244 CHANGES IN THE TRANSCRIPTOME OF THE BOVINE PRE-IMPLANTATION EMBRYO INDUCED BY COLONY-STIMULATING FACTOR-2

Abstract

Colony-stimulating factor-2 (CSF-2) is a cytokine expressed in bovine oviduct and endometrium that has been reported to improve the proportion of embryos that become blastocysts in vitro and survive after transfer to recipients. One effect of CSF-2 that might be related to increased embryonic survival is a preferential increase in the number of cells in the inner cell mass. The objective of the current study was to determine changes in the embryo transcriptome caused by CSF-2 that promote blastocyst formation and establishment and maintenance of pregnancy after transfer. Bovine embryos were produced in vitro and cultured in KSOM-BE2 +10 ng/mL recombinant BoCSF-2 added at Day 5 after insemination. On Day 6 (24 h after treatment), embryos at the morula and early blastocyst stage were harvested and stored in groups of 50 at -80°C. A total of 4 pools of GM-colony-stimulating factor treated blastocysts and 4 control blastocysts were subjected to transcriptional profiling using the Bos taurus 2-color Agilent chip (4 × 44 K format). Before labeling, total RNA starting sample was spiked with control genes (artificial clones) of known concentration provided by Agilent. Labeling was done simultaneously with complimentary RNA (cRNA) amplification. Two rounds of linear RNA amplification were employed. Images were extracted using the Agilent Feature Extraction Software (Agilent Technologies, Santa Clara, CA, USA) and normalized within arrays by the Lowess method. Statistical analysis was performed using the JMP Genomics program (SAS Inst., Cary, NC, USA). The normalized data were log2 transformed, and the quantile normalization method was used to normalize data between arrays. Differences in gene expression were determined using PROC ANOVA (fixed false discovery rate = 0.01). Only genes with a 1.5-fold difference and P < 0.05 were considered differentially expressed. A total of 216 genes were differentially expressed between CSF-2 and control embryos. Of these, 141 could be annotated (61 genes up-regulated and 80 genes down-regulated by CSF-2). These included 13 genes involved in Wnt pathways, including 5 inhibitors of Wnt signaling (FRP, MAB21L2, PCDH24, PDE7, PPPR23A) that were up-regulated by CSF-2 and 5 genes involved in transmission of Wnt signals (WNT16, ROR2, CSNK2B, CELSR2, DTX3) that were down-regulated by CSF-2. Several other genes associated with differentiation were down-regulated by CSF-2 including CXCL12, FEZF1, PLD2, and RGS12. Expression of 1 gene that inhibits apoptosis (PRKAR2B) was increased by CSF-2, whereas expression of 6 genes involved in apoptosis pathways (DAPK1, MADD, NOD2, PIK3IP1, RIPK3, RNF7) were down-regulated. Results indicate that CSF-2 promotes pluripotency and decreases apoptosis in bovine pre-implantation embryos.

This research was supported by USDA-AFRI. B. Loureiro andL. Oliveira were supported by a CAPES (Brazil)/Fulbright Fellowship.