BIOLOGY OF REPRODUCTION 37, 527-538 (1987) 527 The Estrogenic Responses to Clomiphene in the Different Cell Types of the Rat Uterus: Morphometrical Evaluation’ GERMANA GRUNERT, GUILLERMO NEUMANN, MAURO PORCIA, and ANDREI N. TCHERNITCHIN2 Laboratory of Experimental Endocrinology Department of Experimental Morphology University of Chile Medical School Santiago, Chile ABSTRACT The present study was designed to investigate the dose-response of clomiphene on several estrogenic responses in the immature rat uterus and to compare it to available data on estradiol-1 713. A dissociation was demonstrated among the different estrogenic responses induced by clomiphene. Very high doses of clomiphene were needed to induce the 6-h uterine eosinophilia and deep endometrial edema, and maximal response levels were not reached at any dose studied. On the contrary, many genomic responses were induced with much lower doses of clomiphene, and maximal response levels were reached with at least the two highest doses of clomiphene. This dissociation is in agreement with the existence of separate groups of responses that are mediated by multiple and independent mechanisms of estrogen action involving different kind of receptors. Luminal epithelial, glandular epithelial, and myometrial hypertrophies were also found to differ with regard to the dose needed to induce this response in each cell type. The dissociation between genomic responses of the different uterine cell types supports the hypothesis of different estrogen receptors for each kind of cell. Clomiphene induces mitoses in the different cell types, but the proportion of mitoses in the cell types was different from that described for estradiol. It is suggested that these differences are also due to differences between receptors involved in cell proliferation. INTRODUCTION The responses to estrogen in the rat uterus can be classified into three separate groups that are mediated by independent hormone mechanisms (Tchernitchin, 1983). The first group includes several genomic responses, such as estrogen-induced increases in RNA and protein synthesis, increase in the content of some specific enzymes, and biochemical, morphological, and functional differentiation of target cells. Jensen and De Sombre (1972) suggested that these responses are mediated by the “cytosol-nuclear” estrogen receptors through the classical two-step mechanism, involving translocation of cytosol estrogen-receptor Accepted March 19, 1987. Received November 10, 1986. ‘This work was supported by Grant B-1493-8655 of the Departa- mento de Investigacion y Bibliotecas, University of Chile. 2 Reprint requests: Andrei N. Tchernitchin, Laboratory of Experi- mental Endocrinology, Department of Experimental Morphology, University of Chile Medical School, Casilla 21104, Correo 21, Santiago, Chile. complex to the nucleus, its transformation to nuclear estrogen-receptor complex, and its interaction with chromatin. Although recent reports of nuclear localization of unoccupied estrogen receptors have challenged the classical “two-step mechanism” (Gorski et al., 1984;Kingand Greene, 1984;McClellan et a!., 1984), these findings do not substantially affect the principle of the two-step hypothesis. The hormone must still first bind to the receptor; this interaction still results in an increased affinity for nuclear elements and ultimately in gene expression (Knowler and Beaumont, 1985). These responses are considered to be genomic responses to estrogen, since they are blocked by inhibitors of RNA and protein synthesis (Ui and Mueller, 1963; Tchernitchin and Galand, 1982). Recently, the development of techniques for the purification of steroid hormone receptors, and also the availability of defined frag- ments of hormone-inducible genes cloned under different experimental conditions, have permitted a detailed analysis of the interaction of receptors with the genome (Payvar et al., 1981; Jost et a!., 1985).
Transcript
BIOLOGY OF REPRODUCTION 37, 527-538 (1987)
527
The Estrogenic Responses to Clomiphene in theDifferent Cell Types of the Rat Uterus:
Morphometrical Evaluation’
GERMANA GRUNERT, GUILLERMO NEUMANN,
MAURO PORCIA, and ANDREI N. TCHERNITCHIN2
Laboratory of Experimental Endocrinology
Department of Experimental Morphology
University of Chile Medical School
Santiago, Chile
ABSTRACT
The present study was designed to investigate the dose-response of clomiphene on several estrogenic responses
in the immature rat uterus and to compare it to available data on estradiol-1 713. A dissociation was demonstrated
among the different estrogenic responses induced by clomiphene. Very high doses of clomiphene were needed
to induce the 6-h uterine eosinophilia and deep endometrial edema, and maximal response levels were not
reached at any dose studied. On the contrary, many genomic responses were induced with much lower doses of
clomiphene, and maximal response levels were reached with at least the two highest doses of clomiphene. This
dissociation is in agreement with the existence of separate groups of responses that are mediated by multiple
and independent mechanisms of estrogen action involving different kind of receptors. Luminal epithelial,
glandular epithelial, and myometrial hypertrophies were also found to differ with regard to the dose needed to
induce this response in each cell type. The dissociation between genomic responses of the different uterine cell
types supports the hypothesis of different estrogen receptors for each kind of cell. Clomiphene induces mitoses
in the different cell types, but the proportion of mitoses in the cell types was different from that described for
estradiol. It is suggested that these differences are also due to differences between receptors involved in cell
proliferation.
