Perinatal Glucocorticoid Treatment Produces Molecular, Functional, and Morphological Changes in the Anterior Pituitary Gland of the Adult Ma
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《内分泌学杂志》
Department of Cellular and Molecular Neuroscience (E.T., C.D.J., J.C.B.), Division of Neuroscience and Mental Health, Imperial College London, London W12 0NN, United Kingdom
Respiratory Health Services Research Group (S.F.S.), National Heart and Lung Institute, Imperial College London, London W6 8RF, United Kingdom
Department of Human Anatomy and Genetics (H.C.C., J.F.M.), University of Oxford, London OX1 3QX, United Kingdom
Abstract
Stress or glucocorticoid (GC) treatment in perinatal life can induce long-term changes in the sensitivity of the hypothalamo-pituitary-adrenocortical axis to the feedback actions of GCs and, hence, in GC secretion. These changes have been ascribed largely to changes in the sensitivity of the limbic system, and possibly the hypothalamus, to GCs. Surprisingly, the possibility that early life stress/GC treatment may also exert irreversible effects at the pituitary level has scarcely been addressed. Accordingly, we have examined the effects of pre- and neonatal dexamethasone treatment on the adult male pituitary gland, focusing on the following: 1) the integrity of the acute annexin 1 (ANXA1)-dependent inhibitory actions of GCs on ACTH secretion, a process requiring ANXA1 release from folliculostellate (FS) cells; and 2) the morphology of FS cells and corticotrophs. Dexamethasone was given to pregnant (d 16–19) or lactating (d 1–7 postpartum) rats via the drinking water (1 μg/ml); controls received normal drinking water. Pituitary tissue from the offspring was examined ex vivo at d 90. Both treatment regimens reduced ANXA1 expression, as assessed by Western blotting and quantitative immunogold labeling. In particular, the amount of ANXA1 located on the outer surface of the FS cells was reduced. By contrast, IL-6 expression was increased, particularly by the prenatal treatment. Pituitary tissue from untreated control rats responded to dexamethasone with an increase in cell surface ANXA1 and a reduction in forskolin-induced ACTH release. In contrast, pituitary tissue from rats treated prenatally or neonatally with dexamethasone was unresponsive to the steroid, although, like control tissue, it responded readily to ANXA1, which readily inhibited forskolin-driven ACTH release. Prenatal dexamethasone treatment reduced the size but not the number of FS cells. It also caused a marked reduction in corticotroph number and impaired granule margination without affecting other aspects of corticotroph morphology. Similar but less marked effects on pituitary cell morphology and number were evident in tissue from neonatally treated rats. Our study shows that, when administered by a noninvasive process, perinatal GC treatment exerts profound effects on the adult pituitary gland, impairing the ANXA1-dependent GC regulation of ACTH release and altering the cell profile and morphology.
Introduction
WIDESPREAD EVIDENCE NOW supports the view that adverse events in early life exert long-term effects on the physiology of the developing organism and thereby increase its susceptibility to disease in adulthood. Barker et al. (1) first noted the correlation between low birth weight and increased risk of cardiovascular and metabolic disorders in later life. Subsequently studies in rodents, pigs, sheep, and humans linked malnutrition and stress in early life to an array of common adult pathologies, including hypertension (2), coronary heart disease (3), impaired glucose tolerance (4), hyperlipidemia, type 2 diabetes mellitus (5), and central nervous system disturbances including anxiety (6). The molecular mechanisms responsible for this early life programming are a focus of much current research with evidence supporting roles for genetic factors (7), nutrients (8), and mediators such as growth factors, cytokines, and hormones (9). Substantial interest has centered on the potential role of glucocorticoids (GCs). This is partly because these stress hormones are obvious candidates to mediate the effects of stress on development (for review, see Ref.10) but also because GCs have growth-regulating properties, and synthetic GCs, i.e. betamethasone, are used in perinatal medicine to mature the lung in conditions of threatened or actual preterm birth (11).
In rats prenatal dexamethasone treatment, given either throughout gestation or during the week before parturition [embryonic days (E) 14–21], produces metabolic, cardiovascular, neuroendocrine, and behavioral changes in adulthood that are broadly comparable with those engendered by prenatal stress (9, 12, 13). The changes in neuroendocrine function are typified by increased activity of the hypothalamo-pituitary-adrenocortical (HPA) axis (12, 13) and, in particular, augmentation of the HPA responses to stress. Because raised glucocorticoid levels and/or altered tissue sensitivity to glucocorticoids are strongly implicated in the etiology of the adult pathologies, which emerge in rats treated preterm with steroids (for review see Ref.14), considerable interest has centered on the mechanisms by which prenatal GC treatments program the overactivity of the HPA axis.
Several groups have shown that administration of GCs during gestation causes region-specific alterations in the expression of brain mineralocorticoid and glucocorticoid receptors (GRs), which vary in profile according to the timing and/or duration of steroid treatment (for review see Ref.14). For example, when given during the last week of gestation (E14–21), dexamethasone has no effect on GR mRNA expression in the adult hypothalamic paraventricular nucleus but reduces hippocampal GR and mineralocorticoid receptor mRNA expression (15). Because the hippocampus is a key site of GC feedback (14), it has been argued that GCs given in late pregnancy induce hyperglucocorticoidemia in the offspring in adulthood by impairing the negative feedback effects of endogenous GCs at the hippocampal level (14, 15). In accord with these findings, adult rats exposed to dexamethasone in utero (E14–21) show increased expression of CRH mRNA in the hypothalamic paraventricular nucleus at adulthood (14, 16), together with exaggerated ACTH and corticosterone responses to stress (14, 16). On the other hand, glucocorticoids given throughout pregnancy augment GR expression in the basolateral nucleus of the amygdala and may thereby augment the positive influence of this structure on HPA activity (15). Neonatal GC treatment also exerts long-term effects on brain GR expression and HPA function in the adult rat. However, whereas the literature is not entirely consistent (17), it appears that the profile of changes differs from that induced by prenatal GC treatment. In particular, the bulk of evidence suggests that dexamethasone or corticosterone treatment in the first week of postnatal life reduces basal and stress-induced HPA activity in adulthood (18, 19) via mechanisms involving increased sensitivity of the HPA axis to GC feedback at a suprapituitary level (20), particularly the hippocampus in which increased GR expression has been reported (21).
Surprisingly, few studies have considered the impact of early life GC treatment on the functional activity of the pituitary gland, despite substantive evidence that the pituitary is an important site of the negative feedback actions of GCs and a major target for dexamethasone. There is, however, evidence that the pituitary shows some degree of compensation for the central suppression of HPA activity induced by neonatal GC treatment, with evidence of decreased pituitary GR binding (19) and substantially prolonged ACTH and corticosterone responses to exogenous CRH (17). To the best of our knowledge, however, the long-term effects of prenatal GC treatment on pituitary function have not been explored.
The feedback actions of GCs at the pituitary level involve suppression of the gene encoding ACTH, proopiomelanocortin, and more immediate effects that attenuate the release of preformed ACTH from the secretory granules (22). Work in our laboratory has identified a key role for annexin 1 (ANXA1) in mediating the early inhibitory effects of GCs on ACTH release (reviewed in Ref.22). ANXA1 is a well-characterized member of a structurally related family of Ca2+- and phospholipid-binding proteins. It is found in abundance in the anterior pituitary gland, in which it is localized to the nonendocrine S100-positive folliculostellate (FS) cells (23) and its expression (24), phosphorylation status (25, 26), and subcellular localization (27, 28) are regulated by GCs. Several lines of evidence have led us to propose that ANXA1 acts as a paracrine/juxtacrine mediator of the inhibitory actions of GC on ACTH release within the anterior pituitary gland (23, 28, 29). First, GCs cause phosphorylation and exportation of ANXA1 from FS cells at loci adjacent to corticotrophs (25, 29). Second, specific, high-affinity ANXA1 binding sites are expressed on the surface of corticotrophs (30). Third, in vitro the acute inhibitory effects of GCs on the CRH-induced release of ACTH from the corticotrophs (25, 27) are mimicked by ANXA1 and a number of ANXA1-derived peptides, whereas drugs that suppress the synthesis (31), cellular exportation (32, 33 , or actions (27) of ANXA1 overcome the acute regulatory effects of GCs on ACTH secretion.
The importance of ANXA1 in mediating the regulatory effects of GCs on ACTH secretion at the pituitary level, together with the evidence that neonatal GC treatment reduces GC binding in the adult rat pituitary gland, has led us to propose that the programming actions of GCs on the adult HPA axis might include disruption of ANXA1-dependent GC actions in the pituitary gland. In the present study, we tested this hypothesis, using a noninvasive method of steroid administration to explore the effects of prenatal and neonatal dexamethasone treatment on the expression, subcellular localization and function of ANXA1 in the pituitary gland of adult male rats. In addition, we examined the impact of the steroid treatments on the morphology of the FS cells and corticotrophs.
