521 Pituitary mitosis and apoptotic responsiveness following adrenalectomy are independent of hypothalamic paraventricular nucleus CRH input

L A Nolan, C K Thomas and A Levy Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol Royal Infirmary, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK (Requests for offprints should be addressed to L A Nolan; Email: [email protected])

Abstract We have previously identified a series of age-dependent, carries a significant proportion of the pituitary systemic temporally constrained and closely interdependent mitotic supply. When bilateral adrenalectomy and paraventricular and apoptotic events in the male rat that nucleus disconnection were combined, the adrenalectomy- occur in response to timed single and repeated induced increase in anterior pituitary pro-opiomelanocortin hypothalamo–pituitary–adrenal axis stimuli. One of the (POMC) transcript prevalence was abolished, confirming most dramatic of these is the short burst of apoptosis the loss of paraventricular corticotrophin-releasing hor- that occurs 24–48 h after exposure to dexamethasone. If mone (CRH) input. However, the amplitude and pattern bilateral adrenalectomy precedes exposure to dexametha- of the adrenalectomy-induced anterior pituitary mitotic sone by 1–2 weeks, mitotic activity is transiently increased response and enhancement of the apoptotic response to and the subsequent apoptotic response to dexamethasone dexamethasone 1 week later remained completely intact. greatly enhanced. This study was designed to determine These data demonstrate that anterior pituitary trophic whether adrenalectomy-induced augmentation of the responses following bilateral adrenalectomy are more likely apoptotically sensitive pituitary cell population is mediated to be mediated through direct glucocorticoid withdrawal at via glucocorticoid withdrawal at the level of the pituitary, the level of the pituitary rather than via changes in or whether increased exposure to hypothalamo- hypothalamo–hypophyseal releasing factor exposure. This hypophyseal trophic of paraventricular origin is finding highlights the presence of distinct control systems responsible. We used stereotaxic surgery to isolate both for pituitary gene expression and pituitary mitotic paraventricular nuclei without disturbing either median and apoptotic responses. eminence input from the arcuate and supraoptic nuclei, Journal of Endocrinology (2004) 181, 521–529 or the hypothalamo-hypophyseal-portal blood flow that

Introduction pituitary microadenomas (Levy 2002, Levy & Lightman 2003). The pituitary responds to physiological demand in the In addition to hormone synthetic and secretory changes, short term by modifying hormone synthetic and secretory we have previously defined several patterns of timed single activity, and over hours, days and weeks by altering the and repeated hypothalamo–pituitary–adrenal axis stimuli relative proportions of different cellular subtypes that it that induce specific anterior pituitary mitotic and apoptotic contains. The latter is achieved by increased or responses (Nolan et al. 1998, Nolan & Levy 2001, 2003). decreased rates of cell division and cell death, by Of particular interest is a significant but relatively modest differentiation of nascent and previously uncommitted increase in anterior pituitary mitotic activity during the cells to specific secretory subpopulations, and by direct week following surgical bilateral adrenalectomy. Subse- change – real or apparent – from one mature secretory quent exposure to dexamethasone, provided it is delivered phenotype to another. The mechanisms that account within 4 weeks after surgery, induces a short burst of for changes in mitotic and apoptotic activity in response apoptosis that typically begins a day or two after the start to physiological demand and the resolution of such of dexamethasone treatment and lasts for 24 h or less. changes after transient stimuli have passed are of Intact animals demonstrate a similar response to dexa- considerable interest, as they may be implicated in the methasone but of reduced amplitude. Whether the effects propagation and maintenance, if not induction, of of bilateral adrenalectomy on mitotic activity and apoptotic

