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Effect of progesterone treatment on the uptake of estradiol-17β by uterine tissues of ovariectomised rats
Moleculur and CellularEndocrinology, 10 (1978) 319-325 0 El~~er~North-Hound Scientific abashed, Ltd.
EFFECT OF PROGESTERONE TREATMENT ON THE UPTAKE OF ESTRADIOL-17~ BY UTER~E TISSUES OF OVARIECTOM~SEDRATS P.A.T. KELLY, C. MORRISON and B. GREEN Department of Biochemistry, Universityof Strathclyde, The Todd Centre, 31 Taylor Street, GlasgowG4 ONR, U.K. Received 7 November 1977;accepted
10 January 1978
[3H]Estradiol uptake was measured in nuclei isolated from uterine tissues of rats which had earlier been ovariectomised, then primed with estradiol and treated for various periods of time with either progesterone (4 mg/day) or vehicle alone. Whether [3H]estradiol was injected in vivo or the uterine tissues exposed to it in vitro, there was no signiticant depression of uptake into the luminal epitheiium even after periods of progesterone treatment (48-72 h) which suppress the ability of estradiol to cause mitosis in this tissue. Thii strongly suggests that progesterone does not abolish the mitogenic action of estradiol on the luminal epithelium solely by preventing it from gaining access to the cells or their nuclei. Uptake of estradiol by the stroma and myometrium was also little affected by progesterone pretreatment. Keywords: progesterone; estradiol uptake; uterus.
Several studies have indicated that treatment of animals for short (60 min) periods with progesterone alone does not influence subsequent estradid uptake by mouse uteri (e.g., Lee, 1974) or by the epithelia or muscle cells of rat uteri (Trams and Holstein, 1974). Recently Bhakoo and Katzenellenbogen (1977) have shown that longer treatments with progesterone alone have no effect on the levels of cytosol estrogen receptor in the whole rat uterus but, where individual uterine tissues have been examined, the situation is more controversial. In seeking to explain how progesterone diverts the mitogenic action of estradiol from the epithelium to the stroma of the mouse or rat uterus (Martin and Finn, 1969;Clark, 1971, 1973;Tachi et al., 1972) investigators using two different experimental approaches reached differing conclusions. The results of autoradiographical studies suggested that progesterone pretreatment greatly reduced or abolished the (nuclear) uptake of [3H]estradiol by the luminal epithelium but not the stroma of rat uteri when examined 2 h after adm~istration (Stumpf, 1972; Tachi et al., 1972; Stumpf and Sar, 1974). In contrast, when the [3H]estradiol was extracted from the subcellular components of the separated mouse uterine tissues, no such reduction in epithelial uptake of this hormone was observed (Smith et al., 1970). To try to clar319
320
P.A. T. Kell?, zt al.
ify the position, [“Hlestradiol uptake in vivo and in vitro has been measured in the rat uterus by methods basically similar to those employed by Smith et al. (1970) but differing in the following respects: (a) the species used (rat) was the same as that in the autoradiographical studies;(b) estradiol uptake was measured in purified nuclei instead of the total cell particulate material; and (c) incubations in vitro were carried out at 37’C rather than 4’C.
MATERIALS
AND METHODS
Radiochemicals [2,4,6,7-3H] Estradiol (85 Ci/mmol) were purchased from the Radiochemical
and [methyl-3H]thymidine Centre, Amersham.
