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Division of Anatomy, Cell and Human Biology, Division of Physiology (S.R.M.), GKT School of Biomedical Sciences, King’s College London, Guy’s Campus, London, United Kingdom SE1 1UL; Medical Research Council Human Reproductive Sciences Unit (I.A.S.), Edinburgh, United Kingdom EH3 9ET; and AstraZeneca, Central Toxicology Laboratory (A.N.B.), Alderley Park Macclesfield, Cheshire, United Kingdom SK10 4TJ
Address all correspondence and requests for reprints to: Dr. Kevin T. O’Byrne, Division of Anatomy, Cell and Human Biology, Endocrine and Reproductive Research Group, GKT School of Biomedical Sciences, 2.36D, New Hunts House, King’s College London, Guy’s Campus, London, United Kingdom SE1 1UL. E-mail: kevin.o'byrne{at}kcl.ac.uk
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Abstract |
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Introduction |
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The aims of the present study were to examine the effects of two phytoestrogens, the isoflavone, genistein, and the coumestan, coumestrol, on the activity of the hypothalamic GnRH pulse generator and on pituitary sensitivity to GnRH stimulation in vivo. In addition, the effects of these phytoestrogens on GnRH-induced pituitary LH release in vitro were examined using primary pituitary cell cultures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Another group of ovariectomized rats was fitted with an array of four recording electrodes chronically implanted in the mediobasal hypothalamus as described previously (9, 10). After a 4- to 5-day recovery period these animals were fitted with indwelling cardiac catheters as described above.
Pituitary cell preparation and culture conditions
Anterior pituitary glands obtained from adult male Wistar rats
(150–250 g) were used for the preparation of primary cultures of
pituitary cells as described previously (11). The cells
were cultured in DMEM (Life Technologies, Inc., UK)
containing 1% L-glutamine (Sigma, Poole, UK),
10 µg/ml streptomycin, 100 U/ml penicillin, and 12.5%
charcoal-stripped FCS (Sigma) on multiwell culture dishes
at a density of 2 x 105 cells/200
µl/well. The cultures were maintained in a water-saturated atmosphere
of 95% air-5% CO2 at 37 C. They were used for
experimentation between days 3 and 5 of culture.
Experimental design
LH pulse study. Animals were allowed to recover for 2–5
days after catheterization before blood sampling commenced. Rats were
then attached via one of the two cardiac catheters to a
computer-controlled automated blood-sampling system, which allows for
the intermittent withdraw of small blood samples (25 µl) without
disturbing the animals (12). The second catheter was used
for the administration of test compounds. Two phytoestrogens,
coumestrol and genistein, were used. The estrogenic potency of
coumestrol varies considerably depending on the in vivo and
in vitro assay methods used, and ranges from 100- to
10,000-fold less potent than 17
-estradiol (13, 14). The
estrogenic potency of genistein is generally about 10-fold less than
that of coumestrol (14). Animals were given one of four
treatments: 1) a bolus injection (0.3 ml) followed by a continuous iv
infusion of vehicle (0.1% DMSO in 45% cyclodextrin; Fluka Chemie,
Switzerland) at a flow rate of 0.45 ml/h for 8.5 h (n = 6);
2) 17-estradiol (0.14 µg in 0.3-ml bolus injection followed by
continuous iv infusion of 0.21 µg/0.45 ml·h for 8.5 h; n
= 6; Sigma); 3) coumestrol [two doses, low or high, 0.4
or 1.6 mg in 0.3-ml bolus injection followed by continuous iv infusion
of 0.6 mg/0.45 ml·h (n = 6) or 2.4 mg/0.45 ml·h (n = 6),
respectively, for 8.5 h; Acros Organic, Morris Plains,
NJ]; or 4) genistein (1.6 mg in 0.3-ml bolus injection followed
by continuous iv infusion of 2.4 mg/0.45 ml·h for 8.5 h; n
= 6; Apin Chemicals, Oxford, UK).
Automated blood sampling commenced between 0800–0900 h, and blood samples were collected every 5 min for 11 h for the measurement of LH. After 2 h of blood sampling, the test compound was administered as described above, and blood sampling continued. At the end of the infusion all animals were challenged with GnRH (500 ng/kg, iv bolus injection; Sigma), and sampling continued for an additional 30 min. In the case of the estradiol-treated rats, a 0.3-ml blood sample was collected at the end of the experiment for the measurement of estradiol.
