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480 Part VI: The Erythrocyte Chapter 32: Erythropoiesis 481

than more mature erythroid progenitors to form a colony of erythrob- marrow erythroblasts and reticulocytes have shown approximately
lasts (10 to 14 days) and form a large colony approximately 2000 to 50 erythroblasts and approximately 124 reticulocytes for each proery-
3000 cells. BFU-E express low levels of EPO receptors (EPORs). BFU-E throblast (Table 32–1). 40,41 This distribution conforms to the number
mature into colony-forming unit–erythroid (CFU-E), the more mature of cells in a theoretic erythroid pyramid (Table 32–1, Fig. 32–3). In
erythroid progenitor. A CFU-E is identified through more differentiated the pyramid, each erythroblast undergoes five mitotic divisions over
erythroid progenitor that in vitro form smaller colonies (50 to 200 cells) 5 days before the orthochromatic erythroblast loses its nucleus and
that mature in 3 to 5 days with EPOR density and EPO dependency as an immature erythrocyte enters a 2- to 3-day period of matura-
increase gradually as progenitor cells mature, culminating at the level of tion before its release from the marrow. The size and shape of these
the CFU-E. 34,35 BFU-E and CFU-E cannot be identified by microscopy erythroid pyramids undoubtedly vary, but such variations play a role
(Chap. 31), but they can be studied in vitro by their ability to gener- in the physiologic control of red cell production. When production
ate microscopically recognizable hemoglobinized precursors (i.e., ery- is suppressed, as in low EPO as seen in anemia of chronic renal dis-
throblasts) by so-called clonogenic assays on semisolid media. ease, the distribution of erythroblasts appears normal, with no mor-
phologic or ferrokinetic evidence of ineffective erythropoiesis but the
38
number of erythroid progenitors is decreased. When production is
PRECURSOR CELLS increased, as in severe hemolytic anemia, the pyramid of erythroid
In contrast, cells that constitute the latter stages of erythropoiesis can be precursors also appear normal, with no evidence of additional mitotic
identified by light microscopy (Chap. 31). The earliest morphologically divisions but the number of erythroid progenitors is increased. Conse-
recognizable erythroid precursor in the adult marrow is the pronor- quently, the rate of red cell production largely depends on the number
moblast. Pronormoblasts are conspicuously large and they have large of erythroid progenitors formed.
uncondensed nuclei and deep basophilic cytoplasm as a result of the As the erythroblast matures, its synthetic activities increase
presence of numerous RNA-containing polyribosomes. Pronormoblast rapidly, producing all proteins characteristic of mature red blood cells,
has a volume of 900 fL, 10 times the volume of the mature red blood cell. particularly globin. Eventually 95 percent of all protein in the red cell is
With each successive division, the precursor cells give rise to daughter hemoglobin, almost all hemoglobin A (α β ) in adults, with only small
2 2
cells of half their volume. Furthermore, with each division there is an amounts of hemoglobin F (α γ ) and hemoglobin A (α δ ). Hemoglo-
2
2 2
2 2
increase in hemoglobin synthesis and condensation of nucleus. Thus, bin F is unequally distributed and is present only in some erythrocytes,
when the pronormoblasts divide to become basophilic normoblasts, designated as F cells (Chaps. 48 and 49).
the daughter cells have less blue cytoplasm because of hemoglobin EPOR density declines sharply on early erythroblasts, and EPORs
synthesis and also a greater condensation of the nucleus. When the are absent from the more mature erythroblast forms while the number
basophilic normoblasts divide further, they give rise to mature cells of receptors for transferrin increases, reflecting the increased demands
with more cytoplasmic hemoglobin that is stainable with both acid and for iron for heme synthesis.
basic dyes resulting in muddy-colored cytoplasm. These cells are called
polychromatophilic normoblasts. The offspring of polychromatophilic
normoblasts are called orthochromic normoblasts. Their nuclear chro- ERYTHROBLAST ENUCLEATION
matin is completely condensed and cytoplasm is pink from complete The microenvironment may be important for proliferation and matu-
hemoglobinization. These cells do not divide further. Following extru- ration of erythroblasts. However, in situ secreted or circulating growth
sion of the nucleus, the enucleated cells derived from orthochromic factors and cytokines appear to be less important for precursor cells
orthochromatic erythroblasts are termed reticulocytes, named after the than for progenitor cells. Intercellular adhesion molecules secure the
cytoplasmic remnants of the endoplasmic reticulum and the persistence structural integrity of the marrow, and fibronectin is of special impor-
of a few mitochondria and strings of ribosomes seen when stained with tance for erythroblasts. Loss of fibronectin receptors heralds the
42
supravital dyes. Reticulocytes remain in the marrow for 48 to 72 hours translocation of polychromatophilic macrocytes (reticulocytes) into
before being released to the blood. The reticulocytes have an irregular blood, but some newly emerging erythrocytes remain sticky even after
polylobated shape and various membrane-bound organelles. In the release and are temporarily sequestered by the spleen (Chap. 6). Because
36
blood, immature erythrocytes (reticulocytes) undergo further matura- erythroid colonies developed in vitro consist principally of nucleated
tion with the removal of vestiges of organelles and reconditioning of the red cells, enucleation may primarily be induced by marrow stromal cells
membrane to become mature red blood cells with the morphology of a (Chaps. 5 and 31).
biconcave disk. 37 The extrusion of the spent pyknotic nuclei at terminal erythroid
The number of erythroid precursor cells determines to a great maturation is unique to mammals. This process results in the forma-
extent the number of red cells produced. The proerythroblasts also tion of pliable biconcave disc from rigid spheroidal cells. Enucleation
contain EPORs that, in the presence of higher than normal levels of decreases workload of the heart as it reduces one-third of the ery-
EPO, may accelerate their entry into their first mitotic division. This throcyte weight. The retinoblastoma protein and its effector, E2f-2, are
process may lead to a shortened marrow transit time of erythrob- critical for erythroid cells to exit the cell cycle for enucleation to take
lasts and result in release of still immature erythrocytes (polychro- place. During terminal differentiation, the plasma membrane forms
38
43
matophilic macrocytes), so-called stress reticulocytes (Fig. 32–2). an envelope around the nucleus, followed by the formation of the con-
39
Creation of a normal sized and shaped red cells, devoid of organelles, tractile actin ring on the plasma membrane to move the nucleus for
is the end result of an orderly transformation of a proerythroblast with disposal. Rac guanosine triphosphatases (GTPases) and their effec-
44
a large nucleus and a volume of approximately 900 fL to a hemoglo- tor mDia2 are required for the contractile actin ring movement in the
binized anucleate disc-shaped cell with a volume of approximately release of the nucleus. After separation from the cell, the expelled
45
90 fL. Although cytoplasmic maturation is continuous, the inter- nucleus displays phosphatidylserine and is recognized and engulfed
posed mitotic divisions cause a stepwise reduction in cytoplasmic by macrophages. Emp, an erythroblast–macrophage protein initially
46
and nuclear volumes, enabling recognition of proerythroblasts, ery- identified as a mediator of erythroblast–macrophage interactions,
throblasts, and polychromatophilic macrocytes (reticulocytes) with also plays a role in the enucleation. Emp associates with F-actin, an
47
light microscopy (Chap. 31). Direct measurements of the number of interaction important for the normal distribution of F-actin in both

Kaushansky_chapter 32_p0479-0494.indd 481 9/17/15 6:10 PM