Iabeled 1713estradiol yielded 75,000 cpm.
specific binding. The assays were made with two or four replicates at each of three concentration levels of cytosol with a logIO dose interval of 0.3010 for both control and test solutions. The incubation mixtures (total volume 300 f-Ll) were left for 16 to 18 hours at 4° C, after which 0.5 ml of charcoal suspension was added to all of the tubes except those used for protein measurement after in vitro incubation with the lead salt. The tubes were agitated for approximately 2 seconds, allowed to stand at 4° C for 10 minutes, and then centrifuged at 1000 x g for 10 minutes at 4° C. After centrifugation, 0.5 ml of the supernatant was removed, mixed with 4 ml of scintillation solution, and counted for 2 minutes using a Searle Isocap/300 liquid scintillation system (Nuclear Chicago, Chicago, Ill.) A control incubation experiment was carried out in the presence of dihydrotestosterone, 6 nmoles/liter, to exclude the possibility of interference due to binding to sex hormone-binding globulin in the cytosol. No such interference could be detected. Before calculations of the results of the assays, correction for background activity was made by subtracting the counts of the incubations made in the presence of Cl-628. 1713-Estradiol receptor activity was expressed in relation to that of the control per uterus, per uterine wet weight, and per mass of protein in the cytosol. This experiment was performed four Cpm
PbCI2 in vivo
2000 Control
1500
1000
500
Estimation of 1713-Estradiol Cytosol Receptor Activity Lead Addition in Vivo
o
i
1.5
5
protein gIl cytosol
ExperimentA. To 200 f-LI of the cytosol was added 0.1 pmole of 3H-estradiol. Parallel incubations were made throughout the experiment in the presence of O.lnmole of Cl-628 to correct for non-
FIG. 1. Binding of 1713-estradiol to uterine cytosol of day 5 of pregnancy in three different dilutions from one group of control mice and one group oflead-treated mice. Means and ranges are indicated.
Vol. 34, No.5
ESTRADIOL RECEPTOR ACTIVITY IN PREGNANT MICE GIVEN LEAD
505
TABLE 1. 17f3-EstradiolReceptor Activity in Uterine Cytosol from Lead-Treated Animals. 17~·E8tradiol
Experiment no.
Per,uterus Mean
receptor activity" Per amount of protein in cytosol
Per wet weight of uterine tissue 95%CL
Mean
Mean
95%CL
95%CL
%
%
%
%
%
%
2 3 4
169 160 104 137
146-195 142-182 94-111 113-167
161 147 115 164
139-186 130--167 109-123 135-200
116 129 117 167
100--134 114-147 111-125 138-204
Mean
144
135-153
147
138-156
127
119-135
1
aExpressed as percentage of that of controls. Mean values were weighted according to number of observations. bCL, Confidence limits.
times. The results and tests of validity of the assays were calculated according to the method of Gaddum. 7 When results from identical experiments were combined, the mean activity and its 95% confidence limits were calculated from the logarithms of the values. The significance of the differences between control and experimental groups was calculated by using Student's t-test. Experiment B. Pools of cytosol, in one concentration only, from lead-treated and nontreated animals were incubated with different amounts of 3H-labeled 17~-estradiol to measure binding capacity and binding affinity. The incubation and separation procedures were performed as above. The affinity, expressed in terms of K d , and the maximal 3H-estradiol binding capacity of the cytosol were calculated according to the method of Scatchard. 8
Lead Addition in Vitro Different concentrations of lead chloride were added to cytosol from uteri removed on the 5th day of pregnancy from untreated animals. On three occasions the lead chloride was added to give concentrations of 1 mmole/liter and 5 mmoles/liter of incubation mixture. One experiment was performed in which concentrations of PbCl2 were 10 ~moles/liter and 100 ~moles/liter. The 17~ estradiol binding capacity was then measured (as in experiment A) and expressed in relation to that of the control without lead chloride.
Measurements of Protein The protein concentration in the cytosol preparations was measured according to the method of Lowry et a1. 9 This measurement was also performed in the cytosol (original protein concentration approximatelY,4 gm!liter) after incubation in
vitro with the different concentrations of lead chloride and separation of precipitated material by centrifugation at 1000 x g for 10 minutes at 4° C. The possible interference oflead with the assay was investigated using 1, 5,10,50,100, and 200 ~g ofPbCl2 /ml of bovine serum albumin in five different concentrations. In no case did the lead inter- . fere with the results of the protein assay.
Measurements of Lead Levels Lead levels in the nonprecipitated fraction of cytosol after in vitro incubation with different concentrations of lead chloride were measured using . flame atomic absorption spectroscopy.
