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., 24·1 i.u/mg.) was used at a concentration of 5 μg./ml. medium. After 24 hr. of incubation the cultures were pulsed for 4 hr. with [methyl-3H]thymidine (18·3 c/m-mole; Radiochemical Centre, Amersham) at a concentration of 0·5 μc/ml. medium (Stockdale & Topper, 1966). The tissue was removed and weighed, the acid-insoluble material hydrolysed (Mahin & Lofberg, 1966), and
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