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responsiveness to dexamethasone are mediated via gluco- 2 days, together with supplementary dexamethasone in the corticoid withdrawal at the level of the pituitary, or via drinking water (0·75 µg/ml; Sigma). Groups of rats were increased hypothalamo-hypophyseal trophic hormone killed by stunning and decapitation 1 week after surgery effects has remained uncertain (McNicol & Carbajo-Perez and 1 or 2 days after the start of dexamethasone treatment. 1999). Bilateral paraventricular nucleus lesioning has been Preparation of tissue sections shown greatly to reduce corticotrophin-releasing hormone Immediately after decapitation, brains were quickly frozen (CRH) immunoreactivity in the median eminence and to on dry ice and stored at 70 C. A series of coronal block adrenalectomy-induced increase in pituitary pro- sections was cut and stained to check that the position of opiomelanocortin (POMC) transcripts (Bruhn et al. 1984, the lesion included the entire PVN and that the arcuate Dallman et al. 1985, Makara et al. 1986). In the current nucleus was intact. For non-lesioned animals, a series of study, we have used stereotaxic surgery to isolate both 12-µm-thick coronal brain sections was cut through the paraventricular nuclei (PVN) without disturbing either PVN at 18 C, thaw-mounted onto gelatin-coated hypothalamo–hypophyseal–portal blood flow or median slides and stored at 70 C for CRH in situ hybridization eminence input from the arcuate nucleus (Makara et al. histochemistry. Pituitary glands were processed for paraffin 1986). We then accurately quantified the mitotic response wax embedding, as previously described (Nolan & Levy of the anterior pituitary to adrenalectomy and the 2001, 2003). A series of 2-µm-thick axial sections was cut apoptotic response of the anterior pituitary to high-dose from each pituitary for histological analysis and for in situ dexamethasone treatment 1 week later. hybridization histochemistry.

Materials and Methods In situ hybridization histochemistry In situ hybridization histochemistry was performed Animals and treatments exactly as previously described (Nolan & Levy 2003), All procedures were carried out, in accordance with UK using 35S-dATP 3 end-labelled synthetic 48 mer oligo- Home Office animal welfare regulations, on male Wistar nucleotide probes complementary to either CRH rats weighing 180 g. Rats were adrenalectomized between (2105 counts/slide), POMC (105 counts/slide), 0900 and 1100 h and given 0·9% sodium chloride to drink -stimulating hormone (TSH)(2105 counts/ after surgery. Paraventricular disconnection was carried slide) or prolactin (PRL) (105 counts/slide) transcripts. out stereotaxically with a rotating wire knife, as previously Autoradiographs (on Hyperfilm MP; Amersham) were described (Makara et al. 1986). Briefly, the end of a 19 G analysed densitometrically with the program NIH Image needle was ground flat, and three-quarters of the shaft and results expressed as the meanstandard error relative circumference ground away in a section between 0·5 and to controls (%S.E.). 4·5 mm from the distal end. The cut-away needle shaft was then bent out to form an inverted cone approximating Adrenocorticotrophic hormone (ACTH) immunohistochemistry to the profile of a PVN, and a 21 G needle inserted as a Endogenous peroxidases in deparaffinized and rehydrated stent to keep the distal end of the knife in the midline. The pituitary sections were inactivated in phosphate buffer 19 G needle was removed from its hub, and spliced to a containing 20% methanol, 0·2% Triton X-100 and 1·5% further 19 G needle shaft with a 21 G needle, and the hydrogen peroxide for 30 min. Sections were blocked in whole was set to rotate and slide freely inside a wider bore 1% normal horse serum and incubated overnight at 4 C needle attached to a stereotaxic frame. With the tooth bar  with monoclonal anti-ACTH (residues 1–24; 1/200 set at 2 mm, the centre of the knife was positioned in diluted in blocking buffer; Novacastra Labs, Newcastle- the midline, 1·2 mm caudal to the bregma, and lowered upon-Tyne, UK) at 4 C, before room temperature incu- vertically through the sagittal sinus until it touched bation with biotinylated anti-mouse IgG for 4 h (Vector the sphenoid bone (Makara et al. 1986). The knife was Labs, Peterborough, UK; 1/200 diluted in blocking then fully rotated twice before being withdrawn. In buffer). Finally, sections were incubated in Vectastain Elite sham-operated animals, the knife was not rotated before ABC reagent (Cat. no. PK-7100; Vector Labs) for 30 min withdrawal. Bleeding from the sagittal sinus – generally at room temperature and developed in DAB substrate profuse but short-lived – was allowed to stop spon- (Cat. no. SK-4100; Vector Labs) for 8 min before being taneously before closure. Paraventricular disconnection counterstained with haematoxylin. and bilateral adrenalectomy were carried out under the same anaesthetic. After 7 days, groups of rats received daily subcutaneous Image analysis for trophic activity injections of either 50 µg/100 g body weight dexametha- Apoptotic and mitotic event prevalence was analysed sone sodium phosphate in saline (David Bull Labs, on 2-µm-thick haematoxylin and eosin (H&E)-stained Warwick, UK) or vehicle between 1300 and 1400 h for pituitary sections at 1000 magnification (Nolan et al.

