Uterine expression of mucin1 in a line of sheep displaying reduced embryo survival

During the first 30 days of pregnancy, 20-25 % of embryos from ewes with multiple ovulations are lost. This represents a major economic loss to the sheep industry as the affected ewes remain pregnant but have a reduced litter size. Within the AgResearch Booroola flock we have observed differences between two half sibling rams which produce either a reduced embryo survival (RES) or normal embryo survival (NES) phenotype in their daughters. These ewes provide a unique resource for studying the underlying physiology of this reduced embryo survival trait. In this Booroola line of sheep, embryo loss occurs primarily before day 30 of gestation and the uterus rather than the embryo appears to be the major determinant of embryo survival. Consequently this study has focused on the interactions between the uterus and the embryo during the time of implantation. One factor that has been identified as being involved in successful implantation is mucin1 (MUC1). MUC1 is a large transmembrane protein reported to be expressed by the luminal and glandular epithelial cells of the uterus in various species, where it is believed to inhibit the interaction between the trophoblast and the luminal epithelium, and block implantation. It is hypothesised that the presence of an embryo initiates a cascade of events resulting in the down regulation of MUC1, thereby allowing adhesion to occur. Embryos and reproductive tissue were collected from the daughters of the RES sire (n=10) and from the NES sire (n=5) at day 18 of pregnancy. This study utilised Western blotting, immunohistochemistry and in situ hybridisation to evaluate MUC1 mRNA and protein expression. The antibody used was a polyclonal mouse anti-rabbit MUC-CT1 antibody which corresponds to the highly conserved region within the cytoplasmic tail domain of the protein. For in situ hybridisation, sense and anti-sense probes were generated from ovine cDNA encoding a 278 bp fragment corresponding to the cytoplasmic tail region. Western blotting analysis revealed that MUC1 exists as a single 70 kDa band under reducing conditions in ovine uterine tissue. This expression pattern is similar to MUC1 protein expression observed in day 19 pregnant mouse uterine tissue. The doublet (19-23 kDa) often seen in virgin mouse uterine tissue was undetectable in ovine uterine tissue. Specificity of the ovine 70 kDa band was confirmed by blocking with the MUC-CT1 peptide. Semi-quantitative analysis by Western blotting did not reveal any differences in protein levels between the RES and NES groups. Both MUC1 mRNA and protein, as evaluated by in situ hybridisation and immunohistochemistry, showed expression in the luminal epithelial cells, and strong expression in the glandular epithelial cells of the compactum stroma, with a weaker signal observed in the glandular epithelial cells of the spongiosum stroma. There were no differences observed in the expression patterns of both MUC1 mRNA and protein between the RES and NES groups. Blood samples were collected daily from day of mating to day 18 of pregnancy to measure progesterone concentrations by radioimmunoassay (RIA). As progesterone is considered to modulate MUC1 via the progesterone receptors it was hypothesised that there could be a detectable difference in progesterone concentrations if MUC1 expression levels were different between the RES and NES groups. Analysis revealed no difference in progesterone concentrations between the RES and NES groups throughout the first 18 days of gestation. MUC1 mRNA and protein was detected in endometrial tissue in both the RES and NES groups in the Booroola line of sheep, however no discernable differences in expression levels were found. Animal numbers available for the study were low and consequently results were inconclusive as to whether MUC1 plays a significant role in the partial loss of embryos seen in these animals