In the presence of cortisol and prolactin, insulin at concentrations as low as 1 ng/ml significantly stimulates casein synthesis in mammary explants from midpregnant mice; maximal synthesis occurs at 10 ng/ml. However, in the absence of insulin, no detectable immunoprecipitable casein is produced. Insulin also supports enhanced accumulation of casein mRNA in the presence of cortisol and prolactin; neither epidermal growth factor nor somatomedin C has this effect. These inductive actions ofinsulin are not secondary to a general maintenance effect on the mammary epithelial cell; insulin, epidermal growth factor, and somatomedin C can support the accumulation of RNA in rough endoplasmic reticulum equally well. In addition, these effects do not reflect a specific insulin requirement for prolactin sensitivity; epidermal growth factor can support prolactin-induced total RNA synthesis as well as insulin can. The results demonstrate that, although insulin, epidermal growth factor, and somatomedin C can all function as cell maintenance agents, only insulin, together with cortisol and prolactin, can induce casein mRNA accumulation.Casein can be induced in mouse mammary explants in the presence of insulin, cortisol, and prolactin. Placental lactogen (1) and, in some instances, growth hormone (2, 3), can substitute for prolactin in this regard. Other glucocorticoids (4) can substitute for cortisol in this induction. On the other hand, other agents tested as possible substitutes for insulin have been found ineffective. Neither serum (5) nor epidermal growth factor (6) supports the induction of casein synthesis when used in the presence of cortisol and prolactin, although both are active mitogenic agents for mammary epithelial cells. Despite these findings, insulin has not been regarded as essential physiologically for phenotypic expression of casein genes in mammary epithelial cells (for review, see ref. 7). Rather, it has been considered to function in cell maintenance in vitro. In more recent studies, it has been reported that prolactin (8) tained from Calbiochem. The somatomedin C used was derived from the final high-pressure liquid chromatography purification step described previously (11) and was free of both insulin and insulin-like growth factor II. This material was 33% pure, as determined by radioimmunoassay; the amounts given below take this factor into account. Medium 199/Hanks' salts was purchased from GIBCO, Hepes was obtained from Sigma, and rabbit anti-sheep IgG antiserum was from Miles-Yeda. aip1, carrier-free, and [5_3H]uridine (30 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) were purchased from New England Nuclear.Organ Culture. The abdominal mammary glands from C3H/ HeN mice, 10-12 days into theirfirst pregnancy, were removed under sterile conditions and explants were prepared as described (12). The culture medium (medium 199/Hanks' salts/ 20 mM Hepes, pH 7.6) contained cortisol (1 jug/ml), prolactin (1 ug/ml), and insulin, epidermal growth factor, or somatomedin C at 50 ng/ml, unless otherwise stated....

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The relationship between kinase C activity and mammary gland differentiation was investigated by following kinase activity throughout the mouse reproductive cycle and by pharmacologically perturbing the kinase, while monitoring biochemical differentiation. Protein kinase C activity declined during pregnancy and remained low throughout lactation, suggesting an inverse relationship with milk protein expression. This negative association was further supported by the use of quercetin (50-100 mumol/l) and gossypol (50 mumol/l), which are both protein kinase C inhibitors. These compounds doubled alpha-lactalbumin levels in mammary explants cultured with hormones. However, a phorbol ester, known to activate protein kinase C, had no effect on alpha-lactalbumin production, although it did stimulate this milk protein 2.5-fold in the presence of the calcium ionophore, A23187. In the absence of raised calcium levels, protein kinase C activity therefore appeared to be inversely correlated with biochemical differentiation; but, in the presence of increased calcium concentrations, both calcium and the kinase acted synergistically to augment hormone-induced alpha-lactalbumin expression.

Rabbit placental lactogen, a polypeptide hormone functionally related to the growth hormone/prolactin family, was isolated from placenta by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography on DEAE-and CM-cellulose. The hormone was purified to more than 90% homogeneity, as determined by end-group analysis. On disc gel electrophoresis at pH9.0 it migrates as a pair of closely spaced bands with mobilities of 0.489 (minor band) and 0.511 (major band), and its isoelectric point is 6.1. Its mol.wt. is 20600, as determined by sedimentation--equilibrium centrifugation, and 24200, as estimated by gel electrophoresis in sodium dodecyl sulphate. Its amino acid composition resembles that of rabbit growth hormone and rat prolactin, except for a lower glutamic acid and leucine content. Like the prolactins, rabbit placental lactogen has two tryptophan and six cysteine residues, and its N-terminus, valine, is identical with that for human placental lactogen. By radioimmunoassay, it does not cross-react with antisera to either rat growth hormone or rat prolactin; in addition, it does not cross-react with antisera to bovine placental lactogen by double immunodiffusion. The similarity of the biochemical characteristics of rabbit placental lactogen to the other non-primate placental lactogens lends further support to the hypothesis that these molecules occupy a more central position in the growth hormone/prolactin "tree" than do their primate counterparts.

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