petitive inhibitor of concanavalin A such as 0.2M methyl a-D-glucoside must be added to block recombination of concanavalin A with serum glycopro- teins in the column. Myron A. LEON Pathology Research Department, St. Luke’s Hospital, ‘Cleveland, Ohio 44104 References and Notes 1. 8. Nakamura, K. Tanaka, S. Murakawa, Nature 188, 144 (1960). 2. H. Harris and E. B. Robson, Vox Sang. 8, 348 (1963). 3. I. J. Goldstein and R. N. Iyer, Biochim. Biophys, Acta 121, 197 (1966). 4.B. B. L. Agrawal and I. J. Goldstein, Biochem. J, 96, 23C (1965). 3. The serum inhibitor of C’l (first component of complement) esterase was measured by Dr, J. Pensky. 6. I thank Miss Janet Wilson, in Dr. A. Steinberg’s laboratory, for performing the Gm titrations. 7. J. R. Clamp, G. Dawson, L. Hough, Biochem. J. 100, 35C (1966). References to earlier papers on IgG carbohydrate are given here. 8. Dr. G. Bernier, Dr. W. D. Terry, and Dr. G. A. Spengler provided the typed myeloma serums. Supported by NIH grant 5-ROIA103104-07. 18 October 1967 sulin; however, proliferation does not lead to differentiation unless hydrocorti- sone and prolactin are present in the culture medium. The sequence of action of the hormones in relation to the cell cycle in explants of mammary gland from pregnant mice has been described (8). Although the relationships between hormone-dependent proliferation and differentiation have been characterized in the mature gland, the biochemical behavior of the immature gland in vitro has been less clear. We examined the capacity of the immature tissue to differentiate in vitro and compared the requirements for and responses to the hormones with those of the mature tissue. Although the onset of “maturity” is variable, 21-day-old female weanling mice of the C3H/HeN strain are termed immature in our report. The glands of the 21-day-old mouse consist principally of branching ducts several cell layers thick with terminal buds. No alveoli are seen in serial sections, nor have any been seen in whole Hormone-Dependent Differentiation of Immature Mouse Mammary Gland in vitro Abstract. Explants from the mammary glands of 3-week-old mice can be induced to synthesize casein in vitro in the absence of lobuloalveolar development, Maximum biochemical differentiation requires the presence of insulin, hydrocortisone, and prolactin in the culture medium. In contrast to explants from adult mice, the mammary epithelium of immature animals undergoes DNA synthesis and mitosis in vitro in the absence of exogenous insu- lin; however, such proliferation does not lead to the formation of differentiated daughter cells. Insulin acts in at least two ways during the proliferative phases of the cell cycle of differentiating mammary epithelial cells, Mammary gland epithelial cells in explants derived from pregnant or non- pregnant mature mice proliferate, assume a secretory appearance, and be- gin to synthesize certain milk proteins when cultured in the presence of in- sulin, hydrocortisone, and prolactin (/-4). This functiona! differentiation of mounts in other laboratories (9). Mammary epithelial cells in immature animals proliferate into the mam- mary fat; yet, as the animal matures, proliferation virtually ceases and does not resume until pregnancy. We con- firmed this by injecting thymidine-H? (1 pe per gram of body weight) intraperitoneally and preparing autoradiographs of the mammary gland 24 hours later. Only about 1.5 percent of the epithelial cells from 3-month-old mice were labeled, whereas 18 percent of the epithelial cells from 3- week-old animals were labeled. It is unlikely that the results reflect a difference in pool size, because almost all the labeled nuclei had approximately 35 to 40 grains. Synthesis of DNA in vitro by mammary epithelial cells from mature mice is completely dependent on the pres- ence of insulin in the culture medium (5-7). In contrast, our studies indicate that DNA synthesis in vitro by mammary gland explants derived from im- mature mice [as reflected by the amount of thymidine-H? incorporated during 4-hour periods as previously de- scribed (5)] is quantitatively independent of the addition of insulin. Since these explants contain large numbers of the epithelial cells has been found to be necessarily coupled to their prolifera- sulin-independent. data on total incor- sequent cell division require only in- terpreted with caution. To determine tion (5-7). Synthesis of DNA and sub1326 fibroblasts whose DNA synthesis is in- poration of thymidine-H? must be in- whether such incorporation of thymidine-H? into DNA reflected epithelial cell activity, we made autoradiographs of explants exposed to thymidine-H? (1 ype/ml) for 72 hours. Similar ex- plants were cultured in the presence of colchicine (0.05 »g/ml) for 72 hours and were examined for mitotic activity. The duct epithelium, especially the epithelium of smaller terminal branches, was heavily labeled, and mitotic figures were numerous in either the presence or absence of insulin in the cultures. In explants cultured without the addition of hormones, 12 percent of the cells were in metaphase (5000 epithelial cells counted); in those cultured in the presence of insulin, 9 percent were in metaphase (5000 epithelial cells counted); and in those cultured in the presence of hydrocortisone and prolactin, 11.5 percent were in metaphase (1000 epithelial cells counted). In contrast to tissue from mature mice (5-7) then, proliferation of mammary epithelial cells from immature animals occurs in vitro in the absence of exogenous insulin. However, proliferation in the absence of exogenous insulin does not lead to functional differentia- tion. Figure 1 shows that explants of mam- mary glands of immature mice can be induced to synthesize the major casein components when cultured in the presence of insulin, hydrocortisone, and prolactin. During the first 24 hours, the synthesis of casein bands 2, 3, and 4 is virtually undetectable, but by the 4th or 5th day these proteins are synthesized at greatly accelerated rates in ratios similar to those observed in tissue from mature animals (10). Casein band 1, a more heavily phosphorylated component (7/7), is present from the start and is least affected by culture. This component persists longest in ma- ture tissue cultured in incomplete hor- mone systems (J0). Figure 2 shows patterns of casein production during the 5th day of cuiture in several media. The full differentiative response is elicited only when the three hormones are present. Of the incomplete systems, only those con- taining insulin are even partially effective, and insulin alone is as effective as it ts in combination with hydrocortisone or prolactin. In the absence of insulin, not even band 1 is sythe- sized. The hydrocortisone-prolactin system (Fig. 2), in which proliferation occurs SCIENCE, VOL. 158