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Departments of Physiology (D.O., 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
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Introduction |
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We have shown that repeated antenatal synthetic glucocorticoid (sGC) exposure has sex-specific effects on HPA development in the near-term fetal and adult guinea pig (6, 7). The guinea pig provides a model in which fetal brain development profiles approximate more closely those in the human than do profiles from other small animal species (5). Fetal neuroendocrine development in the guinea pig has also been shown to parallel that described in other species that give birth to mature young (8, 9). In the guinea pig, prenatal exposure to sGC modifies hippocampal mineralocorticoid receptor (MR) mRNA and glucocorticoid receptor (GR) mRNA levels in adult offspring (7). GR mRNA levels were reduced in the paraventricular nucleus (PVN), but were elevated in the hippocampus, in adult female offspring that had been exposed to sGC prenatally. In these animals, hippocampal MR mRNA levels were also significantly lower than control values. These changes resulted in adult female offspring that exhibited significantly increased basal and stimulated cortisol levels during the follicular phase of the reproductive cycle as well as significantly increased adrenal/body weight ratios (7). The changes are strongly supportive of a resetting of glucocorticoid negative feedback in these animals. In adult male offspring that had been exposed to sGC in utero, hippocampal MR mRNA levels were significantly elevated, suggestive of increased glucocorticoid negative feedback sensitivity. This was reflected by reduced basal and activated cortisol levels (7).
Although of considerable clinical importance, the mechanisms that underlie these sex-specific effects of repeated prenatal sGC exposure on subsequent HPA function are not known. Previous studies in the rat have focused on the postnatal ontogeny of brain GR and MR mRNA expression (10, 11, 12). Attempts to investigate the prenatal development of brain GR and MR in the rat are limited by its short gestation period (21 d) and its neurological immaturity at birth compared with that in the human and guinea pig (5). Although detected in the fetal human brain, virtually nothing is known about the developmental regulation of GR and MR. There is also little information on the relationship between corticosteroid receptor mRNA and the corresponding protein in development or the impact of prenatal sGC exposure on GR and MR protein levels. In the current study we hypothesized that 1) there are sex differences in prenatal development of the GR and MR systems in the fetal guinea pig brain; 2) there is a close association between mRNA and mature protein for corticosteroid receptors during late gestation and early postnatal life; and 3) prenatal treatment with sGC [betamethasone (BETA); which specifically activates GR] alters the expression pattern of MR and GR development in the fetal guinea pig brain in a sex-specific manner.
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Prenatal BETA treatment
Pregnant guinea pigs were sc injected with BETA (Betaject, Sabex, Boucherville, Canada; 1 mg/kg; 6 mg/ml) or vehicle (VEH; 166 µl/kg) on gd40/41 and 50/51. Animals were euthanized on gd52 by decapitation, and fetuses were collected. Trunk blood was collected, and fetal body and placental weights were recorded. Whole hippocampi were dissected from the right cerebral hemispheres and frozen. Hippocampi were stored at -80 C until processing for Western blot analysis.
In situ hybridization
The method for in situ hybridization has been described in detail previously (13). Coronal cryosections (10 µm) were mounted onto poly-L-lysine coated slides, dried, and fixed in paraformaldehyde (4%). Previously characterized oligonucleotide probes for GR and MR mRNA (8) were labeled using terminal deoxynucleotidyl transferase (Life Technologies, Inc., Burlington, Canada) and [35S]deoxy-ATP (1300 Ci/mmol; Perkin-Elmer, Woodbridge, Canada) to a specific activity of 1.0 x 109 cpm/µg. Labeled probe in hybridization buffer (200 µl) was applied to slides at a concentration of 1.0 x 103 cpm/µl. The antisense probes were complementary to bases 1–45 of the coding sequence of guinea pig GR mRNA and bases 2942–2986 of the coding sequence of human MR mRNA (14, 15); the guinea pig MR gene has not been sequenced. 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), the slides were rinsed and dehydrated in ethanol. The slides were dried and exposed to autoradiographic film (Biomax MR, Kodak, Perkin-Elmer). Films were developed using an automatic processor (exposure: hippocampal, cortical, and paraventricular GR, 28 d; hippocampal and cortical MR, 14 d).
