Hematological Changes in Cold-Acclimated Xenopus laevis.
Authors: Kuramochi Yuko , Nogawa Nami , Ishida Takako , Watanabe Takafumi , Kosaka Nobuyoshi , Kato Takashi 
Published: April 2020

Abstract
While hematopoiesis in individuals is strictly regulated for maintenance of homeostasis, it has been reported that the numbers of peripheral blood cells are modulated in response to environmental temperature in vertebrates including hamsters, rats, squirrels, dogs, and bullfrogs. To date, the physiological and molecular mechanisms have not been elucidated yet. Amphibians are poikilothermic vertebrates exposed to various fluctuations of environmental conditions; therefore they need to exert their capacities to acclimate to such changes. Additionally numerous studies have demonstrated a measurable metabolic reduction in the metabolic rate of cold-acclimated frogs. We examined hematological changes in response to environmental temperature in an aquatic amphibian, Xenopus laevis. Frogs initially maintained at 25°C were acclimated to 10°C, and hematological changes were observed. The numbers of erythrocytes, leukocytes and thrombocytes gradually reduced as the transient phase by 4 weeks, and subsequently reached the steady-state that sustained for more than 4 months. Whereas the reduction in the numbers of white blood cells and thrombocytes were moderate, the number of erythrocytes and the level of hemoglobin at nadir were remarkably low (approximately 40% of the initial values). It is known that oxygen levels may reduce in ice-cold water, and cold-acclimated animals therefore tolerate prolonged severe hypoxia; nevertheless cold-acclimated Xenopus exhibited severe erythrocytopenia. In addition, morphological change of peripheral blood cells and hematopoietic tissues (liver, spleen, kidney, and bone marrow) were examined. There were no remarkable cellular changes in cellular size and shape. However, increased numbers of mature erythrocytes were observed in the bone marrow of the steady state cold-acclimated Xenopus, while mature erythrocytes were not found in the bone marrow in Xenopus at 25 °C. This cold-temperature-induced pancytopenia was reversible when the temperature was put back to 25°C, as all of blood cell counts returned to the normal levels within 4 weeks in a reverse fashion as the transient phase of cold-acclimation. During the recovery phase, immature erythrocytes that were scarcely existed in the normal peripheral blood appeared in the circulation, suggesting that erythrocytes were newly produced at 25 °C after prolonged exposure to cold temperature. The possible explanations for the reduction in the numbers of circulating peripheral blood cells might be due to a number of various reasons such as reduced productions of hamatopoietic progenitors and/or related cytokines, alternation in the storage capacities and/or the life span of blood cells, and systemic suspension of normal activities. To compare the lifespan of erythrocytes between normal and cold-acclimated Xenopus, erythrocytes were covalently labeled with biotin. The surviving biotinylated erythrocytes in the circulation were quantitatively detected as avidin-biotin complex by microscopy and flowcytometry. Furthermore expression levels of several genes responsible for the hematopoietic regulation were comparatively examined. The cold-acclimated Xenopus model developed here may allow for a valuable approach aiming at exploring undiscovered systems in hematopoietic regulation.