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University Research Center for Neuroendocrinology, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom
Address all correspondence and requests for reprints to: Dr. L. Nolan, University Research Center for Neuroendocrinology, University of Bristol, Bristol Royal Infirmary, Lower Maudlin Street, Bristol BS2 8HW, United Kingdom. E-mail: lesley.a.nolan{at}bris.ac.uk.
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Abstract |
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Introduction |
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The cell proliferative response of the rat anterior pituitary following the extreme stimulus of bilateral adrenalectomy without concomitant glucocorticoid replacement is well documented and appears to result in an absolute increase in the number of cells transcribing the proopiomelanocortin (POMC) gene (4, 5, 6, 7, 8, 9). Under these circumstances, our own data and the work of others suggest that the origin of the cells entering the cell cycle is a combination of mature corticotrophs and perhaps other hormone-secreting cells together with a number of more immature or pluripotent stem cells (2, 8, 9, 10). In this model, the elevation of mitotic activity above basal levels is not maintained despite both the continued absence of glucocorticoid feedback and sustained elevation of CRH transcripts, so that by 2 wk after surgery, mitotic rate has returned to preoperative levels (2, 11). The intact rat pituitary also contains a specific subpopulation of cells, the functional identification of which remains elusive, that undergoes apoptosis in response to the in vivo administration of the synthetic glucocorticoid dexamethasone. We have previously shown that the size of this population increases 3-fold between 1 and 2 wk after bilateral adrenalectomy (2, 12).
To further clarify the temporal relationships between physiological change and pituitary trophic responses, we have in the present study determined the precise timing of the changes in size of the dexamethasone-sensitive apoptotic cell population following adrenalectomy and correlated these changes with fluctuations in the rate of mitosis and changes in pituitary POMC transcript levels and paraventricular CRH transcripts during the first 28 d post adrenalectomy. We have also examined the relationship between mitotic activity and apoptotic sensitivity at the single cell level and addressed the possibility that in chronically adrenalectomized animals, pituitary mitotic activity, and apoptotic responsiveness can be restored by using dexamethasone withdrawal as a surrogate for surgical adrenalectomy.
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Materials and Methods |
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To examine the effects of glucocorticoid withdrawal and repeated exposure to dexamethasone on pituitary trophic activity following long-term adrenalectomy, groups of rats that had been adrenalectomized 28 d previously received dexamethasone for 14 d as described above. Dexamethasone was then withdrawn for 14 d before a second dexamethasone exposure was carried out.
To follow cumulative changes in the number of recently divided anterior pituitary cells, additional groups of rats received daily ip injections of bromodeoxyuridine (BrdU; 10 mg/ml in 0.007 M NaOH/0.9% NaCl; Roche, Welwyn Garden City, Hertfordshire, UK) at a dose of 200 mg/kg body weight. Injections were started on the day of adrenalectomy or sham surgery and continued for 8 d. On d 6 of the experiment, a group of animals received dexamethasone treatment for 2 d as described above.
Groups of rats were killed by stunning and decapitation at intervals from 24 h to 10 wk following either surgery or the start or withdrawal of dexamethasone treatments and checked post mortem for remnants of adrenal tissue. Animals were killed immediately after removal from their cages in a separate room adjoining that in which they had been housed. The number of animals at each time point was between 4 and 6.
Preparation of tissue sections
Immediately after decapitation, brains were quickly frozen on dry ice and stored at -70 C. A series of 12-µm-thick coronal brain sections were cut through the paraventricular nucleus at -18 C and thaw mounted onto gelatin-coated slides. Slides were stored at -70 C before in situ hybridization histochemistry. Pituitary glands were carefully removed and fixed in 4% formaldehyde in PBS for 48 h, washed in two changes of fresh PBS and embedded in 1% agar before being processed for paraffin wax embedding. A series of 2-µm-thick axial sections were cut from each pituitary for histological analysis and for in situ hybridization histochemistry and BrdU immunohistochemistry.