Materials and Methods
Animals
All animal work was carried out under license in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Adult male and female Sprague Dawley rats weighing approximately 210 g were purchased for breeding from Harlan Olac, Blackthorn, Bicester, Oxfordshire, UK) and housed in the Comparative Biology Unit (Charing Cross Campus, Imperial College London, UK). Throughout the study, the rats and their progeny were maintained in an environment with controlled lighting (lights on 0800–2000 h) and temperature (21–23 C) with water and food available ad libitum. On arrival at the college, the male and female rats used for breeding were caged separately for approximately a week to allow them to acclimatize to their new environment. Groups of two female and one male rat were then housed together overnight. Mating was confirmed the following morning by the presence of vaginal plugs and, at a later stage (approximately 6 d), pregnancy was confirmed by palpation. Timed pregnant rats were housed five per cage until gestational d 15 when they were isolated in preparation for littering. Near-term pregnant rats were monitored several times per day and, if litters were found, the day of birth was defined as d 0 for that litter.
Perinatal dexamethasone treatments
Batches of pregnant rats and lactating rats were randomly assigned to control or treated groups (n = 4–8 rats per treatment group). Dexamethasone sodium phosphate (David Bull Laboratories, Warwick, UK) was administered via the drinking water (1 μg/ml) to the rats on d 16–19 of pregnancy (E16–19, prenatal treatment) or for 7 d immediately postpartum [(P) 1–7, neonatal treatment]. The developing rats were thus exposed to the steroid in utero via the placenta or as neonates via the mother’s milk. Control dams received normal drinking water throughout pregnancy and lactation. With the exception of routine cleaning, the progeny were left undisturbed until weaning when they were divided according to gender and perinatal treatment and caged in groups of four to five until aged 60–90 d (young adulthood).
Because techniques for measuring the circulating concentrations of dexamethasone in the developing pups (e.g. mass spectrometry) were not available to us, we made estimates of drug delivery to the developing young based on drug intake by the mothers, body mass of the mother, body mass of the neonates, and the following published pharmacokinetic data: bioavailability of the oral dose of approximately 66% (34); volume of distribution of approximately 0.78l/kg (35); a maternal to fetal plasma gradient of approximately 40% (34); a milk to plasma gradient of approximately 40% (36); and an average fluid intake of the neonates of approximately 4.2 ml/d (37). Pregnant rats ingested 50 ± 8 ml drinking water per day during the treatment period. Their intake of dexamethasone sodium phosphate was therefore 50 ± 8 μg/d; using the published pharmacokinetic data, we estimated the plasma concentrations of the steroid in the mother and the developing fetuses to be approximately 105 and 40 ng/ml, respectively. The lactating rats ingested a larger volume of water (54 ± 3 ml/d), i.e. 54 ± 3 μg dexamethasone sodium phosphate per day. This would yield estimated plasma steroid concentrations of approximately 125 and 15 ng/ml, respectively, in the mothers and pups.
Experimental procedures and design
This study employed three experimental approaches, each requiring separate batches of tissue: Western blot analysis of ANXA1 and IL-6 expression; electron microscopy; and in vitro studies. Tissue from a minimum of four litters was always included in each experimental group, and tissue for any given experiment was always collected on the same day to minimize variance. All experiments were conducted on male rats and repeated at least once, using tissue from further cohorts of animals.
Animals were handled regularly for 1 wk before experimental procedures were commenced. When required, they were selected randomly from their cages and killed by decapitation between 0900 and 1000 h to standardize effects associated with the circadian rhythm. After autopsy, the anterior pituitary gland was removed promptly and processed for one of the following: analysis of ANXA1 and IL-6 expression; electron microscopy; or in vitro studies. In some cases the rats were treated with either dexamethasone sodium phosphate (David Bull Laboratories, 20 μg/100 g body weight ip) or an equivalent volume of sterile saline (100 μl/100 g body weight ip) 2.5 h before decapitation (38).
Extraction and detection of annexin 1 and IL-6 by Western blot analysis
ANXA1 was extracted from the pituitary tissue as described previously (38). Briefly, cell surface ANXA1 was removed from the outer cell membranes by washing the tissue gently for 20 min in a solution containing 1 mM EDTA (Sigma Chemical Co., Poole, UK) in PBS [0.05 M (pH 7.4), Oxoid Chemicals Ltd., Hants, UK], which, by chelating Ca2+, releases ANXA1 into the medium from Ca2+-dependent cell surface binding sites. Intracellular ANXA1 was extracted from the remaining tissue by sonication (25 Hz, 20 sec, Soniprep 150; Sanyo Gallenkamp, Leicester, UK) on ice in EDTA (10 mM) containing Triton X-100 [1% (vol/vol); Sigma]. The samples were stored at –80 C until analysis.
To facilitate ANXA1 detection, extracts from four to five animals (littermates from the same cage) were pooled before analysis. At least four separate pooled samples (i.e. samples from four litters) were analyzed per experiment. The protein content of each pooled sample was determined (39), and, in the case of the cell surface EDTA washes, if it fell less than 1 μg/ml, the samples were concentrated to 20 μg total protein per 20 μl using a centrifugal filter microconcentrator with a 10-kDa cut-off (Microcon; Amicon, Inc., Beverly, MA). The samples were then analyzed for ANXA1 by SDS-PAGE and subsequent Western blotting, using the method of Taylor et al. (31) and a well-characterized polyclonal anti-ANXA1 antibody [anti-ANXA1 polyclonal antibody (pAb), raised in sheep against the full-length human recombinant ANXA1 and diluted 1:10,000] (31) as a probe. The blots were scanned using a flatbed scanner [HP Scanjet 5200 (Hewlett Packard Ltd., Bracknell, UK) with Adobe Photodeluxe Business Edition, version 1.1 (Adobe Systems UK, Oxbridge, UK)] and the band intensity analyzed using the TINA software program (TINA version 2.10; Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany). Levels of IL-6 were also examined in the tissue extracts by Western blot analysis, using the same protocol and a commercial anti-IL-6 antibody (R & D Systems, Abingdon, UK) diluted to 1 μg/ml.
Electron microscopy: morphological studies and detection and quantification of ANXA1 by immunogold histochemistry
Pituitary tissue from male rats was collected immediately after decapitation and fixed in 3% (wt/vol) paraformaldehyde, 0.05% (vol/vol) glutaraldehyde (VWR International Ltd., Lutterworth, UK) in PBS (Oxoid Chemicals) (pH 7.2) for 4 h at room temperature. The tissue was then processed exactly as described by Traverso et al. (23), and ultrathin sections were prepared and mounted onto formvar-coated mesh nickel grids (Agar Scientific Ltd., Stanstead, UK) and viewed with a JEOL 1010 transmission electron microscope (JEOL, Peabody, MA).
The corticotrophs were identified by immunogold labeling of ACTH, using rabbit antirat ACTH pAb (diluted 1:250; National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA) as a probe. The FS cells were identified by both their positivity for the FS cell marker, S100 (detected by immunogold labeling using a rabbit antirat S100 pAb, diluted 1:100; Dako Corp., Cambridge, UK) and their morphological characteristics, which typically include an agranular cytoplasm and long cytoplasmic processes that surround and make contact with neighboring endocrine cells.
For analysis of cell morphology by point counting (40), 10 micrographs (magnification, x4000) of corticotrophs and FS cells per animal were taken from three to four sections per pituitary gland. The negatives were scanned into Adobe Photoshop (version 5.5) and printed onto A4 paper for analysis. In all cases the reader was blind to the sample code. To determine total and nuclear cell areas, a 1-cm grid acetate sheet was placed at random over the cell and the number of grid intersections that overlaid the cell was recorded. The cytoplasmic area was determined by subtracting the nuclear area from the total cell area and multiplying the result by an area conversion factor of 1.23 because on each micrograph 1 cm2 represented 1.23 μm2. In corticotrophs, granule diameter, density, and granule margination (the ratio of granules at the perimeter of the cell to those in the cell interior) were also measured. Mean granule diameter was determined by the overlay and match of circles of known diameter onto granules in the cell for 30 granules per cell. To determine granule areal density, a 0.7-cm grid was used: the number of granules that overlaid an intersection was counted, adjusted by the areal conversion factor, and divided by the cytoplasmic area of the cell. The perimeter of each corticotroph was defined as the area from the plasma membrane to a margin 2 μm into the cell interior drawn on each cell around the circumference. The ratio of the number of granules in the cell perimeter (counted as the number of granules that overlaid an intersection on the 0.7-cm grid) to the number within the cell interior was determined. Tissue from four animals per group, each from a separate litter, was examined in each experiment.
The percentages of corticotrophs and FS cells in the pituitary were quantified by counting the number of ACTH and S100 immunogold-positive cells and total number of nucleated cells, dividing the ACTH and S100-positive cell number by the total number of nucleated cells, and converting to a percentage. Six complete grid squares in three sections taken from different depths of the tissue block were counted per animal (i.e. 18 grid squares in total). Sections from four animals per group, each from a separate litter, were examined in each experiment.
