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, Cholecystokinin, and Preproenkephalin Messenger Ribonucleic Acid in the Limbic-Hypothalamic Circuit1
Department of Neurobiology, Laboratory of Neuroendocrinology, Brain Research Institute, Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, California 90095-1763
Address all correspondence and requests for reprints to: Paul Micevych, Ph.D., Department of Neurobiology, UCLA School of Medicine, Box 1763, 10833 LeConte Avenue, Los Angeles, California 90095-1763. E-mail: pmicevych{at}mednet.ucla.edu
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Abstract |
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(ER) mRNA in the nuclei of
the circuit. We examined the possibility that thyroid hormone treatment
would block the estrogen modulation of these messages. Estradiol
benzoate (EB), EB + thyroxine (T4), T4, or oil
were administered to ovariectomized, adult female rats for 10 days.
Isotopic in situ hybridization histochemistry revealed
that within the limbic-hypothalamic nuclei, levels of CCK and
preproenkephalin (PPE) mRNA levels were significantly higher in EB and
EB + T4-treated animals compared with T4 or
oil-treated animals. ER mRNA levels were low in EB treated animals,
elevated in T4 or oil-treated animals and further elevated
in EB + T4-treated animals. In summary, T4
treatment had neither an independent nor an antagonistic effect on
estrogen induced expression of CCK or PPE mRNA in the circuit. However,
T4 did prevent the normal estrogenic decrease of ER mRNA
levels in the nuclei of the limbic-hypothalamic circuit. |
Introduction |
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A number of studies have examined the influence of estrogen and thyroid hormone interactions on reproduction. Studies using a variety of rodent species have demonstrated that food deprivation and cold exposure influence reproductive function as measured by: estrous cyclicity, display of reproductive behavior, litter size, and survival (1, 2, 3, 4, 5, 6). Because serum thyroid hormone levels have been found to be elevated under these unfavorable environmental circumstances, thyroid hormones have been proposed to dampen estrogenic induction of the metabolically expensive process of sexual reproduction. Interactions between environmental stressors that alter thyroid hormone levels and reproduction are species specific (7, 8). For example, the reproductive success of hamsters and guinea pigs is more sensitive to environmental manipulations (2, 6, 7), than that of mice or rats (3, 4, 9).
An important event in the estrogenic regulation of lordosis behavior is
the induction of neuropeptide gene transcription [as measured by
messenger RNA (mRNA) expression] in the limbic-hypothalamic circuit.
This neural circuit is composed of several steroid concentrating,
interconnected cell groups, including the principal portion of the bed
nucleus of the stria terminalis (BSTp), central portion of the medial
preoptic nucleus (MPNc), ventrolateral portion of the ventromedial
nucleus of the hypothalamus (VMHvl), and the posterodorsal part of the
medial amygdaloid nucleus (MeApd) (for review see Ref.10). The
expression of cholecystokinin (CCK) and preproenkephalin (PPE; the
precursor to enkephalin peptides) mRNA in the circuit have been
demonstrated to be estrogen dependent. Ovariectomy reduces and estrogen
replacement increases the expression of CCK and PPE mRNA in nuclei of
the circuit (11, 12). In contrast, estrogen inversely regulates the
expression of the estrogen receptor (ER). Ovariectomy increases
and estrogen treatment decreases the expression of ER mRNA and
protein in the nuclei of the circuit (13, 14, 15, 16). Further, ER, CCK, and
enkephalin peptides all dramatically impact the display of lordosis
behavior. Antagonism of ER with tamoxifen prevents the estrogenic
regulation of reproductively relevant genes, and therefore the
display of lordosis behavior is inhibited (17). In the MPN, CCK
facilitates lordosis behavior but inhibits lordosis in the VMH
(18, 19, 20). Also, enkephalin acting in the VMH and midbrain central gray
has been implicated in regulation of lordosis (21, 22, 23). Because
estrogen normally up-regulates the mRNA of the reproductively relevant
neuropeptide mRNAs and down-regulates ER mRNA, we examined the
possibility that, in the hyperthyroid condition, the estrogenic
modulation of these reproductively important molecules would be
antagonized. Such an antagonism of estrogenic actions in the
limbic-hypothalamic circuit would implicate thyroid hormone as a
transducing agent of environmental effects on reproduction.
