73 PROTEOMIC ANALYSIS OF UTERINE LUMINAL FLUID ON DAY 7 OF PREGNANCY IN CATTLE

Abstract

In vitro fertilization studies have demonstrated that contact with the female reproductive tract is not necessary in order for the embryo to reach the hatched blastocyst stage. Furthermore, the endometrial transcriptome does not differ between cyclic and pregnant cattle before maternal recognition around Days 15 to 16, suggesting that before that time the cow does not know she is pregnant. Nonetheless, embryo quality is significantly improved by exposure to reproductive tract secretions. Therefore, the aims of this study were to (1) characterise the protein content of uterine luminal fluid (ULF) on Day 7 of pregnancy and the oestrous cycle; (2) identify if differences in ULF composition exist between pregnant and cyclic heifers on Day 7; and (3) determine if blastocyst-derived proteins contribute to ULF composition. Following oestrus synchronization, heifers observed in standing oestrus were randomly assigned to either inseminated (AI: n = 12) or cyclic control (C: n = 6) groups. At slaughter, the uterine horn ipsilateral to the corpus luteum was flushed with 10 mL of PBS. In the AI group, the presence of an appropriately developed embryo was noted (n = 8), and the ULF of both confirmed pregnant and cyclic heifers was clarified by centrifugation at 1000 × g for 15 min at 4°C. Blastocyst-conditioned medium was produced by culturing in vitro-produced blastocysts (n = 300) for 24 h in synthetic oviduct fluid (SOF; groups of 50 blastocysts in 500 µL of SOF). After 24 h the conditioned medium (n = 6 pools), along with contemporaneous blanks (n = 6), was collected and snap frozen. Proteomic analysis of ULF as well as blastocyst-conditioned medium was carried out by nano-LC tandem mass spectrometry (nano-LC-MS/MS). A total of 661 proteins were identified in the ULF of heifers on Day 7 with >95% confidence in at least 4 out of the 6 animals analysed per group. The most abundant proteins were myosin-9, cytoplasmic dynein 1 heavy chain 1-like, fatty acid synthase isoform X1, serum albumin, and aminopeptidase N. No differences were detected between pregnant and cyclic ULF. Of the proteins identified, the biological processes of gene expression (23.8%), small molecule metabolic process (22.6%), and cellular protein metabolic process (17.8%) were most overrepresented. Interestingly, 400 proteins were associated with extracellular exosome as well as extracellular space (87 proteins) and extracellular region (58). Analysis of blastocyst-conditioned medium identified 11 proteins that were not detected in the contemporaneous blanks, of which one (α-1-acid glycoprotein) was also detected in Day 7 ULF. In conclusion, no differences in the ULF composition between pregnant and cyclic heifers were observed on Day 7, consistent with studies on the endometrial transcriptome. A large proportion of the proteins detected are associated with the extracellular space region and may represent candidate proteins that can be added to embryo culture media to enhance early development.

Research was funded by Science Foundation Ireland (13/IA/1983).