RESULTS
Lead Addition in Vivo Experiment A. The dose-response relationship in the estradiol receptor assay for uterine cytosol of control and lead-treated animals in one of the four experiments is illustrated in Figure 1. There was no significant difference from parallelism of the slopes of the lines. The calculated receptor activity of the cytosol of the lead-treated animals was higher than that in the control group. Table 1 shows the results obtained in all four experiments, and in these there was significantly (P < 0.05) higher activity in the cytosol of the lead-treated groups. The mean activity per wet weight of uterine tissue oflead-treated animals (expressed as a percentage of that of the controls) was 147, which is highly significantly different from 100 (P < 0.001). The corresponding value for activity expressed per mass of protein in the cytosol was 127% (P < 0.001); per uterus, 144% (P < 0.001).
WIDE AND WIDE
506 ..§. F
0.30
~ r = 0.989 Kd • 8.8"0'0 mot/I
0.20
.£2!!!!2!. r,O,99<
Kd ,6.6,10 '0 ..../1
0.10
.. 0.05
0.10
0.15
0.20
0,25
FIG. 2. Scatchard plot showing the difference in binding of 17p-estradiol to uterine cytosol from one group of control mice and one group of lead-treated mice. r, Correlation coefficient; K d , dissociation constant.
Experiment B. The maximal 1713-estradiol binding capacity (Bmax) of the uterine cytosol was estimated according to the method of Scatchard8 (Fig. 2). In the control group the Bmax was 5.5 ± 0.16 (SD) pmoles/gm wet weight of uterine tissue, or 162 ± 4.6 (SD) pmoles/gm of protein in the cytosol. The corresponding figures for the lead-treated group were significantly higher (P < 0.001) than those of the control group, namely 16.7 ± 0.73 (SD) pmoleslgm wet weight of uterine tissue and 440 ± 20 (SD) pmoleslgm of protein in the cytosol. The dissociation constants were 0.66 nmoleslliter and 0.88 nmoles/liter for the control and leadtreated groups, respectively.
November 1980
plemented with 10 and 100 ILmoles/liter ofthe lead salt, whereas a significant decrease was obtained when concentrations of 1 mmolelliter (P < 0.01) and 5 mmoles/liter (P < 0.001) were used. The addition oflead salts at the two highest concentrations to the incubation mixture caused the appearance of a milky precipitate. At the end of the incubation time the test tubes with the highest concentrations of cytosol were centrifuged, and the protein concentration and the lead concentration in the supernatant were measured. The protein concentrations, expressed in relation to those of the controls, are plotted in Figure 4. The decrease in protein content followed closely the decrease in receptor activity. With 5 mmoles/liter of PbCI 2 , the protein concentration was approximately 37% and the receptor activity was approximately 40% of the controls. The lead content of the supernatant had decreased to 19% and 12% of the original concentrations of 1 mmolelliter and 5 mmoles/liter, respectively. This indicates that part of the cytosol receptors had co-precipitated with the lead salt and lost the ability to bind 1713estradiol. DISCUSSION
There are very few studies on the effect of the in vivo addition of heavy metals on sex steroid receptor activity in uterine tissue. When copper was Cpm
PbCI2 in vitro
2000
1500
Lead Addition in Vitro Control
The effect of the addition oflead chloride (1 and 5 mmoles/liter) to uterine cytosol from nontreated animals on day 5 of pregnancy in one of three experiments is shown in Figure 3. There was a marked decrease in estradiol binding after the addition of 5 mmoles of PbCl 2 lliter, and the binding was measurable only in the highest concentration of cytosol. Also, the 1-mmole/liter concentration caused a significant decrease in estradiol binding. A summary of the results of the effect of the in vitro addition of lead chloride in four different concentrations on estradiol receptor binding is shown in Figure 4. No significant effect on estradiol binding was observed in the cytosol when sup-
1000
1 mmolll
t
500
.5 mmolll
o
I
1.5
5
protein g/l cytosol
FIG. 3. Binding of 17j3-estradiol to uterine cytosol of day 5 of pregnancy in different dilutions from one group of nontreated mice without in vitro addition (control) and with in vitro addition oflead chloride, 1 mmolelliter and 5 mmoles/liter. Means and ranges are indicated.
Vol. 34, No.5
ESTRADIOL RECEPI'OR ACTIVITY IN PREGNANT MICE GIVEN LEAD • Receptor activity
Per cent
o
200
Protein conc.
