Blockade of the in vitro effects of testosterone and erythropoietin on Cfu-E and Bfu-E proliferation by pretreatment of the donor rats with cyproterone and flutamide.

Abstract

Erythropoietin is obligatory to support the terminal proliferation and differentiation of erythroid cells but it is not the only agent in modulating red cell production. Previously, we have shown that Testosterone enhances erythropoiesis, at least in part, by increasing renal erythropoietin production. Testosterone may also influence directly the behavior of the erythroid progenitor cells increasing erythroid stem cell proliferation. To gain further insight into the role of testosterone in regulation of erythropoiesis and its interactions with erythropoietin, we studied the effect of testosterone and erythropoietin, using clonal cultures of erythropietic progenitors, to observe CFU-E and late and early BFU-E colonies proliferation from bone marrow cells of donor rats pretreated for 2 days with the androgen antagonists cyproterone (10 mg/kg/day) and flutamide (160 mg/kg/day). Specific nuclear receptors for testosterone were demonstrated in marrow erythroid cells. Then, erythroid progenitors may come with their androgen receptors blocked by pretreatment. Cultures were prepared using the methylcellulose technique containing a standard dose of erythropoietin (250 mU/ml) or testosterone (10(-7)M). Results obtained demonstrate that testosterone produced a significant stimulation on CFU-E and BFU-E colony formation. A dose effect correlation was apparent. Testosterone enhances proliferation of late BFU-E more than CFU-E and produce only a slight augmentation of early BFU-E. As expected, erythropoietin markedly stimulate all erythroid colony growth, mainly CFU-E. The effects of testosterone were completely abolished in cultures from bone marrow cells of rats pretreated with cyproterone and flutamide. Activation of the specific androgen nuclear receptors in erythroid cells appears to be necessary for testosterone to develop the erythropoietic effect. Surprisingly, the effects of erythropoietin on erythroid colonies proliferation were also completely blocked by pretreatment with flutamide and partially blocked by pretreatment with cyproterone. Right now, we do not have a satisfactory explanation regarding inhibition of the effects of erythropoietin by pretreatment to marrow donor rats with the androgen antagonists. In conclusion, we postulate triggering late BFU-E cells, a marrow erythropoietin responsive cell population, into active cell cycle, as well as by increasing blood erythropoietin concentration.