Abstract:
Macrophage colony-stimulating factor (M-CSF) is a hemopoietic cytokine that plays a primary role in placental physiology. Gene expression of M-CSF in bovine intercaruncular endometrium shows an upward trend in mid-pregnancy. The objective of this study was to determine the plasma M-CSF levels and the M-CSF gene expression levels in maternal peripheral white blood cells (PWBCs) during pregnancy using ELISA and quantitative RT-PCR. In Experiment 1, the plasma M-CSF levels in 112 Japanese Black heifers or cows were determined. Animals were divided into four groups according to pregnancy stage: first- (n = 29), second- (n = 33), third- (n = 26) trimester, and non-pregnant (n = 24). ELISA for bovine M-CSF established by Yoshihara et al. (2003 Vet. Immunol. Immunopathol. 95, 103?111) was used according to their instructions. The absorbance was measured at 405 nm in the Biomek Plate Reader (Beckman Coulter, Fullerton, CA, USA). In Experiment 2, the plasma M-CSF levels and M-CSF gene expression levels in PWBCs during pregnancy were determined. The plasma samples for ELISA were obtained from 8 heifers and 3 cows every 1 and/or 2 weeks. The PWBCs samples for quantitative RT-PCR were obtained from 4 heifers every 1 and/or 4 weeks. All quantitative RT-PCR protocols were carried out according to the previous report (Oshima et al. 2003 Theriogenology 60, 1217?1226). The quantitative PCR assay used an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA). Signals were detected according to the manufacturer's instructions. The relative level of M-CSF expression was calculated on the basis of glyceraldehyde-phosphate-dehydrogenase (GAPDH) quantity (in the method of calculation, the relative level = M-CSF quantity/GAPDH quantity). Data were analyzed by Kruskal-Wallis test. In Experiment 1, the plasma M-CSF level in second-trimester cows was significantly higher than those in other stages (P < 0.05). In Experiment 2, the plasma M-CSF levels were significantly higher in gestational age from ?4 to 1 weeks compared with the last stage of pregnancy (P < 0.05). The levels decreased until 6 weeks, appeared to temporarily increase, and were relatively constant until 35 weeks. Macrophage colony-stimulating factor genes were expressed in all samples examined; the levels were relatively constant in early pregnancy, and then were widely varied until parturition. These results suggest that plasma M-CSF levels may be related to the maternal condition of pregnancy and to a slight extent to M-CSF gene expression in PWBCs. This work was supported in part by a Grant-in-aid from the Recombinant Cytokine Project (RCP2002-2110), provided by the Ministry of Agriculture, Forestry, and Fisheries, Japan.