Effect of mechanical stress o the integrity, signalling mechanisms and function of bovine mammary epithelial cells : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Manawatū, Palmerston North, New Zealand

Abstract

Mammary gland engorgement due to milk accumulation in late lactation leads to changes in cell morphology and has been recognised as a potential key initiator of involution and remodelling of the mammary gland. The physical distension of mammary epithelial cells (MEC), due to udder filling, is likely to result in mechanical tension on cell-cell and cell-matrix interactions. Cell-cell and cellmatrix junctions provide tissue integrity, promote cell polarity, guarantee sufficient communication between cells to ensure synchronised milk secretion and support cell survival. Their disruption may be one of the early initiators of the mammary gland remodelling process. As a consequence, the primary goal of this study was to determine the potential effects of MEC stretch on changes in cell sensing within the mechanical micro-environment in the initiation of bovine MEC involution. During this investigation, particular emphasis was put on three potential mechanosensors: tight junctions (TJ), focal adhesions (FA) and primary cilia (PC), and their regulation in the early stages of involution using in vivo and in vitro experimental approaches. Static, biaxial in vitro cell stretch and acute physical distension in vivo resulted in changes in TJ protein expression levels implying a potential disruption of cell-cell communication as well as communication with the cell‟s cytoskeleton. Furthermore, down-regulation of Akt and pAkt following different periods of mechanical strain applied in vitro and decreased levels of pAkt following acute physical distension in vivo indicated a disruption of β1-integrin-FAK survival signalling through the PI3K-Akt pathway downstream of FA interactions. Increased numbers of ciliated MEC following extended periods of non-milking indicated a dedifferentiation of MEC. Furthermore, increased levels of STAT6 transcription (part of PC signalling following mechanical stimulation) factor indicates the initiation of macrophage accumulation and promotion of tissue remodelling of the bovine mammary gland. In conclusion, this study supports the hypothesis that local factors play an important role during bovine mammary gland involution and that mechanical stimulation may play a part in the initiation of this process.