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Materials and Methods |
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Hormone treatments. To determine whether thyroid hormone treatment affects the estrogenic regulation of reproductively relevant mRNAs in the limbic-hypothalamic circuit, female rats were divided into four groups and injected with: 1) 17ß-estradiol 3-benzoate (EB; 2 µg sc, daily; Sigma, St. Louis MO); 2) EB + L-thyroxine (T4; 250 mg/kg ip, twice daily; Sigma); 3) T4 only or 4) oil vehicle for 10 days and were killed 24 h following the last hormone treatment (n = 6/group). This dose of estrogen was chosen because it is not completely metabolized within 24 h, and therefore, slowly increases total serum estrogen over the course of the 10 day paradigm (11, 24). The thyroid hormone dose was chosen since it has previously been shown to induce a hyperthyroid serum concentration (7, 24).
Hybridization histochemistry
Tissue preparation. At the time the rats were killed, all
animals were transcardially perfused with ice-cold physiological saline
and 4% paraformaldehyde in 0.1 M Sörensen’s
phosphate buffer. Brains were removed and postfixed in paraformaldehyde
solution for 4 h at room temperature, then cryoprotected in 20%
sucrose in PBS at 4 C for 24 h. Serial, coronal, 20-µm-thick
sections through the MPNc, VMHvl, and MeApd were obtained on a cryostat
(Reichert, Deerfield, IL) and mounted onto Superfrost Plus slides
(Fisher, Pittsburgh, PA). Slide mounted sections were desiccated and
stored at -70 C until processing.
cRNA probes. CCK mRNA was localized using an 35S-UTP labeled (Dupont-NEN, Boston, MA) single-stranded antisense complementary mRNA (cRNA) probe to the entire coding sequence of the rat CCK mRNA. The antisense probe was transcribed from a 527-bp CCK complementary DNA (cDNA) template (Dr. J. Dixon, University of Michigan, Ann Arbor, MI) using T7 RNA polymerase.
PPE mRNA was localized using an 35S-UTP labeled single-stranded antisense cRNA probe complimentary to the entire coding sequence of the rat PPE-A mRNA. The antisense probe was transcribed from a 935 bp PPE-A cDNA template (Drs. Yoshikawa and Sabol, NIH, Bethesda, MD) using SP6 RNA polymerase.
ER mRNA was localized using an 35S-UTP labeled
single-stranded antisense cRNA probe complimentary to a portion of the
rat ER mRNA (DNA binding and ligand binding domain coding region).
The antisense probe was transcribed from an 820-bp ER cDNA template
(Dr. Muramatsu, University of Tokyo, Tokyo, Japan) using T7 RNA
polymerase. At the hybridization temperature and wash stringency used
in this study, this cRNA probe recognizes the mRNA for the ER
subtype (Norell and Micevych, unpublished observation).
In situ hybridization. The protocol used in this study was
adapted from Abelson and Micevych, 1991 (25). Alternate brain sections
were processed for CCK, PPE, or ER mRNA in situ
hybridization histochemistry. Briefly, slide-mounted sections were
brought to room temperature, rehydrated in diethylpyrocarbonate
(DEPC)-treated PBS, and sequentially treated with predigested
proteinase K (10 µg/ml in 0.1 M Tris, 0.05 M
EDTA, pH 8.0; 10 min at 37 C), DEPC-PBS, 0.1 M
triethanolamine (TEA, pH 8.0; 2 min), acetic anhydride (0.25% in 0.1
M TEA; 10 min), and 2x SSC (1xSSC = 0.15
M NaCl, 0.04 M
C6H5Na3O7, pH 7.2; 4
min) and were dehydrated in graded ethanols and xylene. Slides were air
dried and incubated for 1 h with prehybridization buffer
containing deionized formamide, 5x hybridization salts (1x
hybridization salts = 0.15 M NaCl, 5 mM
EDTA, 5 mM PIPES, pH 6.8), 0.1 x Denhardt’s buffer
(100 x Denhardt’s = 2% each polyvinylpyrrolidone, Ficoll,
and BSA), 0.2% SDS, 100 mM DTT, and 250 µg/ml each of
denatured salmon sperm and poly (A)+ RNA. For CCK mRNA
in situ, slides were hybridized with 35S-CCK
cRNA probe (2.0 x 106 cpm/3 ng/slide) and 40%
formamide overnight at 60 C, as described (25). For PPE mRNA in
situ, slides were incubated with 35S-PPE-A cRNA probe
(1.5 x 106 cpm/4 ng/slide) and 50% formamide
overnight at 55 C, as described (12). For ER in situ,
slides were incubated with 35S-ER cRNA probe (2.0
x 106 cpm/3 ng/slide) and 40% formamide overnight at 60
C. Following hybridization, slides were immersed in 4x SSC until
coverslips floated off and were rinsed in fresh 4x SSC. Sections were
treated with RNase A (100 µg/ml in 0.5 M NaCl, 10
mM Tris, 1 mM EDTA, pH 8.0) and washed in SSC
solutions of increasingly higher stringencies containing 1
mM DTT to a final stringency of 0.1x SSC (30 min at 55 C).