INTRODUCTION
The responses to estrogen in the rat uterus can be
classified into three separate groups that are mediated
by independent hormone mechanisms (Tchernitchin,
1983).
The first group includes several genomic responses,
such as estrogen-induced increases in RNA and
protein synthesis, increase in the content of some
specific enzymes, and biochemical, morphological,
and functional differentiation of target cells. Jensen
and De Sombre (1972) suggested that these responses
are mediated by the “cytosol-nuclear” estrogen
receptors through the classical two-step mechanism,
involving translocation of cytosol estrogen-receptor
Accepted March 19, 1987.
Received November 10, 1986.
‘This work was supported by Grant B-1493-8655 of the Departa-
mento de Investigacion y Bibliotecas, University of Chile.
2 Reprint requests: Andrei N. Tchernitchin, Laboratory of Experi-
mental Endocrinology, Department of Experimental Morphology,
University of Chile Medical School, Casilla 21104, Correo 21, Santiago,
Chile.
complex to the nucleus, its transformation to nuclear
estrogen-receptor complex, and its interaction with
chromatin. Although recent reports of nuclear
localization of unoccupied estrogen receptors have
challenged the classical “two-step mechanism”
(Gorski et al., 1984;Kingand Greene, 1984;McClellan
et a!., 1984), these findings do not substantially
affect the principle of the two-step hypothesis. The
hormone must still first bind to the receptor; this
interaction still results in an increased affinity for
nuclear elements and ultimately in gene expression
(Knowler and Beaumont, 1985). These responses
are considered to be genomic responses to estrogen,
since they are blocked by inhibitors of RNA and
protein synthesis (Ui and Mueller, 1963; Tchernitchin
and Galand, 1982). Recently, the development of
techniques for the purification of steroid hormone
receptors, and also the availability of defined frag-
ments of hormone-inducible genes cloned under
different experimental conditions, have permitted a
detailed analysis of the interaction of receptors with
the genome (Payvar et al., 1981; Jost et a!., 1985).
528 GRUNERT ET AL.
This has led to the concept that genomic responses to
steroid hormone stimulation are the result of the
interaction of hormone-receptor complexes with
regulatory DNA sequences (Payvar et al., 1981;
Jost et a!., 1985).
The second group includes several nongenomic
responses, such as estrogen-induced uterine eosino-
of histamine, and uterine luminal fluid accumulation.
Tchernitchin (1972, 1983) and Grunert et a!. (1984b)
suggested that these responses are mediated by
eosinophils. The eosinophils migrate from the blood
to the uterus under estrogen stimulation and
degranulate, releasing into the uterine stroma various
agents and enzymes that could play a role in estrogen
action (Tchernitchin, 1973, 1983). The finding of
high-affinity estrogen binding (estrogen receptors) in
the eosinophils (see review in Tchernitchin et al.,
1985) suggested that specific hormone receptors
could mediate eosinophil migration to the uterus
(Tchernitchin, 1983). This suggestion was further
supported by the finding that eosinophil migration to
the uterus was dependent on estrogen levels in the
blood but not in the uterus (Tchernitchin and Galand,
1983). The estrogen-induced migration of eosinophils
to the uterus and the eosinophil-mediated responses
are considered to be nongenomic estrogen responses.
This is based on the fact that they are not blocked by
inhibitors of RNA and protein synthesis (Tchernitchin
and Galand, 1982; Finlay et al., 1983). The findings
supporting this hypothesis of the role of eosinophils
in estrogen action are reviewed elsewhere (Tchernit-
chin, 1983; Tchernitchin eta!., 1985).
