A complete inventory of pituitary trophic responses depends on precise estimates of mitotic activity and apoptotic events, and accurate characterization and quantification of pituitary cell subtypes irrespective of previous and current physiological demand. For a discrete structure that has been so extensively studied, it is disappointing but perhaps not surprising that none of these measures is available and therefore that the relative contributions to changes in the proportions of pituitary cellular subpopulations of trophic activity, differentiation of pluripotent cells and variations in the secretory profiles of apparently committed cells remain almost impossible to determine. To fully appreciate the extent of this dilemma, it should be remembered that conservative estimates of the proportion of corticotrophs in the rat anterior pituitary under basal conditions vary over twofold and that it is still not clear whether the apparent threefold increase in mammotrophs during pregnancy is the result of maturation of uncommitted cells, transdifferentiation of other cells such as somatotrophs, cell division, or a mixture of all three. Equally, while it has been known for some time that adrenalectomy results in a transient increase in anterior pituitary mitotic activity and appropriately timed supraphysiological glucocorticoid replacement with a wave of apoptosis, the precise identity of the cells involved in both of these responses is open to question. Thus, although many of the physiological stimuli associated with apparent changes in the proportions of pituitary cellular subpopulations are known, the precise mechanism of the changes and the consequences of the same remain obscure. This review summarizes the limited literature on pituitary trophic activity and asks what, if anything, analysis of pituitary trophic activity using current technology can tell us.

Glucocorticoid withdrawal, depending on the dose and duration of treatment, results in a transient but sometimes prolonged reduction in hypothalamo-pituitary-adrenal (HPA) axis secretory responsiveness. As the anatomic basis of HPA axis suppression remains uncertain, we have directly examined changes in trophic activity within the rat anterior pituitary gland following dexamethasone withdrawal and re-treatment. Treatment of adrenalectomised, male Wistar rats with dexamethasone results in a discrete, highly significant burst of apoptosis in the anterior pituitary with concurrent suppression of mitosis. Despite a surge in mitotic activity immediately after dexamethasone withdrawal, calculated total anterior pituitary cell populations remain below that seen in untreated adrenalectomised controls. Repeated exposures to dexamethasone show that the dexamethasone-sensitive cell population that is deleted by apoptosis is partially but not completely restored. As the amplitude of apoptotic bursts induced by second and third dexamethasone exposures are similar but smaller than that induced by initial exposure, it appears that the very first exposure to dexamethasone deletes a subset of anterior pituitary cells that are either not restored at all, or are only replaced very slowly. The reduced proportion of corticotrophs contributing to the increase in mitotic index after dexamethasone withdrawal corroborates this. Although continued cell turnover within the pituitary predicts that the absolute cellular deficit would diminish with time, the effects seen may contribute to the delayed recovery of pituitary axis function following cessation of glucocorticoid treatment.

Oestrogen is a powerful mitogen that is believed to exert a continuous, dose-dependent trophic stimulus at the anterior pituitary. This persistent mitotic effect contrasts with corticosterone and testosterone, changes in the levels of which induce only transient, self-limiting fluctuations in pituitary mitotic activity. To further define the putative long-term trophic effects of oestrogen, we have accurately analysed the effects of 7 and 28 days oestrogen treatment on anterior pituitary mitotic activity in ovariectomized 10-week-old Wistar rats using both bromodeoxyuridine (BrdU) and timed colchicine-induced mitotic arrest. An oestrogen dose-dependent increase in mitotic index was seen 7 days after the start of treatment as expected, representing an acceleration in gross mitotic activity from 1.7%/day in ovariectomized animals in the absence of any oestrogen replacement to 3.7%/day in the presence of a pharmacological dose of oestrogen (50 mcg/rat per day: ∼230 mcg/kg per day). Despite continued exposure to high-dose oestrogen and persistence of the increase in pituitary wet weight, the increase in mitotic index was unexpectedly not sustained. After 28 days of high-dose oestrogen treatment, anterior pituitary mitotic index and BrdU-labelling index were not significantly different from baseline. Although a powerful pituitary mitogen in the short term, responsible, presumably, for increased trophic variability in oestrus cycling females, these data indicate that in keeping with other trophic stimuli to the pituitary and in contrast to a much established dogma, the mitotic response to longer-term high-dose oestrogen exposure is transient and is not the driver of persistent pituitary growth, at least in female Wistar rats.

