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Neuroendocrinologie et Physiopathologie Neuronale, INSERM U-422, Lille, France
Address all correspondence and requests for reprints to: Dr. V. Mitchell, INSERM U-422, 1 place de Verdun, 59045 Lille Cedex, France. E-mail: mitchell{at}biserte.lille.inserm.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of galanin on the various functions are mediated by binding of the peptide to specific membrane receptors that have been localized within several regions of the hypothalamus (17, 18, 19). More recently, cloning of the galanin receptor Gal-R1 (20) allowed the localization of Gal-R1-expressing cells in the rat hypothalamus, including the POA (21). Gal-R1 mRNA-expressing cells have been identified in the majority of brain regions containing steroid receptors (22), providing a possible mechanism for genomic regulation of galanin receptor synthesis by steroids as observed in other receptor systems (23, 24, 25). Interestingly, the onset of mammalian puberty, which requires the activation of GnRH-containing neurons and which, in turn, implicates modifications in gonadal steroid secretions, is associated with increases in galanin-like immunoreactivity and galanin gene expression (26, 27) and with changes in the number and/or affinity of galanin-binding sites in several brain regions including the POA (27). It therefore seems probable that steroids regulate transcription of the galanin receptor gene.
To test this hypothesis, the present study examined and compared the patterns of Gal-R1 gene expression throughout the POA. The number and the intensity of Gal-R1 mRNA cells have been analyzed in the POA of intact female animals during different stages of the estrous cycle and compared with those in the male. In addition, to determine the respective roles of estrogen and progesterone in regulating Gal-R1 expression, the effects of short term estradiol (E2) and progesterone (E2P) treatments on Gal-R1 receptor expression in ovariectomized (OVX) rats were examined. The possible existence of circadian variations in Gal-R1 was also evaluated.
The results reveal significant variations in Gal-R1 expression across the estrous cycle. In intact female rats, a peak of Gal-R1 expression occurs after the LH surge. In addition, the number of neurons in which Gal-R1 mRNA can be detected fluctuates throughout the estrous cycle, and peak numbers of Gal-R1-expressing cells vary according to the rostrocaudal level of the preoptic region. In the male, both the number of Gal-R1-expressing cells and the level of expression were low and comparable to the lowest values in the cycling intact female. In OVX rats, Gal-R1 receptor expression significantly declined in E2P-treated rats. These results demonstrate that the expression of Gal-R1 in cells in the POA is influenced by sex steroids during the estrous cycle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A second group of 22 female rats was bilaterally ovariectomized under ether anesthesia. Two weeks later, 12 animals from this group were killed at different times of the day, i.e. 0800 (n = 4), 1200 (n = 4), and 1800 h (n = 4), to control for circadian variations. Ten OVX animals were injected with estradiol benzoate (E2; 30 µg/rat, sc) at 1000 h on day 0. On day 2, half of the E2-treated rats were implanted with progesterone (P; 2 mg, sc) at 1000 h to induce the LH surge in the afternoon. The group of OVX plus E2-treated rats (n = 5) was killed on day 2 at 1000 h. The group of OVX plus E2P-treated rats (n = 5) was killed on day 2 between 1600–1800 h.
Male rats were divided into three groups: four were killed at 0800 h, four were killed at 1200 h, and four were killed at 1800 h. Animals were anesthetized with 20 mg/kg ketamine and 0.2 ml/kg xylazine. Trunk blood was systematically collected into vials containing 0.5 ml 0.3 M EDTA and centrifuged. Plasma was stored at -20 C until LH, estradiol, and progesterone RIAs. They were perfused intracardially with 5–10 ml saline followed by 500 ml 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed and immersed in the same fixative for 2 h. They were then washed overnight in 0.05 M Coon’s veronal buffer (pH 7.4) containing 20% sucrose, embedded in Tissue-Tek (Miles Laboratories, Naperville, CA), and frozen in liquid nitrogen. Fourteen-micron coronal sections were cut on a cryostat at -19 C, and serial sections were collected across the hypothalamus from the decussation of the anterior commissure (coordinates 0.00 mm from Bregma according to Swanson’s rat atlas) (28) to the supraoptic nucleus (coordinates -0.51 mm). The sections were mounted onto gelatin-coated slides and stored at -80 C until used for in situ hybridization.