For detection of ANXA1, grids prepared as above were incubated for 2 h with anti-ANXA1 pAb (diluted 1:200 in 0.1 M phosphate buffer containing 1% egg albumin) and 1 h with protein A-15 nm gold complex (Biocell, Cardiff, UK) and then lightly stained with uranyl acetate and lead citrate (23). Specificity of the antibody staining was confirmed by use of preadsorbed antibody (100-fold excess human recombinant ANXA1) and albumin-containing buffer in place of the primary antiserum. In some cases the sections were double stained for S100 (marker of FS cells) using a specific rabbit antirat antibody (diluted 1:100, Dako) and protein A (5 nm) gold complex (Biocell). For quantification of ANXA1 staining, the number of 15-nm gold particles over each compartment was counted and calculated as particles per square micrometer by dividing the total number of gold particles counted by the relevant area. Six sections per animal were analyzed. For each experiment, tissue from four animals per group, each from a different litter, was used.
In vitro studies
Anterior pituitary tissue from male rats was incubated as described by John et al. (25). Briefly, each gland was divided into four segments of approximately equal size and distributed to the wells of 24-well tissue culture plates (Costar, Cambridge, MA). Each animal contributed one segment per treatment group and each group comprised tissue from several different litters. The tissue was incubated (one segment per well) in 1.5 ml of Earle’s balanced salt solution (Sigma) enriched with a protease inhibitor, 1% aprotinin (Bayer PLC Ltd., Newbury, UK), for 2 h at 37 C in a humidified atmosphere with 95% O2-5% CO2. The segments were then transferred to fresh medium and incubated for a further hour in the presence or absence of the adenyl cyclase activator, forskolin (100 μM; Sigma). The medium was then collected and stored at –20 C for determination of ACTH; the pituitary segments were weighed on a torsion balance and discarded. Where appropriate, dexamethasone sodium phosphate (0.1 μM, David Bull Laboratories) or an ANXA1-derived peptide, ANXA1Ac2–26 (0.2–20 μg/ml), were included throughout the preincubation and final incubations periods.
Determination of ACTH
ACTH was determined in duplicate by RIA according to a published method (41) using a well-characterized primary antibody raised in the rabbit against human ACTH1–39 (code AFP6328031, National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA), human ACTH1–39 as a standard (National Institute of Diabetes and Digestive and Kidney Diseases) and 125I-labeled ACTH1–39 as the tracer. Separation of the bound and free peptide was achieved by the addition of 100 μl goat antirabbit decanting suspension (Pharmacia Upjohn, Buckinghamshire, UK). The inter- and intraassay variations were 11.1 and 10.7%, respectively.
Statistical analysis
For the morphological studies and quantification of ANXA1 by immunogold labeling, Student’s t test was used to assess the differences between normally distributed sets of data with approximately equal variance; the data on individual cells from each animal were pooled into their experimental groups before the averages and SEM were calculated. This is standard practice in morphological analysis to streamline the sampling process without compromising the statistical integrity of the results (42). Granule distribution between the perimeter and interior of cells was assessed by the 2 test, and the odds ratio analysis was used to quantify the change in ratio of peripheral granules to interior granules. Functional studies were analyzed by two-way ANOVA with post hoc comparisons by Duncan’s multiple range test. Because the rate of basal ACTH release in vitro varied between experiments, statistical comparisons were made within experiments only. In all cases, differences were considered to be significant if P < 0.05.
Results
ANXA1 and IL-6 expression in the anterior pituitary gland
ANXA1 was also readily detected in anterior pituitary tissue from control and steroid-treated rats by immunogold labeling. The staining was localized exclusively to the FS cells, which were identified by morphological criteria and S100 labeling (Fig. 2A). Quantitative analysis of the ANXA1 staining by counting the gold particles confirmed the semiquantitative data obtained from Western blot analysis, viz. neonatal dexamethasone treatment reduced the amount of ANXA1 in both the intracellular (cytoplasmic, Fig. 2C, P < 0.05) and the cell surface (Fig. 2D, P < 0.01) pools, whereas the prenatal treatment reduced cell surface (Fig. 2D, P < 0.01) but not intracellular (Fig. 2C, P < 0.01) ANXA1. The data also revealed that the total pool of ANXA1 in the FS cells at adulthood is reduced by both prenatal (P < 0.05) and neonatal (P < 0.01) dexamethasone treatment (Fig. 2B).
Functional studies
These experiments aimed to explore the impact of perinatal dexamethasone treatment on the integrity of the ANXA1-dependent inhibitory actions of dexamethasone on ACTH secretion in adulthood. Figure 3, A and B, demonstrate the effects of prenatal (E16–19) and neonatal (P1–7) dexamethasone treatment on the ability of dexamethasone (20 μg per 100 g body weight ip) in adulthood to translocate ANXA1 to the outer cell surface, a process that is critical to the paracrine mode of ANXA1 inhibition of ACTH secretion (38, 43). In tissue from adult drug-nave rats, administration of dexamethasone 2.5 h before autopsy caused the expected increase in cell surface ANXA1 (Fig. 3, A and B, lanes 3 and 4 vs. lanes 1 and 2). However, in rats treated prenatally or neonatally with dexamethasone, the ability of dexamethasone to cause the translocation of ANXA1 to the cell surface was markedly impaired (Fig. 3, A and B, lanes 7 and 8 vs. lanes 5 and 6). Pituitary tissue from the drug-nave control rats responded in vitro to the adenyl cyclase activator, forskolin, with a significant increase in ACTH release (P < 0.01, Fig. 4, A and B). This response was inhibited by preincubation of the tissue with dexamethasone (100 nM, P < 0.05), which, alone, had no effect on resting ACTH release (Fig. 4, A and B). By contrast, dexamethasone failed to inhibit forskolin-induced ACTH release in pituitary tissue from rats treated prenatally (E16–19, Fig. 4A) or neonatally (P1–7, Fig. 4B) with the steroid, although the tissue responded normally to forskolin.
Like dexamethasone, the N-terminal ANXA1 peptide ANXA1Ac2–26 (2.0 and 20 μg/ml) suppressed the forskolin-induced release of ACTH from pituitary tissue from drug-nave rats without affecting basal ACTH release. A lower concentration of the peptide (0.2 μg/ml) was without effect (Fig. 5). ANXA1Ac2–26 also blocked forskolin-induced ACTH release from pituitary tissue from animals given dexamethasone in pre- or neonatal life and, in contrast to the controls, was effective at all concentrations tested (0.2–20 μg/ml). Paradoxically, however, and also in contrast to the controls, the peptide showed signs of augmenting basal ACTH release in these groups, and, in the case of the neonatal treatment, the increment in ACTH reached significance at a peptide concentration of 2 μg/ml (P < 0.05, Fig. 5).
Pituitary morphology
The effects of perinatal dexamethasone treatment on the morphology and number of FS cells and corticotrophs in the pituitary gland at adulthood are shown in Figs. 6–8. Both prenatal (E16–19) and neonatal (P1–7) dexamethasone treatment produced long-term changes in the morphology (Fig. 6, A–C), but not the number (Fig. 6D), of FS cells. The prenatal treatment reduced the total cell area (Fig. 6A, P < 0.05) and the cytoplasmic area (Fig. 6B, P < 0.01) without affecting the nuclear area (Fig. 6C, P > 0.05). The neonatal treatment also reduced the cytoplasmic area (P < 0.05) but had no significant effect on either the total cell area (Fig. 6A) or the nuclear size (Fig. 6C).
By contrast, neither treatment regimen affected the total cell area, nuclear area, or cytoplasmic area of the corticotrophs (P > 0.05, data not shown). The drug treatments also failed to affect granule diameter and granule density (as a percentage of the cytoplasmic area, P > 0.05, data not shown). However, the prenatal, but not the neonatal, treatment regimen reduced the margination of the granules to the cell membrane (odds ratio 0.765, 95% confidence interval 0.588–0.997, P < 0.05, Fig. 7, A–C). In addition, both treatments produced a significant reduction in the number of corticotrophs (expressed as a percentage of the total population of nucleated cells: prenatal, P < 0.01; neonatal, P < 0.05, Fig. 7). The latter finding was confirmed by a limited screen at the light microscopic level using the anti-ACTH antibody employed for immunogold labeling as a probe (data not shown).
Discussion
The results presented show for the first time that perinatal dexamethasone treatment produces long-term changes in the morphology of the anterior pituitary gland and concomitant disruption of the ANXA1-dependent inhibitory effects of dexamethasone on the secretion of ACTH. They thus support the premise that early life glucocorticoid treatment leads to disturbances of neuroendocrine function in adulthood, which may compromise homeostasis and thereby influence disease susceptibility in adulthood.
Drug treatment regimens
Our study examined and compared the impact of two discrete periods of early life dexamethasone treatment, viz. prenatal and neonatal, on the pituitary gland at adulthood. Both treatment regimens coincided with critical phases of pituitary development. The prenatal treatment (E16–18) started shortly after the initial differentiation of the corticotrophs (E13/14) (44, 45) at the time (E16) when ACTH synthesis (46) and glucocorticoid feedback first emerge (47) and, allowing for drug clearance (t1/2 dexamethasone 36–54 h), the period of maximal proliferative activity of the developing corticotroph population (E17.5–20.5) (48). By contrast, the neonatal treatment (P1–7) encompassed the onset (P5) of the neonatal stress hyporesponsive period, a period during which the HPA axis is refractory to stress and the anterior pituitary gland shows a supersensitivity to the negative feedback actions of GCs on ACTH release (49) and the reported time (P6) at which S100-positive FS cells are first apparent (50).