For in situ hybridization, brain sections were processed simultaneously to allow direct comparison between groups. The sections were exposed together with 14C-labeled standards (American Radiochemicals Inc., 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. Catharines, Canada) (8, 13). Levels of GR mRNA expression were measured in the hippocampus (CA1/2, CA3, and CA4), dentate gyrus, lateral cerebral cortex, and hypothalamic PVN. MR mRNA levels were determined in the hippocampus (as for GR), dentate gyrus, and lateral cerebral cortex.
Western blot analysis
Hippocampi were homogenized in ice-cold RIPA lysis buffer [100–500 µl; 1% Triton X-100, 10% sodium dodecyl sulfate (vol/vol), 0.15 M NaCl, 15.4 mM Tris-HCl, 0.5% deoxycholic acid (wt/vol), 1 µM sodium orthovanadate, and Roche mini-EDTA-free protease inhibitor cocktail, pH 8.0]. The homogenate was centrifuged (4 C, 10,000 x g, 10 min), and the resulting supernatant was recentrifuged. The protein concentration of the resultant supernatant was determined by the Bradford method (16). Laemmli sample buffer (2x; 15 µl; Sigma-Aldrich Corp., Oakville, Canada) 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, Inc., 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 MR antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; H-300, sc-11412) in 5% skim milk PBS-T (1 h, 23 C). Membranes were then washed in PBS-T and incubated with horseradish peroxidase-conjugated goat antirabbit immunoglobulin G (1:1000; 1 h, 23 C; Perkin-Elmer). Blots were washed in PBS-T and exposed to Western Lightning Chemiluminescence Reagent Plus (Perkin-Elmer), and bands were visualized by exposure to Kodak Blue X-OMAT film for 5–30 sec (Perkin-Elmer). Films were developed by an automatic processor. Membranes were stripped in Restore Western Blot Stripping buffer (20 ml, 30 min, 23 C; Pierce Chemical Co., MJS Bioynx, Mississauga, Canada). The blots were blocked overnight in 5% skim milk PBS-T and incubated with an antibody specific for GR
(1:1000; Santa Cruz Biotechnology, Inc.; P-20, sc-1002) as described above. The absolute ODs of MR and GR were analyzed with computerized imaging software. All MR and GR signals were standardized to the signal for the ß-subunit of G protein (Gß; 1:5000; anti-Gß; Santa Cruz Biotechnology, Inc.; M-14, sc-261) or the signal for tubulin (1:5000; antitubulin; Sigma-Aldrich Corp.). The hippocampal expression of Gß did not vary between the ages that we measured, which was consistent with previous studies (17). However, we were concerned that the expression of Gß, a second messenger protein, may be influenced by synthetic glucocorticoids, so we used an alternate internal control (tubulin), in the VEH and BETA-treated animals. 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 expressed as a ratio of GR or MR to Gß/tubulin signal. The small size and anatomical location of the hypothalamic PVN precluded parallel measurement of GR-ir and GR mRNA in this region.
Affinity labeling
Hippocampi were processed for affinity labeling according to the method of Weaver et al. (18). Briefly, hippocampi from gd64 animals were homogenized in ice-cold TEDGM buffer [100–250 µl; 30 mM Tris, 1 mM EDTA, 1 mM dithiothreitol, 10% (wt/vol) glycerol, 10 mM sodium molybdate, and Roche mini-EDTA-free protease inhibitor cocktail, pH 8.3]. The homogenate was centrifuged (100,000 x g, 30 min, 4 C), and the protein concentration was determined by the Bradford method (16). Supernatant (250 µl) was incubated overnight (4 C) with one of the following ligands: 1) 10 nM [3H]dexamethasone mesylate (American Radiochemicals, Inc., St. Louis, MO); 2) 10 nM [3H]dexamethasone mesylate and 100x GR-specific agonist RU28362 (M. J. Meaney, McGill University, Montreal, Canada); and 3) 10 nM [3H]dexamethasone mesylate and 100x unlabeled dexamethasone (Sigma-Aldrich Corp.). Incubates were run through LH-20 columns to remove unbound ligand. The eluate was concentrated by centrifugation (12,000 x g, 30 min, 4 C) in Millipore Ultrafree 0.5 ml centrifugal filters (NLMW 30,000, Fisher Scientific, Whitby, Canada) to a final volume of 15 µl. Concentrated eluate (1 µl, 2 µl) was adjusted to 15 µl with TEDGM buffer. Laemmli sample buffer (2x; 15 µl; Sigma-Aldrich Corp.) was added to concentrated and unconcentrated eluates. Samples were denatured (5 min at 95 C). Protein samples were separated by SDS-PAGE through an 8% resolving polyacrylamide gel. Gels were dehydrated in dimethylsulfoxide and incubated with 22% (wt/vol) 2,5-diphenyloxazole in dimethylsulfoxide (60 min, 23 C). The gels were washed with ultrapure water, dried using a vacuum gel drier at 65 C (Bio-Rad Laboratories, Inc.), and exposed to Kodak MS film (Perkin-Elmer) with a Kodak LE Transcreen (21 d, -70 C).