(45 Ci/mmol)
Hormone treatment of animals Mature virgin female rats were ovariectomised under tribromoethanol (‘Avertin’: Bayer Products) anaesthesia; 2-4 weeks later, all animals in both test and control groups (4-8/group) were given priming injections of 1 yg estradiol/day for two days (1 and 2) then rested for two days (3 and 4). Starting on day 5 the test animals were injected daily with 4 mg progesterone while the control group received vehicle (arachis oil) alone. All these injections were given subcutaneously between 10.30 and 12.00 h. Uterine uptake of / 3H]estradiol (a) In viva After 4548 h progesterone treatment, the rats in the test group (and their vehicle-injected controls) were given a subcutaneous injection of [3H]estradiol (S-lo&i, 0.03-0.10 pug/rat) in 0.5 ml saline. Two h later (8 h in one experiment,) the animals were killed, samples of cardiac blood being taken in some experiments for estimation of plasma radioactivity levels. The uteri were removed, weighed, and the epithelial and stromal fractions separated (Heald et al., 1975) in medium made 10e6 M in unlabelled estradiol to minimise any redistribution of label. This method yields a luminal epithelium fraction, a stroma fraction which also contains glandular epithelium, and a residual fraction of myometrium with some remaining stroma attached. Most separations were checked by rapid microscopic examination, after which nuclei (and cytosol) were prepared from each tissue fraction essentially as described by Lunan and Green (1974), the nuclei being sedimented once through 1.8 M sucrose then washed in 0.2% Triton X-100. The radioactivity present in the nuclei was measured after extraction into ethanol (Lunan and Green, 1974). All tissue manipulation was carried out at 0-4”C. (b) In vitro After test animals had received 48 h progesterone treatment, the uteri were removed, weighed and the horns slit open longitudinally. Usually one uterine horn from each animal in a group was taken and these were incubated together at
Estradiol uptake by rat uteri
321
37’C for 30 min with lo-’ M [3H]estradiol either in Medium 199 (Wellcome Reagents Ltd.) or occasionally in bicarbonate-buffered medium (Krebs and Henseleit, 1932) containing 10 mM glucose. For the progesterone-treated uteri lo-’ M progesterone was included in the incubation medium. As specificity controls, the contralateral horns from one group of animals were incubated in the same medium as the experimental horns but with lo-’ M unlabelled estradiol added. Because the unlabelled estradiol is present in excess, it competes successfully with the labelled hormone for the limited number of specific binding sites, so that this set provides a measure of the extent of nonspecific hormone uptake. Although higher levels of hormone uptake were obtained after a 60-min incubation, the pattern was the same for the 30-min incubations which were adopted to minimize tissue degeneration. After incubation, the uteri were washed 4 times in ice-cold medium containing 10m6M unlabelled estradiol. The epithelial and stromal fractions were separated, fractionated and the radioactivity measured as described for the in vivo work. Uterine DNA synthesis: incorporation of [3H]thymidine
Ovariectomised rats were given the same hormonal regime as those in the r3H]estradiol uptake experiment. After 48-68 h progesterone treatment, the test animals and their controls (given arachis oil only) were injected subcutaneously with 0.2-0.3 pg estradiol; 6.5-l 1.5 h later each animal received an intravenous injection of 50 I.tCi [‘HI thymidine. The animals were killed 90 min after this thymidine injection and their uteri were removed, washed briefly in saline and separated into epithelium, stroma, etc., so that the extent of thymidine incorporation into the DNA of each fraction could be measured. The DNA in the final pellet (Heald et al., 1970) was hydrolysed in 5% trichloroacetic acid for 20 mm at 90°C (Hubbard et al., 1972), portions of hydrolysate being taken for scintillation counting and for DNA estimation (Ceriotti, 1952).
RESULTS Uterine weights
After 48 h of progesterone treatment there was a small increase in uterine weight compared with the controls (ratio = 1.19 + 0.09). This probably reflects the minor proliferation of stroma and myometrium known to occur under the influence of progesterone alone (Martin and Finn, 1968; Marcus, 1974; Glasser and Clark, 1975). Estradiol uptake
All the principal experimental findings concerning the effect of progesterone pretreatment on estradiol uptake by the spayed rat uterus and the subsequent incorporation of [3H] thymidine into DNA are summarized in fig. 1. Each column represents the mean value (+S.E.M.) of the results of 4-6 uptake experiments each
322
2
;i
s
30
60
g
02
2o
40
T
:
10 %? E
T
20
0 30
E a
0
”
60
.g
mvometrium
Fig. 1. Effect of 48-h progesterone pretreatment on estrogen uptake by ovariectomised rat uteri. The left-hand panel (A) shows the amount of [3H]estradiol present in the nuclei of the uterine tissues of rats 2 h after injection in vivo or after 30 mm incubation in vitro with lo-’ M [3H]estradiol. The right-hand panel (B) shows [3H]thymidine incorporation into DNA over a 90min period starting 6.5-11.5 h after injection of 0.2-0.3 ng estradiol; hormone sequence as in (A) except that the estradiol was unlabelled. Open columns = control animals (no progesterone); hatched columns = animals given 4 mg progesterone/day for 45-48 h before the test exposure to estradiol; vertical brackets = *S.E.M.; n = 4-6.
