We have used an extremely accurate, dedicated, real time computerized image analysis system to facilitate the manual quantification of changes in the prevalence of mitotic figures and apoptotic bodies in male rat pituitary following surgical adrenalectomy and, 14 days later, dexamethasone treatment. Under basal conditions, the prevalence of mitotic figures and apoptotic bodies was 0.066+/-0.016% and 0.030+/-0.012% (mean+/-SE) respectively. Dexamethasone treatment reduced the prevalence of mitotic figures and, in adrenalectomized animals, produced a highly significant and reproducible burst of apoptotic activity that peaked 48 h after the beginning of treatment (0.261+/-0.022%) before falling sharply to control levels within a further 8 h. Two weeks after the start of dexamethasone treatment, total pituitary cell numbers continued to decline. The rate of accumulation of mitotic figures in vivo after colchicine treatment indicates that mitosis is histologically overt in 2 microm thick hematoxylin and eosin stained sections under the light microscope for around 80 min; that apoptosis--identified as classical apoptotic bodies--is overt for 44 min and that, on average, a young, adult, male rat anterior pituitary cell either dies or divides as frequently as once every 60-70 days. These data show that transient and apparently trivial fluctuations in the prevalence of apoptotic and mitotic events have a profound effect on pituitary cell population dynamics, and demonstrate that dexamethasone treatment of adrenalectomized rats produces a decline in total anterior pituitary cell numbers that continues for at least 2 weeks after the start of glucocorticoid treatment.
The male rat anterior pituitary responds highly reproducibly to specific hormonal stimuli in terms of the extent and timing of mitotic and apoptotic (trophic) activity. The principal objective of the present study was to define the contribution of hormonally identifiable cells to the trophic responses to bilateral gonadectomy and bilateral adrenalectomy. The patterns of pituitary mitotic responses to adrenalectomy and gonadectomy are similar in amplitude and duration. When adrenalectomy and gonadectomy are combined, the amplitude of the pituitary mitotic response is unchanged. That is, the trophic stimuli are not additive. Dexamethasone-induced apoptosis in nascent cells is amplified not only by recent adrenalectomy, but also, and to an almost identical extent, by gonadectomy. Combining adrenalectomy and gonadectomy does not further enhance the size of the apoptotically-responsive cell population. Dual bromodeoxyuridine and adrenocorticotrophic hormone (ACTH) or luteinising hormone (LH) immunolabelling showed that more than 95% of all dividing cells are not and do not become positive for either of these hormones during the period of peak mitotic response. Following adrenalectomy, most newly-formed ACTH cells are derived from differentiation of pre-existing hormonally undifferentiated cells. Despite an overall increase in mitotic activity, there is no measurable increase in the number of LH immunopositive cells after gonadectomy. The nonadditive pituitary mitotic and apoptotic responses to adrenalectomy and gonadectomy strongly suggest that the same progenitor cell population responds mitotically to both. This weakens the prevailing view that hormonally identifiable cells with specific trophic profiles contribute significantly to pituitary cell subpopulation revision.
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 animalssuppresses 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.
Rapid but often transient changes in mitotic and apoptotic activity are important components of the pituitary response to changes in the hormonal environment. For example, bilateral adrenalectomy and orchidectomy each result in a wave of increased mitosis lasting approximately 1 week, mediated by the same population of trophically active and, to a large extent, endocrinologically inactive cells. By contrast to these tonic inhibitors of pituitary trophic activity, reports of a progressive increase in lactotroph numbers during pregnancy suggest that oestrogen is a potent and persistent pituitary mitogen. By comparing the amplitude and duration of male rat anterior pituitary mitotic responses to oestrogen treatment, to adrenalectomy, and to a combination of the two, the present study aimed to further clarify the characteristics of the oestrogen-induced trophic response, in particular whether lactotrophs are the predominant cell type involved. Adrenalectomy produced a wave of increased mitotic activity, which resolved within 7 days as expected, whereas oestrogen induced a significant increase in mitotic activity, which was sustained for the 14-day duration of the study. The trophic effects of combining adrenalectomy and oestrogen treatment were not additive in that the statistically insignificant upward trend in mitotic index during the first few days compared to oestrogen treatment alone was entirely abolished by oestrogen pre-treatment. The increase in mitotic activity in lactotrophs induced by oestrogen either with or without adrenalectomy did not result in an increase in the relative size of the prolactin-positive compared to prolactin-negative pituitary parenchymal cell numbers by the end of the study. Despite the marked increase in the lactotroph population that is reported during pregnancy, these data indicate that at least the early (i.e. within 2 weeks) mitotic response to pharmacological doses of oestrogen increases mitotic activity in the lactotroph subpopulation by only 5-8% relative to other cellular subpopulations. Unexpectedly, the mitotic response to oestrogen principally occurs in non-prolactin-containing cells and results in the recruitment, amongst other trophically responsive populations, of the entire subpopulation of prolactin-, adrenocorticotrophic hormone- and luteinising hormone-negative cells that respond mitotically to adrenalectomy. Oestrogen therefore has a previously unrecognised non-cell type-specific trophic effect in the pituitary that obscures the relative expansion of the lactotroph population by inducing concurrent increases in numbers of prolactin-negative cells, the nature of which at least in part remains to be determined.
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