Mammary epithelial cells undergo secretory differentiation in cycling virgins but require pregnancy for the establishment of terminal differentiation.

Postnatal development of the mammary gland begins during puberty with ductal proliferation and is completed at delivery with the appearance of secretory alveolar structures. Using endogenous milk protein genes and a WAP-lacZ reporter transgene, we show that the differentiation of alveolar cells is initiated in virgin mice in estrus in a limited number of cells. With the onset of pregnancy, the number of expressing cells and the cellular expression levels increase until full activity is reached at lactation. Milk protein genes are activated in a defined temporal sequence. WDNM1 and beta-casein are expressed early in pregnancy and increase during alveolar proliferation. WAP (whey acidic protein) and alpha-lactalbumin are expressed later near the end of gestation, which is characterized by terminal differentiation of the mammary secretory phenotype. By in situ hybridization, we have established evidence for asynchrony in milk protein gene expression among alveolar cells showing large variations in the intensity of hybridization among adjacent cells. The asynchrony of maturation of epithelial cells within a given alveolus suggests that the genetic program leading to terminal differentiation is subject to local modulation. It is likely that these signals are manifest through various pathways including growth factors, the extracellular matrix or gene products specific to terminal differentiation such as WAP. We extended our analyses to WAP/WAP transgenic mice in which WAP is synthesized precociously and functional differentiation of alveolar cells is impaired. We found an altered expression pattern of milk protein genes, with a strong reduction of alpha-lactalbumin RNA. We conclude that the early production of WAP in WAP/WAP mammary glands disrupts the timing of gene activation leading to a premature termination of the differentiative program.