Pregnancy-induced changes in metabolome and proteome in ovine uterine flushings

Monday, July 21, 2014: 11:00 AM
2103C (Kansas City Convention Center)
Thomas R Hansen , Colorado State University, Fort Collins, CO
Jared J Romero , Colorado State University, Fort Collins, CO
Corey Broeckling , Colorado State University, Fort Collins, CO
Jessica E Prenni , Colorado State University, Fort Collins, CO
Abstract Text:  

The endometrium serves as a primitive placenta by secreting histotroph, which nourishes the developing conceptus (embryo proper and extraembryonic membranes). Modern mass spectroscopy (MS) approaches allow for global analysis of proteins and metabolites in bodily fluids.  We hypothesized that global MS can identify metabolites and proteins that are induced by pregnancy in uterine flushings.  To test this hypothesis, uteri were collected on day 12 of the estrous cycle (n = 5 ewes not exposed to ram) or days 12 (n = 4), 14 (n = 5) or 16 (n = 5) of pregnancy (confirmed by presence of conceptus) and flushed using physiological buffered saline. Pregnancy status and Day were main effects analyzed by GLM-SAS. Metabolites were extracted from uterine flushings using 80% methanol and profiled using UPLC-MS.  The proteome was examined by digestion with trypsin, followed by analysis of peptides with LC-MS/MS.  Metabolite profiling resulted in the detection of 8,510 molecular features, of which 5 were confirmed to be upregulated (>3 fold and P < 0.05) in response to pregnancy by days 14-16 and were not detected on day 12:  acetylcarnitine, carnitine, ecdysteroids, N-acetyl dileucine and valine. These metabolites function in fatty acid transport (carnitines), anti-apoptotic mechanisms (ecdysteroids) and availability of nutrients (amino acids).  Proteome analysis resulted in the detection of 783 proteins that were differentially regulated (P < 0.05) by days 14-16 of pregnancy, 7 of which are described herein:  annexin A1, A2 and A5; calcium binding protein (S100A11); profilin 1, trophoblast kunitz domain protein 1 (TKDP) and interferon tau (IFNT).  These proteins have unique functions in mediating endocytosis, exocytosis, calcium signaling and inhibition of prostaglandins (annexins and associated S100A11); protecting against maternal proteases (TKDP); remodeling cytoskeleton (profilin 1); and altering uterine release of prostaglandin F2 alpha as well as inducing interferon stimulated genes in the endometrium and the corpus luteum (IFNT).  It is concluded that global MS approaches are poweful in delineating the metabolome and proteome in uterine flushings and identifying differential expression in response to the conceptus by days 14-16 of pregnancy. USDA NIFA AFRI 2011-67015-20067.


Pregnancy, Uterus, Conceptus, Proteome, Metabolome