Alkaline phosphatase treatment
Hippocampi from gd64 animals were homogenized as described for Western blotting, but without sodium orthovanadate (phosphatase inhibitor). The hippocampal extracts were incubated with calf intestinal alkaline phosphatase (CIAP; 1000 U, 1 h, 37 C; Sigma-Aldrich Corp.). The protein concentration of incubates was determined by Bradford assay, and the incubates were dispensed into protein aliquots (50 µg) and frozen (-80 C) until analysis by Western blotting for GR protein as described above. Additionally, 1000 U CIAP were incubated in the presence of p-nitrophenyl phosphate (Sigma-Aldrich Corp.) to illustrate that the enzyme was functional.
Plasma ACTH and cortisol determination
Plasma cortisol and ACTH concentrations were measured using RIA kits (ICN Pharmaceuticals, Inc., Costa Mesa, CA) as previously described (6, 19). The ACTH antibody in this kit demonstrates 100% cross-reactivity with human ACTH1–24 and ACTH1–39 and less than 0.1% cross-reactivity with the smaller ACTH-related molecules. We have characterized this assay using guinea pig ACTH1–39 (Peninsula Laboratories, Inc., San Carlos, CA). Human ACTH1–39 and guinea pig ACTH1–39 provided identical standard curves using the assay kit. Cortisol is the predominant endogenous glucocorticoid in the guinea pig (20).
Statistical analysis
Group data are presented as the mean ± SEM and were statistically analyzed using ANOVA, followed by Duncan’s method of post hoc comparison (Statistica, Statsoft, Inc., Tulsa, OK). Statistical significance was set at P < 0.05.
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) and remained unchanged between gd50 and gd64. Plasma ACTH concentrations decreased significantly after birth (term = 68 d) between gd64 and pnd7 (P < 0.01 for both sexes). There was a significant overall time effect on plasma cortisol concentration before birth (P < 0.0001; Fig. 1B). There was also a significant interaction between age and sex on plasma cortisol levels (P < 0.02). Plasma cortisol concentrations were low on gd40 and gd50, but increased dramatically between gd50 and gd64 (P < 0.0005 for both sexes; Fig. 1B). Plasma cortisol levels decreased significantly after birth between gd64 and pnd7 in males only (P < 0.0005). There was a sex difference in postnatal plasma cortisol levels, with males having significantly lower plasma cortisol levels than females (P < 0.008). The ratio of ACTH/cortisol (A:C) represents a crude index of adrenal sensitivity, with a lower ratio denoting higher sensitivity. There was a significant time effect on A:C during gestation (P < 0.002) as well as a significant interaction with sex for A:C (P < 0.03). A:C was low on gd40, but increased significantly between gd40 and gd50 (P < 0.0005; Fig. 1C) in both sexes, although males had a significantly lower ratio than females on gd50 (P < 0.002). A:C decreased dramatically between gd50 and gd64 (P < 0.0001), indicative of increased adrenal sensitivity. The A:C remained very low after birth.