Previous studies on the impact of perinatal GC treatment on adult physiology have involved administration of the steroid by injection of either the pregnant mother or the newborn pups. In preliminary studies, we adopted a similar approach but found that the injection process itself, particularly in the pups, constituted a severe stress and produced changes in the adult tissues that, in some cases, were indistinguishable from those induced by the steroid (data not shown). To circumvent these problems, we administered dexamethasone via the drinking water to pregnant or lactating dams. This noninvasive approach took advantage of the effectiveness of dexamethasone by mouth and ability of the steroid to cross the placenta (51) and enter the milk (52) for delivery to the developing young. Unfortunately, we were not able to measure the concentrations of dexamethasone in the developing pups. However, by using measures of maternal drug intake and body weight together with published pharmacokinetic data (see Materials and Methods), we estimated that the plasma concentrations of the steroid were in the region of 40 ng/ml in the developing fetuses (prenatal treatment) and 15 ng/ml in the pups (neonatal treatment). Given the assumptions in the calculations, the variance in fluid intake in the dams (16%) and other interanimal variables (e.g. milk intake), we would be cautious in drawing any conclusions about the apparent differences in the plasma concentrations between the two groups. However, it is worth noting that dexamethasone is a highly potent steroid (40 times more active than corticosterone); thus, the levels attained would be comparable with the GC levels attained in severe stress. Furthermore, allowing for the route-dependent differences in bioavailability, our dosing regimen is comparable with the standard dose (5 mg im) used in perinatal medicine.
Morphological, molecular, and functional studies
FS cells.
Our morphological studies confirmed reports (53) that the FS cells comprise 5–10% of the total cell population of the adult rat anterior pituitary gland. They also revealed that perinatal dexamethasone treatment has no effect on FS cell number in adulthood but causes a marked reduction in the cytoplasmic area of the cells and, in the case of the perinatal treatment, a reduction in total cell size. It is not clear whether these changes reflect direct actions of the exogenous steroid on the developing pituitary gland or whether they are secondary to other changes induced by the treatment. Because data from immunohistochemical studies suggest that the FS cells do not emerge developmentally until P6 (50), it is possible that dexamethasone targeted a progenitor population of pituitary FS cells and altered its pattern of development. The functional implications of the steroid-induced change in FS cell size require further study. These cells influence multiple facets of pituitary function by releasing mediators (e.g. growth factors, cytokines), which regulate adjacent cells by paracrine or juxtacrine mechanisms. Because release of these mediators occurs by assisted transfer across the cell membrane (54), their release may be compromised by a reduction in cell surface area.
ANXA1.
Our data show for the first time that early life GC treatment has a significant effect on the expression of two FS cell proteins (IL-6 and ANXA1) known to influence the corticotrophs and other pituitary endocrine cells. With regard to ANXA1, Western blot analysis and quantitative immunogold labeling revealed that both pre- and neonatal dexamethasone treatment caused a substantial reduction in cell surface ANXA1; in addition, the neonatal, but not the prenatal, treatment regimen reduced the intracellular content of the protein. These changes could not be attributed to a generalized reduction in protein synthesis because IL-6 expression was increased, particularly by the prenatal treatment regimen. Standardization of the measures of gold particles to unit area also indicated that the reduction in cell surface ANXA1 could not be explained by the reduction in FS cell surface area. Because ANXA1 plays a key role as a paracrine/juxtacrine mediator of the early phase of GC feedback at the pituitary level (22), the reduction in ANXA1 protein raised the possibility that this mode of feedback may be compromised by early-life GC treatment. Our functional studies and analysis of corticotroph morphology support this view. Thus, both perinatal steroid treatment regimens impaired the capacity of dexamethasone to cause the translocation of ANXA1 to the surface and inhibit the forskolin-evoked release of ACTH from adult pituitary tissue. They also caused increased granule margination in the corticotrophs, a morphology that is indicative of a hypersecretory state (55) and is therefore consistent with a state of impaired glucocorticoid feedback. Because the exportation of ANXA1 from FS cells is an essential step in the manifestation of the ANXA1-dependent inhibitory effects of GCs on ACTH release (29, 31), it seems likely that the programming actions of the steroids are effected in part by disruption of this critical mechanism. Exactly how this disruption is achieved is unclear. We did not measure GR expression in the FS cells of the drug treated animals, and we are unaware of evidence perinatal dexamethasone treatment alters pituitary GR in male rats, although there is evidence that such treatment reduces GR expression in the female (56). A reduction in GR would certainly be expected to attenuate GC action, but it would be unlikely to account for the almost total failure of dexamethasone to induce the cellular exportation of ANXA1. Depletion of substrate would also not explain the apparent failure of the ANXA1 export mechanism because, although the neonatal treatment regimen reduced intracellular ANXA1, the prenatal treatment did not.
It thus seems likely that the expression and/or activity of other components of the signaling cascade used by GCs to export ANXA1 may also have been affected by the early life treatment; potential candidates include kinases (26, 27) and other proteins, e.g. ABCA1 (33), and these now require investigation. Interestingly, our in vitro studies show that pituitary tissue from the steroid-treated animals retains its sensitivity to ANXA1, suggesting that the ANXA1 receptors and downstream signaling mechanisms were not down-regulated by the early-life GC treatment. To the contrary, the finding that unlike the controls both steroid-treated groups responded to the lowest concentration of peptide tested (0.2 μg/ml) suggests ANXA1 sensitivity is enhanced, a finding that could reflect early life programming of the signaling system or could be a secondary compensation to the reduction in ANXA1 drive to the receptors. The paradoxical finding that the ANXA1 peptide increased basal ACTH release from pituitary tissue from steroid-treated rats also accords with a state of increased tissue sensitivity to ANXA1 because high concentrations of the peptide facilitate basal, but not stimulated, ACTH release (27).
Corticotrophs.
The finding that perinatal administration of dexamethasone caused a marked reduction in corticotroph numbers was surprising. Corticotroph turnover is dependent in part on the mitogenic actions of CRH (57) and the antiproliferative and proapoptotic actions of GCs (58). However, as discussed above, pituitary GR expression, and therefore GC sensitivity, is reduced by neonatal GC treatment (17). Moreover, although there are reports to the contrary (56), substantial evidence suggests that prenatal, although not neonatal, GC treatment augments the CRH drive to the pituitary gland (15, 20). On the basis of these findings, it might be predicted that perinatal, and particularly prenatal, GC treatment would increase the size of the corticotroph population. In addition, because ANXA1 exerts both antiproliferative (59) and proapoptotic (60) actions, the reduction in the expression and cellular exportation of ANXA1 might also be expected to increase, not decrease, corticotroph numbers. However, other factors derived within the pituitary gland also influence corticotroph proliferation, in particular growth factors (61) and cytokines (62, 63) derived from the FS cells. Of the cytokines, both IL-6 (63) and leukemia inhibitory factor (62) exert proliferative actions, the latter directing progenitor cells toward the corticotroph lineage (62). Because pituitary IL-6 expression is augmented by perinatal dexamethasone treatment, it seems unlikely that the loss of corticotrophs is due to IL-6 withdrawal.
We cannot ascertain from our data when the changes in the corticotroph population first emerged. However, because the prenatal treatment coincided with the period of maximal corticotroph proliferation (E17.5–20.5) (48), it is possible that the steroid acted at this time to halt the differentiation and/or proliferation of progenitor cells, perhaps via a reduction in leukemia inhibitory factor. On the other hand, the adult corticotroph population is surprisingly labile, i.e. acute stress (novel environment) increases the population by 20% (64), and it is conceivable changes reported here are effected in adulthood. The functional consequences of the reduction in the corticotroph population are not clear. The ACTH reserve in the pituitary is substantial (65), and our data indicate that the secretory activity of the cells is enhanced by perinatal GC treatment, as also do other studies, which show exaggerated HPA responses to stress (15) and increased sensitivity of pituitary tissue to CRH (17).
Conclusions
Our study shows that, when administered by a noninvasive process in prenatal and neonatal life, GCs exert profound effects on the adult rat pituitary gland, impairing the ANXA1-dependent GC regulation of ACTH release and altering the cell profile and morphology. The changes caused by the prenatal treatment regime would be expected to contribute to the hyperactivity in HPA function associated with such treatment, which, until now, has been largely attributed to changes in the brain, particularly the hippocampus and hypothalamus (14, 15). However, because the bulk of evidence available indicates that neonatal GC treatment reduces the CRH/arginine vasopressin drive to the pituitary gland (18, 19, 21, 66), the changes in pituitary function reported here may be compensatory. The significance of the changes in IL-6 expression requires further exploration. However, because IL-6 has a positive effect on ACTH secretion and its release from FS cells is modified by GCs (67), the increased expression of the cytokine may serve to augment ACTH release, particularly in conditions of immune stress, i.e. after a bacterial challenge.
Acknowledgments
We are grateful to the National Institute of Diabetes and Digestive and Kidney Diseases for reagents for the ACTH assay, and Colin Rantle for excellent technical support.
Footnotes
This work was supported by the Biotechnology and Biological Sciences Research Council.