In situ hybridization histochemistry
In situ hybridization histochemistry was performed as previously described (13, 14). Briefly, frozen brain sections were warmed to room temperature and allowed to dry for 10 min before fixing in 4% formaldehyde in PBS for 10 min. Paraffin wax-embedded pituitary sections were dewaxed in two changes of xylene, rehydrated through a graded series of alcohols, rinsed in water and incubated with 7.5 µg/ml proteinase K in 50 mM Tris (pH 7.5) for 1 h at 37 C. All sections were then washed in PBS and incubated in 0.25% (vol/vol) acetic anhydride and 1.4% (vol/vol) triethanolamine in 0.9% saline for 10 min at room temperature. Sections were transferred through 70% (1 min), 80% (1 min), 95% (2 min), and 100% (1 min) ethanol; 100% chloroform (5 min); and 100% (1 min) and 95% (1 min) ethanol before being air dried. Hybridization was carried out using 35S-deoxy-ATP 3' end-labeled synthetic 48-oligomer oligodeoxynucleotide probes complementary to either CRH (2 x 105 counts/slide) or POMC (105 counts/slide) mRNA. After hybridization, sections were washed for 1 h in four changes of 1x saline sodium citrate and for an additional 1 h in two changes of 1x saline sodium citrate at room temperature. Slides were briefly dipped in water then air-dried. Dry hybridized sections were opposed to autoradiography film (Hyperfilm MP, Amersham International, Buckinghamshire, UK) and the resulting images were analyzed densitometrically using a Macintosh IIci computer equipped with an image capture board (Scion Corp., Walkersville, MD) running the program Image by Wayne Rasband, NIMH (Bethesda, MD). Results were expressed as the mean deviation from controls (% ± SE).
Image analysis for trophic activity
Apoptotic and mitotic cell counts were performed on 2-µm-thick hematoxylin and eosin-stained rat pituitary sections at x1000 magnification with the aid of a dedicated real-time computer system to tag and tally the coordinates and trophic assignment of manually identified structures within each tissue section (2). The computerized Highly Optimized Microscope Environment [AxioHOME, Carl Zeiss (Jena, Germany) (15)] that was used, projects a virtual image of the computer screen, which appears fractionally above the actual microscope image and allows different markers to be laid down over either apoptotic, mitotic or normal cells by hand. The system retains a cumulative score of the numbers of each cell type counted, together with the coordinates of each individually marked cell irrespective of subsequent movement of the microscope stage or changes in power of the objective lens. Extremely accurate quantification of the various cell types is thus possible for each section studied as areas can be circumscribed at low power (eliminating selection bias), and counted manually at high power without danger of double scoring. The histological markers that were used to identify apoptotic cells were clusters of two or more apoptotic bodies consisting of extremely dense round or oval structures varying in size from approximately 0.7–4 µm and surrounded by normal cells. Earlier stages of apoptosis cannot be visualized using hematoxylin and eosin staining and light microscopy. For each animal, three random areas of approximately 47,000 µm2 were scored for the presence of mitotic and apoptotic figures (Fig. 1
). The sensitivity of detection of trophic events throughout the study was virtually 100%. The error in quantifying the number of normal cells surrounding these events was 2% or less. Thus, the overall error in estimating the prevalence of trophic events was around 0.001%. All slides were coded and counted by one blinded observer.
