146 CONSEQUENCES OF EMBRYONIC EXPOSURE TO COLONY-STIMULATING FACTOR 2 ON TROPHOBLAST ELONGATION, INTERFERON TAU SECRETION, AND GENE EXPRESSION IN THE EMBRYONIC DISC AND TROPHECTODERM

Abstract

This study was designed to evaluate possible mechanisms by which colony-stimulating factor 2 (CSF2) acts during Day 5 to 7 of development to improve embryonic and fetal survival and decrease fetal losses. One hypothesis was that CSF2 causes increased secretion of interferon-τ (IFNT) by the trophoblast of elongated conceptuses. Colony-stimulating factor 2 might also affect elongation of the trophoblast and improve survival of the embryonic disc (ED) because of evidence that treatment of embryo with CSF2 caused differential regulation of a large number of genes involved in developmental processes and a decrease in apoptosis. Bovine embryos were produced in vitro in the presence or absence of 10 ng mL^–1 of CSF2 beginning at Day 5 after fertilization. Embryos were transferred at Day 7 to lactating dairy cows using a timed embryo transfer protocol (n = 20 and 15 for control and CSF2, respectively). At Day 15, embryos were recovered and assessed for length, stage, and presence of an ED. The flushings recovered from the first 60 mL were stored for antiviral activity analysis. A total of 4 filamentous conceptuses were divided into 1) the ED and a small amount of adjacent trophoblast and 2) tissue containing trophoblast only. These tissues were analysed for gene expression using the Bos taurus Two Colour Microarray Chip from Agilent (Palo Alto, CA). Quantitative real time PCR analysis of 11 differentially expressed genes and 1 housekeeping gene (GAPDH) was performed to confirm the microarray results. In addition, quantitative real-time PCR of IFNT was performed in all recovered embryos (7 and 10 for control and CSF2, respectively). Results suggest that higher pregnancy rates at Day 30 represents increased embryonic survival at Day 15 (35% for controls v. 66% for CSF2; P < 0.07), a greater capacity of the embryo to elongate (39 mm for controls v. 62 mm for CSF2) and secrete IFNT (mean IFNT IU = 349 074 for control and 1 883 060 for CSF2; P < 0.07) at Day 15. In addition, the amount of IFNT mRNA was 22 times higher (P = 0.06) in the CSF2-treated embryos. There was no difference in the presence of an ED between groups. Analysis of gene expression between the ED and trophoblast in filamentous embryos indicated no difference in transcription among this subset of embryos. However, more than 500 genes were identified as being preferentially expressed in the ED. These genes represent candidate markers for ED that should prove useful for studying the differentiation of the bovine conceptus through the periattachment period. In conclusion, results support the idea that the increased survival of embryos exposed to CSF2 from Day 5 to 7 of development is the result of increased embryonic survival before Day 15 and a greater capacity of the embryo to elongate and secrete IFNT at Day 15. The reduction in embryonic and fetal loss after Day 30 caused by CSF2 is probably not a direct reflection of altered gene expression at Day 15.