The role of the mammary fat pad during mammogenesis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, New Zealand

Abstract

Development of the female mammary gland involves the proliferation and morphogenesis of epithelial cells within a matrix of adipose and connective tissue which constitutes the mammary fat pad. The objective of this research was to investigate the mechanisms by which this stromal environment locally regulates postnatal mammogenesis. Initial experiments showed that the mouse mammary fat pad liberates a diffusible activity in vitro which stimulates the growth of mouse mammary epithelial cells and enhances their proliferative response to insulin-like growth factor-I, epidermal growth factor and insulin. This effect was specific to these mitogens, and of a variety of cell lines tested was most pronounced for mouse mammary epithelial cells. Subsequent investigations indicated that these responses were likely induced by unsaturated fatty acids, particularly linoleic acid, from mammary adipocytes. Such responses may be effected by increased intracellular signalling via the actions of protein kinase C. The mitogenic capacity of the mouse mammary fat pad was also evaluated across several physiological states. Mammary fat pad-stimulated proliferation during the estrus cycle was increased at estrus concomitant with a phase of ductal elongation in vivo. In certain medium treatments there was evidence for epithelial upregulation of the mitogenic effect of the mammary fat pad, where intact mammary tissue was more stimulatory than mammary fat pad cleared of endogenous epithelium. Further experiments demonstrated that while the mitogenic effect of the mammary fat pad was unaltered by ovariectomy, ovarian function was required for this effect to be increased by exogenous progesterone. The effect of estrogen was independent of ovarian function but was altered by the local epithelial-stromal interaction, where it increased the mitogenic effect of epithelium-free mammary fat pad and decreased that of intact mammary tissue. Mitogenic stimulation by mammary tissues also declined during virginal development to be least in mature virgin and mid-pregnant states. Stimulation by intact mammary tissue increased during lactation, while that from epithelium-free mammary fat pad remained constant in the presence of steroid hormones and increased in the presence of growth factors. Further experiments investigated the stromal regulation of epithelial growth within the ruminant mammary gland. Differences between the ruminant and rodent mammary fat pad were emphasised in vitro where ovine mammary fat pad stimulated the growth of mouse mammary epithelial cells but did not markedly potentiate their growth factor-responsiveness. A subsequent study examined the expression of stroma-derived growth factors within the ruminant mammary gland during postnatal development, and their regulation by several physiological influences. The level of insulin-like growth factor (IGF)-I mRNA in the ovine mammary fat pad was elevated prior to puberty and during late gestation, while IGF-II mRNA was upregulated in mammary parenchyma of virgin ewes in a transcript-specific manner. Abundance of IGF-I mRNA in mammary tissues of prepubertal ewe lambs tended to be increased by exogenous estrogen whereas IGF-II mRNA levels were reduced. Messenger RNA for keratinocyte growth factor (KGF) was detected within the ovine mammary fat pad throughout development as 2.4 and 1.5 kb mRNA transcripts which were expressed by stromal adipocytes and fibroblasts, respectively. The level of KGF mRNA in mammary tissues of prepubertal lambs was increased by ovariectomy and decreased by estrogen, while KGF mRNA expression in cultures of mammary fibroblasts was suppressed by dexamethasone. Messenger RNA for hepatocyte growth factor, a paracrine mitogen and morphogen for mammary epithelial cells, was expressed in the ovine mammary fat pad and by cultured mammary fibroblasts. The abundance of basic fibroblast growth factor (bFGF) mRNA was highest within the ovine mammary fat pad, while in vitro results suggest bFGF may be a paracrine/autocrine mitogen for multiple cell types within the mammary gland. Basic FGF gene expression in mammary tissues of prepubertal ewes was reduced by estrogen treatment. For each of these growth factors there was evidence suggesting that their expression within the mammary fat pad was upregulated by the adjacent mammary epithelium. In conclusion, these findings indicate that the mammary fat pad may stimulate the proliferation of mammary epithelial cells during postnatal mammogenesis by a variety of influences. Such mechanisms may involve the direct stimulation of epithelial growth or the modulation of epithelial responsiveness to other mitogens. These effects may function to mediate the actions of certain mammogenic hormones. Furthermore, strong evidence indicates that mammary growth may be locally regulated by the interaction between epithelial and stromal cells.