100t---t7-r=----1J..1.---'!~--------
, \
\
"
50
o 25
0.01
0.1
5 mmolll PbCI2
FIG. 4. Binding of17~-estradiol to uterine cytosol from three groups of nontreated mice in the presence of four different concentrations oflead chloride, shown together with the values for protein concentrations in the nonprecipitated part of the incubation mixture. Vertical bars indicate 95% confidence limits of calculated mean values.
administered as an intrauterine device to rabbits and rats, Ghosh et al. 10 and Tamaya et al. 11 found that estradiol had a decreased capacity to bind to receptors in the uterus, whereas Adadevoh and Dada12 and Aedo and Zipper13 reported that there was a significant increase in estradiol binding. These discrepancies may be due to differences in experimental procedures, such as time of exposure to the metal. In the present study there was a 1.3- to 3-fold increase in estradiol receptor activity of uterine cytosol on day 5 of pregnancy in mice that had been treated with lead in vivo on the previous day. The binding affinities, expressed as dissociation constants, were very similar for the control and lead-treated groups and were within the range of those found by other investigators for cytoplasmic estradiol receptors in the uterus. 4, 14 There is no obvious explanation for the increased estradiol receptor activity in the leadtreated mice. However, progesterone is known to have an inhibitory effect on the synthesis of estradiol receptors in the uterus, 15 and a decrease in the serum levels of progesterone in lead-treated mice cannot be excluded. In fact, a trend toward lower values in serum, although not statistically significant owing to a large interindividual variation, has been found in two studies. 2, 16 Plasma levels of 17J3-estradiol were almost identical in the nontreated and the lead-treated groups,2 with very
507
small interindividual variation, which makes it seem less likely that the effect of lead observed in the present study was mediated via this hormone. The results from our studies with the addition of lead in vivo may seem to contradict those of previous in vitro studies. A number of different heavy metals, including lead, has been reported to give a decreased binding of 17J3-estradiol and progesterone in uterine Cytosol.4, 5, 17-20 In one study using zinc ions, increased activity was found. 3 The decreased degree of steroid hormone binding was assumed to be due to an interference of the metal with -SH groups on the receptor molecules,5, 17, 19 and the inhibition could be reversed after the addition of ethylenediaminetetracetic acid to the cytosol. 5, 19 In our in vitro addition study there was no effect oflead in concentrations of 10 and 100 ~moles/liter. These concentrations were chosen as they are comparable to those estimated in uterine tissue of mice and under similar esperimental conditions. 21 The higher concentrations (1 and 5 mmoleslliter) caused significant decreases in estimated receptor activity. The most likely explanation for this decrease was an increased precipitation of proteins in the incubation mixture with increased amounts of added lead chloride. As similar high concentrations of lead chloride were used in investigations published previously,3, 4 it seems likely that the effects found in those were also, at least in part, due to coprecipitation oflead chloride and receptor proteins. Thus, the decreased estradiol binding observed in the in vitro studies in which lead was added is thought to reflect a decrease in number of available soluble cytoplasmatic receptors rather than an interference by metal with -SH groups of the binding proteins. This study was conducted to obtain information on how lead chloride causes inhibition of implantation in mice. A decrease in estradiol binding, as might be suspected from previous in vitro studies with lead and uterine receptors, can now be excluded as an explanation for the "lead effect" in mice. In contrast, it is possible that the observed increase in estradiol receptor activity per se causes impaired implantation. An increase in the number of receptors may result in increased estrogen stimulation of the uterus. Such stimulation may be comparable to the situation in which the administration of too much estrogen causes inhibition of implantation. 22 However, another possibility is that a decreased progesterone level both induced an increase in estradiol receptor activity and caused impairment of implantation owing to lack
508
WIDE AND WIDE
of normal gestagenic transformation of the endometrium at the time of implantation. It has been shown that lead can interfere with central neuroendocrine functions. 23 The observed increase in uterine estradiol receptor activity in the present study may represent an indirect effect of lead on the hypothalamus, pituitary, or ovaries. REFERENCES 1. Wide M, Nilsson 0: Differential susceptibility of the embryo to inorganic lead during periimplantation in the mouse. Teratology 16:273, 1977 2. Wide M: Interference of lead with implantation in the mouse. Effect of exogenous oestradiol and progesterone. Teratology 21:187,1980 3. Emanuel MB, Oakey RE: Effect of Zn + + on the binding of oestradiol-17~ to a uterine protein. Nature 223:66, 1969 4. Young PCM, Cleary RE, Ragan WD: Effect of metal ions on the binding of17~-estradiol to human endometrial cytosol. Fertil Steril 28:459, 1977 5. Young PCM, Cleary RE, Ehrlich CE: Effect of metal ions on the binding of progesterone and estradiol by human endometrial cytosol. In Multiple Molecular Forms of Steroid Hormone Receptors, Edited by MK Agarwal. Amsterdam, ElsevierINorth Holland Biomedical Press, 1977, p 215 6. Wide M, Nilsson 0: Interference oflead with implantation in the mouse. A study of the ultrastructure of blastocysts and endometrium. Teratology 20:101, 1979 7. Gaddum JH: Bioassays and mathematics. Pharmacol Rev 5:87,1953 8. Scatchard G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660, 1949 9. Lowry OH, Rosebrough NJ, FaIT AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chem 193:265, 1951 10. Ghosh M, Roy SK, Kar AB: Effect of a copper intrauterine contraceptive device and nylon suture on the estradiol 17~-6, 7_H3 and progesterone 1,2-H3 in the rat uterus. Contraception 11:45, 1975