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BrdU immunohistochemistry
Pituitary sections were processed for BrdU immunohistochemistry according to a previously published protocol with minor modifications (16). Briefly, dewaxed and rehydrated sections were transferred to a hot antigen unmasking solution (0.01 M citric acid in water, pH 6.0) and incubated for 10 min in a microwave oven on a power setting that maintained the solution just below its boiling point. Sections were then cooled in water to room temperature before permeabilizing for 10 min in 0.001% trypsin (Roche) diluted in 0.1% CaCl2/20 mM Tris buffer (pH 7.5). Following three washes in PBS, the slides were denatured in 2 N HCl in PBS at 60 C for 30 min, washed again in PBS, gently agitated for 20 min in blocking serum (3% normal horse serum, 0.5% Triton X-100 in PBS) and incubated overnight at 4 C with monoclonal anti-BrdU (Sigma; 1/1000 diluted in blocking serum). Sections were washed in three changes of PBS, incubated for 1 h at room temperature with biotinylated antimouse IgG (Vector Laboratories, Burlingame, CA; 1/200 diluted in blocking serum), and washed again in fresh PBS before blocking endogenous peroxidases for 30 min with 0.6% (vol/vol) hydrogen peroxide in PBS. Following a further three washes in PBS, sections were incubated with a ready-to-use Vectastain Elite ABC reagent (PK-7100; Vector Laboratories) for 30 min at room temperature, rinsed in PBS and developed for 8 min in DAB substrate according to the manufacturer’s instructions (SK-4100; Vector Laboratories). The resulting brown color reaction was stopped in water and sections counterstained with hematoxylin before being mounted in p-xylene-bis(N-pyridinium bromide) (Fig. 2).
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Statistics
The statistical software package Prism (GraphPad Software, Inc., San Diego, CA) was used to perform statistical calculations. Differences between groups were evaluated using one-way ANOVA followed by Tukey-Kramer multiple comparison post tests. P < 0.05 was considered statistically significant.
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Results |
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) and a 4-fold increase in pituitary POMC transcripts by 14 d after surgery (376 ± 41% of control; n = 6; P < 0.05; Fig. 3B). Ten weeks after adrenalectomy, with no additional interventions, both CRH and POMC transcript levels remained significantly higher than their respective controls.
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As expected, dexamethasone treatment beginning 1 or 2 wk after adrenalectomy induced a discrete, highly significant burst of apoptotic activity in the anterior pituitary within the first 48 h of the start of treatment (2, 12). The effects of a similar duration of dexamethasone exposure commencing at daily intervals during the first week after adrenalectomy and at weekly intervals thereafter for a total of 4 wk on anterior pituitary apoptotic activity (peak prevalence and area under the curve for the first 72 h after the start of dexamethasone treatment) are shown in Fig. 4, A and B.
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). Delaying the start of dexamethasone treatment until either 3 or 4 wk after adrenalectomy resulted in the amplitude of the apoptotic response declining to levels not significantly different from that measured in intact animals.
The temporal relationship between changes in the prevalence of mitotic figures post adrenalectomy and the maximum apoptotic response to dexamethasone exposure is shown in Fig. 4C. Anterior pituitary mitotic activity increased progressively during the week following adrenalectomy to reach a peak after 6 d (0.266 ± 0.02%). By 2 wk post operatively and certainly by 3 and 4 wk, the level of mitosis had spontaneously returned to baseline. It should be noted that the increase in mitotic activity following adrenalectomy preceded the accumulation of the dexamethasone-sensitive apoptotic cell population and that the subsequent decline in mitotic activity, beginning 1 wk after surgical adrenalectomy, preceded the decline in dexamethasone-induced apoptotic sensitivity by approximately 7 d.
The apparent association between recent mitotic activity and apoptotic susceptibility following glucocorticoid treatment was supported by quantifying the change in the proportion of BrdU-labeled cells in the anterior pituitary of adrenalectomized rats treated with dexamethasone for 48 h (Fig. 5). As expected, 8 d after adrenalectomy the BrdU labeling index was significantly higher than that found in sham-operated animals (6.64 ± 0.5% vs. 3.79 ± 0.56%; P < 0.01; Fig. 5). Following 48 h of continuous exposure to dexamethasone in animals adrenalectomized 6 d previously, the BrdU labeling index dropped abruptly to 4.28 ± 0.34%, a level not significantly higher than that measured in control animals.