1 E.T. and C.D.J. contributed equally to the study.
Abbreviations: ANXA1, Annexin 1; E, embryonic day; FS, folliculostellate; GC, glucocorticoid; GR, glucocorticoid receptor; HPA, hypothalamo-pituitary-adrenocortical; P, postpartum day; pAb, polyclonal antibody.
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Respiratory Health Services Research Group (S.F.S.), National Heart and Lung Institute, Imperial College London, London W6 8RF, United Kingdom
Department of Human Anatomy and Genetics (H.C.C., J.F.M.), University of Oxford, London OX1 3QX, United Kingdom
Abstract
Stress or glucocorticoid (GC) treatment in perinatal life can induce long-term changes in the sensitivity of the hypothalamo-pituitary-adrenocortical axis to the feedback actions of GCs and, hence, in GC secretion. These changes have been ascribed largely to changes in the sensitivity of the limbic system, and possibly the hypothalamus, to GCs. Surprisingly, the possibility that early life stress/GC treatment may also exert irreversible effects at the pituitary level has scarcely been addressed. Accordingly, we have examined the effects of pre- and neonatal dexamethasone treatment on the adult male pituitary gland, focusing on the following: 1) the integrity of the acute annexin 1 (ANXA1)-dependent inhibitory actions of GCs on ACTH secretion, a process requiring ANXA1 release from folliculostellate (FS) cells; and 2) the morphology of FS cells and corticotrophs. Dexamethasone was given to pregnant (d 16–19) or lactating (d 1–7 postpartum) rats via the drinking water (1 μg/ml); controls received normal drinking water. Pituitary tissue from the offspring was examined ex vivo at d 90. Both treatment regimens reduced ANXA1 expression, as assessed by Western blotting and quantitative immunogold labeling. In particular, the amount of ANXA1 located on the outer surface of the FS cells was reduced. By contrast, IL-6 expression was increased, particularly by the prenatal treatment. Pituitary tissue from untreated control rats responded to dexamethasone with an increase in cell surface ANXA1 and a reduction in forskolin-induced ACTH release. In contrast, pituitary tissue from rats treated prenatally or neonatally with dexamethasone was unresponsive to the steroid, although, like control tissue, it responded readily to ANXA1, which readily inhibited forskolin-driven ACTH release. Prenatal dexamethasone treatment reduced the size but not the number of FS cells. It also caused a marked reduction in corticotroph number and impaired granule margination without affecting other aspects of corticotroph morphology. Similar but less marked effects on pituitary cell morphology and number were evident in tissue from neonatally treated rats. Our study shows that, when administered by a noninvasive process, perinatal GC treatment exerts profound effects on the adult pituitary gland, impairing the ANXA1-dependent GC regulation of ACTH release and altering the cell profile and morphology.
Introduction
WIDESPREAD EVIDENCE NOW supports the view that adverse events in early life exert long-term effects on the physiology of the developing organism and thereby increase its susceptibility to disease in adulthood. Barker et al. (1) first noted the correlation between low birth weight and increased risk of cardiovascular and metabolic disorders in later life. Subsequently studies in rodents, pigs, sheep, and humans linked malnutrition and stress in early life to an array of common adult pathologies, including hypertension (2), coronary heart disease (3), impaired glucose tolerance (4), hyperlipidemia, type 2 diabetes mellitus (5), and central nervous system disturbances including anxiety (6). The molecular mechanisms responsible for this early life programming are a focus of much current research with evidence supporting roles for genetic factors (7), nutrients (8), and mediators such as growth factors, cytokines, and hormones (9). Substantial interest has centered on the potential role of glucocorticoids (GCs). This is partly because these stress hormones are obvious candidates to mediate the effects of stress on development (for review, see Ref.10) but also because GCs have growth-regulating properties, and synthetic GCs, i.e. betamethasone, are used in perinatal medicine to mature the lung in conditions of threatened or actual preterm birth (11).
In rats prenatal dexamethasone treatment, given either throughout gestation or during the week before parturition [embryonic days (E) 14–21], produces metabolic, cardiovascular, neuroendocrine, and behavioral changes in adulthood that are broadly comparable with those engendered by prenatal stress (9, 12, 13). The changes in neuroendocrine function are typified by increased activity of the hypothalamo-pituitary-adrenocortical (HPA) axis (12, 13) and, in particular, augmentation of the HPA responses to stress. Because raised glucocorticoid levels and/or altered tissue sensitivity to glucocorticoids are strongly implicated in the etiology of the adult pathologies, which emerge in rats treated preterm with steroids (for review see Ref.14), considerable interest has centered on the mechanisms by which prenatal GC treatments program the overactivity of the HPA axis.
Several groups have shown that administration of GCs during gestation causes region-specific alterations in the expression of brain mineralocorticoid and glucocorticoid receptors (GRs), which vary in profile according to the timing and/or duration of steroid treatment (for review see Ref.14). For example, when given during the last week of gestation (E14–21), dexamethasone has no effect on GR mRNA expression in the adult hypothalamic paraventricular nucleus but reduces hippocampal GR and mineralocorticoid receptor mRNA expression (15). Because the hippocampus is a key site of GC feedback (14), it has been argued that GCs given in late pregnancy induce hyperglucocorticoidemia in the offspring in adulthood by impairing the negative feedback effects of endogenous GCs at the hippocampal level (14, 15). In accord with these findings, adult rats exposed to dexamethasone in utero (E14–21) show increased expression of CRH mRNA in the hypothalamic paraventricular nucleus at adulthood (14, 16), together with exaggerated ACTH and corticosterone responses to stress (14, 16). On the other hand, glucocorticoids given throughout pregnancy augment GR expression in the basolateral nucleus of the amygdala and may thereby augment the positive influence of this structure on HPA activity (15). Neonatal GC treatment also exerts long-term effects on brain GR expression and HPA function in the adult rat. However, whereas the literature is not entirely consistent (17), it appears that the profile of changes differs from that induced by prenatal GC treatment. In particular, the bulk of evidence suggests that dexamethasone or corticosterone treatment in the first week of postnatal life reduces basal and stress-induced HPA activity in adulthood (18, 19) via mechanisms involving increased sensitivity of the HPA axis to GC feedback at a suprapituitary level (20), particularly the hippocampus in which increased GR expression has been reported (21).
Surprisingly, few studies have considered the impact of early life GC treatment on the functional activity of the pituitary gland, despite substantive evidence that the pituitary is an important site of the negative feedback actions of GCs and a major target for dexamethasone. There is, however, evidence that the pituitary shows some degree of compensation for the central suppression of HPA activity induced by neonatal GC treatment, with evidence of decreased pituitary GR binding (19) and substantially prolonged ACTH and corticosterone responses to exogenous CRH (17). To the best of our knowledge, however, the long-term effects of prenatal GC treatment on pituitary function have not been explored.
The feedback actions of GCs at the pituitary level involve suppression of the gene encoding ACTH, proopiomelanocortin, and more immediate effects that attenuate the release of preformed ACTH from the secretory granules (22). Work in our laboratory has identified a key role for annexin 1 (ANXA1) in mediating the early inhibitory effects of GCs on ACTH release (reviewed in Ref.22). ANXA1 is a well-characterized member of a structurally related family of Ca2+- and phospholipid-binding proteins. It is found in abundance in the anterior pituitary gland, in which it is localized to the nonendocrine S100-positive folliculostellate (FS) cells (23) and its expression (24), phosphorylation status (25, 26), and subcellular localization (27, 28) are regulated by GCs. Several lines of evidence have led us to propose that ANXA1 acts as a paracrine/juxtacrine mediator of the inhibitory actions of GC on ACTH release within the anterior pituitary gland (23, 28, 29). First, GCs cause phosphorylation and exportation of ANXA1 from FS cells at loci adjacent to corticotrophs (25, 29). Second, specific, high-affinity ANXA1 binding sites are expressed on the surface of corticotrophs (30). Third, in vitro the acute inhibitory effects of GCs on the CRH-induced release of ACTH from the corticotrophs (25, 27) are mimicked by ANXA1 and a number of ANXA1-derived peptides, whereas drugs that suppress the synthesis (31), cellular exportation (32, 33 , or actions (27) of ANXA1 overcome the acute regulatory effects of GCs on ACTH secretion.
The importance of ANXA1 in mediating the regulatory effects of GCs on ACTH secretion at the pituitary level, together with the evidence that neonatal GC treatment reduces GC binding in the adult rat pituitary gland, has led us to propose that the programming actions of GCs on the adult HPA axis might include disruption of ANXA1-dependent GC actions in the pituitary gland. In the present study, we tested this hypothesis, using a noninvasive method of steroid administration to explore the effects of prenatal and neonatal dexamethasone treatment on the expression, subcellular localization and function of ANXA1 in the pituitary gland of adult male rats. In addition, we examined the impact of the steroid treatments on the morphology of the FS cells and corticotrophs.
