The Mechanism of Denucleation in Circulating Erythroblasts

Abstract

Erythroblast denucleation in the peripheral blood was studied by electron microscopy. Blood was used from dogs anemic either by infection with Babesia canis or from injections of phenylhydrazine hydrochloride. One of the earliest stages of denucleation was the migration of nuclei to the plasmalemma. Mitochondria and coalesced vesicles, derived from the cell membrane of the erythroblast, congregated at the underside of the nuclear envelope unapposed by erythroblastic cell membrane. The coalesced vesicles apparently provided the limiting membrane which surrounded the deep circumference of the extruded nucleus and its associated hemoglobin rim, and also furnished a new plasma membrane for the cell in the area where the nucleus, in denucleation, had utilized the original erythroblastic plasmalemma.It is not known whether loss of nuclei from erythroblasts occurs by extrusion, by karyolysis, or both. Most studies to date have used phase-contrast microscopy to determine the mechanism involved, and the predominant opinion seems to favor nuclear expulsion as the method of denucleationThe present study of erythroblast denucleation was made with the electron microscope, with blood from dogs with either induced or spontaneous anemia. MATERIALS AND METHODSThe blood studied was from dogs anemic as a result of severe parasitism with a hematozoic parasite, Babesia canis, and from dogs made anemic by treating them every other day with three subcutaneous injections of phenylhydrazine hydrochloride at a dosage of 16 mg per kg of body weight. Five to six drops of whole blood were collected in fixative when approximately 10% of the erythrocytic cells were nucleated, as determined by examination of Wright-Giemsa-stained blood smears. 1% OsO4 (12) was employed as a fixative for blood obtained from dogs parasitized with B. canis, while blood from phenylhydrazine-treated dogs was fixed in 2% glutaraldehyde and postfixed in 1% Os04. The fixed blood was embedded in Araldite (13), and cut into thin sections with a dimond knife. Sections on grids were stained with uranyl acetate and lead citrate (14), and were examined with a Philips EM200 electron microscope. RESULTS Light MicroscopyNuclei were located centrally, peripherally, or partially protruded from erythroblasts (Fig. 1). Occasionally nuclei were observed extracellularly. Basophilic, polychromatic, and orthochromatic erythroblasts were present in blood smears. Electron MicroscopyThe mechanism of denucleation was the same in both types of anemia, and thus the results were combined for this study.Erythroblasts with centrally located nuclei contained numerous polyribosomes and a scattering of mitochondria as well as open-spaced vesicles throughout the cytoplasm. Such vesicles ap-

Signaling and cytoskeletal requirements in erythroblast enucleation

“…7 A recent study by Keerthivasan et al using primary mouse and human erythroblasts supported the hypothesis that vesicle trafficking and subsequent vacuole coalescence provide additional membrane for the separating nucleus and reticulocyte. 8,9 Actin polymerization was again seen to play a role by Ji et al who found that Rac GTPase deregulation by overexpression of either dominant negative or constitutively active mutants of Rac1 or Rac2 or by pharmacologic inhibition by the Rac-specific inhibitor NSC23766 10,11 decreases enucleation of mouse fetal liver erythroblasts in culture. Moreover, down-regulation of mDia2, a downstream effector of Rho GTPases, by small interfering RNA (siRNA) blocks erythroblast enucleation.…”

Chapter 2 The Erythroblastic Island

“…The loss of the intermediate filaments allows the nucleus to move freely to the periphery of the cell and occupy an acentric position prior to enucleation. A number of studies have proposed that enucleation may be a process similar to cytokinesis (Campbell, 1968;Repasky and Eckert, 1981a,b,c;Simpson and Kling, 1967;Danon, 1967, 1970) and have suggested that F-actin is present in the form of a ring in the constriction, similar to the cleavage furrow of mitotic cells (Perry et al, 1971;Schroeder, 1973). Using splenic erythroblasts from mice infected with the anemia-inducing strain of Friend virus, Koury et al (1989) showed that F-actin is concentrated between the extruding nucleus and incipient reticulocyte in enucleating erythroblasts.…”

Mammalian erythroblast enucleation requires PI3K-dependent cell polarization

“…During enucleation, the erythroblast extrudes its nucleus tightly apposed to the plasma membrane, forming a reticulocyte (Ihle and Gilliland, 2007;Koury et al, 2002;Richmond et al, 2005). Pioneering studies using electron microscopy revealed that at the earliest stage of enucleation the erythroblast nucleus becomes located close to the cell membrane away from the center of the cell (Simpson and Kling, 1967;Skutelsky and Danon, 1967) and that a cytokineticlike furrow is formed in the region between the extruded nucleus and the incipient reticulocyte (Koury et al, 1989;Skutelsky and Danon, 1967). Actin filaments (F-actin) accumulate in the cytokinetic-like furrow (Ji et al, 2008;Koury et al, 1989) and disruption of F-actin (Ji et al, 2008;Koury et al, 1989;Yoshida et al, 2005) or depletion of mDia2, a regulator of actin polymerization (Ji et al, 2008), blocked enucleation, suggesting that actin-based forces drive nuclear extrusion.…”