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Figure 1 Representative stained frozen coronal rat brain section taken approximately 1 mm caudal to the anterior commissure through the site of the paraventricular nucleus (PVN) 7 days after PVN lesioning with the rotating wire knife, showing necrosis and shrinkage of tissue remnants within the transection line and normal tissue immediately outside it. The scale bar is 1 mm in length.

1998). The dedicated real-time computer system used corrections were made for changes in corticotroph cell size (AxioHOME, Zeiss (Brugal et al. 1992)) projects a virtual following adrenalectomy. All slides were coded and image of a computer screen fractionally above the histo- counted by one blinded observer (L A N), and results logical section. Identifier tags manually placed over cells expressed as a percentage of the total cell numbers counted and trophic events remain in registration with the targets for each animal. irrespective of subsequent stage movements and magnifi- cation changes. For each animal, three random areas of Statistics approximately 47 000 µm2 were scored for the presence of  mitotic and apoptotic figures. By defining counting Data were expressed as the mean S.E. GraphPad Prism boundaries at low power and counting events at high (GraphPad Software, San Diego, CA, USA) was used to ff power, selection bias and double scoring were eliminated, perform statistical calculations. Di erences between allowing the error in quantifying the number of normal groups were evaluated by one-way ANOVA followed by cells surrounding these events to be reduced to c2% and Tukey–Kramer multiple comparison post-tests. A value of the overall error in estimating the prevalence of trophic P,0·05 was considered statistically significant. events to be reduced to approximately 0·001%. The histological characteristics used to define apoptotic cells were clusters of two or more extremely dense, round Results or oval structures varying in diameter from approximately Histological analysis of PVN lesions 0·7 to 4 µm and surrounded by normal cells (Nolan & Levy 2003). Earlier stages of apoptosis cannot be visualized Serial 12-µm-thick sections were cut in the coronal plane by H&E staining and light microscopy. before, through and behind the lesioned area and stained ACTH-immunopositive cell counts were performed to check that removal of the PVN was complete and that at 400 magnification. For each animal, three areas of the arcuate nucleus was intact. A representative frozen approximately 75 000 µm2 each, containing an average of section of a 7-day-old PVN lesion is shown in Fig. 1. Data 800 cells, were scored as positive or negative for the were included only from rats showing complete presence of immunoreactive ACTH. In this study, no destruction of the PVN (all but one animal). www.endocrinology.org Journal of Endocrinology (2004) 181, 521–529

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Figure 2 (a) Paraventricular nucleus CRH transcript prevalence changes relative to control. The effects of 2-day dexamethasone treatment (Dex) in age-matched controls and 1 week after sham PVN disconnection (PVNx) or bilateral surgical adrenalectomy (Adx) are shown. Mean deviation from controls is shown as %S.E.; n=4–9; ***P,0·001 compared to controls. (b) Anterior pituitary POMC transcript prevalence changes relative to control. In addition to the groups in panel a, the effects of PVN disconnection (PVNx), bilateral adrenalectomy (Adx) or both, dexamethasone treatment for 24–48 h, 1 week later, are shown. Mean deviation from controls is shown as % S.E.; n=4–11; **P,0·01 and ***P,0·001 compared to controls.

Effect of adrenalectomy with or without PVN lesion on POMC transcript prevalence 7 days after adrenalectomy transcriptional activity was completely blocked by concurrent PVN lesioning (Fig. 2b). This finding, together with the reduction in PVN CRH transcript prevalence significantly increased pituitary TSH transcript prevalence seen in PVN- 7 days after bilateral adrenalectomy and was suppressed to lesioned rats irrespective of their adrenal status (Fig. 3a), a level below that found in age-matched intact controls confirmed the efficacy of PVN disconnection and thus loss after 2 days of dexamethasone exposure (Fig. 2a). The of paraventricular CRH/arginine vasopressin (AVP) and expected and observed increase in anterior pituitary thyroid-releasing hormone (TRH) input to the pituitary.