Materials and Methods
Animals
All animal work was carried out under license in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Adult male and female Sprague Dawley rats weighing approximately 210 g were purchased for breeding from Harlan Olac, Blackthorn, Bicester, Oxfordshire, UK) and housed in the Comparative Biology Unit (Charing Cross Campus, Imperial College London, UK). Throughout the study, the rats and their progeny were maintained in an environment with controlled lighting (lights on 0800–2000 h) and temperature (21–23 C) with water and food available ad libitum. On arrival at the college, the male and female rats used for breeding were caged separately for approximately a week to allow them to acclimatize to their new environment. Groups of two female and one male rat were then housed together overnight. Mating was confirmed the following morning by the presence of vaginal plugs and, at a later stage (approximately 6 d), pregnancy was confirmed by palpation. Timed pregnant rats were housed five per cage until gestational d 15 when they were isolated in preparation for littering. Near-term pregnant rats were monitored several times per day and, if litters were found, the day of birth was defined as d 0 for that litter.
Perinatal dexamethasone treatments
Batches of pregnant rats and lactating rats were randomly assigned to control or treated groups (n = 4–8 rats per treatment group). Dexamethasone sodium phosphate (David Bull Laboratories, Warwick, UK) was administered via the drinking water (1 μg/ml) to the rats on d 16–19 of pregnancy (E16–19, prenatal treatment) or for 7 d immediately postpartum [(P) 1–7, neonatal treatment]. The developing rats were thus exposed to the steroid in utero via the placenta or as neonates via the mother’s milk. Control dams received normal drinking water throughout pregnancy and lactation. With the exception of routine cleaning, the progeny were left undisturbed until weaning when they were divided according to gender and perinatal treatment and caged in groups of four to five until aged 60–90 d (young adulthood).
Because techniques for measuring the circulating concentrations of dexamethasone in the developing pups (e.g. mass spectrometry) were not available to us, we made estimates of drug delivery to the developing young based on drug intake by the mothers, body mass of the mother, body mass of the neonates, and the following published pharmacokinetic data: bioavailability of the oral dose of approximately 66% (34); volume of distribution of approximately 0.78l/kg (35); a maternal to fetal plasma gradient of approximately 40% (34); a milk to plasma gradient of approximately 40% (36); and an average fluid intake of the neonates of approximately 4.2 ml/d (37). Pregnant rats ingested 50 ± 8 ml drinking water per day during the treatment period. Their intake of dexamethasone sodium phosphate was therefore 50 ± 8 μg/d; using the published pharmacokinetic data, we estimated the plasma concentrations of the steroid in the mother and the developing fetuses to be approximately 105 and 40 ng/ml, respectively. The lactating rats ingested a larger volume of water (54 ± 3 ml/d), i.e. 54 ± 3 μg dexamethasone sodium phosphate per day. This would yield estimated plasma steroid concentrations of approximately 125 and 15 ng/ml, respectively, in the mothers and pups.
Experimental procedures and design
This study employed three experimental approaches, each requiring separate batches of tissue: Western blot analysis of ANXA1 and IL-6 expression; electron microscopy; and in vitro studies. Tissue from a minimum of four litters was always included in each experimental group, and tissue for any given experiment was always collected on the same day to minimize variance. All experiments were conducted on male rats and repeated at least once, using tissue from further cohorts of animals.
Animals were handled regularly for 1 wk before experimental procedures were commenced. When required, they were selected randomly from their cages and killed by decapitation between 0900 and 1000 h to standardize effects associated with the circadian rhythm. After autopsy, the anterior pituitary gland was removed promptly and processed for one of the following: analysis of ANXA1 and IL-6 expression; electron microscopy; or in vitro studies. In some cases the rats were treated with either dexamethasone sodium phosphate (David Bull Laboratories, 20 μg/100 g body weight ip) or an equivalent volume of sterile saline (100 μl/100 g body weight ip) 2.5 h before decapitation (38).
Extraction and detection of annexin 1 and IL-6 by Western blot analysis
ANXA1 was extracted from the pituitary tissue as described previously (38). Briefly, cell surface ANXA1 was removed from the outer cell membranes by washing the tissue gently for 20 min in a solution containing 1 mM EDTA (Sigma Chemical Co., Poole, UK) in PBS [0.05 M (pH 7.4), Oxoid Chemicals Ltd., Hants, UK], which, by chelating Ca2+, releases ANXA1 into the medium from Ca2+-dependent cell surface binding sites. Intracellular ANXA1 was extracted from the remaining tissue by sonication (25 Hz, 20 sec, Soniprep 150; Sanyo Gallenkamp, Leicester, UK) on ice in EDTA (10 mM) containing Triton X-100 [1% (vol/vol); Sigma]. The samples were stored at –80 C until analysis.
To facilitate ANXA1 detection, extracts from four to five animals (littermates from the same cage) were pooled before analysis. At least four separate pooled samples (i.e. samples from four litters) were analyzed per experiment. The protein content of each pooled sample was determined (39), and, in the case of the cell surface EDTA washes, if it fell less than 1 μg/ml, the samples were concentrated to 20 μg total protein per 20 μl using a centrifugal filter microconcentrator with a 10-kDa cut-off (Microcon; Amicon, Inc., Beverly, MA). The samples were then analyzed for ANXA1 by SDS-PAGE and subsequent Western blotting, using the method of Taylor et al. (31) and a well-characterized polyclonal anti-ANXA1 antibody [anti-ANXA1 polyclonal antibody (pAb), raised in sheep against the full-length human recombinant ANXA1 and diluted 1:10,000] (31) as a probe. The blots were scanned using a flatbed scanner [HP Scanjet 5200 (Hewlett Packard Ltd., Bracknell, UK) with Adobe Photodeluxe Business Edition, version 1.1 (Adobe Systems UK, Oxbridge, UK)] and the band intensity analyzed using the TINA software program (TINA version 2.10; Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany). Levels of IL-6 were also examined in the tissue extracts by Western blot analysis, using the same protocol and a commercial anti-IL-6 antibody (R & D Systems, Abingdon, UK) diluted to 1 μg/ml.
Electron microscopy: morphological studies and detection and quantification of ANXA1 by immunogold histochemistry
Pituitary tissue from male rats was collected immediately after decapitation and fixed in 3% (wt/vol) paraformaldehyde, 0.05% (vol/vol) glutaraldehyde (VWR International Ltd., Lutterworth, UK) in PBS (Oxoid Chemicals) (pH 7.2) for 4 h at room temperature. The tissue was then processed exactly as described by Traverso et al. (23), and ultrathin sections were prepared and mounted onto formvar-coated mesh nickel grids (Agar Scientific Ltd., Stanstead, UK) and viewed with a JEOL 1010 transmission electron microscope (JEOL, Peabody, MA).
The corticotrophs were identified by immunogold labeling of ACTH, using rabbit antirat ACTH pAb (diluted 1:250; National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA) as a probe. The FS cells were identified by both their positivity for the FS cell marker, S100 (detected by immunogold labeling using a rabbit antirat S100 pAb, diluted 1:100; Dako Corp., Cambridge, UK) and their morphological characteristics, which typically include an agranular cytoplasm and long cytoplasmic processes that surround and make contact with neighboring endocrine cells.
For analysis of cell morphology by point counting (40), 10 micrographs (magnification, x4000) of corticotrophs and FS cells per animal were taken from three to four sections per pituitary gland. The negatives were scanned into Adobe Photoshop (version 5.5) and printed onto A4 paper for analysis. In all cases the reader was blind to the sample code. To determine total and nuclear cell areas, a 1-cm grid acetate sheet was placed at random over the cell and the number of grid intersections that overlaid the cell was recorded. The cytoplasmic area was determined by subtracting the nuclear area from the total cell area and multiplying the result by an area conversion factor of 1.23 because on each micrograph 1 cm2 represented 1.23 μm2. In corticotrophs, granule diameter, density, and granule margination (the ratio of granules at the perimeter of the cell to those in the cell interior) were also measured. Mean granule diameter was determined by the overlay and match of circles of known diameter onto granules in the cell for 30 granules per cell. To determine granule areal density, a 0.7-cm grid was used: the number of granules that overlaid an intersection was counted, adjusted by the areal conversion factor, and divided by the cytoplasmic area of the cell. The perimeter of each corticotroph was defined as the area from the plasma membrane to a margin 2 μm into the cell interior drawn on each cell around the circumference. The ratio of the number of granules in the cell perimeter (counted as the number of granules that overlaid an intersection on the 0.7-cm grid) to the number within the cell interior was determined. Tissue from four animals per group, each from a separate litter, was examined in each experiment.
The percentages of corticotrophs and FS cells in the pituitary were quantified by counting the number of ACTH and S100 immunogold-positive cells and total number of nucleated cells, dividing the ACTH and S100-positive cell number by the total number of nucleated cells, and converting to a percentage. Six complete grid squares in three sections taken from different depths of the tissue block were counted per animal (i.e. 18 grid squares in total). Sections from four animals per group, each from a separate litter, were examined in each experiment.
For detection of ANXA1, grids prepared as above were incubated for 2 h with anti-ANXA1 pAb (diluted 1:200 in 0.1 M phosphate buffer containing 1% egg albumin) and 1 h with protein A-15 nm gold complex (Biocell, Cardiff, UK) and then lightly stained with uranyl acetate and lead citrate (23). Specificity of the antibody staining was confirmed by use of preadsorbed antibody (100-fold excess human recombinant ANXA1) and albumin-containing buffer in place of the primary antiserum. In some cases the sections were double stained for S100 (marker of FS cells) using a specific rabbit antirat antibody (diluted 1:100, Dako) and protein A (5 nm) gold complex (Biocell). For quantification of ANXA1 staining, the number of 15-nm gold particles over each compartment was counted and calculated as particles per square micrometer by dividing the total number of gold particles counted by the relevant area. Six sections per animal were analyzed. For each experiment, tissue from four animals per group, each from a different litter, was used.
