1. Explants of mammary tissue from pseudopregnant rabbits were cultured at 37°C in air for 24–48h in Medium 199 buffered with 20m m -Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The medium contained insulin and corticosterone, or insulin, corticosterone and sheep prolactin in the presence or absence of ouabain, an inhibitor of Na + /K + -dependent adenosine triphosphatase. The responses of explants were assessed histologically, by measuring the tissue concentration of K + , and by rates of synthesis of RNA, protein and fatty acids. The effect of ouabain on Na + and K + concentrations in slices of lactating rabbit mammary-gland tissue incubated for 1h at 37°C in Krebs bicarbonate buffer was also studied. 2. Prolactin increased the concentration of K + in mammary explants, an effect prevented by ouabain. In slices of lactating tissue, there was a linear relationship between the log dose of ouabain (from 0.1 to 10μ m ) and increased Na + and decreased K + concentrations in the tissue. 3. Ouabain at concentrations up to 1μ m did not affect the rate of synthesis of RNA, protein or fatty acids by explants cultured with insulin and corticosterone. By contrast, the stimulatory effect of prolactin on protein synthesis was diminished and the induction of medium-chain fatty acid synthesis by prolactin was almost abolished. RNA synthesis was unaffected. Histological examination showed no tissue damage by 1μ m -ouabain. 4. Explants cultured in the presence of 2μ m -ouabain for 24h retained their ability to respond to prolactin when the ouabain was removed from the culture medium. Between 24 and 48h they showed responses to prolactin of a magnitude similar to those of explants never exposed to ouabain. 5. These results show that a fully functional Na + /K + -dependent adenosine triphosphatase system is necessary for prolactin to promote secretory activity in rabbit mammary gland.
1. Congenitally goitrous thyroid tissue was obtained from South Australian Merino sheep. Ultrastructural studies of the secretory cells in this tissue showed active cells of normal appearance, containing apical protein droplets. 2. 125 I-labelling in vivo of goitre tissue was used to investigate the iodoproteins, in which the major proportion of 125 I appeared in the cell protein fraction soluble in 0.9% sodium chloride (average 62% in goitres from untreated sheep). 3. Ammonium sulphate fractionation showed two clear peaks of iodoprotein precipitation, one at 35–40% saturation and the other at 50–55% saturation. Both iodoprotein fractions contained iodotyrosines and iodothyronines, which were identified chromatographically after enzymic hydrolysis of the protein. 4. Polyacrylamide-gel electrophoresis at pH9.4, at either 7.5 or 5.0% acrylamide concentration, was used to characterize the iodoproteins. Two major fractions were observed, the fastest-migrating fraction coincident with serum albumin, and a slower-migrating, less-well-defined zone. This fraction migrated in 7.5% acrylamide gel, which excluded normal thyroglobulin. 5. Density-gradient (10–40% sucrose) centrifugation was used to determine the approximate sedimentation coefficients of the iodoproteins, which showed major components at s 20,w 8–9S and s 20,w <5S. 6. Immunoprecipitation with rabbit anti-(sheep thyroglobulin) failed to sediment 125 I-labelled proteins from goitre extracts. 7. Ouchterlony-type double diffusion in agar plates demonstrated immunoprecipitation lines between rabbit anti-(sheep thyroglobulin) and both the concentrated goitre extract and its Sephadex G-200-excluded fraction, which were confluent with that obtained on reaction with purified normal thyroglobulin. 8. It was concluded that both major iodoprotein fractions were capable of supplying thyroid hormones to the animal, and that the fraction of s 20,w <5S was iodinated serum albumin. As 125 I-labelled thyroglobulin was not detected in goitre tissue from untreated or thyroxine-treated animals, it was possible that the genetic defect causing goitre resulted in an abnormal thyroglobulin, incapable of being iodinated but immunologically reactive.