R. C. Hallowes

Two monoclonal antibodies, BA16 and BA17, have been developed using a detergent-insoluble extract of human mammary epithelial organoids as immunogen. Indirect immunofluorescent staining of cultured cells showed that the component reacting with the antibodies was filamentous and the intensity of staining was stronger in mitotic cells. Immunoblotting of cell extracts showed that both antibodies react with only one band of 40 X 10(3) molecular weight, which was present in keratin-enriched extracts of cells or organoids. Furthermore, the tissue distribution of the component reacting with the antibodies was that predicted for human keratin 19. The antibodies showed differences in the intensity of staining of cells or tissue sections fixed and prepared in different ways indicating that they reacted with different epitopes. The pattern of expression of the 40 X 10(3) Mr keratin by normal mammary epithelial cells was investigated by immunoperoxidase staining of tissue sections, cultured milk cells, and organoids of different sizes cultured in collagen gels. It was found that basal or myoepithelial cells did not express this keratin. Some heterogeneity of expression of this component was seen in luminal epithelial cells, found almost exclusively in the smaller structures. These cells did, however, express other keratins characteristic of luminal cells. The distribution in the mammary tree of the luminal cells that did not express the 40 X 10(3) Mr keratin appears to be similar to that expected for cells with the proliferative potential to produce new terminal ductal lobular units or an increase in branching of existing terminal ductal lobular units. It is shown that these cells have considerable proliferative potential by the fact that they form large colonies in milk cell cultures.

SUMMARY Explants of mammary glands and of subcutaneous body fat from sexually mature virgin and from 19-day pregnant Sprague-Dawley rats and of mammary gland from 5-day lactating Sprague-Dawley rats, were maintained in organ culture for up to 96 h. The effects of insulin (I), corticosterone (B), prolactin (P) and growth hormone (G) on the rate of fatty acid synthesis were measured by the incorporation of [ 14 C]acetate. The effect of these hormones on the synthesis of various carbon-chain length fatty acids was measured by radio gas-liquid chromatography. Explants from both tissues had a reduced rate of fatty acid synthesis after 24 h in medium 199, but this rate was increased by the addition of insulin. In explants of subcutaneous fat from virgin rats, the rate was further increased by culture in IBP or IBG, but this increase was not blocked by actinomycin D. In explants from subcutaneous fat of 10-day pregnant rats the rate was not increased by the addition of B, P or G to the insulin-containing medium. In mammary gland explants from virgin rats, IBP stimulated a greater rate of fatty acid synthesis than did insulin alone. In mammary gland explants from 10-day pregnant rats, the rate of fatty acid synthesis was increased by both IBP and IBG. In mammary gland explants from rats on the 5th day of lactation, both IBP and IBG increased the rate of fatty acid synthesis compared with insulin or IB. Actinomycin D blocked the increased fatty acid synthesis produced by prolactin or growth hormone but not that produced by insulin alone. Mammary gland explants from rats on the 5th day of lactation were cultured for the first 4 h after excision in medium 199 that contained sodium [ 14 C]acetate. Sixty-eight per cent of the 14 C was incorporated into C 8 -C 12 fatty acids. In explants from subcutaneous fat none of the hormones tested increased 14 C incorporation in these fatty acids. In mammary gland explants from virgin or 10-day pregnant rats, insulin, corticosterone and prolactin increased the incorporation in these fatty acids. Growth hormone was less efficient than prolactin in stimulating C 8 -C 12 fatty acid synthesis.