RIAs
To eliminate animals that might present abnormal values for
estradiol, LH, and progesterone, different measures were performed.
Plasma LH levels were measured using materials supplied by the NIDDK
rat pituitary hormone distribution program (Baltimore, MD), and values
were expressed in terms of the LH pituitary reference preparation RP-3;
the assay sensitivity was 0.02 ng/tube, and intra- and interassay
variabilities were 6% and 8.5%, respectively. Plasma estradiol was
measured using a RIA kit optimized for the direct quantitative
determination of very low concentrations of 17ß-estradiol in human
serum and plasma (e.g. in children), purchased from Sorin
Biomedica (Antony, France). The assay sensitivity was 0.2 pg/tube, and
intra- and interassay variabilities were 5.6% and 7.3%, respectively.
Progesterone levels were measured in plasma samples without extraction,
using a RIA kit purchased from Sorin Biomedica. The assay sensitivity
was 5 pg/tube, and intra- and interassay variabilities were 5.5% and
8.1%, respectively.
Probe labeling
A HindIII/BamHI fragment of approximately
1600 bp, which was isolated from a rat brain complementary DNA library
by Habert-Ortoli (Rhône Poulenc Rorer, Vitry, France) and
contained the entire coding region of the rat Gal-R1 gene, was used to
prepare complementary RNA probes. BamHI plus T3 polymerase
made the antisense probe, and HindIII with T7 polymerase
produced the sense probe. Radioactive probes were generated by labeling
with 200 µCi [35S]CTP (Amersham, Arlington Heights, IL)
using 1 µl of the appropriate RNA polymerase in a 40-µl
transcription mixture containing 8 µl 5 x transcription buffer,
2 µl 0.1 M dithiothreitol (DTT), 2 µl linearized
plasmid (50 ng/µl), 1 µl 5 mg/ml Escherichia coli
transfer RNA, 1 µl RNAsin, and 1 µl of 10-mM stocks of
ATP, GTP, and UTP. The transcription reagents were incubated for 3
h at 39 C. The labeled probes were separated from unincorporated
nucleotides on a Sephadex G-50 column.
In situ hybridization
Sections were removed from storage at -80 C and placed into 0.1
M glycine-0.2 M Tris-HCl (pH 7.4) for 10 min
before treatment with proteinase K (1 µg/ml in 100 mM
Tris, pH 8.0, and 50 mM EDTA) for 15 min at 37 C. Slides
were then rinsed in water, followed by fixation in 4% paraformaldehyde
in 0.1 M phosphate buffer for 15 min at 20 C. Slides were
treated with 0.1 M triethanolamine (pH 8.0) for 10 min
followed by 0.25% acetic anhydride for 10 min. The sections were
rinsed again in water, dehydrated with a graded series of alcohols, and
allowed to air dry. The 35S-labeled riboprobes were diluted
in hybridization buffer to a final concentration of 30,000 dpm/µl.
The hybridization buffer contained 50% formamide, 10% dextran
sulfate, 0.3 M NaCl, 20 mM Tris-HCl (pH 8.0), 5
mM EDTA, 1 x Denhardt’s solution, 0.5 mg/ml E.
coli transfer RNA, and 100 mM DTT. Tissue sections
were apposed to the diluted probe with coverslips and placed in a
hybridization chamber containing Whatman filter paper (Whatman,
Clifton, NJ) moistened with 4 x SSC (standard saline citrate) and
50% formamide. The hybridization boxes were then sealed and placed in
a 55 C oven overnight. The slides were washed twice with 4 x SSC
and 10 mM DTT for 30 min and 1 h, then washed in 0.3
M NaCl, 20 mM Tris-HCl (pH 8.0), 5
mM EDTA, and 50% formamide. After treatment with
ribonuclease A [20 µg/ml in 0.1 M Tris (pH 8.0), 0.5
M NaCl, and 0.5 M EDTA] for 30 min at 37 C,
sections were subsequently rinsed in 2 x SSC for 15 min at 60 C
and 0.1 x SSC for 15 min at 60 C, dehydrated with a graded series
of alcohols in 0.3 M ammonium acetate, and air-dried. Each
slide was dipped in photographic NTB-2 emulsion (Eastman Kodak,
Rochester, NY), dried, and stored at room temperature. All sections
were developed after a 20-day exposure and counterstained with 0.1%
azure blue. They were examined with an Axiophot microscope equipped
with dark- and brightfield condensers (Zeiss, Gottingen, Germany).