In vitro studies
Anterior pituitary tissue from male rats was incubated as described by John et al. (25). Briefly, each gland was divided into four segments of approximately equal size and distributed to the wells of 24-well tissue culture plates (Costar, Cambridge, MA). Each animal contributed one segment per treatment group and each group comprised tissue from several different litters. The tissue was incubated (one segment per well) in 1.5 ml of Earle’s balanced salt solution (Sigma) enriched with a protease inhibitor, 1% aprotinin (Bayer PLC Ltd., Newbury, UK), for 2 h at 37 C in a humidified atmosphere with 95% O2-5% CO2. The segments were then transferred to fresh medium and incubated for a further hour in the presence or absence of the adenyl cyclase activator, forskolin (100 μM; Sigma). The medium was then collected and stored at –20 C for determination of ACTH; the pituitary segments were weighed on a torsion balance and discarded. Where appropriate, dexamethasone sodium phosphate (0.1 μM, David Bull Laboratories) or an ANXA1-derived peptide, ANXA1Ac2–26 (0.2–20 μg/ml), were included throughout the preincubation and final incubations periods.
Determination of ACTH
ACTH was determined in duplicate by RIA according to a published method (41) using a well-characterized primary antibody raised in the rabbit against human ACTH1–39 (code AFP6328031, National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA), human ACTH1–39 as a standard (National Institute of Diabetes and Digestive and Kidney Diseases) and 125I-labeled ACTH1–39 as the tracer. Separation of the bound and free peptide was achieved by the addition of 100 μl goat antirabbit decanting suspension (Pharmacia Upjohn, Buckinghamshire, UK). The inter- and intraassay variations were 11.1 and 10.7%, respectively.
Statistical analysis
For the morphological studies and quantification of ANXA1 by immunogold labeling, Student’s t test was used to assess the differences between normally distributed sets of data with approximately equal variance; the data on individual cells from each animal were pooled into their experimental groups before the averages and SEM were calculated. This is standard practice in morphological analysis to streamline the sampling process without compromising the statistical integrity of the results (42). Granule distribution between the perimeter and interior of cells was assessed by the 2 test, and the odds ratio analysis was used to quantify the change in ratio of peripheral granules to interior granules. Functional studies were analyzed by two-way ANOVA with post hoc comparisons by Duncan’s multiple range test. Because the rate of basal ACTH release in vitro varied between experiments, statistical comparisons were made within experiments only. In all cases, differences were considered to be significant if P < 0.05.
Results
ANXA1 and IL-6 expression in the anterior pituitary gland
ANXA1 was also readily detected in anterior pituitary tissue from control and steroid-treated rats by immunogold labeling. The staining was localized exclusively to the FS cells, which were identified by morphological criteria and S100 labeling (Fig. 2A). Quantitative analysis of the ANXA1 staining by counting the gold particles confirmed the semiquantitative data obtained from Western blot analysis, viz. neonatal dexamethasone treatment reduced the amount of ANXA1 in both the intracellular (cytoplasmic, Fig. 2C, P < 0.05) and the cell surface (Fig. 2D, P < 0.01) pools, whereas the prenatal treatment reduced cell surface (Fig. 2D, P < 0.01) but not intracellular (Fig. 2C, P < 0.01) ANXA1. The data also revealed that the total pool of ANXA1 in the FS cells at adulthood is reduced by both prenatal (P < 0.05) and neonatal (P < 0.01) dexamethasone treatment (Fig. 2B).
Functional studies
These experiments aimed to explore the impact of perinatal dexamethasone treatment on the integrity of the ANXA1-dependent inhibitory actions of dexamethasone on ACTH secretion in adulthood. Figure 3, A and B, demonstrate the effects of prenatal (E16–19) and neonatal (P1–7) dexamethasone treatment on the ability of dexamethasone (20 μg per 100 g body weight ip) in adulthood to translocate ANXA1 to the outer cell surface, a process that is critical to the paracrine mode of ANXA1 inhibition of ACTH secretion (38, 43). In tissue from adult drug-nave rats, administration of dexamethasone 2.5 h before autopsy caused the expected increase in cell surface ANXA1 (Fig. 3, A and B, lanes 3 and 4 vs. lanes 1 and 2). However, in rats treated prenatally or neonatally with dexamethasone, the ability of dexamethasone to cause the translocation of ANXA1 to the cell surface was markedly impaired (Fig. 3, A and B, lanes 7 and 8 vs. lanes 5 and 6). Pituitary tissue from the drug-nave control rats responded in vitro to the adenyl cyclase activator, forskolin, with a significant increase in ACTH release (P < 0.01, Fig. 4, A and B). This response was inhibited by preincubation of the tissue with dexamethasone (100 nM, P < 0.05), which, alone, had no effect on resting ACTH release (Fig. 4, A and B). By contrast, dexamethasone failed to inhibit forskolin-induced ACTH release in pituitary tissue from rats treated prenatally (E16–19, Fig. 4A) or neonatally (P1–7, Fig. 4B) with the steroid, although the tissue responded normally to forskolin.
Like dexamethasone, the N-terminal ANXA1 peptide ANXA1Ac2–26 (2.0 and 20 μg/ml) suppressed the forskolin-induced release of ACTH from pituitary tissue from drug-nave rats without affecting basal ACTH release. A lower concentration of the peptide (0.2 μg/ml) was without effect (Fig. 5). ANXA1Ac2–26 also blocked forskolin-induced ACTH release from pituitary tissue from animals given dexamethasone in pre- or neonatal life and, in contrast to the controls, was effective at all concentrations tested (0.2–20 μg/ml). Paradoxically, however, and also in contrast to the controls, the peptide showed signs of augmenting basal ACTH release in these groups, and, in the case of the neonatal treatment, the increment in ACTH reached significance at a peptide concentration of 2 μg/ml (P < 0.05, Fig. 5).
Pituitary morphology
The effects of perinatal dexamethasone treatment on the morphology and number of FS cells and corticotrophs in the pituitary gland at adulthood are shown in Figs. 6–8. Both prenatal (E16–19) and neonatal (P1–7) dexamethasone treatment produced long-term changes in the morphology (Fig. 6, A–C), but not the number (Fig. 6D), of FS cells. The prenatal treatment reduced the total cell area (Fig. 6A, P < 0.05) and the cytoplasmic area (Fig. 6B, P < 0.01) without affecting the nuclear area (Fig. 6C, P > 0.05). The neonatal treatment also reduced the cytoplasmic area (P < 0.05) but had no significant effect on either the total cell area (Fig. 6A) or the nuclear size (Fig. 6C).
By contrast, neither treatment regimen affected the total cell area, nuclear area, or cytoplasmic area of the corticotrophs (P > 0.05, data not shown). The drug treatments also failed to affect granule diameter and granule density (as a percentage of the cytoplasmic area, P > 0.05, data not shown). However, the prenatal, but not the neonatal, treatment regimen reduced the margination of the granules to the cell membrane (odds ratio 0.765, 95% confidence interval 0.588–0.997, P < 0.05, Fig. 7, A–C). In addition, both treatments produced a significant reduction in the number of corticotrophs (expressed as a percentage of the total population of nucleated cells: prenatal, P < 0.01; neonatal, P < 0.05, Fig. 7). The latter finding was confirmed by a limited screen at the light microscopic level using the anti-ACTH antibody employed for immunogold labeling as a probe (data not shown).
Discussion
The results presented show for the first time that perinatal dexamethasone treatment produces long-term changes in the morphology of the anterior pituitary gland and concomitant disruption of the ANXA1-dependent inhibitory effects of dexamethasone on the secretion of ACTH. They thus support the premise that early life glucocorticoid treatment leads to disturbances of neuroendocrine function in adulthood, which may compromise homeostasis and thereby influence disease susceptibility in adulthood.
Drug treatment regimens
Our study examined and compared the impact of two discrete periods of early life dexamethasone treatment, viz. prenatal and neonatal, on the pituitary gland at adulthood. Both treatment regimens coincided with critical phases of pituitary development. The prenatal treatment (E16–18) started shortly after the initial differentiation of the corticotrophs (E13/14) (44, 45) at the time (E16) when ACTH synthesis (46) and glucocorticoid feedback first emerge (47) and, allowing for drug clearance (t1/2 dexamethasone 36–54 h), the period of maximal proliferative activity of the developing corticotroph population (E17.5–20.5) (48). By contrast, the neonatal treatment (P1–7) encompassed the onset (P5) of the neonatal stress hyporesponsive period, a period during which the HPA axis is refractory to stress and the anterior pituitary gland shows a supersensitivity to the negative feedback actions of GCs on ACTH release (49) and the reported time (P6) at which S100-positive FS cells are first apparent (50).
