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Endocrinology Vol. 145, No. 8 3796-3803
Copyright © 2004 by The Endocrine Society

Glucocorticoids Do Not Alter Developmental Expression of Hippocampal or Pituitary Steroid Receptor Coactivator-1 and -2 in the Late Gestation Fetal Guinea Pig

Elaine Setiawan, Dawn Owen, Lucy McCabe, Alice Kostaki, Marcus H. Andrews and Stephen G. Matthews

Departments of Physiology (E.S., D.O., L.M., A.K., M.H.A., S.G.M.), Obstetrics and Gynecology (S.G.M.), and Medicine (S.G.M.), Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Dr. S. G. Matthews, Department of Physiology, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: stephen.matthews{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroid receptor coactivator (SRC) proteins interact with glucocorticoid receptors in a ligand-dependent manner to enhance transcription. Although glucocorticoids are essential for normal brain maturation, little is known about the presence or regulation of SRC proteins in the developing central nervous system. In the current study we demonstrated that SRC-1 was highly expressed in the fetal limbic system (hippocampal CA3>CA1/2>CA4>dentate gyrus) at gestational d (gd) 40 (term, ~70 d), whereas SRC-2 was undetectable at all time points. Hippocampal SRC-1 mRNA and protein expression were reduced in male and female fetuses with advancing gestation. In contrast, SRC-1 mRNA levels increased significantly in the dentate gyrus near term. Repeated maternal injection (1 or 10 mg/kg on gd 40, 41, 50, 51, 60, and 61) with synthetic glucocorticoid had no effect on fetal limbic SRC-1 expression at gd 62 in either sex. SRC-1 and SRC-2 mRNA expression in the anterior pituitary did not change over the second half of gestation and was unaffected by prenatal exposure to synthetic glucocorticoid. In conclusion, SRC-1 expression undergoes spatial, temporal, and region-specific regulation during development, and limbic and pituitary SRC-1 and SRC-2 are not regulated by glucocorticoids in late gestation. Developmental changes in limbic SRC-1 expression probably have important consequences on steroid receptor signaling, which is known to be critical for brain maturation in late gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL PROTEINS HAVE been found to associate with and to be critical for transcriptional activity of nuclear receptors, such as the glucocorticoid (GC) receptor (GR), estrogen receptor, and thyroid hormone receptor (1, 2). This family of steroid receptor coactivators (SRCs) enhances transcriptional activity of the nuclear receptors through histone acetyltransferase activity and the recruitment of other basal transcription machinery (2).

GCs are critical for normal development of the fetal brain (3). We have shown that there are dramatic region-specific changes in GR and mineralocorticoid receptor expression in the brain of the late gestation guinea pig fetus (4). Given the critical role that the SRC family plays in GC signaling (1), it is important to establish levels of expression in the developing brain as well as to understand how they are regulated at this time.

Investigating the effects of GCs on the fetal brain is especially salient, because synthetic GCs (sGCs) are commonly used to treat women at risk of preterm labor. Fetal exposure to synthetic GC is highly effective in maturing the fetal lung and decreasing neonatal mortality and morbidity (5). Due to the difficulty in diagnosing preterm labor and the effectiveness of the treatment, until recently, multiple course therapies were being widely adopted (6). Despite the positive effects on lung function, several studies have demonstrated profound effects of prenatal exposure to sGC on neuroendocrine function and behavior throughout postnatal life (7, 8, 9, 10, 11). This involves programming of the hypothalamic-pituitary-adrenal (HPA) axis (for review, see Ref. 9). Previous work in our laboratory has shown that fetal exposure to high levels of sGC does not alter hippocampal GR expression through autoregulation. However, HPA function is very significantly attenuated resulting in dose-dependent decreases in CRH mRNA levels in the hypothalamic paraventricular nucleus (PVN) and fetal plasma ACTH and cortisol concentrations (10). Recruitment of SRC-1 and enhancement of transcription via these coactivator interactions are potential mechanisms by which changes in GC signaling might occur in the absence of observable changes in GR expression. Previous studies have indicated that SRC-1 is up-regulated in response to increased endogenous, but is down-regulated in response to exogenous GCs in adult rats; however, there is no information about SRC-1 during development (12, 13).