Sections were then dehydrated in graded ethanols and xylene and air
dried. Slides were dipped in Kodak NTB-2 nuclear emulsion (Kodak,
Rochester, NY) diluted 1:1 with distilled water, and exposed at 4 C for
7 (PPE), 24 (ER), or 30 (CCK) days. Slides were developed using Kodak
D-19 developer and fixer and were counterstained with toluidine blue,
dehydrated, and coverslipped with Permount (Fisher).
Analysis. The boundaries of each nucleus were identified under brightfield optics, and the density of silver grains over each nuclear cell group was measured using darkfield optics and a computer-assisted morphometric system (NIH Image, modified by Karl Beykirch, UCLA). Nonspecific background hybridization levels were determined by measuring the density of silver grains over an adjacent area of neuropil. For each nucleus, four to six sections evenly spaced through the rostro-caudal extent were analyzed from each animal. These values were used to calculate an mRNA labeling ratio (silver grain density of the specified nucleus/background density of silver grains) to express a relative level of CCK, PPE, or ER mRNA within a nucleus; a labeling ratio of one is equal to background levels of hybridization (11, 12). The labeling ratio allows for comparison of hybridization of the same probe between slides within the same hybridization experiment and from different in situ hybridization experiments. If the specific activity of the probes are similar and within the linear range of the emulsion, the labeling ratio is independent of exposure time and emulsion thickness of the autoradiogram (for a detailed discussion see Ref.26). Additionally, labeling ratios of control areas were determined to ensure consistency between in situ hybridization experiments in nonestrogen-sensitive brain regions. For CCK mRNA, parietal cortex at the level of the MPN was examined; and for PPE-A mRNA, the striatum at the level of the VMH was evaluated. Comparisons of the data between treatment groups were made using one-way ANOVA and Student Newman-Keul’s post hoc planned comparisons (Sigmastat, Jandel Scientific). Differences were considered statistically significant if P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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illustrates the CCK mRNA
hybridization pattern in the MPNc after hormone treatment. In the
MPNc of oil-treated (OVX) animals, CCK mRNA levels were low.
T4 treatment alone did not induce CCK mRNA labeling ratios
compared with oil-treated animals (1.14 ± 0.05 vs.
1.09 ± 0.02). EB or EB + T4 treatment significantly
induced CCK mRNA levels compared with oil-treated animals (labeling
ratios: 2.10 ± 0.06 or 2.96 ± 0.22 vs. 1.09
± 0.02; Fig. 2A). Additionally, EB +
T4-treated animals had CCK mRNA levels which were
significantly higher than EB treated animals (labeling ratios:
2.96 ± 0.22 vs. 2.10 ± 0.06). In the
MeApd of oil-treated animals, CCK mRNA labeling ratios were
low. T4 treatment alone did not induce CCK mRNA levels
compared with oil-treated animals (1.55 ± 0.12 vs.
1.20 ± 0.07). EB or EB + T4 treatment
significantly induced CCK mRNA levels compared with oil-treated animals
(2.11 ± 0.28 or 2.61 ± 0.20 vs. 1.20 ±
0.07). In this nucleus, levels of CCK mRNA were not significantly
higher in EB + T4 compared with EB treated animals
(2.61 ± 0.28 vs. 2.11 ± 0.28). In the cortex,
CCK mRNA levels were statistically similar regardless of the treatments
examined indicating a lack of EB, T4 or a combined effect
of these hormones on CCK mRNA levels in this brain region.
