Purification of a stage-specific lectin receptor from the sheep uterus

Selected figures from Chapter 4 (4.5-26) have been rescanned at a higher resolution in a separate file.Prenatal mortality in sheep has been estimated to be as high as 30 % of all conceptions with much of the loss occurring before the eighteenth day of gestation. It is possible that the receptive stage of the endometrium to an implanting conceptus, at day 15 and 16 after mating (Day 0), may be a significant period for this embryonic mortality. The uterine endometrial surface is receptive to attachment of the trophoblast for only a short period in early pregnancy, with attachment in the sheep occurring after 15 to 16 days of pregnancy. If uterine receptivity to the conceptus during the first 48 hours of implantation proves to be an important time for occurrence of prenatal mortality it would suggest that cell surface molecules, taking part in cell recognition and adhesion between the epithelia of the conceptus and the endometrium, have a role in such mortality. Although the expression of a number of candidate molecules on the conceptus and endometrial epithelia correlates with receptivity, the actual molecules that participate in and the molecular forces driving this interaction at receptivity are still unknown. To investigate the biochemical changes associated with this window of receptivity, frozen sections of uterus from pregnant or nonpregnant ewes on Days 11, 13, 15, 16, 17 and 18 after mating were cut and stained with the biotinylated lectins Dolichos biflorus (DBA) and Ulex europaeus-I (UEA) agglutinins. Lectins are proteins which bind specifically to appropriate sugar molecules and have been used to histochemically label cell surface glycoconjugates by attaching the lectin to glycoconjugates containing these sugar residues. Many of the molecules reported to be instrumental in cell recognition and adhesion are glycoproteins. DBA and UEA reacted differentially with the endometrial epithelium of pregnant and nonpregnant ewes. Both lectins stained the endometrial epithelium uniformly and strongly throughout, without staining the stroma. DBA stained the epithelium from pregnant ewes up to Day 16, decreased in intensity on Day 17 and was absent on Day 18; DBA did not stain epithelium from nonpregnant ewes over the corresponding days post mating. UEA stained epithelium on all days examined in both pregnant and nonpregnant ewes, except for a complete absence of staining on Days 15 and 16 of pregnancy. There was no difference in staining between caruncular and intercaruncular regions of the uterus. The UEA receptor isolated from the sheep uterus was purified to apparent homogeneity by lectin-affinity chromatography and preparative gel electrophoresis. SDS-PAGE fractionation, isoelectric focusing and lectin staining after electrotransfer (lectin blotting) of the affinity column fractions revealed a diffuse band in both electrophoresis procedures, suggestive of a heavily glycosylated, heterogeneous, glycoprotein. The purified fractions from the preparative gel ran as a series of discrete bands that together formed a continuum of Mᵣ roughly 90 to 130 kDa on SDS-PAGE and an isoelectric point centred at pI 4.5 on IEF gels. Coomassie Blue or silver staining did not reveal any protein bands. Partial characterization suggests the UEA receptor shares physico-chemical affinities with the mucin-like glycoproteins whose expression has been reported to correlate with receptivity in mice and pigs. The fucose determinant on the UEA receptor is a residue shared with the mucin-like ligands to the selectins, two types of glycoproteins mediating leucocyte adhesion in the inflammatory response of mammals. The downregulation in epithelial expression of the UEA receptor's fucose determinant at receptivity and its continued expression in epithelia from nonpregnant ewes over the corresponding days post mating suggests an ovarian steroid control mechanism but steroid influence and the UEA receptor's association with the stage-specific lectin staining of the epithelium, remains to be established. In conclusion, this study has identified a possible key candidate in the embryo-uterine recognition and adhesion process as being a heavily glycosylated, heterogeneous glycoprotein specifically identified by its putative fucose residues that bind temporally to UEA over the days of pregnancy when implantation commences