Prolactin receptors (PRLr) were detected in anestrous (n=3), winter cycling (n=2), follicular (n=3) and luteal phase (n=3) equine ovaries by IHC. Follicle stages evaluated were primordial, preantral and antral. Receptors were detected in all follicle stages and in CL. PRLr staining was not different (P > 0.05) between primordial and preantral, but was greater (P < 0.001) in antral follicles. Primordial follicles stained weakest in anestrous and follicular phase ovaries, followed by luteal phase ovaries and was most intense in winter cycling. Staining in preantral follicles was weakest in anestrus, followed by follicular phase and highest in winter cycling and luteal phase. Staining was most intense in antral follicles with no difference (P > 0.05) between any of the reproductive states. Oocytes and ovulation fossa also possessed PRLr. In conclusion, concentrations of PRLr are highest in large, antral follicles suggesting a mechanistic role for PRL around the time of ovulation.

Epithelial cell adhesion to the surrounding extracellular matrix (ECM) is necessary for their proper behaviour and function. During pregnancy and lactation mammary epithelial cells (MECs) require signals imparted by specific β1 integrin-laminin interactions for their functional differentiation in response to Prolactin (Prl) and for the correct formation of polarised secretory acini. Downstream of β1 integrin (β1Itg), the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both Prl driven lactational differentiation and the establishment of apico-basal polarity in MECs. ILK is a multifunctional adaptor protein that links integrins to the actin cytoskeleton and Rho GTPases such as Rac1. ILK forms a ternary IPP (ILK-PINCH-Parvin) complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known which of ILKs interacting partners are important for controlling tissue-specific gene expression, or what acts downstream of the IPP complex. In this thesis I have now established that inducible ILK deletion in MECs from ILKfl/flCreER mice, prevents phosphorylation of Stat5 leading to a failure of Prl induced milk expression. In addition I have established a 3-dimensional culture model using the EpH4 mammary epithelial cell line, which respond to Prl treatment and form polarised acini similar to primary cells. In these cells knocking down β1Itg and ILK by lentiviral shRNA delivery was confirmed to have a profound effect on β-Casein production. Expression of ILK mutants that disrupt its protein-protein interactions, showed that mutation of K220 and E359 in the kinase domain also reduced milk production. This means that ILKs kinase domain is important for MEC differentiation, and suggests that Parvin binding (which is disrupted by these mutations) is key in mediating ILKs differentiation functions. Using a complimentary shRNA approach, knockdown of the βParvin binding Rac guanine nucleotide exchange factor αPix also prevented MEC differentiation. This identified for the first time that αPix is required for differentiation and suggests a route by which ILK, via it’s interaction with Parvin, can link integrins to αPix and Rac activity. Interestingly, αPix depletion did not disrupt the IPP complex or polarity, suggesting that αPix represents a differentiation specific bifurcation point in β1Itg-ILK adhesive signalling. Together, this work has helped to establish how ILK is involved in MEC differentiation and has identified a new role for the downstream Rac GEF αPix. In addition, this work contributes to our understanding of the molecular mechanisms by which cell adhesion regulates fundamental cell biological behaviours.

The anterior pituitary gland is the major hormonal regulator in the body. The gland contains five secretory cell types whose emergence during development is defined by a tightly regulated array of transcription factors. In adult life, the gland is plastic with the relative proportions of cells varying depending on physiological context. Tumours of the pituitary gland account for 15% of all intracranial tumours in man and are caused by the selective proliferation of one of the secretory cell types. The majority of these (60%) are prolactinomas which consist of very slowly proliferating lactotroph cells, which produce the hormone prolactin. Pituitary tumours are almost never malignant and do not express common genetic markers for cancer, suggesting endogenous proliferative stimuli in the pituitary are the cause of tumour development. Oestrogen causes lactotroph hyperplasia during pregnancy and increases prolactin secretion. Our group previously showed that Wnt-4 mRNA was upregulated during oestrogen-induced lactotroph hyperplasia in Fischer 344 rats. Wnt molecules are key regulatory proteins controlling differentiation, proliferation and migration in development and adult life. Wnt-4 is involved in the emergence of lactotrophs during development, and has been implicated in pituitary tumour formation. Wnt molecules signal through three pathways. The well studied canonical pathway has been implicated in numerous cancers and centres around gene transcription initiated by translocation of β-Catenin into the nucleus. There are two non-canonical pathways: the Wnt-planar cell polarity (PCP) pathway and the Wnt-calcium pathway which are both poorly understood. In this thesis, the expression of Wnt-4 was studied in the pituitary, and effects of downstream signalling pathways in response to oestrogen were assessed. Wnt-4 was expressed in all secretory cell types of the pituitary, as well as the marginal zone (MZ), a region of the pituitary that may harbour stem cells. Oestrogen upregulated Wnt-4 protein in the somatolactotroph GH3 cell line, though this could not be replicated in primary tissue. A number of approaches (western blotting, immunofluorescence, reporter gene assays and mutant β-Catenin overexpression) were utilised to show that the canonical pathway was not activated in the pituitary. Wnt-4 had a clear inhibitory effect on calcium oscillations in GH3 cells, showing for the first time a non-canonical effect in the pituitary, though the downstream effects are currently unknown. Attempts made to study the activation of the PCP pathway were inconclusive. Efforts focused on the distribution of key structural and regulatory proteins in the anterior pituitary and the MZ. The MZ was characterised by a single layer of cells at the border of the anterior and intermediate lobes of the pituitary, with high expression of E-Cadherin and Sox 9, though no change in distribution was observed with oestrogen treatment. In the anterior lobe, oestrogen treatment decreased N and E-Cadherin expression, which could be an indicator of PCP pathway activation during oestrogen induced-lactotroph hyperplasia. Overall, data suggest that Wnt-4 does not directly cause oestrogen-induced lactotroph proliferation, but is likely to play a role in regulating tissue plasticity in the adult gland, as well as in the pathogenesis of pituitary tumours.

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Reliable observation of ECT-induced hormone release requires that other processes that affect hormone levels remain constant and not obscure it. This article reviews principles and pitfalls in making such observations. Clinical applicability and limitations of measurements of prolactin, cortisol, oxytocin, in vasopressin, and other hormones are described. Applications, include elucidation of ECT physiology and seizure quality, comparison of ECT techniques, and description of illness severity. Accounting for each of these different effects can be needed to characterize any of them. An important but unrealized application of neuroendocrine measurement is prediction of the stability of individual ECT response.

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