Maturation of the reticulocyte in vitro

The maturation of reticulocytes into erythrocytes was demonstrated in vitro. Reticulocytosis was induced in rats by repeated bleeding or by phenylhydrazine injections. Whole blood samples were then incubated for 2 days at 37 degrees C. Reticulocytes in culture changed from polylobulated, monoconcave or triconcave forms to biconcave disks. During the first 12 h in vitro, the average reticulocyte count decreased from 39% to 12%, and the membrane-bound organelles, ribosomes and exocytic figures in the remaining reticulocytes were markedly diminished. In contrast, the number of red cells containing inclusions of denatured haemoglobin (Heinz bodies) in phenylhydrazine-treated blood did not decline. The reduction in reticulocyte count was not the result of differential cell destruction, since little haemolysis occurred in vitro. During red cell maturation three modes of organelle removal were observed particularly well when mitochondria were followed by cytochrome oxidase cytochemistry. First, some mitochondria degenerated, presumably through autolysis, by swelling, losing cristae and forming small single membrane-bound vesicles. Second, individual mitochondria became enclosed in vacuoles that fused with the plasma membrane and expelled their mitochondria by exocytosis. Third, autophagic vacuoles containing mitochondria, cytosol and membrane fragments fused with existing lysosomes. We conclude that all aspects of normal reticulocyte maturation occur in vitro, independent of the spleen, including the removal of organelles and the assumption of the mature biconcave disk shape.