BRIEF COMMUNICATION On the Expulsion of the Erythroid Nucleus and Its Phagocytosis EHUD SKUTELSKY AND DAVID DANON Section of Biological Ultrastructure, T h e W e i z m a n n Institute of Science, Rehovot, Israel Electron microscopical examination of thin sections of guinea pig and rabbit bone marrow indicates that nuclei of their normoblasts undergo ABSTRACT constriction during their expulsion. Late erythroblasts in bone marrow of adult rabbits and guinea pigs were often seen surrounded by cytoplasmic protrusions of macrophages. No such contact was observed between macrophages and erythroblasts that were expelling their nuclei in the lumen of blood sinuses of rabbit bone marrow. It is concluded that macrophages are not necessary for nuclear expulsion. The expulsion of the nucleus from the normoblast is considered by several authors to be a biological phenomenon, very similar to cytokinesis (Bessis and Bricka, ’52; Jones, ’60; Skutelsky and Danon, ’67, ’70). As such, it would not require any assistance from neighboring cells. Seki, Yoneyama and Shirasawa (‘65a,b,c) have attributed a n active role to the reticular cells in removing the nucleus from the normoblast. Campbell (’68) claimed that all normoblasts’ nuclei observed were either attached to the cytoplasmic mass or surrounded by processes of reticular cells, or attached and partially surrounded. No “free” nuclei were observed. This statement and the discussion of the whole paper seem to support a hypothesis that the phagocytic cells are necessary for the detachment of the final connection between the extruded nucleus and the future reticulocyte. Campbell (’68) pointed out another difference between his results and ours (Skutelsky and Danon, ’67). It refers to our description of the deformation and constriction of the nucleus that occurs during its extrusion, which he did not observe in his study. He, therefore, concludes that “this phenomenon is a species difference between the guinea pig and mouse.” It is difficult to conceive that the process of nuclear expulsion is restricted to one or two species of mammals. The present report provides evidence that the nuclei of guinea pig normoblasts, like those of mouse (Skutelsky and Danon, ’67, ’ 7 0 ) , ANAT. REC., 173: 123-126 rabbbit (Skutelsky and Danon, ’69) and m a n (Efrati -personal communication) are constricted during their expulsion and that phagocytic cells are not indispensable for either extrusion of the nucleus or its final detachment. MATERIALS AND METHODS Guinea pig and rabbit bone marrow were prepared for electron microscopical analysis as follows: The tissues were fixed with 3.8% glutaraldehyde in phosphate buffer at pH 7.4 (Millonig, ’62) for two hours at room temperature, washed three times and left overnight in the same buffer at 4°C. They were then postfixed in 1% OsO, in phosphate buffer (Millonig, ’62) for two hours at room temperature, gradually dehydrated with acetone, embedded in Vestopal-W (Ryter and Kellenberger, ’58) and sectioned on a Danon Ultramicrotome (Yeda Research and Development Co., Rehovot, Israel), equipped with glass knives. Sections were mounted on Formvar coated grids reinforced with a thin layer of carbon. They were stained on the grid with uranyl acetate, followed by lead citrate. JEM-7 and JEM-T7 electron microscopes were used. OBSERVATIONS AND DISCUSSION When sections of guinea pig bone marrow were observed i n the microscope, Received June 29, ’71. Accepted Dec. 6, ’71. 1The Patrick E. Gorman Professor of Biological Ultrastructure. 