Epithelial and immune cell dynamics in non-traditional models of mammary postnatal development

Mammary terminal duct lobular units comprise an epithelial bilayer of luminal and myoepithelial cells. The specific contribution of each distinct epithelial compartment to mammary postnatal development and the mammary microenvironment remains to be elucidated. The present study aims to exploit the ovine and rabbit mammary gland to interrogate epithelial proliferation and immune cell dynamics during mammary postnatal development and in mastitis. These species exhibit comparable histo-anatomy to the breast, constituting promising adjunctive non-traditional model systems. Moreover, unlike the male mouse, the male ovine gland persists into postnatal development. This study therefore also aims to evaluate the male ovine mammary microenvironment as a model for male mammary biology. Initially, luminal and myoepithelial cell proliferation was quantified during key stages of ovine mammary postnatal development. This demonstrated that proliferation in both compartments is highest during the neonatal stage, correlating with an allometric prepubescent ovine mammary growth phase. A further reduction in epithelial proliferation was observed between pregnancy and lactation. Similar investigations of the rabbit mammary gland indicated that proliferation remains relatively consistent from gestation to lactation, highlighting species-specific proliferation dynamics which underpin differences in reproductive strategies. Analysis of ovine mammary involution indicated a previously undescribed increase in cell proliferation at approximately 2 months into the involution process. This outlines the timeline for gland establishment for subsequent pregnancies. An epithelial response towards mastitis was observed within both the ovine and rabbit mammary gland. In the ovine gland, epithelial proliferation increases in cases of chronic mastitis, detected during involution, suggesting a compensatory proliferation mechanism within the non-necrotic mammary epithelia to limit tissue damage. In contrast, clinical cases of lactational mastitis within rabbits resulted in a decrease in epithelial proliferation. The epithelial response to mastitis is therefore likely dependent on the severity of the infection and the stage of mammary development at which the mastitis occurs. The role of myoepithelial cells within the ovine mammary microenvironment and their interactions with macrophages were then investigated. Deep tissue 3-D imaging demonstrated that ovine mammary myoepithelial cells undergo morphological changes during development and the microenvironment comprises a spatially and morphologically heterogenous macrophage population, which is closely associated with the myoepithelium. Innovative 2-D and 3-D AI-enabled image analysis pipelines were developed, which attempted to isolate and segregate macrophages based on their association with the myoepithelial compartment. Whilst the difficulties in conducting 3-D image segmentation are exemplified, the 2-D image analysis pipeline analysed a dense macrophage population from an in-situ image and segmented individual cells to quantify their convoluted morphology, demonstrating no significant difference in morphology between myoepithelial-associated and non-myoepithelial-associated macrophages. Thirdly, enzymatic tissue digestion and flow cytometry protocols were successfully optimised for ovine mammary tissues and used to investigate the impact of ovine obesity on the pregnant mammary microenvironment. This investigation demonstrated the viability of using ovine mammary tissue for single cell analytical techniques. Interestingly, the proportions of mammary cell populations were unaffected by ovine obesity. Finally, the male ovine mammary gland was explored to facilitate the understanding of normal male mammary biology in a species in which the male gland persists postnatally. Both intact and neutered male sheep were analysed to highlight how the male-specific sex hormonal milieu impacts the mammary microenvironment. Irrespective of neutered status, the male gland displays limited expression of proliferative and apoptotic markers. Up to 72% of the luminal epithelial cells exhibit androgen receptor expression, which is significantly abrogated by neutering. Macrophages, T-lymphocytes and mast cells were demonstrated to cluster around the mammary ducts, implicating them in a protective immunological role within the male gland. Neutered males also exhibit a reduction in mast cell abundance, when compared to intact males, suggesting that hormone receptor signalling influences mast cell recruitment. In summary, this thesis provides insights into epithelial and immune cell dynamics to develop our understanding of mammary development and the microenvironment. Through utilising sheep and rabbit tissue, the data presented is more translatable to humans and elevates the use of non-traditional mammary gland models for the breast. It further uncovers key information that could improve animal welfare and agricultural production.