Controls
Specificity controls included incubation of sections with the
35S-labeled sense probe, pretreatment with ribonuclease,
and coincubation with a 100-fold excess of unlabeled antisense probe.
Under these conditions, no specific labeling was observed.
Analysis of hybridization results
Sections were selected from two rostrocaudal levels of the basal
forebrain. They included first the rostral preoptic area (rPOA) in the
transition of the diagonal band of Broca and the medial preoptic area
at the level of the organum vasculosum of the lamina terminalis
[coordinates 0.00 to -0.26 mm from the Bregma according to the rat
atlas of Swanson (28)]. The rPOA contains the median preoptic nucleus
and the anteroventral preoptic nucleus; the medial preoptic area (mPOA;
coordinates -0.26 to -0.51 mm from Bregma) contains the anteroventral
preoptic nucleus and the medial preoptic nucleus.
Labeled cells were identified using brightfield microscopy after counterstaining with azure blue, and darkfield microscopy. During the first analysis, Gal-R1 mRNA-expressing cells were counted under darkfield illumination under a x20 objective. The total number of labeled cells per hemisection was calculated for each animal and averaged. During the second analysis, the grain density per labeled cell was determined using the DensiRag computerized program of Biocom (Les Ulis, France). Tissue sections were viewed under a x60 epiillumination darkfield objective. Video images were obtained with a camera attached to the microscope. More precisely, the grain densities overlying each cell were measured by reflected light under darkfield epiillumination using the computerized image analysis system. Areas examined for the counting of labeled cells and for quantification of grain densities included rostrally to caudally the rPOA and the mPOA.
Statistical analysis
In all groups, n refers to the number of animals
within a group. The number of cells counted and the grain density per
cell were used to calculate the mean ± SEM for each
group. Differences among groups in these two parameters were assessed
with a one-way ANOVA. When the ANOVA indicated a significant
difference, the post-hoc Bonferroni’s t test was
used to determine differences between groups across the cycle and the
effect of steroids (OVX vs. OVX plus
E2 and OVX plus E2P) on
Gal-R1 mRNA levels (number of labeled cells and density of labeling).
Differences between the groups were regarded as significant when
P < 0.05.
In addition, the relationships among the number of cells, the grain density, and estradiol or progesterone concentrations during the estrous cycle were measured by the Spearman’s rank correlation coefficient, r. This coefficient falls within the range -1 to +1.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In the mPOA, most Gal-R1 cells were
localized in the anteroventral preoptic nucleus and medial preoptic
nucleus (Fig. 2). Gal-R1 mRNA-expressing
cells extended laterally to the lateral preoptic area.
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Quantification of Gal-R1-expressing cells during the estrous
cycle
The ANOVA indicated significant differences in both the rPOA and
mPOA. In the female, the number of neurons expressing Gal-R1 mRNA
fluctuated significantly (P < 0.05) throughout the
estrous cycle in the rPOA (Figs. 1
and 3)
as well as in the mPOA (Figs. 2 and 3). However, these changes occurred
differentially in the two regions. Moreover, whatever the region, the
peaks in Gal-R1 mRNA cell number did not coincide with maximal plasma
hormone levels.
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and 3). This maximum was abruptly attained and
declined in the same manner to persist equally until 1800 h on
estrus. The number of Gal-R1-labeled cells was significantly greater at
0800 h on proestrus vs. diestrus 0800 and 1200 h
on proestrus (P < 0.05; Fig. 3, a vs.
b). In the male, the mean number of Gal-R1-expressing cells was
significantly lower (Fig. 1d) than that of Gal-R1 cells in the female
across the cycle (P < 0.05; Fig. 3, c
vs. d).