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). EB or EB + T4
treatment significantly induced PPE mRNA levels compared with
oil-treated animals (1.79 ± 0.18 or 2.05 ± 0.07
vs. 1.17 ± 0.05). In the MeApd of
oil-treated animals, PPE mRNA levels were low. T4 treatment
alone did not induce PPE mRNA labeling ratios compared with oil-treated
animals (1.38 ± 0.12 vs. 1.66 ± 0.17). EB or EB
+ T4 treatment significantly induced PPE mRNA levels
compared with oil-treated animals (2.42 ± 0.17 or 2.73 ±
0.29 vs. 1.66 ± 0.17) respectively. Compared with
other areas, unstimulated levels of PPE in the VMH were
high. In the VMHvl, T4 treatment alone did not induce PPE
mRNA labeling ratios compared with oil-treated control animals
(2.84 ± 0.36 vs. 2.79 ± 0.19). EB or EB +
T4 treatment significantly induced PPE mRNA levels compared
with oil-treated animals (4.34 ± 0.48 or 4.46 ± 0.44
vs. 2.79 ± 0.19, respectively). Figures 3 and 4 illustrate the PPE mRNA hybridization pattern in the VMHvl following
hormone treatment. In the striatum, PPE mRNA labeling ratios were
statistically similar among all treatments indicating that these
hormone treatments do not alter PPE mRNA levels in this brain region
(Fig. 2B).
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mRNA mRNA
labeling ratio in oil-treated animals were significantly elevated above
the ratio in EB treated animals (Fig. 2C). In the MPNc,
T4 treatment did not alter ER mRNA expression compared
with oil-treated animals (3.09 ± 0.13 vs. 2.99 ±
0.18). Treatment with EB significantly reduced mRNA levels (2.01
± 0.02 vs. 2.99 ± 0.18), and EB + T4
significantly elevated ER mRNA levels compared ER with
oil-treated animals (3.81 ± 0.09 vs. 2.99 ±
0.18). In the MeApd, T4 treatment did not alter
ER mRNA labeling ratios compared with oil-treated animals (3.32
± 0.13 vs. 3.37 ± 0.10). EB treatment significantly
reduced ER mRNA levels compared with oil-treated animals (2.17
± 0.03 vs. 3.37 ± 0.10). In this nucleus, EB +
T4 treatment did not stimulate ER the mRNA labeling
ratio significantly compared with oil-treated animals (3.52 ±
0.24 vs. 3.37 ± 0.10), but levels were higher than
those of EB-treated animals. In the VMHvl, T4
treatment alone did not change ER mRNA labeling ratios compared with
oil-treated animals (4.01 ± 0.09 vs. 3.86 ±
0.05). EB treatment significantly reduced ER mRNA labeling ratio
(2.54 ± 0.03 vs. 3.86 ± 0.06), and EB +
T4 significantly elevated ER labeling ratio
compared with oil-treated animals (4.75 ± 0.16 vs.
3.86 ± 0.06). Figure 5 illustrates the ER mRNA hybridization
pattern in the VMHvl following hormone treatments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA within
the circuit. ER mRNA levels in the MPNc and VMHvl were higher after
the combined estrogen and thyroid hormone treatment than in the control
group, suggesting a thyroid hormone facilitation of ER mRNA
expression in EB treated rats. 2) Thyroid hormone had neither an
independent nor an antagonistic effect on the estrogenic induction of
CCK and PPE mRNA in the limbic-hypothalamic nuclei examined.
Indeed, thyroid hormone potentiated the estrogenic effect on CCK mRNA
expression in the MPNc. These results indicate that thyroid hormone
would not likely antagonize reproductive behavior through a suppression
of estrogen-induced CCK or PPE mRNA expression in the circuit.
The generally accepted pattern of ER regulation in the brain is that
increasing levels of estrogen down-regulate ER mRNA, producing
decreased levels of protein, binding sites, ER immunoreactive, and
estrogen concentrating cells (13, 27, 28, 29, 30, 31). This pattern is evident
during the estrous cycle: ER mRNA levels are lowest when estrogen
titers are elevated, diestrus and proestrus, and highest when estrogen
titers are lowest, estrus and metestrus (15, 16). The close agreement
between distribution and relative levels of ER mRNA and protein
indicate that measurement of message is a good predictor of ER
levels.
Despite these observations, there have been inconsistencies in the
literature about which ER expressing cell groups respond to estrogen.
