Temporal sequence of prolactin actions on phospholipid biosynthesis in mouse mammary gland explants

Studies were carried out to determine the effect of PRL on the metabolic fate of 32PO4 in cultured mammary gland explants derived from 12 to 14-day pregnant mice. Explants were initially cultured for 24-36 h with 10(-7) M cortisol and 1 microgram/ml insulin. PRL (1 microgram/ml) was then added to certain of the cultures and incubation continued for 2-24 h. Tissues were pulse labeled with 5 microCi/ml 32PO4 during the final 2 h of culture. Tissues were fractionated by the method of Bligh-Dyer. Radioactivity was determined in the organic fraction (containing phospholipids), the aqueous fraction, and an insoluble fraction containing macromolecules. In all these fractions, PRL effected a greater than 2-fold increase in radioactivity content; the onset of the PRL responses was 8-12 h after PRL exposure and PRL effected responses at concentrations of 2.5 ng/ml and above. The enhanced rate of 32P incorporation in the macromolecular fraction was found to occur in both the RNA and phosphoproteins in that fraction. As determined by TLC, PRL was also found to increase 32P incorporation into all phospholipid fractions. This was confirmed by observing that [3H]inositol and [3H]choline incorporation into their respective phospholipids was also increased by PRL; the time sequence of response was similar to that when 32PO4 incorporation was determined. The magnitude of the PRL stimulation of 32PO4 incorporation, however, was about 2-fold higher than either the [3H]inositol or [3H]choline incorporation. The magnified response when 32PO4 was employed may reflect a PRL effect on phosphate uptake into the mammary cells; this is supported by the fact that the radioactive content of the aqueous fraction was significantly elevated in the Bligh-Dyer extract. The effect of PRL on phospholipid synthesis probably reflects the initiation of the packaging process involved in the assimilation of milk products.