In the mPOA, the number of Gal-R1-labeled cells was significantly
greater in the 1800 h estrous group (Figs. 2b and 3) than at all
other time points (P < 0.05; Fig. 3, a vs.
b), except at 0800 h on proestrus. The average number of Gal-R1
cells rose abruptly to a high value at 1800 h on estrus. In the
male, the number of Gal-R1-expressing cells was significantly lower
(Fig. 2d) than all values obtained in the cycling female
(P < 0.05; Fig. 3, c vs. d), except those
at 1800 h on diestrus.
Cellular labeling intensity during the estrous cycle
The ANOVA also indicated significant differences between groups in
the mPOA and rPOA. The density of labeling in Gal-R1 mRNA-expressing
neurons varied significantly across the estrous cycle in the rPOA and
mPOA (Figs. 4 and 5).
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and 5). These observations were significant vs.
those at 1800 h on diestrus and in the male (P <
0.05; Fig. 5, a vs. b). The increase appeared
progressive and began from 0800 h on proestrus. The level of
Gal-R1 expression in the male (Fig. 4a) was as low as the level at
1800 h on diestrus (Fig. 5).
In the mPOA, the cellular labeling intensity in Gal-R1-labeled neurons
fluctuated similarly, but highest values were evident 10 h later
than in the rPOA, i.e. at 1800 h on estrus (Fig. 4f and 5). The expression began to increase in a progressive manner from
proestrus (Fig. 5). The labeling intensity was significantly greater at
1800 h on estrus than at every other time point examined
(P < 0.05; Fig. 5, a vs. b), except
at 0800 h on estrus. In the male, the density of labeling in
Gal-R1-expressing cells in the mPOA (Fig. 4d) was significantly lower
than all values in the female (P < 0.05; Fig. 5).
Quantification of Gal-R1-expressing cells in steroid-treated OVX
rats
The number of Gal-R1-expressing cells was evaluated in each region
of the POA in OVX and steroid-treated OVX animals (Fig. 6). No significant difference
(P > 0.05) could be measured in either rPOA or mPOA.
The mean numbers of labeled cells were comparable to those in female
rats at diestrus in the two regions of the POA.
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, the estradiol plus
progesterone treatments elicited changes in the levels of Gal-R1 mRNA
in cells of the rPOA and mPOA. The labeling densities in cells of
E2P-treated rats declined significantly
(P < 0.05) vs. those in the OVX and OVX
plus E2 rats.
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). The LH levels completely returned to
baseline values by 0800 h on estrus. In each animal in this study,
estrogen (Fig. 9) and progesterone (Fig. 10) levels were typical of their estrous cycle
stage. Average estrogen levels were very high on the day of proestrus
and reached a peak at 1200 h on proestrus (46.62 ± 5.41
pg/ml). The average progesterone levels had two peaks; the first
occurred during the morning of diestrus (50.75 ± 5.07 ng/ml), and
the second occurred during the late afternoon of proestrus (47.66
± 4.91 ng/ml).
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).
The levels of plasma estradiol and progesterone in
E2P-treated OVX rats were significantly higher
than those in OVX animals. The OVX plus E2
animals showed significantly increased levels of estradiol.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study examines variations in Gal-R1 cell number, Gal-R1 gene expression per cell, regional changes in Gal-R1 gene expression in the POA, temporal patterns of periovulatory Gal-R1 gene expression, and finally, Gal-R1 gene expression between the sexes. To valid these comparisons, both male and ovariectomized female rats were killed at different times of the day to determine whether there is a significant circadian pattern of Gal-R1 expression during the light-dark cycle. Gal-R1 mRNA expression did not vary significantly over the times of the day.