involving the combined uteri from 4-8 progesterone-treated animals and a similar number of controls. Uptake is expressed as estradiol concentrations since thin-layer chromatography revealed that, for both sets of experiments, 95% of the radioactivity in epithelial nuclei and 86-90% of that in the stromal nuclei was due to estradiol itself. For in vivo uptake, results are corrected to 0.1 pg estradiol injected/rat and for in vitro work to 10q9 M labelled estradiol in the incubation medium. Only values for the nuclei are shown because most of the cytosol radioactivity levels were too close to background for accurate counting. The luminal epithelium was the tissue of greatest interest and for this tissue the individual results of the in vitro experiments are displayed in fig. 2. The [‘Hlestradiol uptake being measured is specific (presumably receptor-mediated), as indicated by the suppression observed in vitro (fig. 2) in the presence of loo-fold excess of unlabelled estradiol and by the low levels of radioactivity (<7% of those in the uterus) present in spleen nuclei after administration in vivo. A small amount of labelled hormone was released into the medium during the mechanical tissue separation process and if any significant proportion entered the other tissues (especially epithelium) it could seriously distort the final pattern observed. To counteract this, 10e6 M unlabelled estradiol was always present in media used for washing, tissue separation, etc. In a control experiment in which normal rat uteri were put through the entire separation sequence in medium containing [3H]estra-
Estradiol uptake by rat uteri
323
Fig. 2. Nuclear levels of [ 3H]estradiol in luminal epithelium of uteri from ovariectomised rats. Uteri from prqgesterone-pretreated and control rats were incubated in vitro with lo* M [ 3H] estradiol before separation of tissues and isolation of nuclei. Four separate experiments with combined uteri from 4-8 rats/group are shown. Filled columns = specificity controls (uterine horns incubated with [3H]estradiol plus lo- ’ M unlabelled estradiol); others as in fig. 1.
diol at a concentration (350 fmol/ml) some 15 times greater than that actually found during the in vivo uptake experiments, the concentrations in the nuclei were less than 25% of those indicated in fig. 1. Thus, translocation is minimal. The experimental results (figs. 1 and 2) revealed no significant effect of progesterone pretreatment on nuclear estradiol uptake by the uterine tissues of ovariectomized rat. In one of these experiments the [3H]estradiol levels were measured 8 h after dosing; the pattern of distribution of this ‘retained’ estradiol (Anderson et al., 1975; Lan and Katzenellenbogen, 1976) was essentially the same as at 2 h postinjection. Unlike this simple picture of estradiol uptake, the pattern of ensuing DNA synthesis was strongly modified in the uteri of animals given prior progesterone treatment. There was a significant (t-test, P < 0.01) reduction in thymidine incorporation in the epithelium and a similar increase in the myometrium. The pattern in the stroma was less clear cut.
DISCUSSION The results for estradiol uptake by the rat uterine stroma are broadly in agreement with those obtained in the rat by Tachi et al. (1972) using autoradiography and in the mouse by Smith et al. (1970) who extracted the labelled hormone from the cell components. Although some increase in thymidine incorporation into stromal DNA was observed when estradiol was given to those animals which had already received progesterone, the effect was not statistically significant (cf. Smith et al., 1970; Martin et al., 1973). Perhaps this small difference results from the opposing actions of prior progesterone treatment on the two tissues present in the ‘stromal fraction’ studied here, i.e., it leads to stimulation of DNA synthesis in the stroma and suppression in the glandular epithelium (Clark, 1971; Martin et al., 1973). Studies at different intervals after estrogen administration could perhaps resolve this.
324
P.A. T. Keflv ef al.
