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On the expulsion of the erythroid nucleus and its phagocytosis.

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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.
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