ns eel three ways: (i) it is necessary for the initiation of DNA synthesis by epithelial cells of mature tissue as a prelude to functional differentiation (5—7); (ii) it must be present during the post- . mitotic action of prolactin {8) when phenotypic differentiation is expressed; 4 48-72 HRS 7 Fig. 1. Effect of culture on synthesis of the major casein components. Explants weighing 0.5 to 1 mg were prepared and cultured in sterile “Medium 199” (2). The medium contained 5 yg/ml each of crystalline beef insulin (Lilly), ovine prolactin (NIH Endocrinology Study Sec- tion), and hydrocortisone. Explants were exposed to P-labeled inorganic phos- phate (75 wc/ml) during the times indicat- ed and then were homogenized in the presence of mouse carrier casein. Total casein was isolated by precipitation with calcium and rennin and was further characterized with vertical starch-urea gel electrophoresis at pH 8.6 (/3). Bars represent counts in gel sections corresponding to the four major casein bands with appropriate background subtracted (/0@). Ordinate refers to radioactivity in terms of counts per minute per centimeter per 10 mg of tissue. in the absence of insulin, did not synthesize casein. Furthermore, when explants were cultured in such a hydrocortisone-prolactin system for 96 hours and insulin was then added during the final 24 hours, no effect on casein syn- thesis was observed. This suggests that actions of insulin other than postmitotic synergism with prolactin (8) and initiation of DNA synthesis (7) must occur before functional differentiation can be of DNAsynthesis which, atone, cannot comparedto tissue from unprimed mice. Thus, it is possible to dissociate struc- differs from the mature in displaying sue from unprimed immature mice. required during the proliferative phases of immature cells in some capacity other than initiation lead to differentiation. Thus, even though immature tissue insulin-independent DNA syathesis and mitosis, such proliferation apparently does not lead to functional differentiation unless it occurs in the presence of insulin. Although casein bands 2, 3, and 4 are not detectably synthesized by explants of immature mammary gland during the Ist day of culture in the presence of all three hormones, such explants synthesize q-lactalbumin and ~lactoglobulin. Tissue was exposed to Cl4-labeled algal hydrolyzate (10 yc/ ml). Explants were homogenized in mouse skim-milk carrier, and then q- lactalbumin and -lactoglobulin were isolated by ammonium sulfate fractionation and electrophoresis on polyacetate strips as previously described (2). When the culture was exposed to the hydrolyzate from 0 to 24 hours, the rate of synthesis of q-lactalbumin was 310 count/min and of #-lactoglobulin 295 count/min per milligram of tissue. When the period of exposure was 72 milligram of tissue. Thus, the emergence of the capacity to synthesize the various milk proteins is asynchronous in this immature tissue. Ichinose and Nandi reported (9) that 232 AH to 6A FP se. I 43241 Fig. 2. Effect of culture on synthesis of casein components in incomplete hormone systems. Explants were cultured as in Fig. 1 but in the incomplete hormone system shown. Pulse labeling with P*-labeled inorganic phosphate was from 96 to 120 hours in all cases. Because these experi- ments involve systems lacking insulin, the series was repeated with glucose replaced in the medium by D-fructose (100 mg/ml). No difference was noted. NH, no hor- mones; 7, insulin; F, hydrocortisone; P, prolactin. Ordinate refers to radioactivity in terms of counts per minute per centiMeter per 10 mg of tissue. 8 DECEMBER 1967 thymidine-H? incorporation into DNA was approximately doubled [perhaps due to a shortened S-phase (DNA synthesis) as suggested by Banerjee (/2)], and the development of casein produc- it is to 96 hours, the rate of synthesis of BANG alveolar structures develop in vitro. Un- der these primed conditions, the rate of early (iii) a-lactalbumin was 880 count/min and of 8-lactoglobulin 510 count/min per HOIMONE S*STEM mary tissue into medium containing insulin, hydrocortisone, and prolactin, lobuloalveolar development in| mammary explants from immature mice rarely occurs unless the mice have previously been primed with injections of estrogen, progesterone, prolactin, and growth hormone. Our work confirms this observation. Culture of unprimed explants in the presence of insulin, hydrocortisone, and prolactin elicits the biochemical differentiation described above, but there is little or no formation of alveolar structures, If, however, the animals are primed on each of 7 days with estradiol-178 (1 gg), progesterone (I mg), and growth hormone tion was accelerated about 24 hours, tural development from biochemical development in vitro with mammary tis- Our studies in vitro with mammary gland explants from immature mice revealed that: (1) the epithelium differs from that of the mature gland in that DNA synthesis and mitosis occur in the absence of exogenous insulin; (ii) this insulin-independent proliferation does not result in the appearance of differentiated daughter cells; (iii) addition of insulin to the cultures does not quantitatively affect DNA synthesis or mitosis but allows functional differentiation to occur when hydrocortisone and prolactin are present; (iv) emergence of the ability to synthesize casein bands 2, 3, and 4 is not synchronized with the appearance of wheyprotein synthesis; and (v) it is pos- sible in vitro to dissociate the capacity to synthesize secretory proteins from structural development of this tissue. ANTHONYE. VoyTOvICcH YALE J, TOPPER National Institute of Arthritis and Metabolic Diseases, Bethesda, Maryland 20014 References 1. E. M. Rivera and H. A. Bern, Endocrinology 69, 340 (1961). 2, W. G. Juergens, F. E. Stockdale, Y. J. Topper, J. J. Elias, Proc. Nat. Acad. Sci. U.S. 54, 629 (1965). 3. D. H. Lockwood, R. W. Turkington, Y. J. Topper, Biochim. Biophys. Acta 130, 493 (1966). F. E. Stockdale, W. G. Juergens, Y. J. Topper. Develop. Biol. 13, 266 (1966). F. E. Stockdale and ¥Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 56, 1283 (1966). R. W. Turkington and Y. J, Topper, Endocrinology 80, 329 (1967). D. H. Lockwood, A. E. Voytovich, F. E. Stockdale, Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 58, 658 (1967). D. H. Lockwood, F, E. Stockdale, Y. J. Topper, Science 156, 945 (1967). 9. R. R. Ichinose and S. Nandi, ibid. 145, i 4 3 201 0-24 BRS. (50 ,»g) before explantation of the mam- ao , BAN? CASEIN fae Tea peat expressed. Insulin, then, exerts its influence on mammary tissue in at least tO iii orn padi Aated ee a dn 496 (1964). 10. R. W, Turkington, D. H. Lockwood, Y. J. Topper, Biockim. Biophys, Acta, in press. 11. R. W. Turkington and Y. J. Topper, ibid. 127, 366 (1966). 12. M. R. Banerjee and R. J. Walker, J. Cell. Physiol. 69, 133 Topper, (1965). Biochim. (1967). 13. R. W. Turkington, W. G. Juergens, Y. J. Biophys. Acta 121, 20 September 1967 1327 573