This study examines the ontogeny of SRC-1 or SRC-2 and their potential regulation by GCs during fetal development. We have used the guinea pig model, because, like the human and unlike the rat, it gives birth to neuroanatomically mature offspring (14). We hypothesize that 1) the developmental profiles of SRC-1 and SRC-2 expression in the fetal guinea pig brain and pituitary undergo differential spatial and temporal regulation; and 2) SRC expression in the brain and pituitary will be modified by repeated fetal exposure to GC in late gestation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental treatments
Female guinea pigs were mated in our animal facility as described previously (10). This method produces accurately time-dated pregnant guinea pigs. These studies were performed using protocols approved by the animal care committee at University of Toronto and in accordance with the Canadian Council for Animal Care.

Development.
Maternal guinea pigs were euthanized by rapid decapitation on gestational d (gd) 40, 50, and 60, and fetuses were rapidly removed (term, ~70 d). Brains were hemisected, and the left hemisphere was frozen for in situ hybridization. The right hippocampus was dissected and frozen for Western blotting. Pituitaries were dissected and frozen for in situ hybridization. Tissues were frozen and stored at –80 C until further processing.

Prenatal GC treatment.
Pregnant animals received sc injections of 1 mg/kg (n = 5) or 10 mg/kg (n = 7) dexamethasone (dexamethasone-1-phosphate, Sigma-Aldrich Corp., Oakville, Canada), betamethasone (1 mg/kg, 6 mg/ml; Betaject, Sabex, Boucherville, Canada) or vehicle (n = 8) on gd 40, 41, 50, 51, 60, and 61, as previously described (10). Pregnant guinea pigs were euthanized on gd 62, 24 h after the final injection. Fetal brains and pituitaries were removed and frozen with no further dissection for in situ hybridization. Due to limited tissue availability, we could not measure SRC-1 protein levels in these fetuses. The regimen of maternal treatment with sGC used in this study has been shown to down-regulate both maternal and fetal CRH mRNA, plasma ACTH, and cortisol concentrations in a dose-dependent fashion (10).

In situ hybridization
The method for in situ hybridization has been described in detail previously (11). Briefly, coronal cryosections (10 µm) were mounted onto poly-L-lysine-coated slides, dried, and fixed in paraformaldehyde (4%). Antisense oligonucleotide probes for SRC-1 and SRC-2 were labeled using terminal deoxynucleotidyl transferase (Life Technologies, Inc., Burlington, Canada) and [35S]deoxy-ATP (1300 Ci/mmol; PerkinElmer, Woodbridge, Canada) to a specific activity of 1.0 x 109cpm/mg. Labeled probe in hybridization buffer (200 µl) was applied to slides at a concentration of 1.0 x 105 cpm/ml. Oligonucleotide probes were synthesized by Sigma-Genosys (Oakville, Canada). The antisense probe for SRC-1 mRNA was complementary to bases 4250–4294 of human SRC-1 (GenBank reference no. NM_003743) (15), which shows 100% homology with bases 4906–4950 of mouse SRC-1 (GenBank reference no. MMU64828) (16), Similarly, the SRC-2 probe was complementary to bases 2816–2860 of human SRC-2 (GenBank reference no. NM_006540) (17), a region 100% homologous to mouse SRC-2 (GenBank reference no. NM_008678) (18). Slides were incubated overnight in a moist chamber at 42.5 C. After washing in 1x standard saline citrate (20 min at 23 C, then 35 min at 55 C), slides were rinsed and dehydrated in ethanol. Slides were dried and exposed to autoradiographic film (Biomax MR, Kodak, PerkinElmer). Films were developed using an automatic processor (exposure, 8–14 d for SRC-1 hippocampus, 8 wk for SRC-1 PVN, 14 d for SRC-1 anterior pituitary, and 7 d for SRC-2 anterior pituitary). Brain sections were processed simultaneously to allow direct comparison between groups. The sections were exposed together with 14C-labeled standards (American Radiochemical Co., St. Louis, MO) to ensure analysis in the linear range of the autoradiographic film. The relative OD of the signal on autoradiographic film was quantified after subtraction of background values, using a computerized image analysis system (Imaging Research, Inc., St. Catharine’s, Canada). All analyses were undertaken by an operator blinded to age or treatment. Levels of SRC-1 mRNA expression were measured in the hippocampus (CA1, CA2/3, and CA4), dentate gyrus (DG), cingulate and lateral cortex, and anterior pituitary. SRC-2 was detected in the anterior pituitary only. Adult guinea pig brains were probed alongside fetal brains to compare detection of SRC-1 mRNA expression in the PVN. Because SRC-1 was virtually undetectable in the fetal PVN, we did not quantify SRC-1 expression in this region. Sense probes labeled in the same way and incubated with brain and pituitary sections known to contain SRC-1 and SRC-2 mRNA showed no hybridization signal.

