Neuroendocrine regulation of hypothalamo-pituitary function in the fetus
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During fetal development there is a progressive maturation of the hypothalamo- pituitary- adrenal (HPA) axis which is responsible for co- ordinated fetal maturation and the onset of birth. The aim of this thesis is to investigate the neuroendocrine regulation of the developing HPA axis. The first approach was to investigate the role of putative neurotrophic factors in the functional maturation of hypothalamic arginine vasopressin (AVP) neurons. The role of AVP in the release of adrenocorticotropic hormone (ACTH) has been extensively demonstrated but little is known about the factors controlling the development of AVP neurons. The objective of this study was to chart the growth and development of AVP neurons in cell culture in response to various neurotrophic factors. Functional maturation of the neurons was assessed in terms of the secretory response to potassium- induced depolarisation whilst development of neuronal morphology was to be monitored by immunocytochemistry. Fetal rat hypothalamic neurons were cultured in the presence and absence of insulin - like growth factor -1 (IGF -1), which has been implicated as a putative neurotrophic factor, for periods of up to 20 days. IGF -1 had no significant effect on either basal or stimulated levels of AVP secretion, suggesting that AVP neurons do not appear to be the specific target population for IGF -1 within the developing fetal hypothalamus. Despite extensive screening of several different antibodies, we were unable to specifically identify AVP neurons in culture by immunocytochemistry.The second approach to studying the neuroendocrine regulation of the HPA axis utilised the sheep as an animal model as it affords chronic in utero manipulation of the fetus. In the ovine fetus, maturation of the HPA axis is pivotal in the onset of parturition. The hypothesis that POMC- derived peptides of the ovine fetal pituitary are subject to tonic dopamine inhibition throughout gestation was investigated. Removal of this inhibitory tone may induce synthesis and release of adrenal active peptides which in turn elicit cortisol secretion from the fetal adrenal gland. The approach to the hypothesis was to investigate the effects of dopaminergic manipulation of the fetus, on the secretion of POMC- derived peptides and cortisol into the fetal circulation, on pituitary content of POMC -derived peptides and on the level of POMC gene expression. Specifically, fetal sheep at day 131 gestation (gestation =145 days) were treated with a 72 hour intravenous administration of the dopamine agonist bromocriptine, the dopamine antagonist sulpiride or vehicle control. Treatment with bromocriptine significantly decreased the concentration of a -MSH in the fetal plasma, represented by an abolition of the pulsatile mode of secretion of a -MSH. In response to sulpiride infusion, plasma concentrations of both a -MSH and ACTH were significantly elevated, with a -MSH secretion displaying increased pulse amplitude with decreased pulse frequency. By contrast, the pulsatile characteristics of ACTH were unaltered by sulpiride infusion. Despite increased plasma concentrations of a- MSH and ACTH following sulpiride infusion, the concentration of cortisol in the fetal plasma was unaltered.α-MSH and ACTH were identified by immunohistochemistry and determination of peptide content in the pars distalis and pars intermedia of the fetal pituitary. The content of ACTH in the pars intermedia tended to increase in response to bromocriptine and decrease in response to sulpiride treatment. Pituitary a -MSH content was unaffected by either treatment. POMC mRNA levels in the fetal pars intermedia was significantly reduced following bromocriptine treatment and were unaltered by treatment with sulpiride. The level of POMC gene expression in the pars distalis was not significantly altered by either treatment.Studies on the ontogeny of POMC gene expression and the development of α-MSH and ACTH immunoreactivity revealed that POMC gene expression, ACTH and α-MSH immunoreactivity can be detected as early as day 40 gestation. ACTH immunoreactivity was detected in both the pars intermedia and pars distalis of the pituitary. In contrast, α-MSH immunoreactivity was confined to the pars intermedia at most gestational ages examined. However, at day 40 -50 gestation a -MSH immunopositive cells were also identified in the pars distalis. POMC mRNA levels were consistently higher in the pars intermedia compared to the pars distalis and increased progressively from day 40 until day 134 gestation. In the pars distalis, POMC mRNA levels were high at day 40 gestation, decreased at day 50 and then rose progressively until day 134 gestation.Fetal plasma prolactin concentrations were also influenced by treatment with bromocriptine and sulpiride. Secretion of prolactin into the fetal plasma was influenced by season, with concentrations peaking in the summer months and decreasing to very low levels in the winter. Winter prolactin concentrations could be further suppressed by infusion of bromocriptine. Conversely, sulpiride treatment significantly increased prolactin concentrations in fetuses treated in the winter months however, concentrations in sulpiride- treated winter fetuses were still lower than those of vehicle controls in the summer months. The seasonal alteration in plasma prolactin concentrations was mirrored by a significant decline in the number of immunopositive lactotrophs in the winter. The number of immunopositive lactotrophs was unaffected by bromocriptine or sulpiride infusion in either season.These results demonstrate the existence of an endogenous inhibitory system regulating the release of ACTH, α-MSH and prolactin from the fetal pituitary gland. However, these results do not support the hypothesis that peptides released upon removal of the inhibitory dopaminergic tone are capable of eliciting the secretion of cortisol from the fetal adrenal gland.