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The mammary gland, with its hormone-dependent proliferation and development, offers an excellent opportunity to study problems of differentiation. Under hormonal stimulation, this tissue acquires the ability to synthesize specific products characteristic of lactation. Previous workers have demonstrated that insulin added to chemically defined medium maintains the initial histological pattern of midpregnant mammary gland during several days of organ culture. Addition of prolactin to an insulin-hydrocortisone medium elicits an alveolar secretory appearance.1 -Since the histological appearance of the stimulated tissue suggests the alteration of nonsecretory alveolar epithelial cells into highly differentiated secretory cells, we have adapted these organ culture methods to the study of the synthesis of "caseinlike" phosphoproteins by pregnant mouse mammary tissue cultured under various hormonal conditions. Materials and Methods.-Animals: C3H/HeN mice, either virgin or 11-12-day primigravida, were used. After decapitation the abdominal mammary glands were removed bilaterally under sterile conditions and cut into explants ranging in weight from 0.6 to 1.0 mg each. Each experiment was done with tissue from a single animal.Culture methods: For each experiment stock solutions of hormones were made with the following compositions: insulin (Eli Lilly beef zinc insulin, lot 795372), 2.5 mg/ml in 10-3 N HCl; prolactin (NIH-P-S-6, ovine), 2.5 mg/ml in 10-4 N NaOH; hydrocortisone, 8 mg/ml in absolute ethanol. Aliquots from these stock solutions were added to medium 199 (Microbiological Associates) in various combinations to give final concentrations of 5 ug/ml for each hormone. The Pi32 media contained 15 suc/ml, and the C14-amino acid media contained 5 ,uc/ml (Chlorella hydrolysate, specific activity 0.5 mc/mg, obtained from Volk Radiochemical Co.). Sodium penicillin G powder was added directly to the media in most experiments to a final concentration of 35 jug/ml. After all additions, the media were sterilized by filtration through MNillipore filters (0.45 M) into sterile flasks, and stored at 5°if not used immediately.The Fell and Robison culture method as adapted by Chen6 and as previously describedl-3 was used. Two ml of media were added to a watch glass enclosed in a Petri dish. Siliconized lens paper was floated on the media and 3-6 explants were placed on each lens paper (zero time).The Petri dishes were placed in plastic boxes and exposed to 95% air-5% CO2 sufficient to maintain the pH at about 7.4, after which the systems were closed and incubated -at 37°.Nonisotopic media were replaced with isotopic media 4 hr prior to assaying the tissue for "casein." Initial rates of casein synthesis were obtained by placing explants on isotopic media for the first 4 hr of the incubation. Thus, each time point represents the incorporation of isotope during a 4-hr pulse. For systems cultured longer than 2 days, media were replaced with fresh media at 48 hr. D)uplicate culture dishes were used for each determination.Casein assay: At ...

The developmentally and hormonally regulated expression of the mouse whey acidic protein (WAP) gene and a hybrid gene containing the WAP gene promoter and a cDNA for human tissue plasminogen activator (tPA) were studied in a line of transgenic mice. During mammary gland development from the mature virgin state to the seventh day of lactation, the relative concentration of WAP mRNA increased about 10(4)-fold, the increase being most pronounced between days 14 and 16 of gestation. In mammary gland organ culture from virgin and midpregnant animals, the concerted actions of insulin, hydrocortisone, and PRL were required to increase WAP mRNA levels. Steady state levels of transcripts from the WAP-tPA hybrid gene increased about 100-fold during pregnancy; this occurred mainly around day 10 of gestation. Insulin, hydrocortisone, and PRL were necessary to maintain the levels of WAP-tPA RNA in explants from virgin and pregnant animals, but could not further elevate it. The results suggest that the WAP gene promoter and upstream region contains some, but perhaps not all elements conferring developmental and hormone regulated expression of the mouse WAP gene.

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