Electrophysiological study. After 4–5 days of recovery following catheterization the hypothalamic multiunit electrical activity (MUA) was recorded as described previously (9, 15). Blood samples were collected at 5-min intervals, using the automated system described above, for the measurement of LH. GnRH pulse generator activity was assessed by the characteristic increases in hypothalamic MUA (MUA volleys) and by LH pulses in the peripheral blood. After a control period, during which at least six MUA volleys were observed, coumestrol was given by a bolus injection (1.6 mg in 0.3 ml) followed by continuous iv infusion (2.4 mg/0.45 ml·h; n = 3) for 4 h. At the end of the infusion, blood sampling and electrophysiological recording were continued for an additional 2–3 and 4–6 h, respectively. Control animals were administered vehicle as a bolus injection (0.3 ml) followed by a continuous iv infusion of solution containing 0.1% dimethylsulfoxide in 45% cyclodextrin at a flow rate of 0.45 ml/h for 4 h (n = 3); sampling and recording continued as described above.
Pituitary LH in vitro study. Before an experiment the
cultures were washed with freshly prepared medium. The cells were
subsequently incubated for 1, 2, or 4 h with medium containing
17-estradiol (10-11,
10-9, or
10-7 M),
coumestrol (10-9,
10-7, or
10-5 M), or
genistein (10-9,
10-7, or
10-5 M) with
or without antiestrogen (ICI 182,780:
10-7 M; Tocris
Cookson Ltd., Bristol, UK), respectively. Test compounds were
added to the medium from appropriate stock solutions in ethanol. The
final concentration of ethanol was 0.01%. Respective control cultures
were exposed to medium containing the same quantity of ethanol (with or
without antiestrogen, ICI 182,780,
10-7 M), but
without the test compounds (17-estradiol, coumestrol, or genistein).
During the last 1 h of the indicated incubation periods the cells
were stimulated with GnRH
(10-9 M).
Before the cells were stimulated with GnRH they were washed with
medium, which was then aspirated and replaced with medium containing
the appropriate test compounds, as indicated above, and then GnRH was
directly added to the cultures in 20-µl volumes. In the case of the
1-h incubation experiment, GnRH was added at the onset of the
incubation period. At the end of the stimulation period the medium was
aspirated and stored at -20 C before LH levels were determined by RIA.
All treatments were run in triplicate, and experiments repeated three
or four times, with the exception of the 2-h incubation period where
treatment with the lowest dose of 17-estradiol, coumestrol, or
genistein (with and without ICI 182,780) was repeated twice.
Hormone measurements
Estradiol was measured in plasma using an RIA based on reagents
(E2 MAIA rabbit antiestrogen antibody,
[125I]estradiol tracer, and goat antirabbit
-globulin coupled to magnetic particles) supplied in an assay kit
produced by BioChem ImmunoSystems (Bologna, Italy) as described
previously (16). The minimum detectable concentration of
estradiol was 12 pg/ml. The intra- and interassay coefficients of
variance were less than 10% and 9.7%, respectively. LH in the culture
medium and in the 25-µl whole blood samples collected by the
automated blood-sampling system was measured by double antibody RIA
using the reagents provided by the NIDDK, reference preparation rat LH
RP-2. The mean minimally detected concentration of LH was 0.093 ng/ml.
Inter- and intraassay variations were 15.5% and 8.3%,
respectively.
Data analysis
The effect of 17-estradiol, coumestrol, or genistein on
GnRHinduced LH release from cultured pituitary cells was expressed
in terms of the percentage of GnRH-induced LH release in the respective
control cultures (no 17-estradiol, coumestrol, or genistein =
100%). The data obtained in two or more independent experiments
performed in triplicate were analyzed. Each treatment yielded an
average of the triplicate (n = 1) and the mean (±
SEM) LH concentrations for the independent experiments
calculated. The significance of differences in the LH response of
cultured pituitary cells to GnRH stimulation between estradiol-,
coumestrol-, or genistein-exposed cells and their respective
vehicle-treated cells in the 1-, 2-, and 4-h experiments was
established using a one-way ANOVA and Dunnett’s test after a Bartlett
test had shown that variances were homogeneous.