It has been reported (Marcus, 1974) that myometrial proliferation in the rat uterus occurs in response to ovarian hormones, though this tissue has not been as widely investigated as the endometrium. Certainly the greater stimulation of DNA synthesis by estradiol in the progesterone-treated myometrium (fig. 1) could not be ascribed to any alteration in the amount of estradiol entering the tissue. It was the controversy concerning the luminal epithelium, however. which led to the present investigation. Here, in progesterone-treated animals. the well-documented suppression of estradiol-induced proliferation (Martin and Finn, 1969: Clark, 1971; Tachi et al., 1972; Martin et al., 1973) was immediately apparent as a lowering of thymidine incorporation, confirming the results of mitotic counts made in these animals (Hamidi, 1976). In contrast to this marked difference in f3Hfthymidine incorporation, the amount of [3H]estradiol present in the epithelia1 nuclei after exposure to the labelled hormone in vivo or in vitro was quite similar, whether the animals were pretreated with progesterone or not. In other experiments in which the uterine tissues were first separated and then incubated with [3H]estradiol, the results after 48-72 h progesterone treatment agreed with the current findings but the proportion of nonspecific uptake in these conditions was rather large (10-45% for epithelium and even higher for the stroma). This inability to demonstrate any major influence of 48-h progesterone treatment on overall estrogen uptake by the luminal epithelium of the rat uterus is consistent with the findings of Smith et al. (1970). It is difficult to reconcile with the conclusions drawn from autoradiographical studies mainly on progesterone-treated rats held in delayed impl~tatior~ (Stumpf, 1972; Tachi et al., 1972) but also on animals given hormone regimes similar to the present ones (Stumpf and Sar, 1974). It should be emphasized that the variation in estradiol uptake over the separate experiments summarized in fig. 1 is such that only a major (> 65%) decrease due to progesterone would be statistically significant. Nonetheless. if the progesterone treatment used here had resulted in a ‘lack’ of specific estrogen uptake by the luminal epithelium (or marked suppression) as the autoradiographical results implied, then the effect should have been detectable, as in the case of incubation in the presence of excess unlabelled estradiol (specificity controls, fig. 2). It is possible that the discrepancy between these sets of results may depend on the amount of estrogen (e.g., residual priming estrogen or adrenal estrogen) to which the uterus is exposed during the progesterone treatment, since progesterone will inhibit the replenishment of those estrogen receptors which are used up. but otherwise has little effect on them (Bhakoo and Katzenellenbogen, 1977). The hormone regime used in the present experiments is of interest because it leads to the temporary sensitization of the uterus to a decidualizing stimulus (Finn, 1966; Psychoyos, 1967; Hamidi, 1976; Kelly and Green. unpublished). It is believed (O’Grady and Bell, 1977) that suppression of mitosis in the luminal epithelium is necessary if sensitization is to occur. At least in the present experimental situation the results suggest that estradiol may still enter these epithelial cells and
Estradiol uptake by rat uteri
325
migrate to the nuclei but even when it does, it is no longer effective in inducing cellular proliferation. This is already generally accepted for the glandular epithelium of the uterus (Tachi et al., 1972). In different experiments with immature rats (Katzenellenbogen and Ferguson, 1975) estradiol had been demonstrated to be bound apparently normally in uterine nuclei, yet to be ineffective in inducing the expected responses.
ACKNOWLEDGEMENTS We thank Mr. J.E. O’Grady for advice and help with injections, Dr. W.E. Stumpf and Dr. G. Trams for their useful comments, and a referee for suggesting the thymidine experiments. We also thank Professor P.J. Heald for his interest in this work, which was supported by the Medical Research Council and the Ford Foundation.