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). In the PVN (total), GR mRNA levels decreased significantly between gd40 and gd64 in females only (P < 0.05), with levels remaining low postnatally (Table 1). There was a significant overall effect of age on total PVN expression of GR mRNA (P < 0.006). However, there were no sex differences in GR mRNA expression between gd40 and pnd7 in the PVN (Fig. 2B). In the medial parvocellular region of the PVN (mpPVN), GR mRNA levels decreased significantly with advancing gestation in male and female fetuses (P < 0.03; Fig. 2B). In both sexes, GR mRNA levels in the mpPVN were low at the end of gestation and postnatally. There was an overall effect of age on mpPVN GR mRNA levels (P < 0.0001), but no sex difference.
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). There was a significant overall age effect for CA1/2 GR mRNA levels (P < 0.0001; Fig. 3B), but no significant difference between sexes. GR mRNA levels increased significantly between gd40 and gd50 in both sexes (P < 0.001) in this hippocampal region, with levels remaining high to the end of gestation and postnatally. In the CA3 region, GR mRNA levels increased significantly between gd40 and gd50 in females only (P < 0.05), although there was a significant overall age effect (P < 0.04; Table 1
). Similarly, in the CA4 region, GR mRNA expression increased significantly between gd40 and gd50 in females only (P < 0.05). In both sexes levels of GR mRNA remained unchanged after gd50 to the end of gestation and into the postnatal period (Table 1). Males showed no significant change in expression in CA4 at any age. In the dentate gyrus, GR mRNA levels increased significantly on gd64 in females only (P < 0.005) despite an overall age effect on GR mRNA expression in this region (P < 0.0005; Table 1). A significant increase in males was not observed until the postnatal period (P < 0.05; Table 1). There were no sex differences in GR mRNA expression in any of the regions examined.
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). These were eliminated after preabsorption with the antigen peptide (Fig. 4A). The pattern of expression for the 95-kDa band correlated very closely with the GR mRNA levels in the CA1/2 region of the hippocampus, which represents the largest region in the hippocampus. However, GR protein expression lagged behind GR mRNA levels in both sexes (Fig. 3, B and C). Although GR mRNA levels were increased by gd50 in CA1/2, we did not observe an increase in GR protein (95-kDa band) until gd64 (P < 0.005; Fig. 3C). However, we could not pinpoint the length of this lag period, as there was a 14-d interval between our measurements on gd50 and gd64. As expected, there was a significant overall age effect on GR protein expression (P < 0.0001). The pattern of expression for the 115-kDa band did not correspond to that for the 95-kDa band or any of the GR mRNA profiles (data not shown). There were no sex differences in GR protein expression at any point in gestation.
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). Pretreatment with alkaline phosphatase demonstrated that the 115-kDa band is not a phosphorylated form of the 95-kDa GR protein. Preincubation did not change the migration distance in SDS-PAGE (Fig. 4C).
Hippocampal MR expression: development
Levels of MR mRNA were higher on gd40 than at any other time of late gestation or early postnatal life. MR mRNA decreased significantly between gd40 and gd50 in both sexes (P < 0.02) in the CA1/2 region, with levels remaining low to the end of gestation and into postnatal life (Fig. 5A). There was a significant overall age effect on CA1/2 MR mRNA expression (P < 0.0001). In the CA3 region, MR mRNA levels decreased between gd40 and gd50 for both sexes (P < 0.0005), with levels remaining low to the end of gestation and into postnatal life (Table 2). Overall, there was a significant decrease in MR mRNA expression for the CA3 region over time (P < 0.0001). In the CA4 region, MR mRNA decreased significantly on gd64 compared with gd50, but only in females (P < 0.03), with levels of expression unchanged postnatally (Table 2). No significant change in MR mRNA was observed in the CA4 region for male fetuses or neonates. MR mRNA levels in the dentate gyrus were unchanged throughout gestation and postnatally for both sexes (Table 2), although there was a significant interaction with sex (P < 0.05). There were no individual sex differences in MR mRNA expression in any of the other regions examined.
