ERYTHROPOIETIN AND ERYTHROPOIESIS IN FETAL AND NEONATAL SHEEP

Abstract

Erythropoietin (Epo) is the major physiological regulator of red blood cell production. This glycoprotein hormone was thought to be synthesized primarily by the adult kidney and fetal liver. In this thesis, the ontogeny of Epo gene expression was examined in the sheep fetus and neonate, and various factors affecting its regulation in the fetus were investigated. To this end, a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to determine levels of Epo gene expression.

Examination of Epo mRNA levels in the livers and kidneys of fetuses and lambs of several age groups showed that in both liver and kidney, Epo mRNA levels per µg total RNA were highest at 60 days of gestation and decreased toward term (145-150 days of gestation). This decrease occurred earlier and was more pronounced in the liver; in the kidney, Epo mRNA levels were greater than that of liver at 60 days of gestation and levels only decreased after 100 days of gestation, suggesting that the kidney plays a larger role earlier in gestation in the sheep fetus than previously thought. In the lamb, both liver and kidney Epo mRNA levels were very variable in the first three weeks of life, consistent with variable plasma Epo levels observed at this age, and thereafter declined, with a nadir at 4-6 weeks, a period corresponding to the period of physiological anaemia of infancy. Haemorrhage at 75, 110 and 140 days of gestation brought about a significant increase in both liver and kidney Epo mRNA levels at all stages. However, the fold­ increase in kidney Epo mRNA expression was always greater than that in the liver, and increased toward term, although the stimulated level was not different between the groups. Several fetuses were also nephrectomized and haemorrhaged at 110 days of gestation. While basal plasma Epo levels were different from normal values, Epo levels stimulated by haemorrhage were much lower than in intact haemorrhaged fetuses, and liver Epo mRNA levels were not greater than that of intact haemorrhaged fetuses. This suggests that the kidney plays a more significant role in the response to haemorrhage in the young fetal sheep than was previously supposed, and that the liver in nephrectomized fetuses is sufficient to maintain basal levels of plasma Epo but is unable to play a compensatory role, when stimulated, in the absence of the kidneys. The effect of manipulating fetal glucocorticoid status on Epo gene expression in the fetal sheep was investigated. Fetuses adrenalectomized at 120 days of gestation and examined near term had significantly higher renal Epo mRNA levels than age­matched controls, while adrenalectomized fetuses which had been infused with cortisol had kidney Epo mRNA levels similar to control. Exogenous cortisol infusion at 110 days of gestation lowered renal, but not hepatic, Epo mRNA levels. Infusion of dexamethasone into ewes at 60-80 days of gestation decreased fetal, but not maternal, renal Epo mRNA levels. Taken together, these results suggest a possible role of glucocorticoids in the developmental down egulation of the levels of renal Epo gene expression in the later part of gestation in the sheep. In order to see if the Epo gene contained any sites for glucocorticoid receptor binding in the regulatory region, a sheep genomic library was screened. A clone containing the Epo gene was isolated and 1 kb of the upstream region sequenced. This sequence contained a number of putative half sites for steroid receptor response elements. Comparison with the corresponding region of the human and mouse genes showed regions of high homology, suggesting that these sequences may play a role in Epo gene regulation. Two partial cDNA clones of the sheep Epo receptor were isolated by reverse transcription and PCR. These corresponded to the full length receptor and a splice variant whose deduced amino acid sequence would give rise to a longer receptor molecule. Using competitive RT-PCR, the ratios of the two mRNA species were compared in fetal liver and lamb bone marrow, from 60 days of gestation to 10 weeks after birth, and both were found to be expressed at all stages but were not significantly different with age.