Effect of a milk fat globule membrane fraction on cultured mouse mammary cells

Abstract

During lactation mammary cell number is regulated by an intramammary mechanism sensitive to the frequency of milking. Thus, a unilateral increase in cell number is elicited when one gland of lactating goats is milked thrice instead of twice daily [l]. The local increase in cell number i s associated with a higher rate of DNA synthesis in the thrice-milked gland [l]. However, it is also possible that the response is due in part to a reduction in the loss of mammary secretory cells at this stage of lactation. Since the effect i s related to frequency of milk removal from the gland we wished to determine whether goat's milk contains factors which may act as local regulators of mammary cell number. In this study we used mammary cells embedded within collagen gels, a culture system which allows extensive cell proliferation and formation of three-dimensional outgrowths reminiscent of the tissue in vivo [2].Mammary cells prepared from late pregnant mice by collagenase digestion [3] were fractionated on a Percoll gradient. Epithelial cells were suspended in foetal calf serum containing 10% DMSO and stored frozen under liquid N,. A batch of cells was thawed for each experiment. Cells were suspended at 4°C and lo6 cells/ml in rat tail collagen solution (1.6 mg/ml, pH 7.4), and aliquants (1.5 ml) were immediately gelled in 3.5 cm diameter wells at 37°C. Cells were cultured for 2-8 days in medium 199 / Hams F12 (5050) containing 15 mM-Hepes, 10% foetal calf serum, 5 pg insulin/ml, 1 pg hydrocortisoneiml, 0.65 ng triiodothyronine/ml, 10 pg epidermal growth factor/ml, 0.12 pg prolactidml, 27 pg oestradiol/ml and 0.78 ng progesterone/ml, and in the presence or absence of a milk fat globule membrane (MFGM) fraction prepared from goat's milk. Milk fat obtained by centrifugation (2500g, 20 min, 20°C) was vortex-mixed and membranes were sedimented by centrifugation at 100,OOOg for 90 min [4]. The MFGM fraction was incubated in 0.3 pM-glutathione pH 6.0 at 37°C for 24 h , centrifuged again, and the supernatant was dialysed against water and freeze-dried. MFGM was added to culture medium (3 ml/well) at twice its milk concentration, and medium was changed every 2 days. DNA synthesis was measured on day 6 and 8 by incorporation of [methyl-'H]thymidine (2 pCi/ml) during the last 4 h of culture, and cells were harvested by collagenase digestion for assay of DNA content [5].

Transport of Milk Constituents by the Mammary Gland

“…3) In the milk fat route, milk fat globules are extruded from the apex of the secretory cell surrounded by membrane (milk-fat-globule membrane); some cytoplasm is sometimes included (126). Examples are milk fat, lipidsoluble hormones and drugs, some unknown growth factors (in milk-fat-globule membrane) (244), and the product of the ob gene, leptin (214).…”

Inhibitory effect of milk fat on milk secretion in the mouse: a re‐examination

“…It must be inferred that the amount of FIL in the circulation was insufficient to affect the mammary cells or that FIL works through the apical rather than the basolateral membrane of the secretory cell. Similarly, MFGM, which contains a substance that inhibits mammary cell proliferation (see Brandt, Pepperle, Otto, Kraft, Bohmer & Grosse, 1988) and a substance that increases the longevity of mammary cells (Wendrinska et al 1993), had no apparent effect. Heesom, Souza, Ilic & Williamson (1992) have suggested that medium-chain fatty acids in milk could provide a local (i.e.…”

“…In addition, the milk fat globules were fractionated into their components to determine whether a specific inhibitory component could be identified. Milk fat globule membranes (MFGM) were prepared as described by Jarasch, Bruder, Keenan & Franke (1977) and Wendrinska, Addey, Orange, Boddy, Hendry & Wilde (1993). MFGM from 2 ml of milk was injected after resuspension in 0 2 ml 0 154 M NaCl.…”