For example, EB treatment has been reported to induce changes of
estrogen receptors in the MPO but not the VMH, or vice versa
(16, 27, 28, 30, 32). The present study demonstrated a robust estrogen
regulation of ER mRNA throughout cell groups of the
limbic-hypothalamic (15, 33). The high levels of ER mRNA in the
MPNc, MeApd, VMHvl of OVX animals were reduced by treatment with EB
(Fig. 2C). Hence, the estrogen-induced down-regulation of its receptor
message is a marker for estrogen action in the hypothalamic-limbic
system. Because T4 has been hypothesized as one of the
endocrine signals suppressing reproduction in response to an adverse
environment (24), we expected that the estrogen-induced decrease of
ER mRNA levels would be antagonized by T4 treatment. Not
only did T4 prevent the estrogen-induced decrease of ER
mRNA levels from OVX levels, but animals treated with EB +
T4 had higher levels of ER mRNA compared with OVX
animals. These results suggest that in the brain, elevated levels of
T4 potentiate ER mRNA levels in the presence of estrogen.
Such an induction has been reported for ER in pituitary and
pituitary tumors, but not hypothalamus (32, 34). This T4
effect was thought to be mediated through an interaction of thyroid
hormone receptor and ER. T4 alone did not elevate ER
mRNA levels, suggesting that T4 is not acting through a
pathway separate from that activated by estrogen (35, 36). Moreover,
this potentiation of ER mRNA in the MPNc and VMHvl was not observed
in the MeApd, suggesting that EB + T4 interactions may be
mediated through the synthesis and release of neuroactive compounds
that have differential distributions and action in cell groups of the
limbic-hypothalamic circuit. These regulatory compounds could interact
with receptors and induce a cascade of intracellular events that result
in alterations of estrogen-induced gene expression. For example, we
have reported that endogenous opioids acting throught the 
-opioid
receptor attenuate the estrogen-induced expression of CCK mRNA
(37). Other transmitter-receptor systems have been implicated in the
regulation of ER levels (38, 39, 40). T4 influences many
neuromodulatory systems implying a synaptic interaction, rather than an
antagonistic interaction of thyroid receptor with the estrogen response
element (41, 42). For example, T4 attenuates the EB-induced
increase in D-2 dopamine receptors which appears to be secondary to
T4 blockade of estrogen inhibition of dopamine synthesis
and release (43).
Others have reported that T4-induced ER immunoreactivity
in the VMHvl above OVX levels (24). It is difficult to reconcile these
findings with the present report, which demonstrates that
T4 does not alter levels of ER mRNA in OVX animals,
especially because levels of ER immunoreactive cells in OVX
animals reported by these authors was low in distinction to other
reports (e.g. 27, 28). A possible explanation was that the
H222 antiestrogen receptor antibody binds to ER with greater
affinity in the presence of high concentrations of T4. In
fact, the number of ER immunoreactive cells after T4
treatment is similar to what others show in OVX, thyroid intact rats.
Another possibility is that the H222 antibody recognizes both the ER
and the ERß subtypes (44), and the receptor subtypes may be
differentially modulated by T4. Several points mitigate
against this as an explanation for the discrepancy of the results.
First, in situ hybridization using a ERß riboprobe does
not reveal specific hybridization in the VMH or MPN (45, Norell and
Micevych, unpublished results). Second, the in situ
hybridization conditions used in the present study allow identification
of ER mRNA but not ERß mRNA.
In this experiment, the antagonistic effects of T4 on EB
action were specific to ER mRNA levels. T4 did not
interfere with estrogen’s actions on levels of CCK or PPE mRNA,
genes that are regulated transsynaptically and directly, respectively.
CCK mRNA levels in the MPNc and MeApd were elevated by continuous EB
treatment compared with low levels of expression in the absence of EB.
This result is consistent with previous observations of CCK mRNA
expression in the circuit after long-term, as well as acute, EB
exposure (11). T4 treatment alone had no effect on CCK mRNA
levels in these nuclei compared with oil-treated animals.
Interestingly, EB + T4 treatment resulted in elevated
levels of CCK mRNA in the MPNc compared with oil-treated animals, and
hyperelevated levels in the MPNc compared with oil or EB treated
animals. Such T4 hormone potentiation of estrogen’s action
on gene expression has been reported; for example, Rabelo and Tata (46)
have reported that T4 treatment potentiated the EB
activation of the vitellogenin gene in vitro, and in
vivo, this treatment also increased the expression of ER in
hepatocytes.