The third group of estrogen responses includes
estrogen-induced uterine hyperemia (Penney et al.,
1981), increase in glycogen content (Hechter et a!.,
1965; Singhal and Lafreniere, 1972), and mitotic
response (Galand et al., 1983). These responses have
been attributed to mediation by different, in-
dependent, and still unknown mechanisms.
The above groups of responses to estrogen can be
dissociated under a number of different experimental
conditions (for a review, see Tchernitchin et al.,
1985). This dissociation strongly supports the hypo-
thesis of the independence of the different mechan-
isms of estrogen action. Furthermore, the finding that
there are specific estrogens, such as estriol and
nafoxidine, that are potent enough to induce some
estrogenic responses, but not others (Hisaw, 1959;
Shelesnyak, 1959; Hechter and Halkerston, 1965;
Ruh et a!., 1973; Tchernitchin et a!., 1975; Bergink,
1980; Arvidson and Terenius, 1982; Galand et a!.,
1984), and the differences found between the receptor
affinities for some estrogens (Tchernitchin, 1972,
1974; Ruh et a!., 1973; Tchernitchin et al., 1985)
suggest that each kind of estrogen receptor displays
its own affinity/specificity spectrum.
In addition to the differences between cytosol-
nuclear receptors, eosinophil estrogen receptors, and
other kinds of estrogen receptors, differences have
also been detected between the cytosol-nuclear
estrogen receptors and the different cell types
(Tchernitchin et a!., 1985). This suggests the possibility
that the genomic responses from the different cell
types also may be dissociated.
Thus, it is possible to expect that various
compounds exhibiting estrogenic activity may display
stronger affinities for some kinds of receptors than
for others, resulting in the dissociation of the various
estrogenic responses. The objective of this study was
to test this possibility with clomiphene, a weak
estrogen investigated by many authors under different
experimental conditions (Kalra and Prasad, 1967;
Poteat and Bo, 1971; Martin, 1980;ClarkandGuthrie,
1981; Clark and Markaverich, 1982). For this purpose,
we investigated the potency of the drug to induce
different estrogenic responses in the different uterine
cell types of the same animal. We have compared
these responses to previously reported (Grunert
et a!., 1986) data obtained with estradiol-1713 under
similar experimental conditions.
Animals
MATERIALS AND METHODS
Immature, 21-day-old female Sprague-Dawley rats,
weighing between 40 and 50 g, were used in the
present study.
Hormones
Clomiphene citrate (Reca!cine Laboratory,
Santiago, Chile) or estradiol-17$ (Sigma Chemical
Co., St. Louis, MO) were dissolved in absolute ethanol
to obtain various concentrations; these stock ethanol
solutions were diluted in saline 1:19 immediately
before injection.
Experiment
The animals were injected in the jugular vein,
under ether anesthesia, with a single dose of
MORPHOMETRY OF ESTROGENIC RESPONSES TO CLOMIPHENE 529
clomiphene or estradio!-17j3 in ethanol-saline, 1:19, at
doses ranging from 0.037 to 3700 nM/kg. Control
animals were injected with vehicle (ethanol-saline,
1:19). The uterine horns were excised 6 and 24 h
after hormone or vehicle administration. After
determination of uterine wet weight, each uterine
horn was fixed in 10% neutral formalin and processed
for histological studies.
Parameters of Estrogen Stimulation in the Uterus
The following parameters of estrogen stimulation
in the uterus of immature rats were measured: (a)
uterine wet weight, (b) cell density in deep endo-
metrial stroma and in (c) superficial endometrial
stroma, (d) total number of uterine eosinophi!s, (e)
degranulation of uterine eosinophils, (f) lumina! and
(g) glandular epithelial cell height, (h) cell density in
the circular myometrial layer, and (i) the number of
mitotic figures in the different uterine cell types. The
histological measurements were performed at different
levels along the uterine horn to cover the whole
uterine horn in the evaluation of the estrogenic
responses. No differences of estrogenic responses
were detected along the uterine horn. The control
and experimental sections were cut at fixed normalized
distances of total tissue length. In each control or
experimental animal, the uterine eosinophils were
counted in about 20 uterine cross sections per uterine
horn; all other parameters were measured in 8 to 10
uterine cross sections per uterine horn.
Uterine wet weight was evaluated as a fraction of
the body weight and expressed as a percentage of the
control values.