During the estrous cycle, two or three time points for each period were considered. The analysis has also differentiated the two divisions of the POA, i.e. rPOA and mPOA, in keeping with the distribution of GnRH mRNA-expressing cells in this region (29). Indeed, we observed different numbers of Gal-R1 neurons at the rostrocaudal levels of the POA; the mPOA contained 3–4 times more Gal-R1-expressing cells than the rPOA. This observation seems important because, in the mPOA, estrogen receptors are abundant (22) and cytoarchitectonic sexual dimorphisms in the rat have been described (30). Consequently, in the rat POA, as has been observed for GnRH expression (29), regional differences in the number of Gal-R1-expressing cells are also detected.
Significant variations in both the intensity of expression and the number of Gal-R1-expressing cells occur across the cycle. In both regions of the POA, the peaks of these two parameters do not coincide with the GnRH and LH surges. In addition, the variations are not found at the same time points of the cycle in the rostral and medial POA. In the mPOA, the peak number of Gal-R1 neurons and the highest labeling intensity per cell coincided at 1800 h on estrus and, consequently, occurred after the GnRH and LH surges. More rostrally in rPOA, these two parameters did not fluctuate in the same manner (peak number at 0800 h on proestrus and highest grain densities on estrus). The probable existence of more than one population of Gal-R1 neurons in the POA might explain these results. It may be hypothesized that Gal-R1 expression interests more that one cell phenotype in the POA, but it is also possible that the expression of the receptors varies even in the same cell type. To our knowledge, in the POA, the phenotypes of the Gal-R1-expressing cells have not yet been identified. Although a preliminary abstract reports no colocalization of Gal-R1 mRNA in GnRH neurons (31), some GnRH-containing neurons could, nevertheless, express the Gal-R1 gene at some point during the estrous cycle. However, because there are many more Gal-R1-expressing cells than GnRH-expressing cells in the POA, the Gal-R1 labeling cannot be ascribed only to GnRH cells.
In addition, our data demonstrate that the male POA has significantly fewer Gal-R1 mRNA-expressing cells than the female POA at most phases of the estrous cycle. Moreover, the levels of expression of Gal-R1 in the mPOA of the male are significantly lower than at any time point in mPOA cycling females. In the male rat, the numbers and grain densities of Gal-R1 cells are comparable or lower than the nadir values in female rats, i.e. during the diestrous period of the cycle. With the observations on the estrous cycle, these results suggest an involvement of gonadal steroids in the regulation of the Gal-R1 gene expression in the POA. The fact that the distribution of steroid receptors overlaps with that of Gal-R1 cells in the POA supports this hypothesis. However, no data are available concerning the possible colocalization of steroid receptors and Gal-R1 on the same neurons in this region or other brain areas. Only colocalization of sex steroid receptors and galanin has been reported in the POA (9, 32), strongly suggesting an interaction between galanin and gonadal steroids. This hypothesis is reinforced by the recent observation that levels of galanin in the mPOA peak during the proestrous phase when circulating estradiol and progesterone also rise (33).
In the rPOA, the peak of recruited Gal-R1 mRNA-expressing cells on the morning of proestrus occurs at the same time as the large increase in plasma estradiol. Consequently, it seems unlikely that these changes in Gal-R1 mRNA expression are a direct consequence of the estradiol concentration, and this is consistent with our experimental results (see below). However, as the rate of estradiol secretion does slightly rise by late on diestrus (34), it cannot be ruled out that estrogen assists in the induction of the recruitment of Gal-R1 mRNA-expressing cells. In the mPOA, the peak in Gal-R1-expressing cells is observed later, on the afternoon of the estrus day, i.e. when plasma estradiol and progesterone are rather low and constant. Similarly, the peaks of Gal-R1 expression in cells occurred on the day of estrus (morning and afternoon, respectively, in the rPOA and mPOA). Therefore, the mechanisms of gonadal steroid feedback that are involved in the recruitment of cells and expression of receptors per cell might be different in these two areas. In one case the effect might be direct, whereas in another it might involve cross-talk among several components of the hypothalamic circuitry. To address specifically the issue of whether estrogen or progesterone is responsible for the changes in Gal-R1 mRNA during the estrous cycle, groups of OVX and steroid-replaced OVX animals have been included in the study. In our experimental conditions, we found that estrogen plus progesterone replacement reduced the level of expression of Gal-R1 mRNA in the POA below the