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Figure 3 Anterior pituitary TSH (a) and prolactin (b) transcript prevalence relative to control. The effects of 2-day treatment with dexamethasone (Dex) in age-matched controls, PVN disconnection (PVNx) and/or bilateral surgical adrenalectomy (Adx), followed 1 week later by 1 or 2 days’ dexamethasone treatment, are shown. Mean deviation from controls is shown as % S.E.; n=4–11; *P,0·05 and **P,0·01 compared to controls.

The integrity of the arcuate nucleus was confirmed by the higher than that found in intact controls (0·2290·025% absence of changes in the level of anterior pituitary vs 0·1160·023% respectively; n=6; P,0·01; Fig. 4a). prolactin transcripts in any of the PVN-lesioned groups PVN lesioning at the time of surgical adrenalectomy had compared with animals with intact PVNs (Fig. 3b). no effect on the increase in mitotic index (0·2490·03%; n=7; Fig. 4a). Supraphysiological dexamethasone treat- ment beginning 1 week after surgery significantly Effect of PVN lesioning on the anterior pituitary mitotic suppressed mitotic activity in all groups of animals response to adrenalectomy irrespective of whether they were intact controls with or One week after bilateral adrenalectomy, the prevalence of without PVN lesion, or adrenalectomized with or without mitotic figures in the anterior pituitary was significantly PVN lesion. www.endocrinology.org Journal of Endocrinology (2004) 181, 521–529

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Figure 4 Mitotic (a) and apoptotic (b) activity as percentage of total anterior pituitary parenchymal cells. The effects of 2-day treatment with dexamethasone (Dex) in age-matched controls, PVN disconnection (PVNx) and/or bilateral surgical adrenalectomy (Adx), followed 1 week later by 1 or 2 days’ dexamethasone treatment, are shown. Mean deviation from controls is shown as %S.E.; n=4–11; *P,0·05, **P,0·01 and ***P,0·001 compared to controls.

Effect of PVN lesioning on the generation of a dexamethasone- equivalent increase in apoptotic sensitivity to dexa- sensitive cell population in the anterior pituitary methasone with the same time course (0·2950·023%; n=11; Fig. 4b). A small increase in apoptotic activity was The prevalence of apoptotic figures in intact control seen 2 days after the start of dexamethasone treatment, anterior pituitary (0·0550·017%) was not significantly both in intact control animals and in PVN-lesioned, altered by adrenalectomy with or without PVN lesioning, adrenal-intact animals, confirming that PVN lesioning PVN lesioning alone or sham surgery (Fig. 4b). Dexa- alone was not responsible for the significant increase seen methasone treatment beginning 1 week after adrenalec- in PVN-lesioned adrenalectomized rats. tomy resulted in the previously reported (Nolan et al. 1998) increase in apoptotic prevalence which peaked at ACTH immunohistochemistry 0·2740·036% within the first 48 h of treatment (n=9; P,0·001 compared to untreated controls; Fig. 4b). PVN- Under basal conditions, with a standard formaldehyde lesioned, adrenalectomized animals demonstrated an fixation procedure, 5·30·09% of the total parenchymal