The effects of estrogen and thyroid hormone treatment on PPE mRNA expression within the circuit was similar to that seen for CCK mRNA. PPE mRNA levels in the MPNc, MeApd, and VMHvl were elevated by EB treatment consistent with previous observations (12, 47, 48, 49). Met-enkephalin immunoreactive neurons contain ER (50) and the PPE gene has a partial estrogen response element sequence (31), suggesting that estrogen directly regulates the expression of PPE mRNA. Thyroid hormone alone did not enhance PPE mRNA levels above levels in OVX animals and T4 did not block the estrogen induction of PPE mRNA levels. Zhu et al. (51), however, reported an elevation of PPE mRNA in the VMH with T4 treatment compared with OVX control animals, and a reduction of PPE mRNA levels with EB + T4 treatment compared with EB treated animals. Unlike our results, the Zhu et al. (51) findings suggest an antagonism of the EB effect on PPE expression. Currently, it is difficult to explain these inconsistent results. One potential explanation for this inconsistency is the different techniques used in these studies. Zhu et al. used slot blot analysis to examine changes in PPE mRNA levels in the VMH. This technique is dependent upon consistent and discrete tissue microdissection of the VMH from neighboring nuclei and regions that also express PPE mRNA. For example, the arcuate nucleus and a band of PPE mRNA expressing cells extending dorsally from the VMHvl toward the fornix also express PPE mRNA in an estrogen dependent fashion, and therefore are a potential source of confoundance in slot blot analysis. In situ hybridization, in contrast, allows for the specific analysis of mRNA expression in a consistent, anatomically defined region. Therefore, it is particularly well suited in this instance for the analysis of the estrogen-thyroid hormone effect in the VMHvl, which would be difficult to dissect away from the remainder of the nucleus and from neighboring estrogen-sensitive PPE expressing cells, although we did not observe an upregulation of PPE mRNA levels after T4 treatment in either of these adjacent areas.
Thyroid hormone did not alter the estrogen-induced expression of CCK
and PPE mRNA within the circuit. These results do not preclude the
possibility that the hyperinduced ER mRNA and protein levels impact
other molecules involved in the regulation of lordosis behavior.
Therefore, provided other cellular processes such as mRNA stability,
translation, and release are not impacted by T4 treatment,
these neuropeptide mRNA patterns indicate that an estrogen-primed
hyperthyroid female may not be completely compromised in its display of
reproductive behavior. This was demonstrated in a behavioral study done
using the same hormone paradigm (24). EB + T4 animals had
lordosis frequencies higher than the OVX or T4 groups but
lower than EB-treated animals (24). Our results are consistent with
these behavioral data (24): oil or thyroid hormone treatment did not
induce CCK or PPE mRNA and do not facilitate lordosis. Treatment with
EB or EB + T4 stimulates the expression of mRNA coding for
CCK and enkephalin which facilitate lordosis behavior. Because
EB-induced down-regulation of ER mRNA is correlated with behavioral
receptivity, we speculate that the hyperinduction ER mRNA in EB +
T4 animals may offer an explanation for the attenuation of
lordosis behavior. As mentioned above, T4 may affect
cellular processes that impact the translation of the mRNA to
functional molecules or modulate receptors within the circuit.
Alternatively, high levels of ER mRNA (and presumably protein) in
combination with, or resulting from T4 treatment may act to
prevent the estrogenic down-modulation of functionally inhibitory
signals within the circuit, thereby attenuating the display of lordosis
behavior.
Regardless of the mechanisms involved, these experiments do indicate that reproductively relevant molecules in nuclei of the lordosis regulating circuit are modulated by estrogen and thyroid hormone. The finding that thyroxine does not antagonize the estrogen-induced expression of CCK or PPE mRNA, but rather potentiates its effects, and that T4 treatment moderately attenuates the estrogen induced expression of lordosis behavior (24), suggests that thyroid hormone actions at the level of the limbic-hypothalamic circuit are not sufficient to account for the reported effect of thyroid hormone suppression of reproductive events.
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Acknowledgments |
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Footnotes |
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Received August 8, 1997.
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References |
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-, and 
-opioid
receptor agonists selectively modulate sexual behaviors in the female
rat: differential dependence on progesterone. Horm Behav 26:457–473[CrossRef][Medline]
-calcitonin gene-related peptide and
preprocholecystokinin messenger RNA expression in -motoneurons:
effects of testosterone and inactivity induced factors. Neuroscience 51:87–96[CrossRef][Medline]
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indicates values statistically higher than EB
treatment values; 
indicates values significantly lower than oil
treatment control values.