The cc!! density quantification was performed in
several areas chosen at random in deep and superficial
endometria! stroma and in the circular myometrial
layer. The number of cell nuclei was determined in a
standard area of 1290 �zm2 by a circle marked in the
ocular piece of the microscope (at 1000X). The
reciprocal of the value obtained for cell density
(RVCD) in each histological location was calculated
by dividing the area in which cells were counted by
the number of cells in that area. This corresponds to
the intracellular plus extracellular area of one ce!! in
the tissue layer under study (Grunert et a!., 1984a).
The RVCD increases in deep and superficial endo-
metrium were shown to reflect the estrogen-induced
edematous reaction (water imbibition) of these
histological layers, whereas RVCD increases in the
circular myometrium were shown to reflect
myometrial cell hypertrophy, a known genomic
response to hormone stimulation (Grunert et al.,
1984a). The RVCD was expressed as a percentage of
the control values.
The eosinophils were counted and classified in
degranulated or nondegranulated types. Eosinophils
that contained less than half of the normal eosin-
stained granule content, assessed visually, were
considered to be degranulated. Uterine degranulated
eosinophils were expressed as percentage of the total
uterine eosinophils. The total number of eosinophils
was estimated by multiplying the average number of
eosinophi!s per section by a previously calculated
appropriate factor (300), which takes into account
the thickness of the tissue section, and by the length
of the fixed uterine horn expressed in mm (Tchernit-
chin et a!., 1974).
The !uminal and glandular epithelia! cc!! heights
were determined by measuring cell height from the
basement membrane to the apical limit of the cell (at
1000x) using a microscale in the ocular piece of the
microscope. For luminal epithelium, 60 cells chosen
at random from the central area of the uterine lumen
were measured for each animal. For glandular
epithelium, 60 ce!ls per animal were measured at the
deepest portions of the uterine glands (Grunert et a!.,
1984a). The lumina! and glandular epithelia! cell
heights were expressed as percentage of the control
values.
The uterine mitotic response was determined by
counting the number of mitotic figures in the different
uterine cell types 24 h after treatment.
Statistics
The log-transformation was performed on eosino-
phil numbers and mitoses data to normalize their
distributions. Transformed (uterine eosinophils and
mitoses) and nontransformed (all other parameters)
data were subjected to further statistical analysis.
Since multiple comparisons were performed
between the different doses within each hormone and
between experimentals and controls, and since the
dose-response curve of clomiphene was compared to
the previously published dose-response curve of
estradio!-17j3 (Grunert et al., 1986), transformed and
nontransformed data were subjected to the least
significant difference a posteriori (LSD) test. The
common variance needed for this test was estimated
530 GRUNERT ET AL.
TABLE 1. Effect of clomiphene or estradiol-17j3 on the percentage of degranulated eosinophils in the uteri of immature rats 6 h after treatment.
Hormone
treatment
Dose
(nM/kg)Number ofanimals evaluated
Percentage ofdegranulated uterine eosinophils
Vehicle 0 41 48
Clomiphene 3.737
370
1110
3700
9
8
8
97
50
NDa69bc
7gb80b
Estradiol�17!3d 37370
1110
3700
16
16
25
6
48
4960b
67b
aND = Eosinophils were not detected in the uterus of these animals.
b�<005 When compared to controls (x2 test).
c�.<0#{248}5 When compared to estradiol-17a at the corresponding dose (x2 test).
dData from Grunert et al., (1986).
from a one-way unbalanced analysis of variance
(ANOVA) of pooled data of clomiphene (present
results) and estradiol-17j3.
In uterine eosinophil degranulation studies, the x2statistic was used to evaluate differences between the
proportions of degranu!ated and nondegranulated
eosinophi!s.
RESULTS
The dose-response effects of c!omiphene of the
different parameters of estrogen action and their
comparison to previously reported data on estradiol-
1713 (Grunert et a!., 1986), are shown in Figures 1-4
and in Table 1.
Uterine Wet Weight
Six h after treatment (Fig. la), half-maximal
response was achieved with 0.37 nM estradiol-1713/kg
and maximal response levels were obtained with 3.7
to 3700 nM estradiol/kg. No significant response was
detected with 370 nM clomiphene/kg or lower doses,
and maxima! response levels were detected with 1110
and 3700 nM clomiphene/kg only. Although the
maximal response levels detected with the highest
doses of both compounds were similar, about 1000
times more clomiphene than estradiol was required to
achieve half-maximal response levels.
