Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes.

Abstract

Cultured on floating collagen membranes in the presence of lactogenic hormones, dissociated normal mammary epithelial cells from prelactating mice acquire the ultrastructural and biochemical characteristics of differentiated mammary secretory cells in vivo. The cells on floating collagen membranes in medium containing insulin alone have sparse secretory organelles, and a small amount of casein can be detected in these cells with a sensitive radioimmunoassay. These cells resemble counterpart cells in early-pregnant mice. When the cells are exposed to insulin, cortisol, and prolactin, the secretory apparatus is elaborated and significant increases in intracellular and extracellular casein are observed. In this environment, the intracellular casein content is generally four to eight times greater than in freshly dissociated cells or cells cultured in insulin alone. The amount of casein secreted into the medium by floating-collagen-membrane cultures in the three hormones is from 25 to 200 times greater than that secreted by cultures in insulin alone. Cells cultured on plastic substrates in either hormone combination fail to show any increase in intracellular or extracellular casein. On floating collagen membranes, the cells differentiate in response to hormones as they do in vivo and in organ culture. This cell-culture system provides an opportunity to study direct effects of environmental factors on mammary differentiation at the cellular level. Preparation of Floating Collagen Membranes. The collagen solution and collagen gels were prepared according to the method of Michalopoulos and Pitot (11) as modified by us (7).Eighteen-twenty hours after the cells were seeded onto collagen-gel-coated dishes, the gels were removed from the plastic substrate by rimming the gels with a scalpel blade and gently

Mouse mammary epithelial cells on floating collagen gels: Transepithelial ion transport and effects of prolactin

“…The major differences between the previous culture systems used and the mammary system to be described herein are that these are primary cell cultures and not cell lines and that cells are plated and maintained on collagen gels that are subsequently released from petri dishes to float freely in the medium. Cultures of midpregnant cells maintained on detached and floating gels have been shown to have extensive ultrastructural differentiation, secretory polarization of organelles, and appropriate biochemical responses to prolactin (12,13). These features are similar to those seen in vivo and are markedly different from those of primary cultures grown on attached collagen gels or plastic substrates (13).…”

“…Also, it should be noted that in the insulin, cortisol, and prolactin cultures, jTNa (3.55 ,ueq/cm2-hr) was now much larger than Isc (1.94 Aeq/ cm2-hr). With regard to chloride transport, Table 2 We also attempted to answer two additional questions regarding these observed prolactin effects: Were they short-term (12,13).…”

A method for the iodination of insulin and its binding to dissociated mouse mammary cells.

“…The amount of nonspecific binding was less than 10% of that of total binding, probably owing to both good iodoinsulin and undamaged mammary cells. Mammary epithelial cells dissociated by the method used are known to grow in vitro on floating collagen gels (Emerman et al, 1977) and to retain specific prolactin receptors (Sakai et al, 1978). The amount of specific binding to cells from lactating mice was higher than to cells from pregnant mice, suggesting a difference in the number of insulin receptors per cell.…”

Linoleic acid, but not cortisol, stimulates accumulation of casein by mouse mammary epithelial cells in serum-free collagen gel culture.

“…Casein accumulation and secretion in cell culture were demonstrated by Emerman et al in 1977 (14) using cells obtained from midpregnant mice and cultured on top of collagen gel. Casein secretion in this system has been repeatedly shown to be dependent on the presence of insulin, prolactin, and glucocorticoid, when compared to medium containing insulin alone (14)(15)(16). The requirement for the three hormones has been recapitulated in cell culture in a variety of culture systems, using cells obtained from normal virgin or midpregnant animals (17)(18)(19)(20)(21)(22)(23).…”

“…2 A (42,43) and are required for the transcription and stabilization of casein mRNA (8-10, 12, 13 and ovariectomized mice will grow and differentiate in the collagen gel culture system; the tissue used for organ culture requires estrogen and progesterone priming (11) or some undefined ovarian influence (44). Use of cells from adrenalectomized and ovariectomized animals prevents carry-over of adrenal or ovarian steroids into the culture (45 (46,47) and in vitro (20,21 14). We feel that in these previous reports the serum or high concentration of fraction V BSA used in the medium supplied the exogenous linoleic acid required by the mammary epithelial cells.…”