Immunohistochemical staining: brain SRC-1
Frozen sections derived from adult (n = 3) and fetal (n = 6) brains known to contain hippocampus and PVN were thawed, postfixed in paraformaldehyde (4%), and washed in PBS. Before incubation with primary antibody, sections were treated with hydrogen peroxide (0.3%, 30 min). Immunohistochemical staining was performed using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA) with an anti-SRC-1 antibody (sc-8995, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:500 dilution and incubated overnight (4 C). SRC-1 protein was visualized using diaminobenzidine tetrachloride (Sigma-Aldrich Corp.). The reaction with diaminobenzidine tetrachloride (2 min) was stopped by rinsing in PBS. Sections were dehydrated before mounting. The specificity of the antibody was characterized by incubation with preimmune serum, which yielded no specific signal in the PVN, cerebral cortex, or hippocampus.

Western blot analysis
Western blotting was undertaken as described previously (4). Briefly, hippocampi were homogenized in ice-cold radioimmunoprotection assay lysis buffer, the homogenate was centrifuged (4 C, 10,000 x g, 10 min), and the resulting supernatant was recentrifuged. The protein concentration of the supernatant was determined by the Bradford method (19). 2x Laemmli sample buffer (15 µl; Sigma-Aldrich Corp.) was added to each sample (50 µg protein), which was then denatured (boiled for 5 min at 95 C). Samples were separated by SDS-PAGE (8% resolving polyacrylamide gel) and transferred electrophoretically to a nitrocellulose membrane (Bio-Rad Laboratories, Mississauga, Canada).

Nitrocellulose membranes were blocked overnight (4 C) in skim milk (5%, wt/vol) PBS with Tween 20 (PBS-T). Membranes were washed with PBS-T and incubated with SRC-1 antibody (1:1000, 1 h, 23 C; Santa Cruz Biotechnology, Inc.; sc-8995) in 5% skim milk/PBS-T. Membranes were then washed in PBS-T and incubated with horseradish peroxidase-conjugated goat antirabbit IgG (1:5000, 1 h, 23 C; PerkinElmer). Blots were washed in PBS-T and exposed to Western Lightning Chemiluminescence Reagent Plus (PerkinElmer), and bands were visualized by exposure to Kodak Blue X-OMAT film for 30 sec to 1 min (PerkinElmer). Films were developed with an automatic processor. Membranes were stripped in Restore Western Blot Stripping buffer (20 ml, 30 min, 23 C; Pierce, MJS Bioynx, Mississauga, Canada). The absolute OD of SRC-1 was analyzed with computerized imaging software. All SRC-1 signals were standardized to the signal for tubulin (1:5000, anti-tubulin; Sigma-Aldrich Corp.). All Western blots were performed a minimum of four times for each animal. Data were pooled to derive a mean value for each animal. Expression levels are the ratio of SRC-1 to tubulin signal. We have previously shown that tubulin levels do not change over the last half of pregnancy in the fetal guinea pig hippocampus (4).