Twenty-four hours after treatment (Fig. ib), near
maximal to maxima! response levels were achieved
with 3.7 nM estradiol/kg. Similar levels of responses
were obtained with 1110 and 3700 nM clomiphene/
kg and half-maximal response was obtained with 370
nM/kg. About 300 times more clomiphene than
estradiol was required to achieve half-maximal
response levels.
R VCD in Deep Endometrial Stroma
Six h after treatment (Fig. ic), the response was
detected with doses of estradiol as low as 0.37 nM/kg,
but 370 nM clomiphene/kg were needed to elicit a
response. Maxima! response levels detected with the
two highest doses of clomiphene were about half of
the maximal response levels detected with the three
highest doses of estradiol. Thus, 300 to 1000 times
more clomiphene than estradiol was required to
achieve ha!f-maximal response levels.
Twenty-four hours after treatment (Fig. id),
maxima! response !eve!s were obtained with 1110 and
FIG. 1. Dose response of clomiphene (do) and its comparison to
estradiol-17a (e2) on uterine wet weight (a and b), reciprocal value of
cell density (RVCD) in deep endometrium (c and d), and RVCD in
superficial endometrium (e and f), 6 h (a, c, and e) and 24 h (b, d, and
f) after treatment.
Immature rats were injected i.v.with the hormones at doses rangingfrom 0.037 to 3700 nM/kg or with vehicle. The uterine horns were
excised 6 and 24 h after treatment. Thirty-seven to 41 controls, 6 to 9
rats for clomiphene, and 6 to 13 rats for estradiol-17a were used foreach experimental condition.
Values are means ± SEM. For analysis of significance, the least
significant difference a posteriori test using a common variance for allexperimental conditions within each parameter of hormone stimulation
was used. �p<0.05, **p<ool in comparison between clomiphene andestradiol-1 7f3 at the same dose. In comparisons between experimental
and control animals, the limits of significance at the level of p=0.05 are
shown by the horizontal dotted lines and at the level of p=0.01 by the
horizontal broken lines. (Estradiol-1 7j3 data from Grunert et al., 1986.)
180
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MORPHOMETRY OF ESTROGENIC RESPONSES TO CLOMIPHENE 531
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FIG. 2. Dose response of clomiphene (do) and its comparison toestradiol-17�3 (e3) on the total number of uterine eosinophils, 6 h after
treatment. Immature rats were injected i.v. with the hormones at doses
ranging from 0.037 to 3700 nM/kg or with vehicle; one uterine hornwas excised 6 h after treatment. Thirty-seven controls, 7 to 9 rats for
clomiphene, and 6 to 13 rats for estradiol-17a were used for eachexperimental condition, Values are geometric means of log-transformed
data. Each vertical line indicates anti-log of (mean of logs + SEM of
logs) and anti-log of (mean of logs - SEM of logs). For analysis of
significance, the least significant difference test was used: #{149}p<o.O1 in
comparison between clomiphene and estradiol-l 7�3 at the same dose. In
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(Estradiol-17p data from Grunert et al., 1986.)
532 GRUNERT ET AL.
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HORMONE DOSE (nM/kg)
3700 nM c!omiphene/kg and with 37 to 3700 nM
estradio!/kg. Maximal response levels were simi!ar for
both compounds, and about 100 times more
c!omiphene than estradio! was required to obtain the
half-maximal response leve!s.
A comparison of the responses obtained at 6 and
24 h after treatment (data not shown) reveals that the
dose-response curve for clomiphene remains un-
changed. However, for estradiol, the maxima! response
levels at 24 h were about half those obtained at 6 h
after treatment (p<0.01, LSD a posteriori test); 10 to
30 times more estradiol was required to obtain
half-maximal response levels at 24 than at 6 h after
treatment.
R VCD in Superficial Endometrial Stroma
Six h after treatment (Fig. le), the response
induced with estradiol was detected with doses as low
as 0.37 nM/kg, and maxima! response levels were
obtained with 3.7 to 1110 nM/kg. However, the
response induced with clomiphene was detected with
370 nM/kg, and the maxima! response was obtained
with 1110 nM/kg. No differences were observed
between maxima! response levels of both compounds,
but 1000 times more clomiphene than estradiol was
required to reach half-maximal response levels.
