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Profiling equine endometrial gene expression during maternal recognition of pregnancy




Klohonatz, Kristin M., author
Bruemmer, Jason E., advisor
Bouma, Gerrit, committee member
Thomas, Milton, committee member

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In order to maintain a pregnancy in the mare the presence of a conceptus in the uterus must be recognized by the endometrium. This is known as maternal recognition of pregnancy (MRP) and is required to prevent the secretion of prostaglandin F2α (PGF), starting on day 14 post-ovulation, from the endometrium into circulation. The secretion of PGF initiates luteolysis of the corpus luteum, which is secreting progesterone, the hormone needed to maintain a pregnancy. However, little is known about maternal recognition of pregnancy in the mare. It is critical that the embryo is mobile throughout the entire uterine lumen to signal maternal recognition of pregnancy between days 12-14. The embryo ceases mobility on day 16 by fixing at the base of one of the uterine horns, independent of the side of ovulation. Previously, an equine specific microarray analysis was performed on days 14, 16, and 18 post-ovulation comparing endometrial gene expression between pregnant and non-pregnant mares. From this analysis, ten genes: juxtaposed with another zinc finger protein 1-like (JAZF1), secretory phospholipase A2 (sPLA2), S100 calcium binding protein G (S100G), estrogen receptor 1 (ESR1), solute carrier family 36 (proton/amino acid symporter), member 2 (SLC36A2), methyltransferase-like protein 7A-like (METTL7A), retinaldehyde dehydrogenase 1-like (RALDH1), eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3), dickkopf 1 homolog (DKK1), and adrenomedullin (ADM), were identified as having consistently higher or lower expression levels in the endometrium of pregnant mares at all three time points. The goal of this study was to confirm and expand upon the results of the microarray on days 14, 16, and 18, by real time PCR (RT-PCR), and to evaluate differential gene expression on day 12. We hypothesized that the expression of the aforementioned ten genes will be the same on day 12 endometrium from pregnant mares as days 14, 16, and 18 endometrium from pregnant mares because day 12 is the start of maternal recognition of pregnancy. To test this hypothesis, 12 normally cycling mares were utilized in a crossover design. Each mare was assigned to a random collection day (day 12, 14, 16, or 18 post-ovulation) and provided endometrial samples from a pregnant cycle and then a non-pregnant (non-mated) cycle (n=3 per day). Endometrial biopsy samples were snap frozen and stored until total RNA was isolated for RT-PCR. This analysis was consistent with the microarray results for days 14, 16, and 18. On day 12, 6 of the 10 differentially expressed genes had the same pattern of expression as day 14, but 4 of the genes had opposite expression levels on day 12. Endometrial samples were then collected on day 13 post-ovulation (n=3) and processed for protein isolation and immunohistochemical analysis. The specificity of rabbit polyclonal antibodies for sPLA2 and DKK1 for equine endometrium were confirmed by Western Blot analysis. Upon conformation of antibody specificity, immunohistochemistry was used to determine the localization of sPLA2, DKK1, and ESR1. sPLA2 was localized to the endometrial epithelium and glandular epithelium in the endometrium from both pregnant and non-pregnant mares. DKK1 showed a localization difference between endometrial samples from pregnant and non-pregnant mares. In the endometrium from the pregnant mare, DKK1 was localized to the endometrial epithelium and the glandular cells, and in the endometrium from the non-pregnant mare DKK1 was localized throughout the glandular region, but not in the endometrial epithelium. ESR1 also showed differential localization based upon pregnancy status. In the endometrium from the pregnant mare, ESR1 was located in the basal glandular region and not close to the lumen or in the endometrial epithelium. In endometrium from non-pregnant mares it was located throughout the entire glandular region and in the endometrial epithelium. This experiment identified the expression patterns of ten genes, previously identified from a microarray analysis, on days 12, 14, 16, and 18 post-ovulation in the endometrium from pregnant and non-pregnant mares. The expression patterns on days 14, 16, and 18 were consistent across each day. Day 12 revealed mixed results for the expression patterns of these genes, indicating that they were undergoing transcriptional regulation based upon the presence or absence of a mobile conceptus. By determining what signal causes these genes to be higher or lower expressed in the endometrium of pregnant mares, it may lead to the identity of the signal for maternal recognition of pregnancy in the mare.


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maternal recognition of pregnancy


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