November 1980 11. Tamaya T, Nakata Y, Ohno Y, Nioka S, Furuta N, Okada H: The mechanism of action of the copper intrauterine device. Fertil Steril 27:767,1976 12. Adadevoh BK, Dada OA: Effect of intrauterine copper on the uptake of estradiol-C 14 by rat tissues. Fertil Steril 24:54,1973 13. 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 14. Puca GA, Nola E, Sica V, Bresciani F: Estrogen-binding proteins of calf uterus. Partial purification and preliminary characterization of two cytoplasmic proteins. Biochemistry 1:3769, 1971 15. Clark JH, Hsueh AJW, Peck EJ J r: Regulation of estrogen receptor replenishment by progesterone. Ann NY Acad Sci 286:161, 1977 16. Jacquet P, Gerber GB, Leonard A, Maes J: Plasma hormone levels in normal and lead treated pregnant mice. Experientia 33:1375, 1977 17. Jensen EV, Hurst DJ, DeSombre ER, Jungblut PW: Sulfhydryl groups and estradiol-receptor interaction. Science 158:385. 1967 18. Sanborn BM, Rao BR, Korenman SG: Interaction of 17~ estradiol and its specific uterine receptor. Evidence for complex kinetic and equilibrium behaviour. Biochemistry 10:4955, 1971 19. Kontula K, J anne 0, Luukainen T, Vihko R: Progesteronebinding protein in human myometrium. Influence of metal ions on binding. J Clin Endocrinol Metab 38:500, 1974 20. LOvgren T, Pettersson K, Lundberg B, Punnonen R: Effect of Cu2 + ions on the binding of estrogen to the human myometrial estrogen binding protein. Contraception 18:181, 1978 21. Wide M: The effect of inorganic lead on the mouse blastocyst in vitro. Teratology 17:165, 1978 22. Sartor P, Dupont H, Dupont MA, Duluc AJ, Mayer G: The action of high doses of oestradiol on implantation and decidual reaction in the rat. Anim Reprod Sci 1:93, 1978 23. Petrusz P, Weaver CM, Grant LD, Mushak P, Krigman MR: Lead poisoning and reproduction: effects on pituitary and serum gonadotrophins in neonatal rats. Environ Res 19:383, 1979
Vol. 34, No.5, November 1980 Printed in V.SA.
ESTRADIOL RECEPTOR ACTIVITY IN UTERI OF PREGNANT MICE GIVEN LEAD BEFORE IMPLANTATION
MARIANN WIDE, PH.D,* LEIF WIDE, M,D.
Institute of Zoology and Department of Anatomy, University of Uppsala, and Department of Clinical Chemistry, University Hospital, Uppsala, Sweden
Lead chloride given intravenously to mice on day 4 of pregnancy has been shown previously to inhibit implantation that normally occurs on the following day. The effect of such lead treatment on the estradiol receptor activity in uterine cytosol from day 5 of pregnancy was investigated. The quantity of estradiol receptors, expressed per uterus, per wet weight of uterine tissue, and per amount of protein, was significantly (P < 0.001) larger in lead-treated animals than in control animals. The estradiol receptor binding affinities were similar for both groups. A possible relationship between the increased estradiol receptor activity and implantation failure in the lead-treated mice is discussed. In vitro addition of 1 and 5 mmoles of lead chloride/liter of cytosol resulted in reduced estradiol binding, which is in accord with previous reports. However, our study indicates that this reduction is due to co-precipitation of lead and receptor proteins. Fertil Steril 34:503, 1980
cytosol was reported by Emanuel and Oakey3 in cows and by Young et a1. 4 in humans. No such decrease was observed for the binding ofprogesterone. 5 In the present investigation the effect of the in vivo addition of lead (intravenously injected on day 4 of pregnancy) on the capacity of uterine cytosol to bind 17[3-estradiol was studied in mice on day 5 of pregnancy. An increase in estradiol receptor activity was found. Since this finding seemed to contradict the results of the abovementioned in vitro addition experiments, our study was extended to include the in vitro addition of different amounts oflead to mouse uterine cytosol.
Implantation of mouse blastocysts has been shown to be inhibited by 1 mg of inorganic lead given intravenously to mothers on the 4th day of pregnancy, the day before implantation. 1 This interference of implantation could be overcome by the administration of 17[3-estradiol and progesterone to the lead-treated mice. 2 These results suggested that the lead had interfered with the synthesis and/or action of ovarian steroid hormones. Since the serum levels of 17[3-estradiol were found to be almost identical in lead-treated and control animals, 2 the investigation of a possible effect of lead on the uterine estradiol receptors seemed ofimportance. In fact, it is well known that heavy metals may affect the binding of estradiol and progesterone to their receptors in uterine cytosol after either in vivo or in vitro. addition of the metals. The effect of lead has been studied only in experiments with in vitro addition of a lead salt. A decrease in the binding of estradiol in endometrial
MATERIALS AND METHODS
Animals and Preparation of Cytosol Albino mice approximately 8 weeks old and weighing 25 to 28 gmreceived intravenous injections of lead chloride (analytic grade, Mallinckrodt, St. Louis, Mo), 75 f.J.g/gm of body weight, in
Received February 12, 1980; revised June 26, 1980; accepted July 7,1980. *Reprint requests: Mariann Wide, Ph.D., Institute of Zoology, Box 561, S-751 22 Uppsala, Sweden.