In the in vivo experiments detection of LH pulses was established by use of the algorithm ULTRA (17). Values for LH pulse frequency and amplitude for each animal were obtained from the ULTRA analysis and were divided into five 2-h blocks; the first 2-h corresponded to pretreatment values. To determine whether estradiol, coumestrol, or genistein had an effect on LH pulse frequency or amplitude, the pretreatment values were compared with the posttreatment values using a one-way ANOVA and Dunnett’s test. Differences in the mean peripheral LH concentrations before and after the GnRH challenge (mean of samples taken during the 30 min before vs. the 30 min after challenge) were analyzed using a one-way ANOVA and Dunnett’s test. The effect of coumestrol on the electrophysiological correlates of GnRH pulse generator activity was calculated by comparing the mean MUA volley frequency before treatment with the mean MUA volley frequency during treatment; the significance of differences between these values was assessed by one-way ANOVA and Dunnett’s test. Controls were examined in an identical manner.
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Results |
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and 2). 17-Estradiol (0.14-µg bolus,
followed by 0.21 µg/h; final plasma estradiol concentration,
165.5 ± 20.4 pg/ml) resulted in a reduction in LH pulse frequency
and amplitude evident at 4–6 h that was maintained for the remaining
duration of the experiment (Fig. 2). Two of the six rats showed a more
immediate response to 17-estradiol, and an example is shown in Fig. 1. The inhibitory effect of coumestrol on LH pulse frequency was
greater than that of estradiol (Fig. 2). Genistein at the same dose
(1.6-mg bolus followed by 2.4 mg/h), coumestrol at the lower dose
(0.4-mg bolus followed by 0.6 mg/h; data not shown), and vehicle were
without effect on pulsatile LH secretion (Figs. 1 and 2).
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-estradiol, coumestrol, genistein, or vehicle are shown in Fig. 3. 17-Estradiol attenuated the GnRH-
induced LH response. Coumestrol at the higher dose completely
blocked the GnRH-induced LH response, whereas the lower dose of
coumestrol, genistein, or vehicle was without effect.
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confirming the results presented above (Figs. 1
and 2). After
termination of the coumestrol infusion, the frequency of the MUA
volleys and attendant LH pulses increased to control values within 1–2
h (Fig. 4). Administration of vehicle (Fig. 4) was without effect on
GnRH pulse generator frequency (interval between volleys, 17.25 ±
0.98 vs. 18.24 ± 2.1 min; mean ±
SEM; before vs. during administration
of vehicle; P > 0.05).
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-estradiol on GnRH-induced
release of pituitary LH in vitro-estradiol
at 10-11,
10-9, or
10-7 M showed
a significantly reduced LH response to GnRH
(10-9 M) at
the two higher concentrations over 1, 2, or 4 h (Fig. 5). In the presence of
10-7 M
antiestrogen (ICI 182,780) this effect was completely blocked,
resulting in an LH response identical to that obtained by vehicle
treatment (Fig. 5). When pituitary cells were treated for 1, 2, or
4 h with coumestrol at
10-9,
10-7, or
10-5 M or with
genistein at 10-9,
10-7, or
10-5 M and
stimulated with GnRH (10-9
M), the LH response was significantly inhibited only by
coumestrol at the highest concentration
(10-5 M) over
2 or 4 h (Fig. 5). In the presence of
10-7 M
antiestrogen (ICI 182,780) the effect of coumestrol on GnRH-induced LH
release was completely blocked (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and ER, which are both present in the
gonadotrophs (28, 29, 30), to the action of coumestrol remain
unknown.
In addition to the pituitary, the hypothalamus is a site of negative
feedback regulation by estradiol, as indicated by a reduction in GnRH
pulse generator frequency (22, 31). The present study
provides the first evidence that the phytoestrogen, coumestrol, has a
similar central action. As with estradiol it is not presently known
whether coumestrol exerts this influence directly on GnRH neurons or
via estrogen-sensitive interneurons. The evidence suggesting the
presence of ER and ER in GnRH neurons (32, 33, 34)
raises the possibility that phytoestrogens may act directly on the GnRH
neurons; this is currently under investigation.