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). The pattern of expression for the MR band correlated well with MR mRNA data from the CA1/2 and CA3 regions of the hippocampus. MR protein levels decreased significantly between gd40 and gd50, but only in males (P < 0.02; Fig. 5B). There was a significant age effect on MR protein expression (P < 0.0001), but no individual changes in MR protein expression were observed in females in late gestation. However, MR protein levels decreased significantly for both sexes after birth, between gd64 and pnd7 (P < 0.04). There were no sex differences in MR protein expression at any point in gestation.
Cortical GR and MR mRNA expression: development
No significant changes in either GR or MR mRNA levels were observed in the cerebral cortex in late gestation or postnatal life (Tables 1 and 2, respectively). There were no sex differences in cortical GR and MR mRNA expression at any age of gestation.
Effect of prenatal betamethasone on fetal brain corticosteroid development
There was no significant effect of BETA treatment or sex on mean litter size (mean ± SEM: BETA, 3.4 ± 0.21 pups; VEH, 4.1 ± 0.37 pups), fetal body weight (BETA female, 36.25 ± 0.99 g; BETA male, 38.01 ± 0.79 g; VEH female, 37.21 ± 0.70 g; VEH male, 39.39 ± 1.42 g), placental weight (BETA female, 4.28 ± 0.18 g; BETA male, 4.31 ± 0.11 g; VEH female, 4.38 ± 0.15 g; VEH male, 4.58 ± 0.22 g), or brain weight (BETA female, 1.65 ± 0.02 g; BETA male, 1.67 ± 0.02 g; VEH female, 1.63 ± 0.02 g; VEH male, 1.66 ± 0.04 g). However, there were significant independent effects of treatment and sex on fetal brain/body weight ratio (BETA female, 0.0458 ± 0.0009; BETA male, 0.0442 ± 0.0009 g; VEH female, 0.0440 ± 0.0006 g; VEH male, 0.0423 ± 0.0008; P < 0.03 and P < 0.05, respectively). There was also a significant interaction with sex on fetal body weight (P < 0.04).
Repeated prenatal BETA exposure markedly suppressed both fetal plasma ACTH and cortisol concentrations on gd52 (P < 0.0005 for both ACTH and cortisol; Fig. 6, A and B). As expected, there was a very significant overall effect of treatment on both plasma ACTH and cortisol levels in these fetuses (P < 0.0001). Interestingly, male fetuses delivered from VEH-treated mothers exhibited a strong tendency for lower plasma cortisol than female fetuses (P = 0.055; Fig. 6B). There was an overall effect of treatment on A:C (P < 0.007; Fig. 6C). There was a significant decrease in A:C (P < 0.006) in BETA-exposed male fetuses compared with VEH males, but no similar effect in females. There was a significant effect of BETA treatment on MR protein expression by ANOVA (P < 0.03). There was also a tendency for MR protein to be down-regulated by prenatal BETA in males (P = 0.06; Table 3). BETA administration did not acutely alter GR protein expression on gd52 (Table 3).
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Discussion |
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The development of GR and MR in the fetal brain is critical for normal maturation of the brain as well as for activation of the fetal HPA axis and the initiation of parturition and organ maturation (3). Many species, including humans, guinea pigs, and sheep, experience a surge in plasma ACTH and cortisol in late gestation (3, 8, 20, 22, 23, 24, 25). This is important for the maturation of several organ systems, including lungs, kidneys, and brain (26, 27). In the current study we observed that plasma cortisol concentrations lagged behind a surge in plasma ACTH levels. This delay is probably a function of increased adrenal sensitivity. In this regard the responsiveness of the fetal guinea pig adrenal to ACTH injections in utero increases dramatically after gd45 (20). Similar increases in adrenal sensitivity have been reported in other species (3, 28, 29). In the fetal sheep, adrenal responsiveness is partly driven by ACTH, which up-regulates key steroidogenic enzymes, and by an increase in ACTH receptor expression (3, 30). There is also evidence for a modification of pituitary ACTH processing, such that levels of bioactive ACTH1–39 are increased in the late gestation fetal sheep (31). Similar changes probably account for increased adrenocortical activity the fetal guinea pig near term.
