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1.
Biol Open ; 12(8)2023 08 15.
Article in English | MEDLINE | ID: mdl-37421150

ABSTRACT

Xenopus liver maintains erythropoietic activity from the larval to the adult stage. During metamorphosis, thyroid hormone mediates apoptosis of larval-type erythroid progenitors and proliferation of adult-type erythroid progenitors, and a globin switch occurs during this time. In addition, the whole-body mass and the liver also change; however, whether there is a change in the absolute number of erythroid progenitors is unclear. To isolate and evaluate erythroid progenitors in the Xenopus liver, we developed monoclonal ER9 antibodies against the erythropoietin receptor (EPOR) of Xenopus. ER9 recognized erythrocytes, but not white blood cells or thrombocytes. The specificity of ER9 for EPOR manifested as its inhibitory effect on the proliferation of a Xenopus EPOR-expressing cell line. Furthermore, ER9 recognition was consistent with epor gene expression. ER9 staining with Acridine orange (AO) allowed erythrocyte fractionation through fluorescence-activated cell sorting. The ER9+ and AO-red (AOr)high fractions were highly enriched in erythroid progenitors and primarily localized to the liver. The method developed using ER9 and AO was also applied to larvae and froglets with different progenitor populations from adult frogs. The liver to body weight and the number of ER9+ AOrhigh cells per unit body weight were significantly higher in adults than in larvae and froglets, and the number of ER9+ AOrhigh cells per unit liver weight was the highest in froglets. Collectively, our results show increased erythropoiesis in the froglet liver and demonstrate growth-dependent changes in erythropoiesis patterns in specific organs of Xenopus.


Subject(s)
Erythroid Cells , Liver , Animals , Xenopus , Liver/metabolism , Larva/metabolism , Aging , Erythroid Cells/metabolism , Cell Separation , Receptors, Erythropoietin/metabolism , Humans , HEK293 Cells , Cell Differentiation , Erythropoietin/metabolism
2.
Sci Rep ; 8(1): 16245, 2018 11 02.
Article in English | MEDLINE | ID: mdl-30390005

ABSTRACT

Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.


Subject(s)
Blood Cells , Cell Separation/methods , Flow Cytometry/methods , Staining and Labeling/methods , Xenopus laevis/blood , Acridine Orange/chemistry , Animals , Animals, Inbred Strains/blood , Animals, Wild/blood , Blood Cell Count/methods , Fluorescent Dyes/chemistry , Models, Animal , Species Specificity
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