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Downloaded from Bioscientifica.com at 09/29/2021 07:34:47AM via free access Pituitary trophic responses · L A NOLAN and others 527 cells in the anterior pituitary were immunohistochemically et al. 1995). Other studies have been unable to confirm a identifiable corticotrophs. Paraventricular disconnection direct mitogenic role for CRH in normal anterior pituitary alone had no effect on ACTH-immunopositive cells after cells in vitro (McNicol et al. 1990, Gulyas et al. 1991), but, 7 days (5·20·22%; n=6). One week after surgical not surprisingly, technical difficulties have prevented clear adrenalectomy, the number of immunohistochemically identification of mitogenic effects within specific cellular identifiable corticotrophs increased to 8·80·46% subpopulations such as corticotrophs. (P,0·001; n=4–5). Loss of hypothalamic paraventricular The present work is distinct from previous studies in CRH/AVP input had no significant effect on this change, that, firstly, we have examined the trophic consequences as 1 week after surgery the number of corticotrophs in of reduced rather than increased exposure to CRH. adrenalectomized, PVN-lesioned animals was 8·10·36% Second, rather than using immunohistochemical markers (n=6). of apparent changes in anterior pituitary cell populations Cell counts were not corrected for the increase in cell as surrogate measures of mitotic activity, we have size that occurs following surgical adrenalectomy, and will quantified trophic events directly. CRH cannot be therefore be an overestimate of the absolute increase in eliminated in isolation, however, as CRH is co-localized corticotroph cell numbers (Floderus 1944). with AVP, and CRH/AVP neurosecretory neurons are interspersed with AVP magnocellular cells that are also deleted by PVN lesioning. AVP derived from the Discussion supraoptic nuclei accounts for 90% of the AVP found in portal blood (Antoni 1993), and, predictably, PVN Elimination of circulating corticosterone by bilateral lesioning alone does not significantly decrease the concen- adrenalectomy affects the pituitary either directly, or via tration of AVP measured in portal blood. However, the hypothalamus and hypothalamic–hypophyseal portal the increase in portal blood AVP induced by bilateral system. While changes in trophic activity may well adrenalectomy is abolished by PVN lesioning (Antoni et al. depend on complex paracrine interactions within the 1990), suggesting that modulation of pituitary trophic pituitary, the aim of the present study was simply to responsiveness to bilateral adrenalectomy after PVN dis- determine whether changes in hormones of paraventricu- connection is not mediated via increased hypothalamic lar origin are responsible for mediating the transient AVP. Furthermore, AVP is believed to act principally as increase in anterior pituitary mitotic activity following an ACTH secretogogue in CRH-primed corticotrophs, bilateral adrenalectomy and the increased amplitude of the and not to have any significant trophic effects (Antoni burst of pituitary apoptotic activity elicited by exposure to 1993). high-dose dexamethasone 1 week later. In this study, we have shown that the short-lived A large body of evidence supports a central role for proliferative response that occurs within a week or two paraventricular CRH in the synthesis of ACTH and for of surgical bilateral adrenalectomy in the male rat both CRH and AVP in controlling changes in the release anterior pituitary is independent of hypothalamic drive of ACTH from anterior pituitary corticotrophs. Evidence derived from CRH/AVP of PVN origin. Bilateral PVN for involvement of these factors in the induction of a lesioning had no effect on the wave of increased anterior pituitary cell proliferative response is, however, much less pituitary mitosis during the week following adrenalec- clear (Antoni 1993, McNicol & Carbajo-Perez 1999). tomy, and no effect on the size of the dexamethasone- Intact rats given a daily intraperitoneal injection of CRH sensitive apoptotic cell population generated over the for 2 or 7 days show an increase in the number of mitotic same time period that depends on recent mitotic activity corticotrophs in the anterior pituitary without an overall (Nolan & Levy 2003). In contrast, the marked increase increase in mitotic index (McNicol et al. 1988). More in anterior pituitary POMC transcripts that normally prolonged administration of CRH via osmotic mini-pump follows adrenalectomy was, as expected, completely has also been shown to increase the number of ACTH- abolished by PVN lesioning. positive cells (Gertz et al. 1987), and rare case reports of There are several explanations of the apparent discrep- ectopic CRH secretion in humans have been linked to ancies between studies that suggest a trophic role for CRH ACTH hypersecretion and corticotroph hyperplasia (Asa and the data presented here. CRH may behave as a et al. 1984, Carey et al. 1984). An animal model of the significant stimulus to corticotroph cell proliferation when same, in which a CRH-secreting tumour cell line was administered at supraphysiological concentrations or implanted into adrenal-intact rats, demonstrated an when administered in vitro away from modulating influ- increase in both the size and percentage of immunohisto- ences encountered in vivo or for a prolonged interval. chemically identifiable corticotrophs 10–15 weeks later The latter explanation, that CRH’s effects on mitosis (Asa et al. 1992). Studies with a corticotroph-enriched depend on longer term exposure, seems unlikely, as cell-culture system have suggested that both CRH and/or the proliferative response that occurs after bilateral epidermal growth factor (EGF) can be mitogenic for adrenalectomy is complete within 14 days, after which a corticotrophs in vitro within a limited dose range (Childs new equilibrium is reached despite the persistence of www.endocrinology.org Journal of Endocrinology (2004) 181, 521–529