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) compared with 0.082 ± 0.023% (n = 6; NS) respectively]. Irrespective of whether the first exposure to dexamethasone was started 14 d after adrenalectomy (when a large apoptotic response is produced) or 28 d after adrenalectomy (when the apoptotic response is similar to that seen in intact animals), withdrawal of dexamethasone after 2 wk treatment induced a similar, statistically significant mitotic response (P < 0.01 at 5 d after withdrawal, Fig. 6). Furthermore, 2 wk after this point, reexposure to dexamethasone produced a similar apoptotic response in both (0.157 ± 0.025%; n = 6; Fig. 6, compared with 0.147 ± 0.025; n = 6). The amplitude of this apoptotic response was smaller than the first response in the group initially treated 2 wk after adrenalectomy, but larger than the first response in the group in which treatment was delayed until 28 d—an observation that again emphasizes the correlation between the amplitude and timing of mitotic activity and subsequent apoptotic responsiveness.
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Discussion |
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Following adrenalectomy, there is a progressive reduction in pituitary corticotroph CRH-binding capacity and CRH-stimulated cAMP production, which is sustained for at least 9 wk after surgery (18, 19, 20). The majority of studies have demonstrated that the level of CRH receptor transcripts is only transiently reduced, however, and although augmented by the absence of glucocorticoids in adrenalectomized rats, returns to control levels between 4 and 14 d after surgery (21, 22, 23). These studies suggest that CRH receptor down-regulation is related to the high rate of receptor occupancy and internalization in the presence of high levels of hypothalamic CRH and AVP rather than decreased receptor synthesis. The molecular mechanism responsible for the recovery of CRH receptor transcripts to basal levels remains unknown but may be a reflection of the continued absence of glucocorticoids (22) or exposure to altered ratios of CRH and AVP with time (23). The timing of the recovery of CRH receptor transcripts to basal levels appears to just precede the reduction in proliferative activity in the anterior pituitary following adrenalectomy.
The anterior pituitary contains a population of cells that undergo apoptosis in response to in vivo administration of dexamethasone (2). The size of this population in intact 3-month-old rats is approximately 150,000 cells. Within a week of bilateral adrenalectomy, the size of this population has increased more than 3-fold and remains at this level for at least a further week (12). In this study, we have shown first that the adrenalectomy-induced increase in dexamethasone-sensitive cells is, like the increase in adrenalectomy-induced mitotic cells, temporally constrained as the response declines to the level seen in intact animals if first exposure to dexamethasone is delayed until 4 wk after surgery. Secondly, we have shown that the increase in the number of dexamethasone-sensitive cells gradually accumulates over the first week following surgery, paralleling, after a short delay, the increase in mitotic cells which peaks 6 d after surgery. Subsequently, the decline in numbers of dexamethasone-sensitive cells lags behind the fall in mitotic cells by approximately 1 wk, suggesting that apoptosis only occurs in cells that have entered the cell cycle within the previous 2 wk. The close association between stimulation of mitotic activity and generation of apoptotically vulnerable cells is also suggested by the increase in size of the apoptotically sensitive cell population following the mitotic response induced by dexamethasone withdrawal when first dexamethasone treatment is delayed until 1 month after adrenalectomy.
To further confirm the association between mitosis and apoptotic sensitivity at the single cell level, cells entering S-phase during the week following adrenalectomy were labeled by daily injection of BrdU. Comparison of the anterior pituitary BrdU labeling indices before and after 2 d of continuous exposure to glucocorticoid demonstrated the expected abrupt step-down in the labeling index to levels indistinguishable from control levels, indicating that apoptosis occurs largely in cells that had undergone recent mitosis. BrdU administered at the dose used in this study is not thought to be inherently toxic and potential dilution of the label was avoided by the use of daily injections (24). In addition, the mitotic and apoptotic indices measured in adjacent hematoxylin and eosin-stained pituitary sections from the animals used in these experiments were identical to those taken from animals not given BrdU. Our data do not yet allow us to directly determine either the number of newly formed cells that die following adrenalectomy before dexamethasone administration or due to basal cell turnover, or the precise number of cells that actively contribute to the dividing cell population.
