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 ...

A.BSTRACTA comparison was made of changes in the morphology of fat cells in the intact mammary gland, de-epithelialized mammary fat pad, and the parametrial fat pad of female C3H mice during lactation. Mammary fat pad not in contact with actively secretory epithelium shows partial loss of fat during lactation (systemic effect) while in the presence of such epithelium there is much greater loss of fat (systemic plus local effect). In the same animal the parametrial fat pad shows greater depletion during lactation than the de-epithelialized mammary fat pad. The extent of fat depletion is dependent on the number of nursing young and the duration of lactation. In the intact mammary gland and parametrial fat pad, marked change ,in fat cell structure is not seen in pregnancy except possibly at the very end; after weaning, replenishment of lactation-depleted fat cells is conspicuous by 48 hours. The basic ultrastructure of mammary fat cells and the kinds of changes they undergo during depletion do not differ from those described for other mammalian adipose tissues.In the female mouse the epithelial component of the mammary gland lies embedded .in a pad of white adipose tissue that occupies most of the mass of the gland i n the non-pregnant state. Although changes in the mammary epithelium during pregnancy and lactation have been described extensively (e.g., Cole, '33; Turner and Gomez, '33), little attention has been given to the interactions of the parenchyma with the adipose stroma. Hass ('3:3) felt that the growing and active parenchyma of pregnancy and lactation exerted a local effect directly on the surrounding fat cells and caused their depletion, but he did not consider the degree to which depletion might be induced by systemic factors. A local interaction is also suggested by the observation that transplanted mammary parenchyma grows only if placed i n adipose tissue sites (Faulkin and DeOme, '58; DeOme et al., '59; Hoshino, '62; Slavin, '66).The questions raised by such observations warrant systematic study. In this paper we have attempted to analyze the question of a possible local action of mamm ry parenchyma on the surrounding fat cel B s during lactation by examining mor-ANAT. REC., 177: 533-548.

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Human breast epithelial cells derived from various sources (fibroadenoma, reduction mammoplasty, and mastectomy tissues from premenopausal patients) have been cultured in collagen gel matrix using serum-free medium. Response to various additives has been analyzed for growth-promoting effect when added to a basal medium containing insulin, cholera toxin, and BSA. A consistent observation has been the effect of EGF and cortisol in growth stimulation of human breast epithelial cells, while separately, each additive elicited only a small response. Under this condition, employing EGF and cortisol combinations, these cells gave rise to organized colonies consisting of clusters of cells, usually spherical, without any duct-like extensions. Ultrastructural and immunocytochemical studies, using a panel of monoclonal and polyclonal antibodies, have shown that cell types and features that can be identified in the original breast tissue can also be delineated in the progeny populations. The topographical feature, consisting of lumina surrounded by a single inner layer of epithelial cells and an outer layer of basal/myoepithelial cells, can be re-created in the collagen gel system starting from small clumps of cells.

Pectin, carragheenan, agar gum arabic, cellulose and wheat bran were each fed to rats at a level of 5 to 7% to examine their effect on serum, liver and tissue cholesterol levels. Diets (casein-sucrose diet containing 10-15% soybean oil, or skim milk-wheat flour diet containing 10-15% soybean oil) supplemented with either 0, 0.2, or 0.5% cholesterol were used to test the possibly dietary interactions. Among the fibers tested, pectin displayed the most hypocholesterolemic effect. In some experiments, pectin lowered the level of cholesterol in the serum, liver, and aorta, but it elevated body cholesterol levels. Carragheenan was inconsistent in lowering serum cholesterol levels and tended to increase liver and carcass cholesterol levels. These results probably suggest that pectin and carragheenan can affect the distribution of cholesterol within the body. Gum arabic and agar did not lower serum cholesterol levels and in one case gum arabic elevated them. Furthermore, in some experiments they elevated liver body cholesterol levels. It appears that feeding of gum arabic and agar probably resulted in an expansion of the whole body cholesterol pool. Feeding of wheat bran or cellulose had no significant effect on either serum or liver cholesterol levels. The study indicates that the effect of dietary fiber is dependent on the composition of the diet. Furthermore, while some fibers such as pectin may exhibit a hypocholesterolemic effect in rats, other fibers such as gum arabic and agar may actually elevate serum or tissue cholesterol levels.