BioProject

Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. More...

Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern pre-implantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil (CO) vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many of the differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of pre-implantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and pre-implantation blastocyst growth and development in sheep. Overall design: Ewes were mated at estrus to intact Suffolk rams and then assigned randomly to receive daily intramuscular (i.m.) injections from days 1.5 to 9 of either corn oil vehicle (CO) or 25 mg progesterone (P4) (Sigma Chemical Co., St. Louis, MO) in CO vehicle, and hysterectomized on either day 9 or day 12 of pregnancy (n=5 per day and treatment). Microarray hybridization was conducted with a bovine whole genome spotted oligonucleotide array. This 24,000-element array was developed by the Bovine Oligo Microarray Consortium at the University of Missouri in collaboration with Texas A&M University and Iowa State University. The array includes 16,846 probes designed from expressed sequence tags (ESTs) that were aligned to homologous vertebrate proteins and to the 6X bovine genome assembly. The probe set was supplemented with oligos designed from 703 predicted RefSeq genes, and 5943 reproductive tissue ESTs. A link to probe sequences and annotations is available at http://www.bovineoligo.org. The annotation included assigning a human gene symbol based on homology using BLAST. The oligos were synthesized by Illumina and spotted onto aminosilane-coated glass slides by Dr. David Galbraith's laboratory at the University of Arizona (http://www.ag.arizona.edu/microarray/BOM.html). Hybridizations were performed with the Genisphere Array 350 kit (Genisphere Inc., Hatfield, PA) using dual channel design with dye-swapping. Preparation of cDNA was performed according to the manufacturer's instructions. Briefly, endometrial total RNA (9 µg) from each ewe (n=5 per day and treatment) was reverse-transcribed with Superscript II (Invitrogen, Carlsbad, CA) with the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labeling reagents. Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was co-hybridized with a cDNA sample from the opposite treatment for direct comparison of differences between treatments. Less...