Estimates of the estrogenic potency of phytoestrogens vary markedly depending on the in vivo and in vitro assay methods used. We have shown that coumestrol is only 100- to 1,000-fold less potent than estradiol at increasing uterine vascular permeability (14), which is similar to its potency at inhibiting stimulus-induced bone resorption in the fetal rat (13). We (unpublished observations) and others (13) have shown equivalent effects on the classical in vivo rat uterotropic assay when coumestrol is given at a dose 10,000-fold higher than that of estradiol. In the present study, however, a similar comparison of these compounds (i.e. when coumestrol is given at a dose 10,000-fold higher than that of estradiol) showed coumestrol to have a greater effect than estradiol in attenuating the LH response to GnRH. However, the response of the pituitary gonadotrophs in vitro suggests a lower potency of coumestrol compared with estradiol of about 4 orders of magnitude. The apparently greater effect of coumestrol in vivo in this context may be a consequence of actions within both the pituitary and hypothalamus. We previously found that coumestrol induced a steep dose response in terms of increased uterine vascular permeability, with minimum to maximum achieved with a 10-fold increase in the coumestrol concentration (14). In the present study a dose of coumestrol only 4-fold lower than that observed to inhibit completely the GnRH-induced LH response in vivo was without effect on pituitary responsiveness to GnRH or LH pulses. These results with our lower dose of coumestrol, which was still within the milligram range, are at variance with the findings of Hughes (7), who demonstrated an attenuated GnRH-induced LH response in the presence of coumestrol in the nanogram dose range. Although it is difficult to reconcile these differences, Hughes (7) used a 10-fold lower dose of GnRH, and it is possible that the higher dose of secretagogue used in the present study may have overridden the inhibitory actions of coumestrol. Nevertheless, the absence of effect on spontaneous LH pulses with the lower milligram dose of coumestrol in the present study tempers this argument. The mechanism underlying the observed dose-response relationships between coumestrol and LH secretion in vivo remains to be established.
In contrast to the effects of coumestrol, we found that administration
of the same high dose of genistein did not affect pulsatile LH release
or attenuate GnRH-induced LH release in vivo. This is
perhaps not surprising given that genistein has a lower (about 10-fold)
in vivo estrogenic potency (14), and a
considerably lower affinity for both ER and ER compared with
coumestrol (35). In contrast, Hughes and colleagues
(8) reported a complete blockade of GnRH-induced LH
release with doses of genistein comparable to that used in the present
study and suggested that genistein was more potent than estradiol.
However, these researchers used a 10-fold lower dose of GnRH; this
might explain the different results, because the lower dose of
secretagogue may be more easily negated by genistein. However, other
methodological differences, such as single injection vs.
bolus injection followed by infusion of genistein, and 2-h
vs. 8.5-h pretreatment with genistein before the GnRH
challenge, may also underlie the difference between our results and
those reported by Hughes and colleagues (8). In addition,
Hughes and colleagues (8) suggested that the dose-response
relationship of genistein may be far from simple, as only their lowest
dose (0.1 mg/kg BW) suppressed basal LH levels, whereas 10- and
100-fold higher doses were without effect. This discontinuity of
the dose-response pattern for genistein’s effects on LH secretion is
an interesting phenomenon, which remains unexplained.
It must be emphasized that the aim of this study was to investigate potential mechanisms of action of phytoestrogens on the reproductive hypothalamic-pituitary axis. The doses of estradiol, genistein, and coumestrol used were based on previous estimates of their estrogenic potency. The materials were administered iv to bypass confounding variables such as bioavailability and first pass metabolism. Consequently, any extrapolation to human dietary exposure to these compounds needs careful consideration.
In summary, these data indicate that coumestrol can act on the hypothalamus and pituitary gland to inhibit gonadotropin secretion and, in doing so, provide an understanding of how phytoestrogens may disrupt normal reproductive function.
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Acknowledgments |
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Footnotes |
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Received August 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and a gonadotrophin-releasing hormone in ewes affected
with clover disease. Neuroendocrinology 11:57–66[CrossRef][Medline]
and
in the rat pituitary gland. Endocrinology 139:3976–3983 (ER) and
(ER-) mRNA in the rat pituitary, gonad, and reproductive tract.
Steroids 63:498–504[CrossRef][Medline]
and in rat pituitary gland
detected by immunohistochemistry. Endocrinology 141:615–620 and messenger ribonucleic acids in adult
gonadotropin-releasing hormone neurons. Endocrinology 140:5195–5- immunoreactivity in
gonadotrophin-releasing hormone-expressing neurones. J
Neuroendocrinology 11:331–335[CrossRef][Medline]
.
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) followed by continuous
0.45 ml/h infusion for 8.5 h] of 17