levels in the OVX and OVX plus E2-treated animals. Estradiol therapy alone to OVX animals did not change Gal-R1 expression in the POA. Administration of estrogen with or without short term progesterone treatment also had little effect on the number of Gal-R1-expressing cells in the POA of OVX rats. If these results are apparently inconsistent with the changes in Gal-R1 expression observed during the estrous cycle, in that Gal-R1 expression did not decrease at the moment of the LH surge when progesterone is high, we found a strong negative correlation between Gal-R1 expression during the estrous cycle and progesterone concentration. This last result is consistent with the decrease in Gal-R1 expression in OVX plus E2P animals. Thus, it seems that progesterone is necessary to induce changes in Gal-R1 mRNA expression. Taking into account all of the results, it seems probable that in addition to a possible direct genomic effect of steroid hormones on Gal-R1 expression, steroids might act through interneuronal systems to affect Gal-R1 mRNA levels. Consequently, in addition to hormonal regulation, transsynaptic modulations probably occur for the regulation of Gal-R1 expression. The differences between Gal-R1 expression in OVX plus E2P and during the estrous cycle are also likely to be due to the experimental conditions that cannot fully replace the changes that occur in physiological conditions. Also, it cannot be ruled out that other ovarian factors might directly or indirectly influence the expression of these Gal-R1 receptors. However, these explanations for differences between expression of Gal-R1 in OVX plus E2P animals and during the estrous cycle remain speculative.
Little is known about the roles of steroids in the regulation of galanin receptors. Some observations report variations in galanin receptor occupancy by endogenous ligand in association with puberty (27, 35). In particular, concerning the female POA, a significantly enhanced iodinated galanin binding has been observed during the period of increased gonadal secretion triggered by puberty. In this same study, the researchers did not observe differences between adult males and females in the density of iodinated galanin-binding sites in the POA. However, their female rats were not chosen at different time points of the cycle and regions, but were examined as a single group, making it difficult to compare with our results. Although the literature concerning the influence of sex steroids on the expression of Gal-R1 is sparse, the action of sex steroids on hypothalamic galanin is well documented. However, in contrast with the clear-cut effects observed in the pituitary, the action of sex steroids on hypothalamic galanin is still a matter of debate. Some researchers have reported increased hypothalamic galanin (16) and galanin mRNA after estrogen treatment (11, 12, 14), whereas others did not observe any effect (36, 37). Very recently, Leibowitz et al. (33) observed a clear relationship between the galanin peptide in the mPOA and the gonadal steroids. More particularly, they found that progesterone produced an enhancement of galanin specifically in the POA. Consequently, the increase in Gal-R1 expression 24 h after this peak of galanin might be a consequence of this high concentration. Concerning sex differences in galanin expression, Planas et al. (38) found no evidence of sex differences in galanin gene expression in either considered brain region despite the solid evidence for the regulation of these pathways by gonadal steroids. The most dramatic sex differences in galanin expression were also found in the anterior pituitary (36) and the median eminence (26).
In conclusion, we observed effects of estrous cyclicity on the number and expression of Gal-R1 receptors in the rat POA. It is therefore probable that gonadal steroid hormones contribute to the modulation of POA sensitivity to galanin. The nature of the interactions between gonadal steroids and Gal-R1 expression is still unclear. It appears that variations in progesterone concentration have more effect on the expression of Gal-R1 mRNA than variations in estradiol. We suggest that the steroid effect on Gal-R1 mRNA involves more than one mechanism; a direct induction of gene expression might alter the rate of gene expression, but neurotransmitters or neuropeptides acting via transsynaptic mechanisms are probably also implicated. Among these neuropeptides, some might be themselves influenced by steroids. Galanin is a good candidate because galanin concentrations in the mPOA vary during the estrous cycle (33), and an estrogen receptor-binding site within the human galanin gene has recently been identified in cells of the adenohypophysis (39).
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Acknowledgments |
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Footnotes |
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Received January 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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