At 24 h after treatment (Fig. if), maximal response
levels were similar for both compounds; they were
obtained with 1110 and 3700 nM clomiphene/kg and
37 to 3700 nM estradiol/kg. About 30 times more
c!omiphene than estradiol was required to obtain the
half-maximal response level.
A comparison of the responses obtained at 6 and at
24 h after treatment (data not shown) revealed that
the responses obtained with c!omiphene at 24 h after
treatment remained unchanged. On the contrary,
response levels obtained with all doses of estradiol at
24 h of treatment were decreased (p<0.01, LSD a
posteriori test).
Uterine Eosinophilia
Figure 2 shows that clomiphene induced a weak
response 6 h after treatment that was detected at the
doses of 1110 and 3700 nM/kg. At these doses,
uterine eosinophilia induced with c!omiphene is much
weaker than that induced by estradiol.
Table 1 shows that clomiphene induced an increase
in the percentage of degranulated eosinophi!s at the
three highest doses, whereas estradiol increased it
only at the two highest doses. Clomiphene was more
potent than estradiol in inducing eosinophil
degranulation at the dose of 370 nM/kg.
Luminal Epithelial Cell Height
Six h after treatment (Fig. 3a), both compounds
induced a decrease in luminal epithe!ial cell height.
While this decrease was detected with 3.7 to 1110 nM
estradiol/kg, this response could be detected with
1110 and 3700 nM clomiphene/kg only. About 300
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cell height (c and d), and the reciprocal value of cell density (RVCD) in circular myometrium (e andj) 6 h (a, c, and e) and 24 h (b, d, andf) after
treatment.Immature rats were injected i.v. with the hormones at doses ranging from 0.037 to 3700 nM/kg or with vehicle, and the uterine horns were
excised 6 and 24 h after treatment. Thirty-three to 43 controls, 4 to 9 rats for clomiphene, and 6 to 13 rats for estradiol-1 713 were used for eachexperimental condition.
Values are means ± SEM. For analysis of significance, the least significant difference test was used: #{149}p<O.05, p<O.O1 in comparison betweenclomiphene and estradiol-17f3 at the same dose. In comparisons between experimental and control animals, the limits of significance at the level of
p=0.05 are shown by the horizontal dotted lines and at the level of p=O.01 by the horizontal broken lines. (Estradiol-1 7j3 data from Grunert eta).,
1986.)
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534 GRUNERT ET AL.
times more clomiphene than estradiol was needed to
induce half-maxima! response levels.
Twenty-four hours after treatment (Fig. 3b), both
compounds induced an increase in lumina! epithelial
cell height. While the responses detected with the
highest doses (1110 nM/kg) of both compounds were
similar, about 20 times more clomiphene than
estradio! was required to induce half-maxima! response
levels.
Glandular Epithelial Cell Height
Six h after treatment (Fig. 3c), clomiphene induced
a decrease in glandular epithelia! cell height with the
doses of 370 and 1110 nM/kg; estradiol, in turn,
induced a decrease in cell height at the doses of 3.7
and 37 nM/kg. Twenty-four hours after treatment
(Fig. 3d), both compounds induced increases in the
glandular epithelial cell height. While estradiol induced
a maximal response with the doses of 37 to 1110
nM/kg, clomiphene induced a maxima! response with
1110 to 3700 nM/kg; maximal response levels were
similar for both compounds and about 30 times more
clomiphene than estradio! was needed to obtain
half-maxima! response levels.
R VCD in Circular Myometrium
Six h after treatment (Fig. 3e), clomiphene induced
a very weak increase in circular myometrial RVCD at
the doses of 1110 and 3700 nM/kg, whereas estradiol
induced a very weak increase in RVCD at doses of
0.37 to 3700 nM/kg.
Twenty-four hours after treatment (Fig. 3f), the
response induced with estradiol was detected with
doses as low as 0.37 nM/kg, and maximal response
levels were detected with 37 to 3700 nM/kg. How-
ever, the response induced with clomiphene was
detected with 370 nM/kg, and the maxima! response
level similar to that of estradiol was achieved with
1110 nM clomiphene/kg. About 100 times more
clomiphene than estradiol was required to induce
half-maximal response levels.
