The study of growth and differentiation of mammary epithelium has been hampered by the difficulty of maintaining these functions in vitro. We describe a system for the primary culture of rat mammary epithelium on an acellular matrix derived from whole rat mammary glands that maintains growth and differentiation for months. Cultures plated on this complex substratum produce 50 times the ei-lactalbumin of those on tissue culture dishes and 5 times the a-lactalbumin of those on floating collagen gels as determined by radioimmunoassay. Unlike cultures grown on floating collagen gels, which rapidly lose the ability to secrete the milk sugar lactose, mammary cells on this matrix retain this ability for over 30 days in culture. The organ specificity ofthis mammary extracellular material is shown by the failure of extracellular matrix prepared from rat liver to support mammary differentiation. Within a given culture dish, cells on the surface of mammary extracellular matrix are more differentiated than those on the adjacent plastic. This is demonstrated by their increased a-lactalbumin content as shown by indirect immunofluorescence, and by their increased ability to bind fluorescein-conjugated peanut lectin. Cells on the surface ofthe matrix continue to synthesize DNA as determined by [3H]thymidine incorporation and autoradiography. Even when mammary epithelial cells are plated at low density, cell division continues until the matrix is covered with a confluent layer. We propose that the limited growth, differentiation, and survival of mammary cells in previously described in vitro systems may have been due to substrata that were inadequate to support these functions.The study of mammary growth and differentiation has been hampered by the lack of a suitable system that is capable of maintaining these functions in vitro. When normal mammary epithelial cells from rodents or humans are cultured on tissue culture plastic surfaces they undergo only a few rounds of cell division and rapidly lose differentiated function (1-3). Sometimes, continuous cell lines that are easy to manipulate in vitro can be established from these cultures (4, 5). However, because these cells are highly selected to proliferate under artificial conditions, their control mechanisms may have little relevance to those ofmammary cells in vivo. Organ culture has the advantage of maintaining more normal tissue orientations. However, these systems have limited viability and the presence of stromal cells makes quantitation of epithelial growth difficult (6, 7).It has been appreciated for some time that cell behavior in vitro may be influenced by placing cells on matrices of stromal collagen (8,9). More recently, Emerman and Pitelka described a system for the culture ofmouse mammary cells on floating gels of stromal collagen. Mammary epithelial cells isolated from midpregnant mice produced considerably more ofthe milk protein casein when plated on these floating collagen gels than when plated on attached collagen gels or tissue culture plastic dishe...

The potential involvement of macrophages, T lymphocytes, and the cytokine tumor necrosis factor (TNF) in regression of the corpus luteum was investigated at different stages of pseudopregnancy and pregnancy by use of immunocytochemical methods and a TNF bioassay. Few macrophages (11 +/- 6 per high power field of 8-microns frozen sections of corpus luteum, Day 10 of pseudopregnancy) were observed until the very end of pseudopregnancy, when the number of macrophages increased greatly (176 +/- 42 per high power field, Day 19 of pseudopregnancy). Pregnancy, of 32 days duration, delayed large-scale macrophage accumulation until 3 days after parturition (154 +/- 30 per high power field). Low TNF activity (approximately 1.0 U/mg protein) was detected in incubations of luteal tissue at all stages; in response to lipopolysaccharide, TNF values in medium increased 10- to 30-fold at times of luteal regression and macrophage accumulation (1 day postpartum and Day 19 of pseudopregnancy). Class II-positive T lymphocytes were observed in luteal tissue, but unlike macrophages, the number of lymphocytes did not increase at the time of regression of the corpus luteum. These data are consistent with the hypothesis that involution of the corpus luteum is promoted through the interactions of inflammatory cells and action of TNF, although the action of TNF has not been determined in this luteal tissue. Through unknown mechanisms, pregnancy postpones the accumulation of macrophages in the corpus luteum, in association with the prolongation of luteal function until the time of parturition.

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Utilizing immunocytochemistry numerous macrophages were localized in regressing corpora lutea. In contrast, few macrophages were observed in young corpora lutea. Regressing corpora lutea readily produced TNF-a in vitro in response to lipopolysaccharide, whereas young corpora lutea produced significantly less TNF-a. T lymphocytes were identified in young corpora lutea preceding the appearance of macrophages. These observations suggest that cells of the immune system and cytokines could be important participants in physiological regression of the corpus luteum.

Alterations in the basal lamina (BL) of developing follicles were studied by immunofluorescent microscopy using antibodies against type IV collagen, laminin, and fibronectin, and by electron microscopy. Ovarian development was induced in immature rats by sequential administration of estradiol, follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). A continuous BL was observed in healthy follicles treated with estradiol and FSH. As determined by immunofluorescence, laminin, type IV collagen, and fibronectin were restricted to the BL and the theca but not to the granulosa. When follicles were allowed