|Abstract or Summary
- This research investigated gene expression in the canine placenta during the peripartum period. Previous studies have recognized molecular changes that occur in the placenta around the time of placental release in other species, but no study has looked at gene expression in the late gestation canine placenta. Of particular significance for this thesis work is the groundwork laid for future studies modeling placental abnormalities in dogs (e.g. subinvolution of placental sites) and humans (e.g. preeclampsia, placenta accreta). Despite years of research in multiple species, the exact mechanisms and processes regulating trophoblast invasion and placental release remain unclear. Therefore, the specific objective of this research was to characterize gene expression changes that occur during the peripartum period in the dog using microarray and real-time RT-PCR. Following total RNA isolation, the microarray analysis was performed by hybridizing total RNA to the Canine 2.0 Array (Affymetrix, Santa Clara, CA). Microarray analysis was carried out using the limma and affy packages through the Bioconductor software in the R statistical environment. Differential expression was defined as p ≤ 0.05, FDR p ≤ 0.10 and a log fold change of ≤ 1.2. Following cDNA synthesis, real-time RT-PCR was performed using TaqMan primer and probes that were pre-made and pre-optimized for canine tissues (Applied Biosystems, Carlsbad, CA). Microarray analysis showed differential expression in 18 genes between pre-term and pre-labor sample groups, 38 genes that were differentially expressed between pre-term and parturient samples and no genes that were differentially expressed between pre-labor and parturient samples. Microarray analysis led to the identification of several candidate genes for closer investigation using real-time RT-PCR. These genes included MMP-1, MMP-2, MMP-9, TIMP-2, VEGF-A, Flt-1, CD44, DAG-1, IL-6 and CXCL10. All of these genes have been linked to trophoblast invasion or regression or placental release in a number of species including humans, cattle and rodents. Using real-time RT-PCR, there was a significant difference in MMP-9 mRNA expression in pre-term samples compared to pre- labor and parturient samples (p<0.05). However, there was no significant difference in mRNA expression of MMP-2, TIMP-2, VEGF-A, Flt-1 CD44, DAG-1, IL-6 or CXCL10. Future studies may focus on additional candidate genes identified by microarray that play a role in tissue remodeling at the end of canine gestation such as IL-8, EPHX2, PI3 and SERPINE1.