We have used a direct, non-immunochemical and highly accurate method to quantify the effects of testosterone and oestrogen on mitotic and apoptotic activity in the young, male rat anterior pituitary in vivo. Surgical gonadectomy resulted in a 3-fold increase in mitotic activity by the fourth post-operative day, which returned gradually to levels seen in intact animals over the subsequent 3–4 weeks. Both a single dose of Sustanon, a mixture of long-acting testosterone esters in arachis oil, and the same dose divided over 7 days (starting 6 days after gonadectomy), initially suppressed mitotic activity to levels seen in intact animals, but was associated after 48–96 h with a wave of increased mitotic activity. The latter was blocked by co-administration of Sustanon with the non-steroidal aromatase inhibitor letrozole and was not seen when the non-aromatisable androgen dihydrotestosterone was substituted for Sustanon. Oestrogen alone in gonadectomised and intact rats produced a marked increase in mitosis as expected. With the exception of a transient increase in response to a single high-dose injection of Sustanon in gonadectomised animals, apoptotic activity was unaffected by all of the above. This study suggests that pituitary mitotic activity is tonically inhibited by gonadal hormone production (at least in the short term) in adult male rats. The study also suggests that supraphysiological testosterone treatment – while unable to reduce anterior pituitary mitotic activity in untreated, intact animals –suppresses the early increase in mitotic activity induced by gonadectomy. Oestrogen, either exogenous or generated locally by aromatisation, stimulates anterior pituitary mitotic activity in a time-dependent manner.

ABSTRACT

The temporal effect of orally administered bromocriptine on pro-opiomelanocortin (POMC) and prolactin gene expression in male Sprague–Dawley rats was examined using in-situ hybridization histochemistry. Messenger RNA in the anterior and intermediate lobes could be clearly delineated in each section. Administration of bromocriptine resulted in a reduction in hybridization of ³⁵S-labelled cDNA probe to prolactin mRNA from 0·69 × 10¹² to 0·29 × 10¹² copies bound/g after 150 h. POMC mRNA in the anterior lobe remained unchanged with 0·08 × 10¹² copies of probe bound/g for the duration of the experiment, while in the intermediate lobe it decreased from 2·44 × 10¹² to 0·44 × 10¹² copies of probe bound/g at 150 h. The rate of reduction in intermediate lobe POMC mRNA was similar to that of prolactin mRNA for the first 24 h but was subsequently more rapid and more profound, falling to 20% of the control value at 84 h and to 18% at 150 h.

J. Endocr. (1988) 118, 205–210

The anterior pituitary is active mitotically and apoptotically under basal conditions and in response to a variety of physiological and pathophysiological stimuli. Hypothyroidism in man is associated with a modest but very occasionally dramatic increase in overall pituitary size. The mechanisms underlying this reversible phenomenon remain obscure. In the present study we have examined young adult rat anterior pituitary following surgical thyroidectomy and subsequent thyroid hormone treatment and withdrawal using an extremely accurate system for quantifying directly identified mitotic and apoptotic events. Despite the expected increase in the number and/or proportion of immunohistochemically identifiable thyrotrophs three weeks after thyroidectomy, mitotic and apoptotic activity remained unchanged, as did pituitary wet weight, in comparison with sham-operated and intact controls. In contrast, mitotic but not apoptotic activity was enhanced by treatment of thyroidectomized animals with thyroid hormones (triiodothyronine (T3) and thyroxine (T4) 1.8 microg and 3.6 microg/100 g body weight per day respectively), and once again declined to levels seen in intact animals within 72 h of subsequent thyroid hormone withdrawal. Thyroid hormone-induced enhancement of mitotic activity was also seen in intact rats treated with similar doses of thyroid hormones for 7 days and in thyroidectomized rats treated for a similar period with very low dose thyroid hormone replacement at a level that had no effect on raised hypothalamic TRH- or pituitary TSHbeta-transcript prevalence (0.018 microg T3 plus 0.036 microg T4/100 g body weight per day). Thus changes in mitotic and apoptotic activity are unlikely to be the principle mechanism for the apparent increase in thyrotrophs up to 4 weeks after thyroidectomy. In contrast, the data indicate that thyroid hormones have a permissive effect on anterior pituitary mitotic activity in thyroidectomized male rats. Thyroid hormone-induced enhancement of mitotic activity in intact rats further suggests that in euthyroid rats, ambient thyroid hormone levels are a limiting factor for anterior pituitary mitotic activity. In summary, this time course study of young, male rats has shown for the first time that thyroidectomy, thyroid hormone replacement and subsequent withdrawal has no significant effect on anterior pituitary apoptotic activity. Secondly, it has shown that the anterior pituitary mitotic response to thyroidectomy is blocked by complete thyroid hormone deprivation, but can be restored by very low level thyroid hormone replacement, and thirdly that in intact animals thyroid hormone levels significantly limit anterior pituitary mitotic activity.

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

We have used in situ hybridization histochemistry to investigate the effects of maternal thyroidectomy and chronic maternal iodine deficiency on basal neuroendocrine function in rat pups. Specifically, we have measured hypothalamic thyrotrophin-releasing hormone (TRH) and pituitary thyroid-stimulating hormone (TSH) expression together with circulating levels of tri-iodothyronine (T₃) in rat pups delivered from and suckled by thyroidectomized or iodine-deficient dams. Because of the close interaction between the thyroid, adrenal and growth hormone axes, we have also examined hypothalamic corticotrophin-releasing hormone (CRH) and growth hormone-releasing hormone (GRH) transcripts at the same time points: birth, 1 month and 2 months of age.