REFERENCES Anderson, J.N., Peck, E.J. and Clark, J.H. (1975) Endocrinology 96,160-167. Bhakoo, H.S. and KatzenelJenbogen, B.S. (1977) Mol. Cell. Endocrinol. 8,121-134. Ceriotti, G. (1952) J. Biol. Chem. 198,297-303. Clark, B.F. (1971) J. Endocrinol. 50,527-528. Clark, B.F. (1973) J. Endocrinol. 56,341-342. Finn, C.A. (1966) J. Endocrinol. 36,239-248. Glasser, S.R. and Clark, J.H. (1975) In: The Developmental Biology of Reproduction, 33rd Symp. Sot. Dev. Biol. (Academic Press Inc., New York - San Francisco - London) pp. 311-345. Hamidi, H. (1976) Ph. D. Thesis, University of Strathclyde. HeaJd, P.J., Govan, A.D.T. and O’Grady, J.E. (1975) J. Reprod. Fertil. 42,593-595. Heald, P.S., OGrady, J.E. and Howie, A.J. (1970) J. Reprod. Fertil. 21,481-488. Hubbard, R.W., Matthew, W.T. and Moulton, D.W. (1972) Anal. Biochem. 46,461-472. Katzenellenbogen, B.S. and Ferguson, E.R. (1975) Endocrinology 97,1-12. Krebs, H.A. and Henseleit, K. (1932) HoppeSeyler’s Z. Physiol. Chem. 210,33-66. Lan, N.C. and Katzenellenbogen, B.S. (1976) Endocrinology 98,220-227. Lee, A.E. (1974) J. Endocrinol. 60,167-174. Lunan, C.B. and Green, B. (1974) Clm. Endocrinol. 3,465-480. Marcus, G.J. (1974) Biol. Reprod. 10,447-452. Martin, L. and Finn,C.A. (1968) J. Endocrinol. 41,363-371. Martin, L. and Finn, C.A. (1969) J. Endocrinol. 44,279-280. Martin, L., Finn, C.A. and Trinder, G. (1973) J. Endocrinol. 56,303-307. O’Grady, J.E. and Bell, S.C. (1977) In: Development in Mammals, Vol. 1, Ed.: M.H. Johnson (North-Holland Publ. Co., Amsterdam - New York - Oxford) pp. 165-243. Psychoyos, A. (1967) Adv. Reprod. Physiol. 2,257-277. Smith, J.A., Martin, L., King, R.J.B. and Vertes, M. (1970) Biochem. J. 119,773-784. Stumpf, W.E. (1972) Acta Histochem. Cytochem. 5,209-211. Stumpf, W.E. and Sar, M. (1974) cited in Prasad, M.R.N., Sar, M. and Stumpf, W.E. (1974) J. Reprod. Fertil. 36,75 -81. Tachi,C.,Tachi, S. and Lindner, H.R. (1972) J. Reprod. FertiJ. 31,59-76. Trams, G. and Holstein, A.F. (1974) J. Steroid Biochem. 5,338-339.
EFFECT OF PROGESTERONE TREATMENT ON THE UPTAKE OF ESTRADIOL-17~ BY UTER~E TISSUES OF OVARIECTOM~SEDRATS P.A.T. KELLY, C. MORRISON and B. GREEN Department of Biochemistry, Universityof Strathclyde, The Todd Centre, 31 Taylor Street, GlasgowG4 ONR, U.K. Received 7 November 1977;accepted
10 January 1978
[3H]Estradiol uptake was measured in nuclei isolated from uterine tissues of rats which had earlier been ovariectomised, then primed with estradiol and treated for various periods of time with either progesterone (4 mg/day) or vehicle alone. Whether [3H]estradiol was injected in vivo or the uterine tissues exposed to it in vitro, there was no signiticant depression of uptake into the luminal epitheiium even after periods of progesterone treatment (48-72 h) which suppress the ability of estradiol to cause mitosis in this tissue. Thii strongly suggests that progesterone does not abolish the mitogenic action of estradiol on the luminal epithelium solely by preventing it from gaining access to the cells or their nuclei. Uptake of estradiol by the stroma and myometrium was also little affected by progesterone pretreatment. Keywords: progesterone; estradiol uptake; uterus.
Several studies have indicated that treatment of animals for short (60 min) periods with progesterone alone does not influence subsequent estradid uptake by mouse uteri (e.g., Lee, 1974) or by the epithelia or muscle cells of rat uteri (Trams and Holstein, 1974). Recently Bhakoo and Katzenellenbogen (1977) have shown that longer treatments with progesterone alone have no effect on the levels of cytosol estrogen receptor in the whole rat uterus but, where individual uterine tissues have been examined, the situation is more controversial. In seeking to explain how progesterone diverts the mitogenic action of estradiol from the epithelium to the stroma of the mouse or rat uterus (Martin and Finn, 1969;Clark, 1971, 1973;Tachi et al., 1972) investigators using two different experimental approaches reached differing conclusions. The results of autoradiographical studies suggested that progesterone pretreatment greatly reduced or abolished the (nuclear) uptake of [3H]estradiol by the luminal epithelium but not the stroma of rat uteri when examined 2 h after adm~istration (Stumpf, 1972; Tachi et al., 1972; Stumpf and Sar, 1974). In contrast, when the [3H]estradiol was extracted from the subcellular components of the separated mouse uterine tissues, no such reduction in epithelial uptake of this hormone was observed (Smith et al., 1970). To try to clar319
320
P.A. T. Kell?, zt al.
ify the position, [“Hlestradiol uptake in vivo and in vitro has been measured in the rat uterus by methods basically similar to those employed by Smith et al. (1970) but differing in the following respects: (a) the species used (rat) was the same as that in the autoradiographical studies;(b) estradiol uptake was measured in purified nuclei instead of the total cell particulate material; and (c) incubations in vitro were carried out at 37’C rather than 4’C.
