SUMMARY: Mammary glands are enzymatically dissociated and the resulting tissue digest enriched for epithelial cells by isopycnic banding on a density gradient of Percoll. The cells are embedded within a rat tail collagen gel matrix and fed with the appropriate medium. Growth and differentiation are superior in such a system when compared to culture on plastic, using identical media.
Experiments were undertaken to demonstrate and characterize specific receptors for epidermal growth factor (EGF) in mammary glands of female BALB/c mice in various physiological states. The results of an in vitro desaturation technique are also presented which allow estimation of the total EGF-binding sites per mg membrane protein. Binding of the ligand [125I]iodo-EGF is both time and temperature dependent. Maximum binding to the membrane is achieved after 6 h of incubation with [125I]iodo-EGF at 23 C. Scatchard analysis of equilibrium binding using membrane preparations of mammary glands from virgin mice yields two classes of high affinity receptors with Kd values of 0.8 +/- 0.1 and 5.0 +/- 0.4 X 10(-10) M and receptor concentrations of 10 +/- 1.2 and 23.5 +/- 2 fmol/mg protein, respectively. Membrane preparations of mammary tissues from cycling, gestating, and lactating mice were used to correlate cellular receptor levels to the physiological state of the animal. Beginning at weaning, there is a constant decrease in high affinity receptor level with increasing age, as well as through the early stages of both gestation and lactation. On day 10 of gestation, receptor levels increase, reaching 15.2 +/- 1.6 fmol/mg protein, followed by a decrease to 3.8 +/- 0.9 fmol/mg protein on day 10 of lactation. We conclude that membrane preparations from the mouse mammary gland contain specific high affinity receptors for EGF, and that receptor levels are characteristic of the physiological state.
The effect of collagenase dissociation of virgin mouse mammary glands on the level of mammary epithelial cytosolic estrogen receptors (ER) and progesterone receptors (PR) was assessed. After cell dissociation, ER was present in mammary epithelial cells at concentrations similar to those found in the whole gland. However, PR appeared to be affected by the collagenase treatment. The regulation of ER and PR in mouse mammary epithelial cells isolated by collagenase dissociation and grown within collagen gels was then determined. After 7 days in culture under serum-free conditions inside a collagen gel, PR and, to a lesser extent ER, as characterized by high affinity binding and specificity, were present in the epithelial cells. Although at a low level, the ER were determined to be functional, since estradiol (E2) was able to promote nuclear accumulation of ER and to induce PR. PRL was able to increase cytosolic ER and PR concentrations. The combination of progesterone (P) and PRL was more effective than PRL or P alone in increasing PR. The induction of PR by P and PRL was inhibited when epidermal growth factor was present in the culture medium. Previous studies have shown that P, PRL, and epidermal growth factor, but not E2 (either alone or in combination with these factors) are able to stimulate cell proliferation in vitro. We conclude that the effects of E2 on protein synthesis and proliferation are dissociated in vitro. The difference between the effect of E2 and PRL or P on growth may be related either to the initial concentrations of their respective receptors or estrogen may stimulate growth indirectly.
We investigated the effect of somatomedin C (SM-C) on the growth of mouse mammary ductal epithelial cells in collagen gel culture. Epithelial cells, isolated by collagenase digestion of whole glands, were placed into primary serum-free collagen gel cell culture for 10-12 days, during which SM-C was added alone or in combination with other growth-promoting factors. Previous work has shown that these cells require a superphysiological concentration of insulin (10 micrograms/ml) for optimum growth in serum-free medium (a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's medium) containing epidermal growth factor (EGF). When SM-C (1-250 ng/ml) alone was added to serum-free basal medium containing EGF, it stimulated growth (at concentrations greater than 25 ng/ml) to at least the same extent as insulin at 10 micrograms/ml. There was no additive stimulation of growth when optimal concentrations of insulin and SM-C were added together. The nonadditive stimulation at optimal concentrations of these hormones may indicate that the previous requirement for a superphysiological concentration of insulin for maximum growth was due to low affinity binding of insulin to the SM-C receptor. Rat insulin-like growth factor II (Collaborative Research) at 50-200 ng/ml did not stimulate growth in the presence or absence of insulin. SM-C could not stimulate growth alone. The presence of EGF or mammogenic hormones (progesterone and PRL) was required.
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