Statistical analysis
Group data are presented as the mean ± SEM and were statistically analyzed using two-way ANOVA, followed by Duncan’s method of post hoc comparison (Statistica, Statsoft, Inc., Tulsa, OK). Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In situ hybridization detected significant expression of SRC-1 mRNA throughout the brain in all gestational age groups (Fig. 1Go, A–C). Relative expression was the highest in the hippocampus and cerebral cortex. SRC-2 mRNA expression was undetectable in the fetal guinea pig brain (Fig. 1DGo). SRC-1 mRNA and protein were essentially undetectable in the fetal guinea pig PVN by in situ hybridization and immunohistochemical staining, respectively (Fig. 2Go, A and C). However, the adult guinea pig PVN demonstrated significant levels of both SRC-1 mRNA and protein (Fig. 2Go, B and D).



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  		FIG. 1. Representative images of SRC-1 mRNA expression in the fetal hippocampus on gd 40 (A), gd 50 (B), and gd 60 (C). D, Representative images of fetal brain SRC-2, sense probe (E), and expression of pituitary SRC-1 (F) and SRC-2 (G) are shown. Subfields of the hippocampus are indicated by arrows. Bar, 6 mm (A–E) and 0.5 mm (F and G).

 


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  		FIG. 2. Representative images of fetal (A) and adult (B) expression of SRC-1 mRNA in the PVN. Immunohistochemical staining for SRC-1 protein in fetal (C) and adult (D) PVN and fetal hippocampal CA1 pyramidal layer (E) and after incubation with preimmune serum in a section containing hippocampus (F). Bar, 0.5 mm (A and B) and 50 µm (C–F).

 
Ontogeny of hippocampal and pituitary SRC-1 and SRC-2 expression
SRC-1 mRNA expression was analyzed in regions CA1/2, CA3, and CA4 in the hippocampus; the DG; and the lateral and cingulate cerebral cortices. All gestational age groups showed detectable expression of SRC-1 transcripts in all hippocampal subfields and the cortex. On gd 40, the overall hippocampal expression pattern was CA3>CA1/2>CA4>DG. This distribution was similar in the gd 50 fetuses. On gd 60, there was a significant increase in SRC-1 levels in the dentate gyrus (F2,31= 36.494; P < 0.0001) in both male and female fetuses, and the relative hippocampal expression of SRC-1 mRNA became CA3>DG>CA1/2>CA4.

There was a significant effect of age on SRC-1 mRNA expression in all hippocampal subfields (CA1/2, F2,31= 7.97; CA3, F2,31= 5.40; CA4, F2,31= 4.15; P < 0.03) after two-way ANOVA. SRC-1 mRNA levels decreased significantly from gd 40 to gd 50 in all hippocampal regions (P < 0.03) in male fetuses (Fig. 3Go, A–C) after post hoc analysis. Females showed a comparable trend in SRC-1 expression, but this did not reach statistical significance (P = 0.08). Conversely, in the dentate gyrus, SRC-1 mRNA levels increased dramatically on gd 60 (F2,31= 36.49; P < 0.0001) in both sexes (Fig. 3DGo). In the cingulate and lateral cerebral cortex (Fig. 3Go, E and F), SRC-1 mRNA levels decreased significantly with advancing gestation (cingulate cortex, F2,32= 19.83; lateral cortex, F2,32= 15.41; P < 0.05) in both sexes. There was also a significant effect of sex on SRC-1 mRNA expression in the lateral cortex (F1,32= 4.88; P < 0.04), with a similar trend observed in the cingulate cortex (F1,32= 3.61; P = 0.07) after two-way ANOVA. Hippocampal SRC-1 protein levels correlated most closely with mRNA expression in CA3, the region of highest SRC-1 expression in the hippocampus (F1,32= 6.98; r = 0.423; P < 0.02); the correlation with CA1/2 approached significance (F1,32= 3.12; r = 0.29; P < 0.08). SRC-1 mRNA and protein levels were reduced in male fetuses on gd 50 (F2,30= 10.24; P < 0.005; Figs. 3AGo and 4Go), with both sexes showing a significant decrease by gd 60 (F2,30= 10.24; P < 0.005). SRC-1 and SRC-2 mRNA expression did not change during late gestation in the anterior pituitary (Fig. 1Go, F and G, and Table 1Go). There were no significant sex differences in the expression of SRC-1 in the hippocampus and anterior pituitary or in SRC-2 levels in the anterior pituitary at any gestational age.