MATERIALS
AND METHODS
Radiochemicals [2,4,6,7-3H] Estradiol (85 Ci/mmol) were purchased from the Radiochemical
and [methyl-3H]thymidine Centre, Amersham.
(45 Ci/mmol)
Hormone treatment of animals Mature virgin female rats were ovariectomised under tribromoethanol (‘Avertin’: Bayer Products) anaesthesia; 2-4 weeks later, all animals in both test and control groups (4-8/group) were given priming injections of 1 yg estradiol/day for two days (1 and 2) then rested for two days (3 and 4). Starting on day 5 the test animals were injected daily with 4 mg progesterone while the control group received vehicle (arachis oil) alone. All these injections were given subcutaneously between 10.30 and 12.00 h. Uterine uptake of / 3H]estradiol (a) In viva After 4548 h progesterone treatment, the rats in the test group (and their vehicle-injected controls) were given a subcutaneous injection of [3H]estradiol (S-lo&i, 0.03-0.10 pug/rat) in 0.5 ml saline. Two h later (8 h in one experiment,) the animals were killed, samples of cardiac blood being taken in some experiments for estimation of plasma radioactivity levels. The uteri were removed, weighed, and the epithelial and stromal fractions separated (Heald et al., 1975) in medium made 10e6 M in unlabelled estradiol to minimise any redistribution of label. This method yields a luminal epithelium fraction, a stroma fraction which also contains glandular epithelium, and a residual fraction of myometrium with some remaining stroma attached. Most separations were checked by rapid microscopic examination, after which nuclei (and cytosol) were prepared from each tissue fraction essentially as described by Lunan and Green (1974), the nuclei being sedimented once through 1.8 M sucrose then washed in 0.2% Triton X-100. The radioactivity present in the nuclei was measured after extraction into ethanol (Lunan and Green, 1974). All tissue manipulation was carried out at 0-4”C. (b) In vitro After test animals had received 48 h progesterone treatment, the uteri were removed, weighed and the horns slit open longitudinally. Usually one uterine horn from each animal in a group was taken and these were incubated together at
Estradiol uptake by rat uteri
321
37’C for 30 min with lo-’ M [3H]estradiol either in Medium 199 (Wellcome Reagents Ltd.) or occasionally in bicarbonate-buffered medium (Krebs and Henseleit, 1932) containing 10 mM glucose. For the progesterone-treated uteri lo-’ M progesterone was included in the incubation medium. As specificity controls, the contralateral horns from one group of animals were incubated in the same medium as the experimental horns but with lo-’ M unlabelled estradiol added. Because the unlabelled estradiol is present in excess, it competes successfully with the labelled hormone for the limited number of specific binding sites, so that this set provides a measure of the extent of nonspecific hormone uptake. Although higher levels of hormone uptake were obtained after a 60-min incubation, the pattern was the same for the 30-min incubations which were adopted to minimize tissue degeneration. After incubation, the uteri were washed 4 times in ice-cold medium containing 10m6M unlabelled estradiol. The epithelial and stromal fractions were separated, fractionated and the radioactivity measured as described for the in vivo work. Uterine DNA synthesis: incorporation of [3H]thymidine
Ovariectomised rats were given the same hormonal regime as those in the r3H]estradiol uptake experiment. After 48-68 h progesterone treatment, the test animals and their controls (given arachis oil only) were injected subcutaneously with 0.2-0.3 pg estradiol; 6.5-l 1.5 h later each animal received an intravenous injection of 50 I.tCi [‘HI thymidine. The animals were killed 90 min after this thymidine injection and their uteri were removed, washed briefly in saline and separated into epithelium, stroma, etc., so that the extent of thymidine incorporation into the DNA of each fraction could be measured. The DNA in the final pellet (Heald et al., 1970) was hydrolysed in 5% trichloroacetic acid for 20 mm at 90°C (Hubbard et al., 1972), portions of hydrolysate being taken for scintillation counting and for DNA estimation (Ceriotti, 1952).