To sustain the elevated output of plasma cortisol and persistent activation of the HPA axis, negative feedback in the late gestation fetus is decreased. As expected, GR in the mpPVN decreased in the fetal guinea pig to reduce feedback on the axis; a similar trend has been reported in the fetal sheep near term (3). In the hippocampus, GR levels increased in parallel with a decrease in MR expression in late gestation. These changes are consistent with the model that activation of the hippocampal MR results in increased tonic inhibition of the HPA axis, while the hippocampal GR participates in facilitating activation of the HPA axis (4, 32, 33, 34). Thus, the complement of hippocampal corticosteroid receptors was shifted in favor of augmenting HPA output.
We also observed differential developmental patterns of GR and MR mRNA expression in the hippocampal subfields. GR mRNA levels in females increased in all hippocampal subfields before birth, whereas the only increase observed in males, other than in CA1/2, was an increase in the dentate gyrus during the postnatal period. In contrast, MR mRNA levels were decreased in CA1/2 and CA3 for both sexes with advancing gestation, but in CA4, MR mRNA was decreased only in female fetuses. These changes in MR mRNA are highly region specific, as there was no change in the dentate gyrus throughout gestation. In summary, female fetuses experience a greater increase in GR mRNA levels and a greater decrease in MR mRNA in the limbic system in late fetal/early embryonic life. This sex-specific pattern of hippocampal corticosteroid receptor mRNA development may underlie the elevated basal plasma cortisol observed in neonatal and adult female guinea pigs (7).
Although two previous studies have colocalized GR and MR mRNA with the corresponding receptor protein expression in the prenatal rat brain (35, 36), the present study is the first to have quantified the relative levels of mRNA and protein during development. There was very close correlation between MR and GR mRNA and corresponding protein in the hippocampal CA1/2 region. However, the increase in protein lagged behind the rise in mRNA. Unfortunately, the relatively long interval between sampling does not allow us to accurately determine the duration between changes in mRNA and protein.
Two GR-ir bands were revealed on Western blotting, but only the 95-kDa species bound to [3H]dexamethasone mesylate on affinity labeling, indicating that only this band represented a functional GR (18). The inability of the 115-kDa band to competitively bind the GR-specific ligand and an expression profile that did not match GR mRNA levels support this conclusion. When GR is bound to an agonist, there is receptor hyperphosphorylation, which can affect trans-activation activity, autoregulation of the receptor, and receptor half-life (37, 38). We initially hypothesized that the 115-kDa band may be phosphorylated and then targeted for ubiquitinylation and proteosome-mediated protein degradation (37). Further investigation of the 115-kDa band showed that it was not a phosphorylated form of the 95-kDa GR, providing further evidence that the 115-kDa band is not a modified form of the functional GR protein.
The rise in hippocampal GR expression in late gestation, when plasma cortisol concentrations are high, is somewhat counterintuitive, as in adults, increased endogenous GCs down-regulate the receptor in vivo and in vitro (39, 40, 41). However, recent studies indicate that ligand-bound MR may tonically inhibit GR biosynthesis in the hippocampus, possibly by binding to the glucocorticoid response element. There is evidence that spironolactone (MR antagonist) administration in adult rats increases GR mRNA expression in the CA1 region (39). Our results might be consistent with this model, as the rise in hippocampal GR mRNA is coincident with a very significant decline in hippocampal MR mRNA in the CA1 region. Perhaps decreased MR expression allows for elevated GR expression in the hippocampus. GR may also be less vulnerable to autoregulation, as it is relatively less occupied than the higher affinity MR in the basal HPA state (3).
The failure of fetal GR to autoregulate in the presence of high glucocorticoid concentrations may also be due to the immaturity of the autoregulatory machinery itself. GR ubiquitinylation and proteasomal degradation are a key pathway for the clearance of existing GR protein (38). The failure of GR to be ubiquitinylated and degraded decreases GR autoregulation and activates GR transcription (37). A recent study demonstrated that endogenous GR in primary hippocampal neurons derived from embryonic rats cannot be down-regulated because the GR degradation machinery is not fully functional in rat embryonic life (42). This is less likely in the guinea pig, as we have previously shown that sGC can reduce GR mRNA in late embryonic hippocampal neurons in this species (43) and because the guinea pig is so much more neurologically mature in late gestation than the rat.
