Erythropoietin mRNA expression in pig embryos (original) (raw)
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Quantitation of the mRNA levels of Epo and EpoR in various tissues in the ovine fetus
Molecular and Cellular Endocrinology, 2002
A partial cDNA of the sheep erythropoietin receptor (EpoR) was obtained and used in real-time PCR to quantitate mRNA levels in placenta, liver and kidney throughout development (term =150 days). This was compared with Epo mRNA levels in the same tissues. Both Epo and EpoR mRNA were present in the placenta throughout gestation at low levels from 66 days onwards and these did not vary throughout gestation. Compared with the expression levels in the placenta, the levels of EpoR gene expression in the liver at 66, 99 and 140 days were, median (range)-288 (120-343), 278 (63-541) and 7 (3 -15), respectively, reflecting the disappearance of erythropoiesis after 130 days. Low levels of EpoR gene expression were seen in the kidney at 3 (2-5), 5 (2-7), and 7 (2-10) times that in the placenta at 66, 99, and 140 days, respectively. By hybridization histochemistry the EpoR mRNA was located in the proximal tubular cells of the mesonephros and metanephros at 42 days. Epo mRNA levels in the kidney were 215 (116-867), 528 (113-765) and 46 (15-204) times those in the placenta at 69, 99, and 140 days, respectively. In the liver at the same ages the concentrations of mRNA were lower than in the kidney, the liver/placenta ratios being 50 (11-90), 17 (3-39), 9 (5-14). At 130 days Epo/EpoR levels in the hippocampus were 6 9 3 and 8 9 3 times that in the term placenta, respectively. These studies demonstrate that the ovine placenta expresses the Epo gene from at least 66 days of gestation. However, gene expression levels are very low compared with those in the liver and kidney, and even the hippocampus.
Porcine erythropoietin receptor: molecular cloning and expression in embryonic and fetal liver
Domestic Animal Endocrinology, 2000
The full coding sequence for porcine erythropoietin receptor (EPOR) was elucidated using reverse transcription polymerase chain reaction (PCR) (rtPCR) and 3Ј and 5Ј rapid amplification of cDNA ends (RACE) procedures. Total RNA collected from Day 30 fetal liver was used as starting material. A 1843 bp sequence was obtained from which could be inferred a 509 amino acid protein which was 79-85% identical to the amino acid sequence of erythropoietin receptor from other species. Total RNA samples collected from white crossbred intact, white crossbred UHO and Meishan gilts on Days 24, 30 and 40 of gestation were subjected to Northern blotting using porcine EPOR cDNA as probe. Results indicated that (1) a major and two minor forms of mRNA are present, (2) fetal liver mRNA concentrations for EPOR are low on Day 24 of gestation and increase dramatically by Day 30 and (3) mRNA concentrations for EPOR tended to be decreased by intrauterine crowding.
Tissue distribution of erythropoietin and erythropoietin receptor in the developing human fetus
Early Human Development, 1998
Objective: Erythropoietin receptors (Epo-R) have been demonstrated on several nonhematopoietic cell types in animal models and in cell culture. Our objective was to determine the tissue distribution and cellular specificity of erythropoietin (Epo) and its receptor in the developing human fetus. Study design: The expression of Epo and Epo-R mRNA was ascertained by RT-PCR for organs ranging in maturity from 5 to 24 weeks postconception. The cellular location of protein immunoreactivity was then determined using specific antiEpo and antiEpo-R antibodies. Antibody specificity was established by Western analysis. Results: mRNA for Epo and Epo-R was found in all organs in the first two trimesters. Immunolocalization of Epo was limited to the liver parenchymal cells, kidney interstitial cells and proximal tubules, neural retina of the eye, and adrenal cortex. As development progressed, immunoreactivity in the kidney became more prominent. In contrast, immunoreactivity for Epo-R was widespread throughout the body, in cell types including endothelial cells, myocardiocytes, macrophages, retinal cells, cells of the adrenal cortex and medulla, as well as in small bowel, spleen, liver, kidney, and lung. Conclusions: The distribution of Epo and its receptor is more widespread in the developing human than was initially postulated. Epo-R is expressed on many cell types during early fetal development, leading us to speculate that Epo acts in concert with somatic growth and development factors during this period. Further investigation is required to understand the nonhematopoietic role of Epo during human development.