RESULTS Uterine weights
After 48 h of progesterone treatment there was a small increase in uterine weight compared with the controls (ratio = 1.19 + 0.09). This probably reflects the minor proliferation of stroma and myometrium known to occur under the influence of progesterone alone (Martin and Finn, 1968; Marcus, 1974; Glasser and Clark, 1975). Estradiol uptake
All the principal experimental findings concerning the effect of progesterone pretreatment on estradiol uptake by the spayed rat uterus and the subsequent incorporation of [3H] thymidine into DNA are summarized in fig. 1. Each column represents the mean value (+S.E.M.) of the results of 4-6 uptake experiments each
322
2
;i
s
30
60
g
02
2o
40
T
:
10 %? E
T
20
0 30
E a
0
”
60
.g
mvometrium
Fig. 1. Effect of 48-h progesterone pretreatment on estrogen uptake by ovariectomised rat uteri. The left-hand panel (A) shows the amount of [3H]estradiol present in the nuclei of the uterine tissues of rats 2 h after injection in vivo or after 30 mm incubation in vitro with lo-’ M [3H]estradiol. The right-hand panel (B) shows [3H]thymidine incorporation into DNA over a 90min period starting 6.5-11.5 h after injection of 0.2-0.3 ng estradiol; hormone sequence as in (A) except that the estradiol was unlabelled. Open columns = control animals (no progesterone); hatched columns = animals given 4 mg progesterone/day for 45-48 h before the test exposure to estradiol; vertical brackets = *S.E.M.; n = 4-6.
involving the combined uteri from 4-8 progesterone-treated animals and a similar number of controls. Uptake is expressed as estradiol concentrations since thin-layer chromatography revealed that, for both sets of experiments, 95% of the radioactivity in epithelial nuclei and 86-90% of that in the stromal nuclei was due to estradiol itself. For in vivo uptake, results are corrected to 0.1 pg estradiol injected/rat and for in vitro work to 10q9 M labelled estradiol in the incubation medium. Only values for the nuclei are shown because most of the cytosol radioactivity levels were too close to background for accurate counting. The luminal epithelium was the tissue of greatest interest and for this tissue the individual results of the in vitro experiments are displayed in fig. 2. The [‘Hlestradiol uptake being measured is specific (presumably receptor-mediated), as indicated by the suppression observed in vitro (fig. 2) in the presence of loo-fold excess of unlabelled estradiol and by the low levels of radioactivity (<7% of those in the uterus) present in spleen nuclei after administration in vivo. A small amount of labelled hormone was released into the medium during the mechanical tissue separation process and if any significant proportion entered the other tissues (especially epithelium) it could seriously distort the final pattern observed. To counteract this, 10e6 M unlabelled estradiol was always present in media used for washing, tissue separation, etc. In a control experiment in which normal rat uteri were put through the entire separation sequence in medium containing [3H]estra-
Estradiol uptake by rat uteri
323
Fig. 2. Nuclear levels of [ 3H]estradiol in luminal epithelium of uteri from ovariectomised rats. Uteri from prqgesterone-pretreated and control rats were incubated in vitro with lo* M [ 3H] estradiol before separation of tissues and isolation of nuclei. Four separate experiments with combined uteri from 4-8 rats/group are shown. Filled columns = specificity controls (uterine horns incubated with [3H]estradiol plus lo- ’ M unlabelled estradiol); others as in fig. 1.
diol at a concentration (350 fmol/ml) some 15 times greater than that actually found during the in vivo uptake experiments, the concentrations in the nuclei were less than 25% of those indicated in fig. 1. Thus, translocation is minimal. The experimental results (figs. 1 and 2) revealed no significant effect of progesterone pretreatment on nuclear estradiol uptake by the uterine tissues of ovariectomized rat. In one of these experiments the [3H]estradiol levels were measured 8 h after dosing; the pattern of distribution of this ‘retained’ estradiol (Anderson et al., 1975; Lan and Katzenellenbogen, 1976) was essentially the same as at 2 h postinjection. Unlike this simple picture of estradiol uptake, the pattern of ensuing DNA synthesis was strongly modified in the uteri of animals given prior progesterone treatment. There was a significant (t-test, P < 0.01) reduction in thymidine incorporation in the epithelium and a similar increase in the myometrium. The pattern in the stroma was less clear cut.