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increased paraventricular CRH/AVP drive (Gulyas et al. Acknowledgements 1991, Taniguchi et al. 1995, Nolan & Levy 2001). As pituitary cells taken from adrenalectomized rats were We thank the Wellcome Trust for funding this study and apparently able to proliferate in culture under conditions Dr Gabor Makara for his advice and help with the design that failed to produce a proliferative response in cells taken and use of the rotating wire knife. from adrenal-intact animals (Gulyas et al. 1991), it is possible that ambient circulating glucocorticoid levels may qualitatively modify the response to CRH. In our References own unpublished study, we were unable to demonstrate a trophic effect of 12 days’ continuous systemic CRH Antoni FA 1993 Vasopressinergic control of pituitary adrenocorticotro- infusion in adrenalectomized rats receiving physio- pin secretion comes of age. Frontiers in 14 76–122. logical corticosterone replacement despite a greater than AntoniFA,FinkG&ShewardWJ1990Corticotrophin-releasing sixfold increase in both pituitary POMC transcripts and peptides in rat hypophysial portal blood after paraventricular lesions: a marked reduction in the concentration of corticotrophin-releasing circulating ACTH compared to vehicle (data not factor-41, but no change in vasopressin. Journal of Endocrinology 125 shown). 175–183. Estimates of trophic activity that depend on changes in Asa SL, Scheithauer BW, Bilbao JM, Horvath E, Ryan N, Kovacs K, the relative populations of different cell types classified on Randall RV, Laws ERJ, Singer W, Linfoot JA et al. 1984 A case for hypothalamic acromegaly: a clinicopathological study of six the basis of pituitary hormone immunohistochemistry are patients with hypothalamic gangliocytomas producing growth extremely difficult to interpret, as the size, shape and hormone-releasing hormone. Journal of Clinical Endocrinology and hormone content of cells, all of which change during Metabolism 58 796–803. manipulation of the hypothalamo–pituitary–adrenal axis, Asa SL, Kovacs K, Hammer GD, Liu B, Roos BA & Low MJ 1992 independently affect the sensitivity of the technique and its Pituitary corticotroph hyperplasia in rats implanted with a medullary ff ff thyroid carcinoma cell line transfected with a corticotropin-releasing quantitative utility. Potential e ects of transdi erentiation, hormone complementary deoxyribonucleic acid expression vector. the onset of hormone secretion by previously endocrino- Endocrinology 131 715–720. logically inactive cells and changes in hormone content Brugal G, Dye R, Krief B, Chassery J-M, TankeH&Tucker JH across the immunohistochemical detection threshold can- 1992 HOME: highly optimized microscope environment. Cytometry ff 13 109–116. notbedi erentiated from trophic events. Importantly, the Bruhn TO, Sutton RE, Rivier CL & Vale WW 1984 Corticotropin- criteria used to define trophic activity in the present study releasing factor regulates proopiomelanocortin messenger ribonucleic (direct identification of mitotic figures and clusters of acid levels in vivo. Neuroendocrinology 39 170–175. apoptotic bodies) were very specific, and the computer- Carey RM, Varma SK, Drake JCR, Thorner MO, Kovacs K, assisted manual system used for quantification is well RivierJ&ValeW1984Ectopicsecretion of corticotropin-releasing factor as a cause of Cushing’s syndrome; a clinical, morphologic validated and highly sensitive (Nolan et al. 1998, 1999, and biochemical study. New England Journal of Medicine 311 Nolan & Levy 2001). Owing to the technical obstacles 381–388. described above, attempts to analyse the subtypes of cells Childs GV, Rougeau D & Unabia G 1995 Corticotropin-releasing involved in pituitary trophic responses immunohisto- hormone and epidermal growth factor: mitogens for anterior pituitary corticotropes. Endocrinology 136 1595–1602. chemically are likely to be misleading. Indeed, our current Dallman MF, Makara GB, Roberts JL, LevinN&BlumM1985 work suggests that trophic responses are most likely to Corticotrope response to removal of releasing factors and occur in a subpopulation of cells that do not express any corticosteroids in vivo. Endocrinology 117 2190–2197. pituitary hormones. Floderus S 1944 Untersuchungen über den Bau der menschlichen In conclusion, bilateral adrenalectomy-induced mitotic Hypophyse mit besonderer Berucksichtigung der quantitativen mikromorphologischen Verhä ltnisser. Acta Pathologica Microbiologica changes in the anterior pituitary and short-term enhance- Scandinavica Supplement 53 276. ment of pituitary apoptotic responsiveness are entirely Gertz BJ, Contreras LN, McComb DJ, Kovacs K, Tyrrell JB & independent of paraventricular CRH/AVP input. These Dallman MF 1987 Chronic administration of corticotropin-releasing findings suggest that if immunohistochemically identified, factor increases pituitary corticotroph number. Endocrinology 120 381–388. CRH-induced changes in relative cell populations in the Gulyas M, Pusztai L, RappayG&Makara GB 1991 Pituitary anterior pituitary occur, they result from maturation of corticotrophs proliferate temporarily after adrenalectomy. hormonally undifferentiated cells into the corticotroph Histochemistry 96 185–189. phenotype, transdifferentiation from other dedicated cell Levy A 2002 Physiological implications of pituitary trophic activity. types or relative changes in hormone synthetic activity in Journal of Endocrinology 174 147–155. LevyA&LightmanSL2003Molecular defects in the pathogenesis of bi- or polyhormonal pituitary cells, rather than from pituitary tumors. Frontiers in Neuroendocrinology 24 94–127. enhancement of mitotic activity in the corticotroph sub- Makara GB, Stark E, Kapocs G & Antoni FA 1986 Long-term effects population or increased apoptosis of other cell subtypes. of hypothalamic paraventricular lesion on CRF content and Independent control of pituitary secretory and trophic stimulated ACTH secretion. American Journal of Physiology 250 E319–324. responses potentially allows a persistent stimulus to elicit a McNicol AM & Carbajo-Perez E 1999 Aspects of anterior pituitary short-term trophic adjustment to accompany a longer term growth, with special reference to corticotrophs. Pituitary 1 secretory response. 257–268.

