The syntheses and secretion of casein and alpha-lactalbumin were examined in organ culture of midpregnancy mouse mammary glands using serum-free medium supplemented with various combinations of insulin, hydrocortisone, PRL, and L-T3. Using highly specific antibodies raised against mouse caseins and alpha-lactalbumin, we demonstrate a selective enhancement of alpha-lactalbumin and lactose synthesis and secretion when all four hormones are present in the culture medium. Production of casein was not modified by the presence of L-T3. Hydrocortisone at concentrations of 10(-9)-10(-6) M inhibited the secretion of both casein and alpha-lactalbumin into the culture medium. The addition of L-T3 to the medium selectively overcame the inhibition of alpha-lactalbumin secretion by hydrocortisone. Extracts of tissue cultured in the presence of L-T3 contained two distinct forms of alpha-lactalbumin, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of L-T3, synthesis of a single form of alpha-lactalbumin prevailed. In the culture medium, predominantly one form of alpha-lactalbumin was detected regardless of the presence of L-T3 during culture. These data clearly indicate that thyroid hormones play an important regulatory role in functional differentiation of the mouse mammary gland.

The prolactin, or lactogenic hormone, receptor has been purified (approximately 80%) from lactating mouse liver and human term placenta by the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate and a prolactin affinity column. The isolated "core-binding unit" has a molecular weight of 37,000 +/- 2,000 daltons. It retains the specificity for lactogenic hormones and binds prolactin with an affinity (Ka = 2 to 6 X 10(9) M-1) similar to that of the receptor as it occurs in its membranous environment (Ka = 3 to 5 X 10(9) M-1). Whether this "core-binding unit" exists on the cell surface in a cryptic or active form is influenced greatly by its association with other membrane proteins and the concentration of phosphatidylcholine within its local membranous environment.

Mammary glands from second generation vitamin D-deficient mice and rats were examined for their ability to make the major milk proteins, casein and a-lactalbumin, both in vivo and in vitro. The glands from the rachitic animals were morphologically indistinguishable from those of age-matched controls. When placed in explant culture, glands from vitamin D-deficient mice and rats underwent DNA synthesis at a rate comparable to that of glands from the vitamin D-replete controls. However, the hormonally induced synthesis of casein and a-lactalbumin was significantly reduced in explants of glands from rachitic us. control animals. The reduction in caseinsynthesizing ability by mouse mammary gland explants was not reversed by the addition of 10~8 or 10~6 M 1,25-dihydroxycholecalciferol to the culture medium, but could be reversed by pretreating the vitamin D-deficient mice with the D metabolite in vivo for 10 days before the onset of culture. The decrease in milk protein synthesis in culture is paralleled in vivo by a decrease in milk protein content in the milk and lactating mammary glands of vitamin D-deficient mice and rats. Both in vivo and in vitro, it is the two highest mol wt caseins that are most affected by the lack of vitamin D in the diet. These data suggest that vitamin D does not play a fundamental role in growth and morphological development of the normal mammary gland, but, rather, it is important in maintenance of full hormonally induced functional differentiation of the mature gland. (Endocrinology 121:865-874,1987)