503
504
November 1980
WIDE AND WIDE
0.15 ml of an aquaeous solution on the morning of day 4 of pregnancy (day 1 = day of vaginal plug). The dose of lead was the same as that which had been found in previous studies to inhibit implantation. 2 , 6 The mice were killed by cervical dislocation 24 hours after the lead injection, and the uteri were excised between the oviducts and the cervix. Uteri from three animals in each experimental and each control group were pooled, weighed, and cut into 1- to 2-mm pieces. These were homogenized, and 1 ml of cold Tris-HCI buffer was added per 80 mg of uterine tissue. The homogenate was centrifuged at 1000 x g for 10 minutes, after which the supernatant was centrifuged again at about 105,000 x g for 60 minutes to yield the cytosol. The entire preparation procedure was performed at temperatures below 4° C. The average (± standard deviation) wet weights of uteri from control mice and lead-treated mice were 95.7 ± 17.2 mg and 87.7 ± 10.6 mg, respectively. The average (± standard deviation) protein concentrations in the cytosol, expressed per gram uterine wet weight, were 46.7 ± 7.4 mg and 53.9 ± 10.2 mg/gm for the two groups of mice, respectively. These differences between control and leadtreated mice were not significantly different.
Reagents Tris-HCI buffer (0.01 M, pH 7.4) was used as a diluent in the assays. 2,4,6,7 -3H-Labeled 1713estradiol (96.0 Ci/mmole) was obtained from New England Nuclear Corporation, Boston, Mass. An "antiestrogen" compound, Cl-628, was supplied by Parke-Davis, Morris Plains, N. J. Dilutions of PbCl2 (analytic grade, Mallinckrodt) were made in two times glass-distilled water. A charcoal suspension of 0.125% Norit A (Serva, Fein Biochemica, Heidelberg, West Germany) and 0.0125% dextran T70 (Pharmacia Fine Chemicals, Piscataway, N. J.) in the buffer was used in the separation of bound from unbound steroid after the incubation procedure. Aqualuma (Lumac) was used in the scintillation counting; 1 pmole of 3H-Iabeled 1713estradiol yielded 75,000 cpm.
specific binding. The assays were made with two or four replicates at each of three concentration levels of cytosol with a logIO dose interval of 0.3010 for both control and test solutions. The incubation mixtures (total volume 300 f-Ll) were left for 16 to 18 hours at 4° C, after which 0.5 ml of charcoal suspension was added to all of the tubes except those used for protein measurement after in vitro incubation with the lead salt. The tubes were agitated for approximately 2 seconds, allowed to stand at 4° C for 10 minutes, and then centrifuged at 1000 x g for 10 minutes at 4° C. After centrifugation, 0.5 ml of the supernatant was removed, mixed with 4 ml of scintillation solution, and counted for 2 minutes using a Searle Isocap/300 liquid scintillation system (Nuclear Chicago, Chicago, Ill.) A control incubation experiment was carried out in the presence of dihydrotestosterone, 6 nmoles/liter, to exclude the possibility of interference due to binding to sex hormone-binding globulin in the cytosol. No such interference could be detected. Before calculations of the results of the assays, correction for background activity was made by subtracting the counts of the incubations made in the presence of Cl-628. 1713-Estradiol receptor activity was expressed in relation to that of the control per uterus, per uterine wet weight, and per mass of protein in the cytosol. This experiment was performed four Cpm
PbCI2 in vivo
2000 Control
1500
1000
500
Estimation of 1713-Estradiol Cytosol Receptor Activity Lead Addition in Vivo
o
i
1.5
5
protein gIl cytosol
ExperimentA. To 200 f-LI of the cytosol was added 0.1 pmole of 3H-estradiol. Parallel incubations were made throughout the experiment in the presence of O.lnmole of Cl-628 to correct for non-
FIG. 1. Binding of 1713-estradiol to uterine cytosol of day 5 of pregnancy in three different dilutions from one group of control mice and one group oflead-treated mice. Means and ranges are indicated.
Vol. 34, No.5
ESTRADIOL RECEPTOR ACTIVITY IN PREGNANT MICE GIVEN LEAD
505
TABLE 1. 17f3-EstradiolReceptor Activity in Uterine Cytosol from Lead-Treated Animals. 17~·E8tradiol
Experiment no.