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McNicol AM, Kubba MAG & McTeague E 1988 The mitogenic Nolan LA, Lunness HR, Lightman SL & Levy A 1999 The effects of effects of corticotrophin-releasing factor on the anterior pituitary age and spontaneous adenoma formation on trophic activity in the gland of the rat. Journal of Endocrinology 118 237–241. rat pituitary gland: a comparison with trophic activity in the human McNicol AM, Murray JE & McMeekin W 1990 Vasopressin pituitary and in human pituitary adenomas. Journal of stimulation of cell proliferation in the rat pituitary gland in vitro. Neuroendocrinology 11 393–401. Journal of Endocrinology 126 255–259. Taniguchi Y, Tamatani R, YasutakaS&Kawarai Y 1995 Proliferation Nolan LA & Levy A 2001 Anterior pituitary trophic responses to of pituitary corticotrophs following adrenalectomy as revealed by dexamethasone withdrawal and repeated dexamethasone exposures. immunocytochemistry combined with bromodeoxyuridine-labeling. Journal of Endocrinology 169 263–270. Histochemistry 103 127–130. Nolan LA & Levy A 2003 Temporally sensitive trophic responsiveness of the adrenalectomized rat anterior pituitary to dexamethasone challenge: relationship between mitotic activity and apoptotic sensitivity. Endocrinology 144 212–219. Received 20 November 2003 Nolan LA, Kavanagh E, Lightman SL & Levy A 1998 Anterior pituitary cell population control: basal cell turnover and the effects Accepted 9 March 2004 of adrenalectomy and dexamethasone treatment. Journal of Made available online as an Neuroendocrinology 10 207–215. Accepted Preprint 17 March 2004

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