Mito tic Response
Figure 4a shows that the c!omiphene-induced
increase in mitotic figures 24 h after treatment was
detected with 370 nM/kg, and the maximal response
levels were achieved with 1110 nM/kg. At the dose of
3700 nM/kg, the number of mitoses shows a
significant decrease (p<0.0i, LSD a posteriori test)
from the maximal response level. A comparison
with estradiol reveals that 20 to 50 times more
clomiphene than estradio! was required to induce a
similar number of mitotic figures.
The comparison of the mitotic responses in the
different uterine cell types (Fig. 4 b-g), reveals that
estradio! induced mitoses preferentially in lumina!
epithelium and much less in other cell types. In
contrast, the number of mitoses per cross section
after clomiphene treatment were only slightly
different for luminal epithelium, superficial stroma,
deep stroma, and circular myometrium.
Only a few mitoses were detected in glandular
epithelium and longitudina! myometrium in rats
treated with estradiol or c!omiphene. Furthermore,
while the maximal response obtained in luminal
epithelium with c!omiphene was about half of the
maximal response induced by estradiol, maximal
responses induced by both hormones were similar in
superficial stroma, deep stroma, and circular
myometrium.
DISCUSSION
This report describes a differential expression of
estrogenic responses induced by clomiphene in the rat
uterus. Very high doses of clomiphene are needed to
induce 6-h uterine eosinophilia and deep endometrial
edema, and maximal response levels were not reached
in our experimental conditions. On the contrary,
several genomic responses were induced with much
lower doses of clomiphene, and maxima! response
FIG. 4. Dose response of clomiphene (do) and its comparison to
estradiol-1713 on the total number of mitoses per uterine cross section
considering all cell types (a) and the number of mitoses per uterine
cross section in luminal epithelium (b), glandular epithelium (c),
circular myometrium (d), longitudinal myometrium (e), deep endo-
metrial stroma (f), and superficial endometrial stroma (g) 24 h after
treatment.
Immature rats were injected iv. with the hormones at doses rangingfrom 0.037 to 3700 nM/kg or with vehicle, and one uterine horn was
excised 24 h after treatment, Eleven controls, 6 to 9 rats for clomi-
phene, and 6 to 13 rats for estradiol-1 713 were used for each experi-
mental condition.
Values are geometric means of log-transformed data. Each vertical
line indicates anti-log of (mean of logs + SEM of logs) and anti-log of
(mean of logs - SEM of logs).For analysis of significance, the least significant difference test was
used: sp<o.o5, * *p<O�Ol in comparison between clomiphene and
estradiol-1 713 at the same dose.
In comparisons between experimental and control animals, thelimits of significance at the level of p=0.05 are shown by the horizontaldotted lines and at the level of p=0.01 by the horizontal broken lines.(Estradiol-1713 data from Grunert et al., 1986.)
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536 GRUNERT ET AL.
levels were reached with at least the two highest doses
of clomiphene.
This dissociation of estrogenic responses is in
agreement with our hypothesis of the existence of
separate groups of responses that are mediated by
multiple and independent mechanisms of estrogen
action: (a) genomic responses mediated by cytosol-
nuclear receptors (Jensen and De Sombre, 1972), (b)
some nongenomic responses mediated by eosinophils
(Tchernitchin, 1972, 1983), and (c), some misce!-
laneous responses for which the mechanisms of
hormone action are still unknown. Furthermore,
additional receptor systems that are possibly involved
in some still unidentified responses were reported:
Type II cytoplasmic and nuclear receptors (Clark et
al., 1978; Eriksson et a!., 1978), cytoplasmic
membrane-associated receptors possibly involved in
some still unidentified responses (Szego and Pietras,
1981), and antiestrogen binding sites (Sutherland et
al., 1980).
The hypothesis that estrogen-induced deep endo-
metrial edema is mediated by eosinophils agrees with
the finding of both a moderate edema and a very low
number of eosinophils in the uterus in animals treated
with high doses of clomiphene. In fact, according to
the previously reported logarithmic correlation
between eosinophil numbers in the uterus and
eosinophil-mediated responses (Tchernitchin and
Galand, 1983), one should expect very low eosinophil
numbers in animals responding with a moderate
edematous response. According to that correlation, it
was shown that very few eosinophils are required to
induce a significant water imbibition response, and
that much higher eosinophi! numbers are needed to
increase the response. It was further proposed that
maxima! edema is limited by the amount of uterine
ground substance molecules susceptible to depoly-
merization under the action of enzymes released from
degranulating eosinophi!s (Tchernitchin and Galand,
1983). In agreement with previously reported data on
eosinophil degranulation in progesterone-treated rats
(Grunert et al., 1984b), we conclude that the increased
eosinophil degranulation in the uterus in clomiphene-
treated rats might contribute to increased edema in
the presence of low eosinophil numbers.
