A biochemical comparison of the lactogenic effect of ovine prolactin and of bovine growth hormone on pregnant mouse mammary gland in organ culture was made. No qualitative differences were observed; both hormones (in the presence of insulin and corticosterone) stimulated the synthesis of casein and RNA in mouse mammary gland explants. The inhibition of RNA synthesis with actinomycin D was associated with a decrease in casein synthesis. However, quantitatively, prolactin was more efficient than growth hormone in stimulating casein synthesis.The synthesis of casein in mouse mammary gland explants incubated in the presence of various combinations of hormones gave results which suggested that prolactin and growth hormone are operating on the same sites of action.Analysis of, and purification by, polyacrylamide gel electrophoresis of the bovine growth hormone showed that the lactogenic effects were not due to the presence of prolactin as an impurity, but were an intrinsic property of the growth hormone.

The rates of synthesis of DNA, RNA and protein of mouse mammary gland explants in organ culture have been determined. Stimulation with insulin resulted in maximal rates of synthesis of these components, all occurring between 18 and 22 h of culture.The use of metabolic inhibitors of DNA, RNA or protein synthesis showed that after insulin stimulation, inhibition of any one of these processes was associated with a reduction in the synthesis of the other two components. Also the maximal rate of protein synthesis is governed by the net amount of RNA formed throughout the period of culture. Evidence is presented that the stimulation of DNA, RNA or protein synthesis by insulin is not due to increased transport of amino acids and that insulin appears to act rapidly on processes which subsequently lead to enhanced synthetic activity.

In experiments on the effect of insulin on the stimulation of incorporation of radioactive thymidine into the DNA of pregnant mouse mammary gland explants in culture, considerable variation between the results of successive experiments was noticed. Dependence of the extent of incorporation on the stage of pregnancy was investigated as a possible cause.Cultures of explants of mammary gland were made (Trowell, 1959) from Schneider mice at various stages of their first pregnancy. The length of pregnancy was taken from the time of appearance of a vaginal plug. Insulin (Sigma Chemical Co., 24\m=.\1 i.u/mg.) was used at a concentration of 5 \g=m\g./ml. medium. After 24 hr. of incubation the cultures were pulsed for 4 hr. with [methyl-3H]thymidine (18\m=.\3c/m-mole; Radiochemical Centre, Amersham) at a concentration of 0\m=.\5 \g=m\c/ml. medium (Stockdale & Topper, 1966). The tissue was removed and weighed, the acid-insoluble material hydrolysed (Mahin & Lofberg, 1966), and the radioactivity determined in a liquidscintillation counter (Packard Model No. 3003). Since the degree of quenching was similar for all samples, the radioactivity was expressed as counts/min./mg. wet weight of tissue without correction for quenching. Control expiants were treated in the same way except that insulin was omitted. Fig. 1 shows the levels of incorporation of thymidine (expressed exponentially) into mammary gland expiants from animals at various stages of pregnancy in the absence or presence of insulin. The incorporation of thymidine increases significantly as the length of pregnancy increases (P < 0-001 and < 0-001, with and without insulin, respectively).It was noticed that the scatter in DNA synthesis by mammary gland expiants ob¬ tained from mice in late pregnancy was less when insulin was present in the medium.This led to an examination of the relationship between the levels of thymidine incor¬ poration in the presence or absence of insulin in expiants from mice pregnant 16-18 days. The results are shown in Fig. 2 where DNA synthesis in the absence of insulin has been plotted against the logarithm of the ratio of insulin-stimulated DNA syn¬ thesis to DNA synthesis without insulin. The relationship is highly significant (P < 0-001). It is probable that the increase in the synthesis of DNA in vitro seen throughout pregnancy reflects cellular proliferation in vivo during this period (