Previous studies on the impact of perinatal GC treatment on adult physiology have involved administration of the steroid by injection of either the pregnant mother or the newborn pups. In preliminary studies, we adopted a similar approach but found that the injection process itself, particularly in the pups, constituted a severe stress and produced changes in the adult tissues that, in some cases, were indistinguishable from those induced by the steroid (data not shown). To circumvent these problems, we administered dexamethasone via the drinking water to pregnant or lactating dams. This noninvasive approach took advantage of the effectiveness of dexamethasone by mouth and ability of the steroid to cross the placenta (51) and enter the milk (52) for delivery to the developing young. Unfortunately, we were not able to measure the concentrations of dexamethasone in the developing pups. However, by using measures of maternal drug intake and body weight together with published pharmacokinetic data (see Materials and Methods), we estimated that the plasma concentrations of the steroid were in the region of 40 ng/ml in the developing fetuses (prenatal treatment) and 15 ng/ml in the pups (neonatal treatment). Given the assumptions in the calculations, the variance in fluid intake in the dams (16%) and other interanimal variables (e.g. milk intake), we would be cautious in drawing any conclusions about the apparent differences in the plasma concentrations between the two groups. However, it is worth noting that dexamethasone is a highly potent steroid (40 times more active than corticosterone); thus, the levels attained would be comparable with the GC levels attained in severe stress. Furthermore, allowing for the route-dependent differences in bioavailability, our dosing regimen is comparable with the standard dose (5 mg im) used in perinatal medicine.
Morphological, molecular, and functional studies
FS cells.
Our morphological studies confirmed reports (53) that the FS cells comprise 5–10% of the total cell population of the adult rat anterior pituitary gland. They also revealed that perinatal dexamethasone treatment has no effect on FS cell number in adulthood but causes a marked reduction in the cytoplasmic area of the cells and, in the case of the perinatal treatment, a reduction in total cell size. It is not clear whether these changes reflect direct actions of the exogenous steroid on the developing pituitary gland or whether they are secondary to other changes induced by the treatment. Because data from immunohistochemical studies suggest that the FS cells do not emerge developmentally until P6 (50), it is possible that dexamethasone targeted a progenitor population of pituitary FS cells and altered its pattern of development. The functional implications of the steroid-induced change in FS cell size require further study. These cells influence multiple facets of pituitary function by releasing mediators (e.g. growth factors, cytokines), which regulate adjacent cells by paracrine or juxtacrine mechanisms. Because release of these mediators occurs by assisted transfer across the cell membrane (54), their release may be compromised by a reduction in cell surface area.
ANXA1.
Our data show for the first time that early life GC treatment has a significant effect on the expression of two FS cell proteins (IL-6 and ANXA1) known to influence the corticotrophs and other pituitary endocrine cells. With regard to ANXA1, Western blot analysis and quantitative immunogold labeling revealed that both pre- and neonatal dexamethasone treatment caused a substantial reduction in cell surface ANXA1; in addition, the neonatal, but not the prenatal, treatment regimen reduced the intracellular content of the protein. These changes could not be attributed to a generalized reduction in protein synthesis because IL-6 expression was increased, particularly by the prenatal treatment regimen. Standardization of the measures of gold particles to unit area also indicated that the reduction in cell surface ANXA1 could not be explained by the reduction in FS cell surface area. Because ANXA1 plays a key role as a paracrine/juxtacrine mediator of the early phase of GC feedback at the pituitary level (22), the reduction in ANXA1 protein raised the possibility that this mode of feedback may be compromised by early-life GC treatment. Our functional studies and analysis of corticotroph morphology support this view. Thus, both perinatal steroid treatment regimens impaired the capacity of dexamethasone to cause the translocation of ANXA1 to the surface and inhibit the forskolin-evoked release of ACTH from adult pituitary tissue. They also caused increased granule margination in the corticotrophs, a morphology that is indicative of a hypersecretory state (55) and is therefore consistent with a state of impaired glucocorticoid feedback. Because the exportation of ANXA1 from FS cells is an essential step in the manifestation of the ANXA1-dependent inhibitory effects of GCs on ACTH release (29, 31), it seems likely that the programming actions of the steroids are effected in part by disruption of this critical mechanism. Exactly how this disruption is achieved is unclear. We did not measure GR expression in the FS cells of the drug treated animals, and we are unaware of evidence perinatal dexamethasone treatment alters pituitary GR in male rats, although there is evidence that such treatment reduces GR expression in the female (56). A reduction in GR would certainly be expected to attenuate GC action, but it would be unlikely to account for the almost total failure of dexamethasone to induce the cellular exportation of ANXA1. Depletion of substrate would also not explain the apparent failure of the ANXA1 export mechanism because, although the neonatal treatment regimen reduced intracellular ANXA1, the prenatal treatment did not.
It thus seems likely that the expression and/or activity of other components of the signaling cascade used by GCs to export ANXA1 may also have been affected by the early life treatment; potential candidates include kinases (26, 27) and other proteins, e.g. ABCA1 (33), and these now require investigation. Interestingly, our in vitro studies show that pituitary tissue from the steroid-treated animals retains its sensitivity to ANXA1, suggesting that the ANXA1 receptors and downstream signaling mechanisms were not down-regulated by the early-life GC treatment. To the contrary, the finding that unlike the controls both steroid-treated groups responded to the lowest concentration of peptide tested (0.2 μg/ml) suggests ANXA1 sensitivity is enhanced, a finding that could reflect early life programming of the signaling system or could be a secondary compensation to the reduction in ANXA1 drive to the receptors. The paradoxical finding that the ANXA1 peptide increased basal ACTH release from pituitary tissue from steroid-treated rats also accords with a state of increased tissue sensitivity to ANXA1 because high concentrations of the peptide facilitate basal, but not stimulated, ACTH release (27).
Corticotrophs.
The finding that perinatal administration of dexamethasone caused a marked reduction in corticotroph numbers was surprising. Corticotroph turnover is dependent in part on the mitogenic actions of CRH (57) and the antiproliferative and proapoptotic actions of GCs (58). However, as discussed above, pituitary GR expression, and therefore GC sensitivity, is reduced by neonatal GC treatment (17). Moreover, although there are reports to the contrary (56), substantial evidence suggests that prenatal, although not neonatal, GC treatment augments the CRH drive to the pituitary gland (15, 20). On the basis of these findings, it might be predicted that perinatal, and particularly prenatal, GC treatment would increase the size of the corticotroph population. In addition, because ANXA1 exerts both antiproliferative (59) and proapoptotic (60) actions, the reduction in the expression and cellular exportation of ANXA1 might also be expected to increase, not decrease, corticotroph numbers. However, other factors derived within the pituitary gland also influence corticotroph proliferation, in particular growth factors (61) and cytokines (62, 63) derived from the FS cells. Of the cytokines, both IL-6 (63) and leukemia inhibitory factor (62) exert proliferative actions, the latter directing progenitor cells toward the corticotroph lineage (62). Because pituitary IL-6 expression is augmented by perinatal dexamethasone treatment, it seems unlikely that the loss of corticotrophs is due to IL-6 withdrawal.
We cannot ascertain from our data when the changes in the corticotroph population first emerged. However, because the prenatal treatment coincided with the period of maximal corticotroph proliferation (E17.5–20.5) (48), it is possible that the steroid acted at this time to halt the differentiation and/or proliferation of progenitor cells, perhaps via a reduction in leukemia inhibitory factor. On the other hand, the adult corticotroph population is surprisingly labile, i.e. acute stress (novel environment) increases the population by 20% (64), and it is conceivable changes reported here are effected in adulthood. The functional consequences of the reduction in the corticotroph population are not clear. The ACTH reserve in the pituitary is substantial (65), and our data indicate that the secretory activity of the cells is enhanced by perinatal GC treatment, as also do other studies, which show exaggerated HPA responses to stress (15) and increased sensitivity of pituitary tissue to CRH (17).
Conclusions
Our study shows that, when administered by a noninvasive process in prenatal and neonatal life, GCs exert profound effects on the adult rat pituitary gland, impairing the ANXA1-dependent GC regulation of ACTH release and altering the cell profile and morphology. The changes caused by the prenatal treatment regime would be expected to contribute to the hyperactivity in HPA function associated with such treatment, which, until now, has been largely attributed to changes in the brain, particularly the hippocampus and hypothalamus (14, 15). However, because the bulk of evidence available indicates that neonatal GC treatment reduces the CRH/arginine vasopressin drive to the pituitary gland (18, 19, 21, 66), the changes in pituitary function reported here may be compensatory. The significance of the changes in IL-6 expression requires further exploration. However, because IL-6 has a positive effect on ACTH secretion and its release from FS cells is modified by GCs (67), the increased expression of the cytokine may serve to augment ACTH release, particularly in conditions of immune stress, i.e. after a bacterial challenge.
Acknowledgments
We are grateful to the National Institute of Diabetes and Digestive and Kidney Diseases for reagents for the ACTH assay, and Colin Rantle for excellent technical support.
Footnotes
This work was supported by the Biotechnology and Biological Sciences Research Council.
1 E.T. and C.D.J. contributed equally to the study.
Abbreviations: ANXA1, Annexin 1; E, embryonic day; FS, folliculostellate; GC, glucocorticoid; GR, glucocorticoid receptor; HPA, hypothalamo-pituitary-adrenocortical; P, postpartum day; pAb, polyclonal antibody.
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