Responses of plasma Epo and kidney and liver Epo mRNA to hemorrhage in perinatal pigs
Domestic Animal Endocrinology, 2002
Despite the fact that pig fetuses in late gestation have extensive erythropoiesis, low blood pO 2 and low hemoglobin concentrations, piglets are born without detectable concentrations of plasma erythropoietin (Epo). In the present study, we have examined the hypothesis that long-term hypoxic stimuli are less efficient than short-term stimuli in stimulating Epo production in perinatal pigs. From fetuses collected by hysterectomy 5 days before term, new-born piglets and piglets 2 and 5 weeks old, blood in amounts corresponding to 2% of body weight was withdrawn from the jugular vein. Twenty-four hours later the animals were killed and their kidney and liver Epo mRNA analysed by a competitive RT-PCR assay. Plasma Epo concentration was estimated by a solid-phase, two-site sequential chemiluminescent enzyme immunometric assay. We found that in nearly fully developed fetuses and in new-born piglets, the concentration of Epo mRNA did not increase upon bleeding. This is in contrast to earlier findings in sheep. In 2-and 5-week-old piglets, bleeding was associated with a 12-15-fold increase in kidney Epo mRNA. In the 2-and 5-week-old piglets, bleeding evoked increased translation of Epo mRNA into the protein hormone. Also in new-born piglets, increased plasma levels of Epo accompanied bleeding, whereas significant changes in gene Epo expression were not observed.
The porcine erythropoietin gene: cDNA sequence, genomic sequence and expression analyses in piglets
Domestic Animal Endocrinology, 2001
The porcine erythropoietin (EPO) gene and its cDNA have been cloned and characterized. The cDNA encodes a protein of 194 amino acids. The gene structure and sequence show a high degree of homology to the corresponding human and murine gene. Steroid hormone receptor binding sites are present both in the promoter and in the 3Ј flanking region of the gene, which also contains an oxygen-sensing sequence. The promoter lacks classical promoter elements such as TATA and CAAT boxes. Expression analyses using a competitive RT-PCR assay showed that the kidneys contain about ten times more erythropoietin mRNA than the liver in five-week-old piglets, thus indicating that the shift from mainly hepatic to mainly renal EPO production has taken place at this age. The testes showed a higher ratio of EPO mRNA to total RNA than the liver. Spleen showed very low levels of expression, while no expression of erythropoietin mRNA was detected in brain tissue, bone marrow, lung, lymph nodes, and ovaries.
Erythropoietin gene expression in fetal and adult sheep kidney
British Journal of Haematology, 1995
The exact location of the cells which express erythropoietin (Epo) in the kidney is still controversial, with conflicting reports suggesting that both peritubular interstitial cells or proximal tubular epithelial cells are possible sites of Epo expression. In the present study we have examined the location of Epo-expressing cells in fetal and adult sheep kidneys after Epo expression was stimulated by anaemia. In situ hybridization histochemistry was performed using synthetic oligonucleotide probes complementary to part of the sheep Epo cDNA sequence or a riboprobe (cRNA) for ovine Epo of 520 bases. Epo expression was confined to peritubular cells of the kidney cortex, in general in the area close to the cortico-medullary junction. In some severely anaemic adult sheep kidneys, Epo-expressing cells were also found in the outer cortex. In addition we located Epoexpressing cells in the kidneys not only of anaemic fetuses (89-140d of gestation, term = 150d) but also in kidneys from normal fetuses 60-110d of gestation. Again, Epo expression was seen only in peritubular cells of the kidney cortex. These findings confirm that the kidney is an important site of Epo production, in the sheep, from at least 0.4 gestation, but also show that there is no ontogenic change in the cellular site of production within the kidney.
Erythropoietin (EPO) is the major hormone that controls the erythropoiesis and tissue oxygenation. EPO has been getting interest to the treatment of anemias also during pregnancy. Erythropoietin con not pass through the placenta; however its receptors are present in placenta especially on trophoblasts. Endogen EPO level can increase under various pathological conditions of pregnancy, in the present study we aimed to test the effects of EPO treatment on fetal-placental development during rat pregnancy period. We used 16 SD rats that have never been pregnant before. Rats were divided into control (vehicle treated) and EPO (100 IU/kg) treated groups (n=8/group). Vaginal smear method was used for diagnosis of pregnancy. During the pregnancy; 3 days/week 100 i.u. /kg EPO was injected s.c, pregnancy was completed with cessarian section at the 21th day, fetuses, placentas and blood samples were taken for measurements. Both fetus length and weights decreased in EPO group. In pathological ex...
