Release of Mammotrophic Activity by the Rat Placenta

Abstract

Release of mammotrophic activity into the culture medium by placenta fragments in vitro could be demonstrated with the aid of an organ culture of mammary explants. Maintenance of the placenta fragments was optimal with less than 8 fragments per ml medium. Release of activity was already high after one hour of culture, but continued for at least four days if the medium was favourable. To obtain this latter effect the presence of salts and glucose in the medium was sufficient, amino acids, serum and insulin being of no or minor importance. The activity observed can be explained partly by a slow release of hormone(s) already present in the fragments. Activity was released by freezing and thawing the placenta fragments. If placenta fragments were kept at + 4°C or + 20°C release of activity continued for several days. Moreover, the release at + 37°C was not inhibited by cycloheximide. On the other hand intact fragments released more activity than frozen fragments, even after one day of culture, while the release continued at a high level for several days in intact fragments and stopped after one day in frozen fragments. The placenta fragments had an active metabolism with glucose consumption and lactate production. When virgin rat serum was incubated with placenta fragments in vitro, its mammotrophic activity became equal to that of pregnant rat serum. Pregnant rat serum seemed to inhibit the release of mammotrophic activity from placenta fragments in some experiments, but the assay method does not permit a definite conclusion. The medium activated by incubation with placenta fragments showed synergism with cortisol as regards secretion, and with progesterone as regards proliferation. An effect of cortisol, progesterone, oestradiol, oestrone or oestriol on the release of mammotrophic activity from the placenta fragments could not be demonstrated. Rat placental giant cells seem to be involved in the production of rat chorionic mammotrophin (rCM). Activity appeared in the maternal serum at day 8-9 of pregnancy when mainly the trophoblastic giant cells adjacent to the foetal membranes are in contact with the maternal circulation. Those placenta fragments which on organ culture released activity into the culture medium contained giant cells. Undifferentiated giant cells and/or basal zone cells also may be capable of producing rCM. The giant cells could be maintained viable in vitro. They incorporated thymidine, uridine and leucine. The giant cells did not stain with acidophilic staining for pituitary hormones. Electron microscopy suggested that if the giant cells do in fact produce rCM, the Golgi apparatus may not be involved in its secretion. Secretion granules could not be demonstrated in the giant cells. Sephadex G-100 gel chromatography fractions were obtained from pregnant rat serum, placenta extract and placenta culture medium. The fractions were tested for the presence of mammotrophic activity. In 13 and 19 days pregnant rat serum the fraction with molecular weight of approximately 35 000 showed high activity. In placenta extracts obtained from 13 and 19 days pregnant rats activity was present in the fraction with molecular weight of approximately 18 000, but other fractions with lower and higher molecular weight also possessed high mammotrophic activity. Media collected from a placenta culture similarly contained multiple fractions with activity.