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  		FIG. 3. SRC-1 mRNA expression in hippocampal region CA1/2 (A), CA3 (B), CA4 (C), DG (D), lateral cortex (Lat Cx; E), and cingulate cortex (Cing Cx; F). Each bar represents the mean ± SEM relative OD (ROD). Numbers in bars represent the number of fetuses in each group. *, P < 0.05 vs. gd 40. {square}, Males; {blacksquare}, females.

 


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  		FIG. 4. Hippocampal SRC-1 protein levels; the inset is a representative Western blot. Each bar represents the mean ± SEM SRC-1/tubulin ratio. Numbers in bars represent the number of fetuses in each group. *, P < 0.05 vs. gd 40. There were no significant sex differences. {square}, Males; {blacksquare}, females.

 

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  		TABLE 1. Ontogeny of SRC-1 and SRC-2 mRNA expression in the anterior pituitary

 
Effects of repeated GC treatment
Repeated exposure to different GC regimens failed to alter hippocampal SRC-1 mRNA levels in the hippocampus (Table 2Go). However, female fetuses that were exposed to 1 mg/kg dexamethasone showed a strong trend toward higher SRC-1 expression in region CA4 than the vehicle-treated group, although this did not reach significance (F1,37= 0.96; P = 0.052). There was no significant treatment effect in region CA1/2, CA3, or the DG (Table 2Go). No significant sex differences were observed in any of the hippocampal subfields. In the pituitary there were no significant treatment or sex differences in SRC-1 or SRC-2 mRNA expression on gd 62 (Table 3Go). Due to limited tissue availability, we were unable to assess protein levels after prenatal GC treatment.


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  		TABLE 2. SRC-1 mRNA expression in hippocampal regions CA1/2, CA3, and CA4 and the DG of gd 62 fetuses after repeated vehicle (saline), dexamethasone, or betamethasone treatments on gd 40/41, 50/51, and 60/61

 

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  		TABLE 3. SRC-1 and SRC-2 mRNA expression in the anterior pituitary of gd 62 fetuses after repeated vehicle (saline), dexamethasone, or betamethasone treatments on gd 40/41, 50/51, and 60/61

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we examined the expression and developmental profile of SRC-1 and SRC-2 in the fetal guinea pig hippocampus, hypothalamus, and pituitary. We also determined the effects of prenatal GC exposure on fetal SRC-1 and SRC-2 expression in the hippocampus and pituitary. Our results indicate spatial and temporal specificity of SRC-1 and SRC-2 expression during fetal development. The CA3 region of Ammon’s horn showed the highest expression of SRC at all gestational ages. SRC-1 mRNA and protein expression decreased in parallel in the hippocampus with advancing gestational age. In contrast, in the DG, SRC-1 transcript levels were relatively low before rising significantly on gd 60 in both male and female fetuses. SRC-1 mRNA and protein were present in the adult, but not in the fetal, hypothalamic PVN. SRC-1 and SRC-2 mRNA were present in the fetal anterior pituitary by gd 40, but levels remained unchanged in late gestation. There were no significant effects of repeated sGC exposure on SRC-1 or SRC-2 expression in the fetal hippocampus or pituitary.