Understanding the spatial and temporal development of GR and MR in the fetal brain provides insight into critical windows of vulnerability to exogenous prenatal glucocorticoid exposure. Synthetic glucocorticoids are routinely prescribed in women with threatened preterm birth in order to mature the fetal lungs (44, 45). Although a single course is recommended, recent surveys in Britain and Australia indicate that many obstetricians are prescribing multiple doses of glucocorticoids to treat threatened preterm birth (45, 46). Unfortunately, there is no evidence that multiple doses are more effective than a single dose regimen (47). In our laboratory we have demonstrated that repeated exposure to prenatal glucocorticoids permanently alters HPA function and brain corticosteroid receptor expression in adult life (6, 7). In the current study, we have shown that two courses of prenatal BETA dramatically suppresses fetal HPA function. There was a significant treatment effect on hippocampal MR protein expression, with the greatest effect in BETA-treated males. These results differ from previous findings where a similar, but extended, treatment regimen produced no significant treatment effect on either GR or MR mRNA on gd62. The acute effects of sGC on hippocampal MR protein observed in the present study may indicate time- and sex-dependent effects of prenatal glucocorticoid administration. BETA administration had no effect on hippocampal GR protein expression in either sex on gd52. This adds further evidence for failure of the normal GR autoregulatory machinery in the fetus.
In conclusion, the changes in corticosteroid receptor are consistent with and probably critically linked to activation of the fetal pituitary-adrenocortical system near term. In addition, GR and MR exhibit subtle sex differences in their temporal and spatial expression profiles during fetal and early postnatal life. These differences may account for the differences in adrenocortical activity in early postnatal life as well as the gender-specific differences in HPA function and behavior that have been reported in adult offspring after manipulation of the fetal environment. Fetal exposure to synthetic glucocorticoid significantly affected overall hippocampal MR protein expression, and this effect was greatest in males. Both endogenous and exogenous synthetic glucocorticoid fail to autoregulate GR expression in the fetus, indicating that regulation of brain corticosteroid receptors is fundamentally different in the fetus compared with that in adult life.
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Acknowledgments |
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Footnotes |
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Abbreviations: A:C, Ratio of ACTH/cortisol; BETA, betamethasone; CIAP, calf intestinal alkaline phosphatase; gd, gestational day; Gß, ß-subunit of G protein; GR, glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; mpPVN, medial parvocellular region of the paraventricular nucleus; MR, mineralocorticoid receptor; pnd, postnatal day; PBS-T, PBS with Tween 20; PVN, paraventricular nucleus; sGC, synthetic glucocorticoid; VEH, vehicle.
Received December 16, 2002.
Accepted for publication March 24, 2003.
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2 adrenoreceptor coupling with GTP-binding proteins in the rat telencephalon. J Neurochem 53:249–257[CrossRef][Medline]
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 O. Chan, K. Inouye, E. M. Akirav, E. Park, M. C. Riddell, S. G. Matthews, and M. Vranic Hyperglycemia does not increase basal hypothalamo-pituitary-adrenal activity in diabetes but it does impair the HPA response to insulin-induced hypoglycemia Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2005; 289(1): R235 - R246. [Abstract] [Full Text] [PDF]
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R. Catalano, T. Bruckner, J. Gould, B. Eskenazi, and E. Anderson Sex ratios in California following the terrorist attacks of September 11, 2001 Hum. Reprod., May 1, 2005; 20(5): 1221 - 1227. [Abstract] [Full Text] [PDF]
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 E. Setiawan, D. Owen, L. McCabe, A. Kostaki, M. H. Andrews, and S. G. Matthews Glucocorticoids Do Not Alter Developmental Expression of Hippocampal or Pituitary Steroid Receptor Coactivator-1 and -2 in the Late Gestation Fetal Guinea Pig Endocrinology, August 1, 2004; 145(8): 3796 - 3803. [Abstract] [Full Text] |
, Female fetuses and neonates; 
, male fetuses and neonates. Data are expressed as the mean ± SEM. The broken line indicates term (
gd68). Numbers in each bar denote the number of animals in each group. Letters that are the same indicate significant differences (P < 0.05).
