Glycosylation dependent cell adhesion molecule 1-like protein and L-selectin expression in sheep interplacentomal and placentomal endometrium.

Glycosylation dependent cell adhesion molecule 1 (GlyCAM-1), a mucin component of sheep histotroph produced by glandular epithelium (GE) during early pregnancy, is hypothesized to function in implantation. However, GlyCAM-1 is present in uterine tissues subsequent to implantation suggesting additional functions of this l-selectin-binding ligand. This study focused on uterine GlyCAM-1 expression during placentome development in sheep. Western blot analysis of day 50 pregnant sheep identified 45, 40, and 25 kDa bands in interplacentomal endometrium, 40 and 25 kDa bands in placentomes, and 80 and 40 kDa bands in chorioallantois. The GlyCAM-1 proteins in interplacentomal regions were comparable to those detected in day 15-19 pregnant sheep, however, the 80 kDa form was unique to chorioallantois, and the absence of the 45 kDa GlyCAM-1 in placentomes indicated differences between interplacentomal and placentomal endometrium. Immunofluorescence identified GlyCAM-1 in lumenal epithelium (LE), stromal fibroblasts, and vascular smooth muscle cells. To better define its cellular distribution, GlyCAM-1 was co-localized with either epithelium-specific cytokeratin, smooth muscle-specific alpha-smooth muscle actin (alpha SMA), or stromal-specific vimentin. In interplacentomal endometrium, GlyCAM-1 co-localized with cytokeratin in LE but not in GE. GlyCAM-1 did not co-localize with alpha SMA, and was localized in the extracellular matrix of vimentin-positive stroma. In placentomes, GlyCAM-1 did not co-localize with cytokeratin, but did co-localize with alpha SMA and vimentin. Thus, in contrast to interplacentomal regions, GlyCAM-1 in placentomes was predominantly localized in vasculature rather than epithelial cells. Further, leukocytes expressing L-selectin were localized to the endothelial surface of GlyCAM-1-expressing vessels within placentomes. These data suggest that GlyCAM-1 assumes distinct functions in compartment-specific regions of the sheep uterus.

Interferon stimulated gene 15 conjugates to endometrial cytosolic proteins and is expressed at the uterine-placental interface throughout pregnancy in sheep.

Interferon-stimulated gene 15 (ISG15) is a ubiquitin homolog expressed in uteri of ruminants in response to interferon (IFN)-tau and is also induced during pregnancy in the uteri of mice, pigs, humans, and baboons. This study examined expression of ISG15 and its conjugation to target proteins in the ovine uterus beyond the period of IFNtau secretion by the conceptus. Although steady-state levels of ISG15 mRNA decreased after d 25 of pregnancy, ISG15 persisted in endometrium through d 120. In situ hybridization and immunocytochemistry localized ISG15 across the entire uterine wall through d 25, after which expression was restricted to endometrial stroma along the maternal-placental interface. Western blots revealed ISG15 and ISG15-conjugated proteins in endometrium. Treatment of ovariectomized sheep with progesterone and IFNtau increased both free and conjugated ISG15. These results are the first to show in vivo regulation of ISG15 function (i.e. conjugation to target proteins) by a type I IFN in the uterus of any species and that ISG15 is expressed at contacts between the placenta and uterus when trophectoderm no longer produces IFNtau. Interestingly, mRNA for the type II IFNgamma was present in the endometrial stromal compartment on d 15-50, which may stimulate the synthesis of ISG15 through later pregnancy. We hypothesize that ISG15 is not merely a consequence of an antiviral state induced by trophoblast IFNtau but represents a critical component of the microenvironment at the uterine-placental interface during the progressive events of conceptus development, implantation, and placentation in sheep and perhaps other mammalian species.