Per,uterus Mean
receptor activity" Per amount of protein in cytosol
Per wet weight of uterine tissue 95%CL
Mean
Mean
95%CL
95%CL
%
%
%
%
%
%
2 3 4
169 160 104 137
146-195 142-182 94-111 113-167
161 147 115 164
139-186 130--167 109-123 135-200
116 129 117 167
100--134 114-147 111-125 138-204
Mean
144
135-153
147
138-156
127
119-135
1
aExpressed as percentage of that of controls. Mean values were weighted according to number of observations. bCL, Confidence limits.
times. The results and tests of validity of the assays were calculated according to the method of Gaddum. 7 When results from identical experiments were combined, the mean activity and its 95% confidence limits were calculated from the logarithms of the values. The significance of the differences between control and experimental groups was calculated by using Student's t-test. Experiment B. Pools of cytosol, in one concentration only, from lead-treated and nontreated animals were incubated with different amounts of 3H-labeled 17~-estradiol to measure binding capacity and binding affinity. The incubation and separation procedures were performed as above. The affinity, expressed in terms of K d , and the maximal 3H-estradiol binding capacity of the cytosol were calculated according to the method of Scatchard. 8
Lead Addition in Vitro Different concentrations of lead chloride were added to cytosol from uteri removed on the 5th day of pregnancy from untreated animals. On three occasions the lead chloride was added to give concentrations of 1 mmole/liter and 5 mmoles/liter of incubation mixture. One experiment was performed in which concentrations of PbCl2 were 10 ~moles/liter and 100 ~moles/liter. The 17~ estradiol binding capacity was then measured (as in experiment A) and expressed in relation to that of the control without lead chloride.
Measurements of Protein The protein concentration in the cytosol preparations was measured according to the method of Lowry et a1. 9 This measurement was also performed in the cytosol (original protein concentration approximatelY,4 gm!liter) after incubation in
vitro with the different concentrations of lead chloride and separation of precipitated material by centrifugation at 1000 x g for 10 minutes at 4° C. The possible interference oflead with the assay was investigated using 1, 5,10,50,100, and 200 ~g ofPbCl2 /ml of bovine serum albumin in five different concentrations. In no case did the lead inter- . fere with the results of the protein assay.
Measurements of Lead Levels Lead levels in the nonprecipitated fraction of cytosol after in vitro incubation with different concentrations of lead chloride were measured using . flame atomic absorption spectroscopy.
RESULTS
Lead Addition in Vivo Experiment A. The dose-response relationship in the estradiol receptor assay for uterine cytosol of control and lead-treated animals in one of the four experiments is illustrated in Figure 1. There was no significant difference from parallelism of the slopes of the lines. The calculated receptor activity of the cytosol of the lead-treated animals was higher than that in the control group. Table 1 shows the results obtained in all four experiments, and in these there was significantly (P < 0.05) higher activity in the cytosol of the lead-treated groups. The mean activity per wet weight of uterine tissue oflead-treated animals (expressed as a percentage of that of the controls) was 147, which is highly significantly different from 100 (P < 0.001). The corresponding value for activity expressed per mass of protein in the cytosol was 127% (P < 0.001); per uterus, 144% (P < 0.001).
WIDE AND WIDE
506 ..§. F
0.30
~ r = 0.989 Kd • 8.8"0'0 mot/I
0.20
.£2!!!!2!. r,O,99<
Kd ,6.6,10 '0 ..../1
0.10
.. 0.05
0.10
0.15
0.20
0,25
FIG. 2. Scatchard plot showing the difference in binding of 17p-estradiol to uterine cytosol from one group of control mice and one group of lead-treated mice. r, Correlation coefficient; K d , dissociation constant.
Experiment B. The maximal 1713-estradiol binding capacity (Bmax) of the uterine cytosol was estimated according to the method of Scatchard8 (Fig. 2). In the control group the Bmax was 5.5 ± 0.16 (SD) pmoles/gm wet weight of uterine tissue, or 162 ± 4.6 (SD) pmoles/gm of protein in the cytosol. The corresponding figures for the lead-treated group were significantly higher (P < 0.001) than those of the control group, namely 16.7 ± 0.73 (SD) pmoleslgm wet weight of uterine tissue and 440 ± 20 (SD) pmoleslgm of protein in the cytosol. The dissociation constants were 0.66 nmoleslliter and 0.88 nmoles/liter for the control and leadtreated groups, respectively.