The present report also confirms the previous
finding of a decrease in !uminal epithelial and
glandular epithelial cell height 6 h after treatment
(Williams and Rogers, 1980; Grunert et al., 1984a).
Based on the finding that this decrease can be observed
at 6 h at those doses of estradiol-17j3, which by
themselves do not induce cellular hypertrophy at 24
h after treatment, it has been suggested that this
response is not an initial step of the hypertrophy
observed at later times (Grunert et al., 1986). In
contrast with the previously described estradiol
effects, we have found that the decrease in luminal
and glandular epithe!ial cell height induced by
clomiphene 6 h after treatment can be observed only
at those doses inducing cellular hypertrophy at 24 h.
The present results show that high doses of
clomiphene induce hypertrophy on luminal and
glandular epithelium and in circular myometrium 24
h after treatment. However, there are doses at which
glandular cell hypertrophy is near to the maximal
response levels, luminal cell hypertrophy is not
detected at all, and hypertrophy in myometrium is at
an intermediate level between both epithelia. This
dissociation of genomic responses in the different
uterine cell types is in agreement with the hypothesis
of the existence of different receptors in these cell
types (Tchernitchin et a!., 1985; Grunert et al.,
1986).
The above responses are different from those
reported by Clark and Guthrie (1981) after 4 days of
treatment with c!omiphene. They found that
clomiphene stimulated epithelial cells of the endo-
metrium while having little effect on the stroma or
myometrial layer. This discrepancy in results may be
explained by the estrogenic-antiestrogenic properties
common to various triphenylethylene antiestrogens
that act as estrogens during the first 24 h of treat-
ment and present antiestrogenic properties thereafter
(Clark et a!., 1985). Accordingly, it is suggested that
the antiestrogenic properties of clomiphene during a
4-day treatment is expressed in some cell types only,
blocking estrogen action in stroma or myometrial
layers but not in epithelia. Work is in progress to
investigate the antiestrogenic properties of clomiphene
in different uterine cell types.
It has been shown previously that clomiphene as
well as nafoxidine stimulated DNA synthesis in the
rat uterus to a significant degree (Clark et aL, 1974;
Baundendistel et al., 1978; Martin, 1980; Markaverich
et al., 1981), and that the majority of hyperplasias
observed takes place in the stroma (Martin, 1980).
These reports have been confirmed and extended by
the present findings. We found that while the maximal
mitotic response obtained in luminal epithelium with
clomiphene is about half of that obtained with
MORPHOMETRY OF ESTROGENIC RESPONSES TO CLOMIPHENE 537
estradiol, the maxima! response levels obtained in
superficial and deep endometria! stroma and in
circular myometrium with clomiphene are similar to
those obtained with estradiol. The difference between
the different uterine cell types regarding the maxima!
response levels and the doses needed to obtain half-
maxima! response are in agreement with previously
reported differences between mitoses-mediating
receptors in the different uterine cell types (Grunert
et a!., 1986).
The dissociation between the different estrogenic
responses induced by clomiphene and in particular
the dissociation between the different genomic or
mitotic responses found in the different uterine cell
types with the morphometric approach (Grunert et
al., 1984a, 1986), may have clinical implications. It
may mean that it is possible to deve!op new estrogenic
or antiestrogenic compounds that may selectively
induce or block some estrogenic responses without
interference with other responses. This would be of
value in several c!inical situations where it is
convenient to selectively inhibit dangerous or un-
desired effects, such as cell proliferation in cancerous
patients, and/or only induce such responses to
hormone stimulation that are needed in these clinical
situations.
ACKNOWLEDGMENTS
We are indebted to Laboratorio Recalcine, Santiago, Chile, for
providing clomiphene. We thank Mr. Dagoberto S#{227}ezand Mr. Eugenio
Astudillo for excellent technical assistance and Mrs. Elena Zanelli for
expert secretarial assistance.
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