Technical difficulties have so far made it impossible to identify the specific nature of apoptotic cells in pituitary tissue sections using quantification of transcript and/or protein markers, as unequivocal phenotypic signs of apoptosis only appear when nuclear and cytoplasmic contents have been degraded. The susceptibility of a specific subpopulation of cells to dexamethasone-induced apoptosis is likely to be determined not only by its metabolic state, but by its specific receptor complement and activated cell signaling pathways.
Glucocorticoid-mediated apoptosis has been extensively studied in lymphocytes in which glucocorticoids are able to induce both G1 cell cycle arrest and apoptosis, although the molecular mechanisms remain poorly understood and no single glucocorticoid-regulated genes have yet been implicated (25, 26). It has been suggested that dexamethasone susceptibility of human T cells to dexamethasone-induced apoptosis is also cell cycle dependent (27).
It is possible that a change in the number of intracellular type II glucocorticoid receptors (GR), which in the presence of ligand result in altered patterns of gene expression and/or repression in a cell-specific context, is one of the factors necessary to induce an apoptotic response (28, 29). Colocalization of GR protein and transcripts with individual pituitary hormones has demonstrated that GR is expressed in virtually all ACTH, GH, and folliculostellate cells together with two-thirds of TSH cells and a smaller minority of FSH, LH, and PRL cells (30, 31, 32). No overall changes in anterior pituitary GR transcripts following adrenalectomy have been reported (33), but dexamethasone exposure in adrenalectomized animals resulted in an overall increase in GR transcripts. Positive autoregulation of GR expression, although not common, has also been shown to be a requirement for glucocorticoid-induced cell death in sensitive T cells (29, 34). GR status was not examined in the current study as it would be impossible to specifically quantify any changes within the tiny fraction of cells that undergo apoptosis in response to dexamethasone, not least because these cells cannot be identified before disclosing their dexamethasone-susceptibility by dying.
Thymocytes from adult GR-knockout mice that express a truncated, nonfunctional dexamethasone-binding fragment of the GR are resistant to dexamethasone-induced apoptosis, data that suggest that functional GR is necessary for this particular apoptotic pathway (35). Conversely, increased apoptotic sensitivity of primary thymocytes was observed in response to dexamethasone in mice with an increased GR gene dosage together with significantly reduced levels of CRH, POMC, and basal corticosterone (36).
We have previously shown that at least partial restoration of a trophically sensitive population of cells occurs after dexamethasone withdrawal, as successive exposures to dexamethasone induce successive bursts of apoptosis (12). The amplitudes of these subsequent apoptotic bursts are reduced suggesting either that a population of dexamethasone-sensitive cells is irrevocably deleted or that the pituitary requires a longer time period in which to fully restore its complement of cells. If the first exposure to dexamethasone is delayed until 4 wk after adrenalectomy, the amplitude of the apoptotic response is similar to that seen in intact animals despite the fact that there is no change in the number of immunocytochemically identified corticotrophs present between 7 and 28 d after adrenalectomy (12). However, withdrawal of dexamethasone is able to prime the formation of a larger trophically sensitive population of cells, which is identical in nature to that seen following reexposure in rats treated with dexamethasone for the first time either 1 or 2 wk after surgery. These data also suggest that an apoptotic response is somehow limited to cells that have recently entered the cell cycle, and that the timing of a stimulus or repeated stimuli such as glucocorticoid treatment(s) may be as important as the dose or route of administration in governing the long term consequences for pituitary functionality.
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Acknowledgments |
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Footnotes |
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Abbreviations: AVP, Arginine vasopressin; BrdU, bromodeoxyuridine; GR, glucocorticoid receptor; POMC, proopiomelanocortin.
Received February 28, 2002.
Accepted for publication October 8, 2002.
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