Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland

Abstract

Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 micrograms/ml), aldosterone (1 micrograms/ml), growth hormone (5 micrograms/ml), cortisol (5 micrograms/ml), and prolactin (80 ng/ml, present as a contaminant in 5 micrograms/ml growth hormone). The alveolar growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA probe (cDNAcsn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum mammary gland in vivo.

Vanadate disrupts mammary gland development in whole organ culture

“…5). Importantly, this culture system promotes mammary epithelial cell differentiation as exhibited by the development of lobuloalveoli and the expression of milk proteins, such as ␤-casein (Terry et al, 1977;Mehta et al, 1980;Ganguly et al, 1981;Plaut et al, 1993). The extent of morphological and functional differentiation exhibited by the control mammary glands in our experiments (Figures 2, 5, and 7) are similar to previous reports (Terry et al, 1977;Mehta et al, 1980;Ganguly et al, 1981;Binas et al, 1992;Plaut et al, 1993;Li et al, 1995).…”

“…Importantly, this culture system promotes mammary epithelial cell differentiation as exhibited by the development of lobuloalveoli and the expression of milk proteins, such as ␤-casein (Terry et al, 1977;Mehta et al, 1980;Ganguly et al, 1981;Plaut et al, 1993). The extent of morphological and functional differentiation exhibited by the control mammary glands in our experiments (Figures 2, 5, and 7) are similar to previous reports (Terry et al, 1977;Mehta et al, 1980;Ganguly et al, 1981;Binas et al, 1992;Plaut et al, 1993;Li et al, 1995). In addition to proliferation and differentiation, mammary glands in whole organ culture can be induced to involute, a process mediated by apoptosis (Atwood et al, 1995;Casey et al, 1996), by the removal of the lactogenic hormones and culturing only with insulin (Topper et al, 1975).…”

The Insulin-Like Growth Factors (IGF) and IGF Type I Receptor during Postnatal Growth of the Murine Mammary Gland: Sites of Messenger Ribonucleic Acid Expression and Potential Functions**This work was supported, in part, by NIH Grant DK-48103 (to T.L.W.)

“…To test whether the IGFs could promote ductal growth in glands in vitro, we used a whole-organ culture system similar to that previously established by other investigators (13,(21)(22)(23). Although these results do not address levels or localization of protein expression for these genes, they do provide support for the hypothesis that locally synthesized IGFs are an important component of postnatal development of the mammary gland.…”

“…Because previously published conditions used to promote growth of mammary epithelium in whole-gland organ culture included micromolar levels of insulin (13,(21)(22)(23), we tested whether IGF-I in the presence of physiological levels of insulin (nanomolar range) could promote epithelial growth in cultured glands. Four-week-old C57Bl6/J female mice were primed with estrogen and progesterone for 9 days, and the mammary glands were removed and cultured in hormone-supplemented media containing one of the following treatments: 1) 50 ng/ml insulin; 2) 50 ng/ml insulin plus 100 ng/ml IGF-I; or 3) 50 ng/ml insulin plus 60 ng/ml EGF.…”

“…IGF-I and IGF-II are mitogenic peptides that have essential growth promoting actions during embryonic development (15,16). Moreover, conditions for optimal growth of mammary gland explant cultures use micromolar concentrations of insulin (13,(21)(22)(23). In vitro, both IGF-I and IGF-II are potent mitogens for normal and tumorigenic mammary epithelial cells (17)(18)(19)(20).…”