Biology of Reproduction, 1998
In this study we investigated erythropoiesis and fetal liver protein secretion during late embryonic (Day 24 and Day 30) and early fetal (Day 40) development in pigs from domestic white crossbred (WC) gilts with a normal (intact; INT) or crowded (unilateral hysterectomized/ovariectomized; UHO) uterine environment, or from prolific Chinese Meishan (MS) gilts. Increased fetal weight, fetal liver weight, placental weight, total red blood cells, hematocrit, blood hemoglobin content, and maternal plasma erythropoietin (EPO) levels were observed as gestation advanced. Cultured fetal liver secretion of transferrin and a protein of M, 12 500 and p 7.5 also increased as gestation advanced. Fetal plasma EPO declined between Day 30 and Day 40. Differential counts of circulating erythroid precursors revealed a decline in basophilic erythroblasts and polychromatic erythroblasts between Day 24 and Day 40, an increase in orthochromatic erythroblasts on Day 30 followed by a drop on Day 40, and an increase in the percentage of reticulocytes/ erythrocytes from < 1.0% to approximately 90% of circulating red blood cells between Day 24 and Day 40. Differences among the treatment groups included a lower fetal survival percentage in UHO (vs. INT or MS) on Day 40, and higher maternal hematocrits, fetal weights, fetal hematocrits, fetal EPO levels, and liver transferrin secretion in WC vs. MS pigs. MS pigs had a lower percentage of polychromatic erythroblasts overall and a higher percentage of orthochromatic erythroblasts on Day 24 followed by a higher percentage of erythrocytes on Day 40 than WC pigs, suggesting a more mature erythron (circulating red blood cells plus erythropoietic tissue) in the MS pigs. Covariate analysis indicated that MS had larger placentae per unit of body weight than did WC. Conclusions were that 1) Days 24-40 of gestation is a critical time for fetal erythropoiesis in pigs as well as survival in a crowded uterine environment, 2) the MS breed may differ in the development of the fetal erythropoietic system because of altered fetal or uterine physiology, and 3) the UHO procedure did not significantly affect erythropoiesis in the fetuses studied but did alter fetal survival and the relationship between fetal weight and both hematocrit and hemoglobin on Day 40.
Effect of hemorrhage and nephrectomy on erythropoietin gene expression in the ovine fetus
Molecular and Cellular Endocrinology, 1996
The purposes of this study were to determine whether (1) erythropoietin (Epo) gene expression could be stimulated, by severe hemorrhage, in both kidney and liver, at three stages of gestation (75-80, 106-l 12. and 140-142 days; term z 150 days) in chronically-cannulated ovine fetuses and (2) whether the liver would compensate for the lack of kidneys at I IO days. Blood was removed (20% of estimated blood volume) at each of 0, I. 19 h, and tissues collected at 24 h. In hemorrhaged fetuses (H) the liver mRNA levels were 2.7-, 5-and 3-times that of control fetuses (C) at 78, 110 and 141 days, respectively. The kidney H:C ratios, for Epo mRNA. were 5. 6.4 and 43. respectively, at these three stages of gestation. In six fetuses at 108 days, nephrectomized 5-7 days before hemorrhage (HN) Epo mRNA increased 5-fold in the liver, and plasma Epo values were significantly lower (P < 0.05) than in intact (H). Thus hemorrhage stimulates increased Epo gene expression in both liver and kidney from mid-gestation. in the ovine fetus. but the liver does not compensate for the lack of kidneys at 110 days. ~S~~~w*orrl\: Erythropoietin: mRNA; Hemorrhage: Competitive RT-PCR (fetal sheep) 0303-7207'96,'$15.00 <s 1996 Elsevier Science Ireland Ltd. All rights reserved SSDl 0303-7207(95)03736-Q
Erythropoietin (Epo) and EpoR expression and 2 waves of erythropoiesis
Blood, 2001
Erythropoiesis occurs in 2 distinct waves during embryogenesis: the primitive wave in the extra-embryonic yolk sac (YS) followed by the definitive wave in the fetal liver and spleen. Even though progenitors for both cell types are present in the YS blood islands, only primitive cells are formed in the YS during early embryogenesis. In this study, it is proposed that erythropoietin (Epo) expression and the resultant EpoR activation regulate the timing of the definitive wave. First, it was demonstrated that Epo and EpoR gene expressions are temporally and spatially segregated: though EpoR is expressed early (embryonic days 8.0-9.5) in the yolk sac blood islands, no Epo expression can be detected in this extra-embryonic tissue. Only at a later stage can Epo expression be detected intra-embryonically, and the onset of Epo expression correlates with the initiation of definitive erythropoiesis. It was further demonstrated that the activation of the EpoR signaling pathway by knocking-in a ...