Global proteomic characterization of uterine histotroph recovered from beef heifers yielding good quality and degenerate day 7 embryos

Abstract

16The objective was to analyse the proteomic composition of uterine flushes collected from 17 beef heifers on Day 7 post-insemination. Oestrus was synchronised in cross-bred beef heifers 18 using a Controlled Intravaginal Drug Releasing device (CIDR) protocol. Heifers detected in 19 standing oestrus (within 24-48 h post CIDR removal) were inseminated (oestrus=Day 0) with 20 frozen-thawed semen from a single ejaculate of a bull with proven fertility. Heifers from 21 which an embryo was recovered (following slaughter on Day 7) were classified as either 22 having a viable embryo (morula/blastocyst stage) or a degenerate embryo (arrested at the 2-23 to 16-cell stage). The overall recovery rate (viable and degenerate combined) was 64%. 24Global LC-MS/MS proteomic analysis of the histotroph collected identified 40 high 25 confidence proteins present on Day 7; 26 proteins in the viable group, 10 in the degenerate 26 group and four shared between both groups. Five proteins (Platelet-activating factor 27 acetylhydrolase IB subunit gamma (PAFAH1B3), Tubulin alpha-1D chain, Tubulin beta-4A 28 chain, Cytochrome C and Dihydropyrimidinase-related protein-2) were unique or more 29 abundant in the histotroph collected from animals with a viable embryo and one protein 30 (S100A4) was more abundant in the histotroph collected from animals with a degenerate 31 embryo. Of interest, PAFAH1B3, detected only in histotroph from the group yielding viable 32 embryos, belongs to the group of platelet activating factors that are known to be important for 33 the development of the pre-implantation embryo in other species. To our knowledge this is 34 the first report of PAFAH1B3 in relation to bovine early embryonic development. 35 36

S100 protein family and embryo implantation

“…After collecting the samples and identifying the proteins, it was found that in the degenerate embryo group, it was only one protein which had high expression in comparison with the normal group, and it was S100a4 protein. Consequently increasing the frequency of S100a4 in the degenerative embryo group indicates a negative effect of S100a4 on early embryo development …”

Proteomics of the periconception milieu

“…It is interesting to note that the majority of studies conducted to understand the global holistic uterine proteome in large animals have been performed in the last 5 years . Cows seem to have been the species of choice in these investigations . The sample most used for the global analysis of the uterine proteome has been based on the uterine fluid or flushings of uterus .…”

“…Cows seem to have been the species of choice in these investigations . The sample most used for the global analysis of the uterine proteome has been based on the uterine fluid or flushings of uterus . Nearly all these studies are concerned with defining, characterising and identifying the proteins present in the uterine fluid or tissue at different stages of the reproductive cycle or during the early stages of establishment of pregnancy.…”

Effect of endometrial biopsy on uterine health in tropically adapted beef cattle

“…These invasive methods allow the collection of epithelial and inflammatory cells (CB, CT, UL), luminal secretions (UL) and uterine tissue (EB). The samples obtained can be used to accurately diagnose the uterine status by using a single or combined assessment with cytological smears (Kasimanickam et al, 2004, Pascottini et al, 2015, bacteriological culture (Bonnett et al, 1991a), protein analysis (Beltman et al, 2014), histopathology (Chapwanya et al, 2010), and gene expression (Munoz et al, 2012) These tests are better indicators of uterine health than the subjective non-invasive methods. Here I describe three common approaches with different degrees of invasiveness which I used experimentally.…”

“…However, the yield of endometrial cells recovered from uterine fluid is less and this affects subsequent cytological analysis and this is worse than in samples recovered from CB (Kasimanickam et al, 2005b, Barlund et al, 2008, Cocchia et al, 2012. As mentioned before, the composition of UF is a complex mixture of molecules and substances derived from genes expressed in the endometrium or transported from maternal blood (Beltman et al, 2014). The proteome profile of the histotroph has been widely studied in cattle.…”