Although we observed differential expression of SRC-1 among the hippocampal subfields in the fetus, SRC-1 mRNA appears to be equivalently expressed in all regions in the adult rat hippocampus (20, 21). In fetal life, GR expression increases in hippocampal subfields CA1 and CA3 and the DG as fetal plasma cortisol levels rise in late gestation (4, 22). The present study demonstrated that SRC-1 has an expression profile opposite that of GR. Higher SRC-1 levels in CA1/2, CA3, and CA4 may confer higher GR sensitivity when hippocampal GR expression is low. Decreasing SRC-1 levels in late gestation may contribute to increased fetal HPA activity at the end of gestation by decreasing GC signaling in the hippocampus. The significance of the parallel increase in GR and SRC-1 in the DG near term is less clear, because the precise role of the DG in GC feedback regulation is not known.

Glucocorticoids are also important in limbic development and neurogenesis. High GCs tend to inhibit the growth and migration of granule cells in the DG (23). The ontogenic profile we report in the DG supports a mechanism by which SRC-1 expression is low during the periods of neurogenesis (gd 40) and peak brain growth (gd 50). This may reduce GR action and the negative effects of endogenous GCs on these processes. The increase in SRC-1 and GR in the DG on gd 60 supports evidence indicating that GC action is critical for myelination in this region (24).

The GR is also highly expressed in the fetal PVN; expression decreases dramatically near term to facilitate activation of the fetal HPA axis (22). Previous studies have localized a splice variant, SRC-1a, to the PVN in the adult rat (20). With the probe in the current study, which was designed to detect total SRC-1 mRNA, we have shown that SRC-1 is highly expressed in the adult PVN, but not in the fetal PVN. We have confirmed this by immunohistochemical staining. This may indicate fundamental differences in GR signaling and coactivator recruitment between the fetus and the adult.

In the current study we have also shown that SRC-1 and SRC-2 mRNA are expressed in the fetal anterior pituitary. This is consistent with reports in the embryonic mouse pituitary and adult rat pituitary (8, 20, 25, 26). There were no changes in SRC-1 or SRC-2 mRNA in the anterior pituitary in late gestation. We have previously shown that GR is down-regulated in the anterior pituitary near term (22). Decreased GR in the anterior pituitary is thought to reduce GC negative feedback on the corticotroph in the approach to parturition, facilitating maintained HPA drive (22). The decrease in GR expression may circumvent the requirement for reduced GR signaling via altered coactivator expression.

Constitutively high SRC expression in the anterior pituitary throughout development may be essential given that several other steroid hormone endocrine systems interact with SRC-1, such as the thyroid and gonadal hormone axes (25, 27, 28, 29, 30) Thyroid hormone, estrogen, and androgen are also endocrine regulators of brain development (31, 32, 33, 34). SRC-1-null mice have been shown to be thyroid hormone resistant via the alteration of thyroid hormone-targeted genes in the pituitary (35, 36, 37). These knockout mice also have mildly impaired reproductive function, as the up-regulation of SRC-2 compensates for the loss of SRC-1 (38). Furthermore, the expression of SRC-1 in the cerebral cortex correlates with high expression of thyroid receptor {alpha}1 and estrogen receptor {alpha} and ß in this area, suggesting that SRC-1 may play a role in thyroid receptor- or estrogen receptor-mediated neuronal differentiation (24, 39, 40). Removal of thyroid hormone in early life can also alter SRC-1 expression and modify sensitivity to circulating thyroid hormone levels, whereas removal of estrogen can alter the distribution and level of brain SRC-1 in a region-specific manner (29, 41). Thus, insight into the ontogeny of the SRC system has implications for signaling of several other nuclear receptors.

In adult rats, hippocampal and anterior pituitary SRC-1 are expressed at higher levels in males than in females (12). We did not observe any gender differences in SRC-1 expression in the fetal hippocampus or the anterior pituitary. In adult male rats, estrogen has been shown to down-regulate SRC-1 in the pituitary (25). In juvenile female rats, SRC-1 levels were found to peak at the onset of puberty in the hypothalamus and to undergo estrous cycle regulation (29). SRCs have also been shown to be critical for the expression of reproductive behaviors in adult rats (38, 42, 43). Perhaps, the onset of detectable SRC-1 mRNA in hypothalamic nuclei occurs postnatally, when sex-specific expression also becomes apparent due to circulating gonadal steroids.