DISCUSSION The results for estradiol uptake by the rat uterine stroma are broadly in agreement with those obtained in the rat by Tachi et al. (1972) using autoradiography and in the mouse by Smith et al. (1970) who extracted the labelled hormone from the cell components. Although some increase in thymidine incorporation into stromal DNA was observed when estradiol was given to those animals which had already received progesterone, the effect was not statistically significant (cf. Smith et al., 1970; Martin et al., 1973). Perhaps this small difference results from the opposing actions of prior progesterone treatment on the two tissues present in the ‘stromal fraction’ studied here, i.e., it leads to stimulation of DNA synthesis in the stroma and suppression in the glandular epithelium (Clark, 1971; Martin et al., 1973). Studies at different intervals after estrogen administration could perhaps resolve this.
324
P.A. T. Keflv ef al.
It has been reported (Marcus, 1974) that myometrial proliferation in the rat uterus occurs in response to ovarian hormones, though this tissue has not been as widely investigated as the endometrium. Certainly the greater stimulation of DNA synthesis by estradiol in the progesterone-treated myometrium (fig. 1) could not be ascribed to any alteration in the amount of estradiol entering the tissue. It was the controversy concerning the luminal epithelium, however. which led to the present investigation. Here, in progesterone-treated animals. the well-documented suppression of estradiol-induced proliferation (Martin and Finn, 1969: Clark, 1971; Tachi et al., 1972; Martin et al., 1973) was immediately apparent as a lowering of thymidine incorporation, confirming the results of mitotic counts made in these animals (Hamidi, 1976). In contrast to this marked difference in f3Hfthymidine incorporation, the amount of [3H]estradiol present in the epithelia1 nuclei after exposure to the labelled hormone in vivo or in vitro was quite similar, whether the animals were pretreated with progesterone or not. In other experiments in which the uterine tissues were first separated and then incubated with [3H]estradiol, the results after 48-72 h progesterone treatment agreed with the current findings but the proportion of nonspecific uptake in these conditions was rather large (10-45% for epithelium and even higher for the stroma). This inability to demonstrate any major influence of 48-h progesterone treatment on overall estrogen uptake by the luminal epithelium of the rat uterus is consistent with the findings of Smith et al. (1970). It is difficult to reconcile with the conclusions drawn from autoradiographical studies mainly on progesterone-treated rats held in delayed impl~tatior~ (Stumpf, 1972; Tachi et al., 1972) but also on animals given hormone regimes similar to the present ones (Stumpf and Sar, 1974). It should be emphasized that the variation in estradiol uptake over the separate experiments summarized in fig. 1 is such that only a major (> 65%) decrease due to progesterone would be statistically significant. Nonetheless. if the progesterone treatment used here had resulted in a ‘lack’ of specific estrogen uptake by the luminal epithelium (or marked suppression) as the autoradiographical results implied, then the effect should have been detectable, as in the case of incubation in the presence of excess unlabelled estradiol (specificity controls, fig. 2). It is possible that the discrepancy between these sets of results may depend on the amount of estrogen (e.g., residual priming estrogen or adrenal estrogen) to which the uterus is exposed during the progesterone treatment, since progesterone will inhibit the replenishment of those estrogen receptors which are used up. but otherwise has little effect on them (Bhakoo and Katzenellenbogen, 1977). The hormone regime used in the present experiments is of interest because it leads to the temporary sensitization of the uterus to a decidualizing stimulus (Finn, 1966; Psychoyos, 1967; Hamidi, 1976; Kelly and Green. unpublished). It is believed (O’Grady and Bell, 1977) that suppression of mitosis in the luminal epithelium is necessary if sensitization is to occur. At least in the present experimental situation the results suggest that estradiol may still enter these epithelial cells and
Estradiol uptake by rat uteri
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migrate to the nuclei but even when it does, it is no longer effective in inducing cellular proliferation. This is already generally accepted for the glandular epithelium of the uterus (Tachi et al., 1972). In different experiments with immature rats (Katzenellenbogen and Ferguson, 1975) estradiol had been demonstrated to be bound apparently normally in uterine nuclei, yet to be ineffective in inducing the expected responses.
ACKNOWLEDGEMENTS We thank Mr. J.E. O’Grady for advice and help with injections, Dr. W.E. Stumpf and Dr. G. Trams for their useful comments, and a referee for suggesting the thymidine experiments. We also thank Professor P.J. Heald for his interest in this work, which was supported by the Medical Research Council and the Ford Foundation.
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