title: Regulation of gene expression in the bovine blastocyst by colony stimulating factor 2.
abstract: BACKGROUND
  Colony stimulating factor 2 can have multiple effects on the function of the preimplantation embryo that include increased potential to develop to the blastocyst stage, reduced apoptosis, and enhanced ability of inner cell mass (ICM) to remain pluripotent after culture. The objective of the current experiment was to identify genes regulated by CSF2 in the ICM and trophectoderm (TE) of the bovine blastocyst with the goal of identifying possible molecular pathways by which CSF2 increases developmental competence for survival. Embryos were produced in vitro and cultured from Day 6 to 8 in serum-free medium containing 10 ng/ml recombinant bovine CSF2 or vehicle. Blastocysts were harvested at Day 8 and ICM separated from TE by magnetic-activated cell sorting. RNA was purified and used to prepare amplified cDNA, which was then subjected to high-throughput sequencing using the SOLiD 4.0 system. Three pools of amplified cDNA were analyzed per treatment.
  
  
  RESULTS
  The number of genes whose expression was regulated by CSF2, using P < 0.05 and >1.5-fold difference as cut-offs, was 945 in the ICM (242 upregulated by CSF2 and 703 downregulated) and 886 in the TE (401 upregulated by CSF2 and 485 downregulated). Only 49 genes were regulated in a similar manner by CSF2 in both cell types. The three significant annotation clusters in which genes regulated by ICM were overrepresented were related to membrane signaling. Genes downregulated by CSF2 in ICM were overrepresented in several pathways including those for ERK and AKT signaling. The only significant annotation cluster containing an overrepresentation of genes regulated by CSF2 in TE was for secreted or extracellular proteins. In addition, genes downregulated in TE were overrepresented in TGFβ and Nanog pathways.
  
  
  CONCLUSIONS
  Differentiation of the blastocyst is such that, by Day 8 after fertilization, the ICM and TE respond differently to CSF2. Analysis of the genes regulated by CSF2 in ICM and TE are suggestive that CSF2 reinforces developmental fate and function of both cell lineages.
authors.affiliation: Dept. of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, PO Box 110910, Gainesville, FL, 32611-0910, USA.|Laboratory of Developmental Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Ozawa
authors.affiliation: Kyushu-Okinawa Agricultural Research Center, National Agriculture and Food Research Organization, Kumamoto, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Sakatani
authors.affiliation: Dept. of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, PO Box 110910, Gainesville, FL, 32611-0910, USA.|Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA, 92083, USA.
authors.forename: Kyle B
authors.initials: KB
authors.lastname: Dobbs
authors.affiliation: Dept. of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, PO Box 110910, Gainesville, FL, 32611-0910, USA.
authors.forename: Jasmine
authors.initials: J
authors.lastname: Kannampuzha-Francis
authors.affiliation: Dept. of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, PO Box 110910, Gainesville, FL, 32611-0910, USA. Hansen@animal.ufl.edu.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hansen
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D019070:Cell Lineage / Q000187:drug effects / Q000235:genetics; D046149:Embryo Culture Techniques / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D013997:Time Factors
chemical_list: D016178:Granulocyte-Macrophage Colony-Stimulating Factor
keywords: Blastocyst; Bovine; Colony stimulating factor-2; Inner cell mass; Transcriptome; Trophectoderm