In adults, SRC-1 expression can be regulated by GCs. Acute studies in the adult rat have shown that a single high dose of dexamethasone (5 mg/kg) can cause a transient down-regulation of SRC-1 mRNA in peripheral tissues and the cerebrum (13). Daily injections of high dose dexamethasone (5 mg/kg, 8 d of injections) in adult rats decreased total cerebral SRC-1 mRNA over the first 3 d, but SRC-1 levels were restored to pretreatment values by d 8 (13). These studies did not assess hippocampal, hypothalamic, or pituitary SRC-1 levels after GC exposure. However, another study demonstrated increases in hippocampal SRC-1 mRNA and protein after restraint stress in adult female rats (12), perhaps indicating that increases in circulating GCs can modulate SRC-1 expression.

It is possible that circulating GCs can also affect prenatal expression of SRC. sGCs are routinely administered to pregnant women at risk of preterm delivery to mature the fetal lungs (5). However, many of these women have received multiple doses of GCs without clinical evidence to support such practice (6). Previous studies in rodents and sheep have shown that prenatal exposure to exogenous GCs can alter the trajectory of neuroendocrine development (4, 8, 10, 11). In the current study repeated prenatal exposure to synthetic GC failed to significantly alter hippocampal or pituitary SRC-1 or SRC-2 mRNA expression in near-term fetuses when assessed 24 h after the final treatment with sGC. However, given the time course of GC exposure on SRC-1 expression in the adult rat brain (13), we cannot exclude the possibility that there were transient effects of sGC on SRC-1 mRNA that had recovered 24 h later, on gd 62. It is also possible that regulation of SRC expression in the fetus is quite different from that in adulthood.

Previously, we have shown that repeated prenatal exposure to sGC has no acute effect on hippocampal, hypothalamic, or anterior pituitary GR mRNA levels (10). Our current report of unaltered SRC expression after the same prenatal GC regimen may indicate that fetal GR and its signaling components are more resistant to GC regulation than in the adult (44). However, prenatal exposure to GCs may potentially alter the trajectory of SRC-1 development postnatally, with multiple effects on steroid receptor transactivation in the stress, thyroid, and reproductive axes. Our laboratory has shown that prenatal exposure to sGC alters HPA and gonadal function in adult offspring and that these changes are associated with changes in hippocampal GR and mineralocorticoid receptor (45). Thus, the effects of prenatal sGC exposure on SRC-1 expression may not be immediately observable in fetal life. Longitudinal studies of SRC-1 expression after prenatal exposure to sGC are required.

In summary, we have observed distinct spatial and temporal regulations of SRC-1 at critical points in neuroendocrine development. In the hippocampus, SRC-1 mRNA and protein decreased significantly in late gestation, but SRC-1 and SRC-2 expression in the anterior pituitary was unchanged during late fetal development. Interestingly, although present in the adult, SRC-1 expression is extremely low or absent in the fetal PVN, indicating that SRC-1 may not be critical for GR modulation of PVN function in fetal life. Additionally, our data indicate that hippocampal SRC-1 and pituitary SRC-1 and SRC-2 are not directly regulated by GCs in the fetus, suggesting differential regulation between fetal life and the adult. Developmental changes in the expression of limbic SRC-1 may have important consequences on steroid receptor signaling, which is known to be critical for brain maturation in late gestation.


   Acknowledgments
 
We thank Sonja Banjanin for her assistance with all of the experiments.


   Footnotes
 
This work was supported by Canadian Institute for Health Research Grant MOP-49511 and Premier’s Research Excellence Award (to S.G.M.), M.D./Ph.D. Studentship (to D.O.).

E.S. and D.O. are joint first authors.

Abbreviations: DG, Dentate gyrus; GC, glucocorticoid; gd, gestational day; GR, glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; PBS-T, PBS with Tween 20; PVN, paraventricular nucleus; sGC, synthetic glucocorticoid; SRC, steroid receptor coactivator.

Received December 18, 2003.

Accepted for publication April 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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