123 124 EHUD SKUTELSKY AND DAVID DANON Fig. 1 Electron micrograph of guinea pig bone marrow. A normoblast nucleus is seen constricted i n the process of expulsion. x 22,000. several normoblasts in the process of expelling their nuclei reveal clearly a constriction in the process of expulsion (fig. 1). This evidence shows that guinea pig normoblasts’ nuclei, like those of mouse, rabbit and man, are constricted during their expulsion. In rabbit bone marrow phagocytic cells are in contact with extruded nuclei. Moreover, pseudopods of macrophages surround erythroblasts or reticulocytes without finally phagocytizing them, as evidenced by the fact that they have never been observed within vacuoles intact or partially digested. Extruded nuclei can be seen not engulfed in close proximity (fig. 2). Furthermore, erythroblasts expelling their nuclei can often be seen within sinuses or blood vessels of rabbit bone marrow where no phagocytic cells are nearby (fig. 3 ) . Nuclear expulsion was observed in suspended peripheral blood without phagocytic cells in the vicinity by phase contrast microscopy of human blood (Bessis and Bricka, ’52) and rabbit marrow ( M brecht, ’51; Bessis and Bricka, ’52) and by electron microscopy of circulating dog blood ( Simpson and Kling, ’67). In view of our data and the cited supporting literature, we believe that the phagocytic cell plays a role in “distinguishing” the expelled nucleus as a candidate for phagocytosis from the other cells in the bone marrow (Skutelsky and Danon, ’69); it may temporarily engulf, but not phagocytize the other cells. The nucleus may be phagocytized either before it is completely expelled or thereafter. LITERATURE CITED Albrecht, M. 1951 Studien zur Frage der Erythroblastenentkernung von Meerschweinchenknochenmark. Acta Haematol., 6 : 83-91. Bessis, M., and M. Bricka 1952 Aspect dynamiques des cellules du song. Son etude par la microcinematographie e n contrast de phase. Rev. Hematol., 7: 407-435. Campbell, F. R. 1968 Nuclear elimination from the normoblast of fetal guinea pig liver, as ’ NUCLEAR EXPULSION AND PHAGOCYTOSIS 125 Fig. 2 Electron micrograph of rabbit bone marrow. Three late erythroblasts (EB) are seen in close contact with a macrophage ( M ) and partially surrounded by its pseudopods. One erythroblast (top) is in the late stage of nuclear expulsion. A macrophage surrounds the extruded nucleus. Several other erythroblasts (EB’) and one erythroid nucleus ( N ) are seen not i n contact with any macrophage. Erythroid nuclei at various stages of digestion can be seen within the macrophage (bottom left). X 7,500. studied in electron microscopy and serial sectioning techniques. Anat. Rec., 160: 539-554. Efrati, P. Personal Communication. Jones, 0. P. 1960 Electron microscope studies of fetal erythropoiesis. Proceedings of the Seventh Congress of the European Society for Haematology, London, 1959. Karger, Basel, Part IT: 79-81. Millonig, G. 1962 Further observations on a phosphate buffer for osmium solutions. In: Electron Microscopy. S. S. Breese, Jr. ed. Academic Press Inc., New York, 2: p. 8. Ryter, A., and E. Kellenberger 1958 L’inclusion au polyester pour l’ultramicrotomie. J. Ultrastr. Res., 2: 200-214. Seki, M., T. Yoneyama and H. Shirosawa 1965 Role of the reticular cells during maturation process of the erythroblasts. I. Denucleation of erythroblasts by reticular cell; electron microscopic study. Acta Path. Jap., 15: 295-301. 126 EHUD SKUTELSKY AND DAVID DANON Fig. 3 A capillary in a rabbit bone marrow, containing erythroblasts and erythrocytes One erythroblast (EB) is in the late stage of nuclear expulsion. Some other hematopoietic cells are seen outside the capillary wall. No phagocytic cells or their pseudopods are visible i n the lumen. x 7,500. 1965 Role of the reticular cells during maturation process of the erythroblasts. 11. Further observation on the denucleation process of erythroblasts. Acta Path. Jap., 15: 303-316. 1965 Role of the reticular cells during maturation process of the erythroblasts. 111. The fate of phagocytized nucleus. Acta Path. Jap., 15: 387-405. Simpson, C. F., and J. M. Kling 1967 Mechanism of denucleation in circulating erythroblasts. J. Cell Biol., 35: 237-245. - Skutelsky, E., and D. Danon 1967 An electron microscopical study of nuclear elimination from the late erythroblast. J. Cell Biol., 33: 625-635. 1969 Reduction in surface charge as a n explanation of the recognition by macrophages of nuclei expelled from normoblasts. J. Cell Biol., 43: 8-15. 1970 Comparative study of nuclear expulsion from the late erythroblast and cytokinesis. Exptl. Cell Res., 60: 427-436.