Prolactin Signalling in the Median Eminence in the Mouse

Abstract

The median eminence represents the interface between the hypothalamus and the portal blood, allowing access to the anterior pituitary and thus regulation of its secretion. The structure of the blood brain barrier at the median eminence is highly modified in order to allow the passage of these hypophysiotrophic factors from nerve terminals into the blood. These modifications also permit blood borne hormones to access the median eminence thus potentially modulating its function. This thesis will examine the potential activity of one such hormone, prolactin, at the level of the median eminence. Prolactin is a multifunctional peptide hormone produced in the anterior pituitary gland. Its secretion is primarily regulated by an inhibitory input by dopamine released into the median eminence by the tuberoinfundibular dopaminergic (TIDA) neurons of the hypothalamus and transported to the pituitary. Prolactin secretion is elevated during a number of physiological events including pregnancy, lactation, stress and sexual activity. Following its secretion, prolactin targets many different cell-types around the body to perform a wide variety of functions. Immunohistochemical observations in this thesis demonstrated that treatment of adult male mice with ovine prolactin resulted in the rapid phosphorylation (phospho-) of Signal Transducer and Activator of Transcription (STAT)5 in the median eminence thus indicating prolactin receptor activation in this tissue. Phospho-STAT5 labelling within the median eminence was present in two forms: nuclear and non-nuclear. Phospho-STAT5, being a transcription factor, typically translocates to the nucleus to regulate gene expression. Observations of non-nuclear phospho-STAT5 have rarely been described and its function remains unknown. The expression of non-nuclear phospho-STAT5 was confirmed through a number of validation experiments which utilised various antibodies, labelling methods and a STAT5b knockout mouse. Immunohistochemistry utilised dual-labelling and an array of knockout mice to investigate the cellular location of prolactin-induced phospho-STAT5 within the median eminence. These experiments revealed nuclear phospho-STAT5 was predominantly within a tanycytes, and that the non-nuclear phospho-STAT5 was present primarily within neuronal processes. The nature of these neuronal processes has remained elusive but they do not appear to be TIDA, CRH, oxytocin or glutamatergic neurons or indeed, based on fluorogold studies, other neuroendocrine neurons, suggesting that they were non-neuroendocrine neurons. The physiological relevance of prolactin actions in the median eminence was investigated using restraint stress, which is known to elevate circulating prolactin levels. These results demonstrated that this elevation of prolactin caused a similar phospho-STAT5 response in the median eminence as that observed following exogenous prolactin stimulation. The effect of prolactin on the permeability of the median eminence was evaluated using Evans Blue. It was found that 1 h treatment with prolactin reduced the permeability of the median eminence. This suggested that during periods of elevated prolactin, the permeability of the median eminence may be modulated. This action of prolactin may have downstream effects on the activity of the median eminence and the regulation of the pituitary. This thesis has identified the median eminence as a novel target of prolactin signalling and in doing so has demonstrated that the median eminence has both secretory and sensory roles.