November 1980
plemented with 10 and 100 ILmoles/liter ofthe lead salt, whereas a significant decrease was obtained when concentrations of 1 mmolelliter (P < 0.01) and 5 mmoles/liter (P < 0.001) were used. The addition oflead salts at the two highest concentrations to the incubation mixture caused the appearance of a milky precipitate. At the end of the incubation time the test tubes with the highest concentrations of cytosol were centrifuged, and the protein concentration and the lead concentration in the supernatant were measured. The protein concentrations, expressed in relation to those of the controls, are plotted in Figure 4. The decrease in protein content followed closely the decrease in receptor activity. With 5 mmoles/liter of PbCI 2 , the protein concentration was approximately 37% and the receptor activity was approximately 40% of the controls. The lead content of the supernatant had decreased to 19% and 12% of the original concentrations of 1 mmolelliter and 5 mmoles/liter, respectively. This indicates that part of the cytosol receptors had co-precipitated with the lead salt and lost the ability to bind 1713estradiol. DISCUSSION
There are very few studies on the effect of the in vivo addition of heavy metals on sex steroid receptor activity in uterine tissue. When copper was Cpm
PbCI2 in vitro
2000
1500
Lead Addition in Vitro Control
The effect of the addition oflead chloride (1 and 5 mmoles/liter) to uterine cytosol from nontreated animals on day 5 of pregnancy in one of three experiments is shown in Figure 3. There was a marked decrease in estradiol binding after the addition of 5 mmoles of PbCl 2 lliter, and the binding was measurable only in the highest concentration of cytosol. Also, the 1-mmole/liter concentration caused a significant decrease in estradiol binding. A summary of the results of the effect of the in vitro addition of lead chloride in four different concentrations on estradiol receptor binding is shown in Figure 4. No significant effect on estradiol binding was observed in the cytosol when sup-
1000
1 mmolll
t
500
.5 mmolll
o
I
1.5
5
protein g/l cytosol
FIG. 3. Binding of 17j3-estradiol to uterine cytosol of day 5 of pregnancy in different dilutions from one group of nontreated mice without in vitro addition (control) and with in vitro addition oflead chloride, 1 mmolelliter and 5 mmoles/liter. Means and ranges are indicated.
Vol. 34, No.5
ESTRADIOL RECEPI'OR ACTIVITY IN PREGNANT MICE GIVEN LEAD • Receptor activity
Per cent
o
200
Protein conc.
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, \
\
"
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o 25
0.01
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FIG. 4. Binding of17~-estradiol to uterine cytosol from three groups of nontreated mice in the presence of four different concentrations oflead chloride, shown together with the values for protein concentrations in the nonprecipitated part of the incubation mixture. Vertical bars indicate 95% confidence limits of calculated mean values.
administered as an intrauterine device to rabbits and rats, Ghosh et al. 10 and Tamaya et al. 11 found that estradiol had a decreased capacity to bind to receptors in the uterus, whereas Adadevoh and Dada12 and Aedo and Zipper13 reported that there was a significant increase in estradiol binding. These discrepancies may be due to differences in experimental procedures, such as time of exposure to the metal. In the present study there was a 1.3- to 3-fold increase in estradiol receptor activity of uterine cytosol on day 5 of pregnancy in mice that had been treated with lead in vivo on the previous day. The binding affinities, expressed as dissociation constants, were very similar for the control and lead-treated groups and were within the range of those found by other investigators for cytoplasmic estradiol receptors in the uterus. 4, 14 There is no obvious explanation for the increased estradiol receptor activity in the leadtreated mice. However, progesterone is known to have an inhibitory effect on the synthesis of estradiol receptors in the uterus, 15 and a decrease in the serum levels of progesterone in lead-treated mice cannot be excluded. In fact, a trend toward lower values in serum, although not statistically significant owing to a large interindividual variation, has been found in two studies. 2, 16 Plasma levels of 17J3-estradiol were almost identical in the nontreated and the lead-treated groups,2 with very
507
small interindividual variation, which makes it seem less likely that the effect of lead observed in the present study was mediated via this hormone. The results from our studies with the addition of lead in vivo may seem to contradict those of previous in vitro studies. A number of different heavy metals, including lead, has been reported to give a decreased binding of 17J3-estradiol and progesterone in uterine Cytosol.4, 5, 17-20 In one study using zinc ions, increased activity was found. 3 The decreased degree of steroid hormone binding was assumed to be due to an interference of the metal with -SH groups on the receptor molecules,5, 17, 19 and the inhibition could be reversed after the addition of ethylenediaminetetracetic acid to the cytosol. 5, 19 In our in vitro addition study there was no effect oflead in concentrations of 10 and 100 ~moles/liter. These concentrations were chosen as they are comparable to those estimated in uterine tissue of mice and under similar esperimental conditions. 