Search

Second thoracic mammary glands of immature BALB/c female mice were stimulated to pregnancy-like lobuloalveolar (LA) development aftr 6 days of incubation in a corticosteroid-free step I culture medium containing insulin, prolactin, estradiol, progesterone, and growth hormone. A low basal level (0.0009%) of casein mRNA (mRNAcsn) sequences was detectable in the LA glands by a specific cDNA probe. Subsequent incubation of the LA glands for 3 days in medium containing insulin and prolactin or insulin and cortisol failed to elicit mRNAcsn above the basal level, indicating that neither prolactin nor cortisol alone can support casein gene expression. However, an increase in mRNAcsn levels was observed when the 3-day incubation with insulin and cortisol or insulin and prolactin was followed by 3 days of culture in presence of insulin, prolactin, and cortisol. When a 3-day incubation with insulin and prolactin was followed by 3 days in insulin and cortisol medium, mRNAcsn levels in the gland remained similar to the basal level. However, a 20-fold increase in the mRNAcsn levels ensued when the LA glands were sequentially incubated for 3 days in insulin and cortisol and then for another 3 days in insulin and prolactin medium. After a preincubation in insulin and cortisol medium, the LA glands retained residual cortisol during subsequent incubation in insulin and prolactin medium, and the mRNAcsn levels in these glands were related to the level of residual cortisol present. When mRNAcsn and the residual cortisol level reached a minimum, addition of fresh cortisol to the medium caused a 20-fold increase in the mRNAcsn levels. This indicates that cortisol is a limiting factor in insulin and prolactin medium and its presence is absolutely required for casein gene expression.

The influence of cortisol and prolactin on casein gene expression in the mammary gland of lactating BALB/c mice was measured by using a specific cDNA probe to 15S casein mRNA (cDNAcsn). Casein mRNA (mRNAcsn) level in the mammary gland was decreased by 85% 5 days after adrenal ablation, but then was increased 4.4-fold 12 hr after a single injection of hydrocortisone-21-acetate. An 80% decrease in serum prolactin level, induced by the prolactin inhibitor 2-bromo-alpha-ergocryptin (CB-154), did not alter the level of mRNAcsn in the gland. Specific transcription of the casein gene in nuclei isolated from lactating mammary glands was measured by cDNAcsn hybridization to the in vitro synthesized Hg-CTP-containing RNA (Hg-RNA), which was purified by SH-agarose chromatography. The level of the mRNAcsn in Hg-RNA synthesized in the isolated nuclei was 0.09% and this was decreased 85% by alpha-amanitin, indicating that the mRNAcsn sequences in the Hg-RNA were the products of RNA polymerase II-directed DNA-dependent RNA synthesis. Transcription of the mRNAcsn in isolated nuclei was decreased by 70% 5 days after adrenalectomy and a single injection of the glucocorticoid then increased the transcription level 2-fold at 6 hr. Essentially no alteration of the level of transcription was detectable in mammary nuclei isolated from lactating mice with 80% decreased serum prolactin level, induced by CB-154 treatment. The results thus demonstrate a glucocorticoid involvement on the modulation of casein gene expression at the transcriptional level of control.

A cell-free protein synthesis system derived from Ehrlich ascites tumor cell ribosomes (S30) plus rabbit reticulocyte tRNA was developed and the activity of the system was dependent on rabbit reticulocyte ribosomal salt (0.5 M KC1) wash factors, The exogenous mRNAs from BALB/c mouse liver and the mammary gland were translated with a high efficiency in this heterologous cell-free system. Furthermore, the RNA from the lactating mammary gland faithfully directed the synthesis of casein. The presence of mouse casein in the reaction product was identified by radioimmunoprecipitation with mouse casein antiserum, co-electrophoresis of the reaction product and mouse casein the urea-polyacrylamide gel and by electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. The major portion of the lactating mammary gland RNA directed synthesis of the milk protein in the cell-free system appeared to be analogous to alphas casein,

A new indirect radioimmunoassay was developed for detection of casein in mouse milk and in mammary tissue extract. Preincubation of rabbit gamma globulin to mouse milk casein (Ca2+-rennin precipitate) with unlabeled casein, milk, extracts of mammary tissues of late pregnancy and lactation, virtually blocked subsequent binding of 125I-labeled mouse milk casein to the antibody. Preincubation with mouse serum, bovine serum albumin, rennin, extracts of liver or immature mammary tissue had little effect on [125I]casein binding to the antibody. The inability of [125I]casein to bind to the antibody after preincubation with protein samples, which are likely to contain casein, is indicative of a specific antigen-antibody reaction. The assay is capable of detecting 0.2 mug casein, 1 mug milk proteins and 10 mug lactating mammary tissue extract. The application of the assay was also demonstrated using organ culture of the entire mammary gland. The glands treated with the lactogenic hormones, insulin + prolactin + cortisol, showed a saturation level of antibody-antigen reaction, indicating hormonal induction of casein; whereas, no reaction was observed with the non-treated gland.

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.