Three weeks after surgical thyroidectomy, adult female Sprague–Dawley rats proved unable to carry pups to term and lactate successfully. Pups delivered from thyroidectomized dams given a small replacement dose of T₃ during pregnancy were significantly lighter than controls (84 ± 3%) and had markedly depressed plasma T₃ levels (36 ±6% of control). Hypothalamic CRH and GRH transcript levels were significantly decreased in pups at birth (to 8±2·5% and 24 ± 8% of control respectively) but had returned to normal by 1 month after delivery. Pituitary TSH transcript levels and hypothalamic levels of TRH transcripts, however, were similar to those of controls.

Only one of seven dams fed a low-iodine diet for 6 months produced live pups, and these were too few in number to produce significant data. Dams fed a lowiodine diet from 4 months before mating, however, did produce live pups and although they were not significantly lighter than control pups at birth, by 1 month after birth, they were significantly lighter (72 ± 3% of controls). Circulating T₃ levels were not significantly different from control at any time point examined. Hypothalamic TRH levels were significantly elevated at birth (451 ± 138% of control), but this difference was not maintained at 1 or 2 months after birth despite the lactating dams being maintained on the low-iodine diet. Pituitary TSH levels showed an upward trend at all time points that reached significance at 1 month after birth (204 ± 19%; P<0·05). Hypothalamic CRH and GRH transcript levels were not different from controls at any time point.

In summary, chronic iodine deficiency or thyroidectomy with low-level T₃ replacement in Sprague–Dawley rats markedly impaired fertility and the ability to carry pups to term, and produced an unexpectedly modest up-regulation of the hypothalamo–pituitary–thyroid axis and down-regulation of the hypothalamo–pituitary–adrenal axis.

Journal of Endocrinology (1997) 152, 423–430

In the present study, we compared rat uterine contractility and myometrial inositol phosphate (InsP) production in response to activation of muscarinic and oxytocin receptors during pregnancy and at term. The level of myometrial phospholipase (PL) Cβ was also determined by Western blotting at different stages of pregnancy and following administration of oestradiol, progesterone or vehicle. The results showed an increased potency of carbachol (CCh), a cholinergic muscarinic agonist, and oxytocin (OT) to enhance myometrial InsP production at term. This correlated with an increased potency of both agonists to induce contraction of the circular but not the longitudinal muscle. For both InsP production and contractile activity, the maximal response of CCh was unaltered, while that of OT was significantly increased. Interestingly, the increased responsiveness to CCh and OT was associated with an up-regulation of PLCβ1 and PLCβ3 enzymes. Such regulation is under the control of oestradiol since administration of this steroid to pregnant rats increased the amount of both enzymes by 200–260%. In contrast, progesterone administration was without effect. The present study presents the first evidence that the expression of rat myometrial PLCβ1 and PLCβ3 is under the positive control of oestradiol. This could participate in the enhancement of myometrial InsP accumulation and uterine contraction at term in response to CCh and OT. Based on contraction studies, we also propose that the longitudinal and circular uterine muscles differ in the regulation of the PLC pathway during pregnancy.

Abstract

The rapid detection of gene activation is important for our understanding of gene regulation. We have therefore studied heteronuclear (i.e. nascent) RNA (hnRNA) by using ³⁵S-labelled corticotrophin-releasing hormone (CRH) riboprobes and arginine vasopressin (AVP) oligonucleotide probes directed against intronic and exonic sequences of both CRH and AVP transcripts for in situ hybridization studies of transcriptional changes during acute stress. CRH and AVP intronic signals (found in newly synthesized transcripts) were confined to the nuclei of the parvocellular cells in the paraventricular nucleus (PVN) whilst CRH and AVP exonic signals (found in both newly formed and mature transcripts) were primarily located in the cytoplasm of these cells. AVP hnRNA and mRNA were also present at high levels in the magnocellular PVN. The levels of CRH hnRNA and parvocellular AVP hnRNA in the PVN were significantly increased 1 and 2 h after the onset of restraint. The levels of CRH mRNA on the other hand were not significantly increased until 4 h after the onset of restraint. The number of AVP mRNA-expressing neurons in the medial parvocellular cells of the PVN significantly increased at 2 h and peaked at 4 h after the onset of stress. In contrast, densitometric analysis indicated that the increase in AVP mRNA levels in these cells did not reach significant difference from control until 4 h after the onset of restraint. There were no significant changes in AVP hnRNA or AVP mRNA levels in the magnocellular subdivision of PVN at any time point after the onset of restraint.

Journal of Endocrinology (1997) 152, 81–89