21 The higher concentrations (1 and 5 mmoleslliter) caused significant decreases in estimated receptor activity. The most likely explanation for this decrease was an increased precipitation of proteins in the incubation mixture with increased amounts of added lead chloride. As similar high concentrations of lead chloride were used in investigations published previously,3, 4 it seems likely that the effects found in those were also, at least in part, due to coprecipitation oflead chloride and receptor proteins. Thus, the decreased estradiol binding observed in the in vitro studies in which lead was added is thought to reflect a decrease in number of available soluble cytoplasmatic receptors rather than an interference by metal with -SH groups of the binding proteins. This study was conducted to obtain information on how lead chloride causes inhibition of implantation in mice. A decrease in estradiol binding, as might be suspected from previous in vitro studies with lead and uterine receptors, can now be excluded as an explanation for the "lead effect" in mice. In contrast, it is possible that the observed increase in estradiol receptor activity per se causes impaired implantation. An increase in the number of receptors may result in increased estrogen stimulation of the uterus. Such stimulation may be comparable to the situation in which the administration of too much estrogen causes inhibition of implantation. 22 However, another possibility is that a decreased progesterone level both induced an increase in estradiol receptor activity and caused impairment of implantation owing to lack
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of normal gestagenic transformation of the endometrium at the time of implantation. It has been shown that lead can interfere with central neuroendocrine functions. 23 The observed increase in uterine estradiol receptor activity in the present study may represent an indirect effect of lead on the hypothalamus, pituitary, or ovaries. REFERENCES 1. Wide M, Nilsson 0: Differential susceptibility of the embryo to inorganic lead during periimplantation in the mouse. Teratology 16:273, 1977 2. Wide M: Interference of lead with implantation in the mouse. Effect of exogenous oestradiol and progesterone. Teratology 21:187,1980 3. Emanuel MB, Oakey RE: Effect of Zn + + on the binding of oestradiol-17~ to a uterine protein. Nature 223:66, 1969 4. Young PCM, Cleary RE, Ragan WD: Effect of metal ions on the binding of17~-estradiol to human endometrial cytosol. Fertil Steril 28:459, 1977 5. Young PCM, Cleary RE, Ehrlich CE: Effect of metal ions on the binding of progesterone and estradiol by human endometrial cytosol. In Multiple Molecular Forms of Steroid Hormone Receptors, Edited by MK Agarwal. Amsterdam, ElsevierINorth Holland Biomedical Press, 1977, p 215 6. Wide M, Nilsson 0: Interference oflead with implantation in the mouse. A study of the ultrastructure of blastocysts and endometrium. Teratology 20:101, 1979 7. Gaddum JH: Bioassays and mathematics. Pharmacol Rev 5:87,1953 8. Scatchard G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660, 1949 9. Lowry OH, Rosebrough NJ, FaIT AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chem 193:265, 1951 10. Ghosh M, Roy SK, Kar AB: Effect of a copper intrauterine contraceptive device and nylon suture on the estradiol 17~-6, 7_H3 and progesterone 1,2-H3 in the rat uterus. Contraception 11:45, 1975
November 1980 11. Tamaya T, Nakata Y, Ohno Y, Nioka S, Furuta N, Okada H: The mechanism of action of the copper intrauterine device. Fertil Steril 27:767,1976 12. Adadevoh BK, Dada OA: Effect of intrauterine copper on the uptake of estradiol-C 14 by rat tissues. Fertil Steril 24:54,1973 13. 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 14. Puca GA, Nola E, Sica V, Bresciani F: Estrogen-binding proteins of calf uterus. Partial purification and preliminary characterization of two cytoplasmic proteins. Biochemistry 1:3769, 1971 15. Clark JH, Hsueh AJW, Peck EJ J r: Regulation of estrogen receptor replenishment by progesterone. Ann NY Acad Sci 286:161, 1977 16. Jacquet P, Gerber GB, Leonard A, Maes J: Plasma hormone levels in normal and lead treated pregnant mice. Experientia 33:1375, 1977 17. Jensen EV, Hurst DJ, DeSombre ER, Jungblut PW: Sulfhydryl groups and estradiol-receptor interaction. Science 158:385. 1967 18. Sanborn BM, Rao BR, Korenman SG: Interaction of 17~ estradiol and its specific uterine receptor. Evidence for complex kinetic and equilibrium behaviour. Biochemistry 10:4955, 1971 19. Kontula K, J anne 0, Luukainen T, Vihko R: Progesteronebinding protein in human myometrium. Influence of metal ions on binding. J Clin Endocrinol Metab 38:500, 1974 20. LOvgren T, Pettersson K, Lundberg B, Punnonen R: Effect of Cu2 + ions on the binding of estrogen to the human myometrial estrogen binding protein. Contraception 18:181, 1978 21. Wide M: The effect of inorganic lead on the mouse blastocyst in vitro. Teratology 17:165, 1978 22. Sartor P, Dupont H, Dupont MA, Duluc AJ, Mayer G: The action of high doses of oestradiol on implantation and decidual reaction in the rat. Anim Reprod Sci 1:93, 1978 23. Petrusz P, Weaver CM, Grant LD, Mushak P, Krigman MR: Lead poisoning and reproduction: effects on pituitary and serum gonadotrophins in neonatal rats. Environ Res 19:383, 1979