(169) Cell-type specific changes in the expression of MUC-1 and PAGs during early conceptus attachment for implantation in beef cows

Cell-type specific changes in the expression of MUC-1 and PAGs during early conceptus attachment for implantation in beef cows

Gabriela Melendes Schneider¹; Thainá Minela^1,2; Michael F. Smith³, Julie A. Walker⁴ Thomas W. Geary⁵, Ky G. Pohler¹; George Perry¹; Gregory A. Johnson²

1. Department of Animal Science, Texas A&M University, College Station, TX

2. Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX

3. Department of Animal Sciences, University of Missouri, Columbia, MO

4. Department of Animal Science, South Dakota State University, Brookings, SD 

5. Fort Keogh Livestock and Range Research Laboratory, USDA-ARS, Miles City, MT

Abstract Text:

Understanding of the process of conceptus (embryo and its associated extraembryonic trophectoderm and endoderm) trophectoderm attachment to the endometrium for implantation and placentation is limited for cattle. In sheep, pigs, and mice, mucin 1 (MUC-1), a transmembrane glycoprotein, is downregulated at the apical surface of the endometrial luminal epithelium (LE), and it is proposed that this is an essential first step in conceptus attachment because its extensive glycocalyx structure extending from the LE would otherwise interfere with conceptus attachment through steric hindrance. In cattle and sheep, pregnancy-associated glycoproteins (PAGs) were expressed by trophoblast mononucleated cells (TMCs) and trophoblast giant cells (TGCs) on day 20 of gestation, and some of these PAG-positive cells invade into the endometrial LE during early placentation. The function(s) of PAGs have not been elucidated, but they may have a role in modulating expression of proteins associated with tissue remodeling, chemokine production, and prostaglandin release. Investigation of cell-type specific changes in expression of MUC-1 and PAGs in the conceptus trophectoderm and endoderm and the uterine endometrium should improve overall understanding of early placentation in cows. This study utilized immunofluorescence microscopy to characterize conceptus and endometrial MUC-1 and PAG proteins expression on days 20 and 26 of bovine gestation.

Beef cows (n=12) received artificial insemination (AI) following estrus (n=4), GnRH-induced ovulation (n=4), and GnRH+E₂-induced ovulation (n=4). Slaughter and collection of reproductive tracts were performed on days 20 (D20; n=6) and 26 (D26; n=6) post-AI. Intact cross sections of utero-conceptus interface were dissected, fixed in 4% PFA, embedded in paraffin, and 5µm sections treated with boiling sodium citrate for antigen retrieval. Immunofluorescence microscopy was performed using antibodies directed against MUC-1 and PAGs. Captures were performed using an Axio Imager.M2 microscope.

On D20, MUC-1 was expressed at the apical surface of all endometrial glands and at the apical surface of LE not adjacent to the attaching conceptus trophectoderm. In contrast, MUC-1 was not expressed at the apical surface of the LE at sites of conceptus implantation. MUC-1 was also sporadically localized to the endoderm. By D26, small intermittent extents of the utero-conceptus interface exhibited MUC-1 expression, but surprisingly MUC1 was highly expressed by many of the trophoblast TGCs.

As expected, PAG expression was observed in the trophoblast TGCs and a small subpopulation of trophoblast TMCs on D20, where initial regions of trophoblast invasion into the LE were already evident. In these regions, PAG-positive TGCs were predominant in the trophoblast layer, and limited numbers of these cells were present in the LE. Interestingly, the mononucleate trophoblast cells in these regions of initial invasion expressed PAGs within the cytoplasm of their apical domains, suggesting potential secretion into the interface between the trophoblast and LE layers. Present in the initial sites of TGC invasion. On D26, PAGs were highly expressed in most TGCs within implantation sites.

Immunolocalization of MUC-1 and PAG revealed novel expression patterns as pregnancy progressed. The apical expression of PAGs in the trophoblast TMCs on D20 may indicate that PAG secretion alters the maternal interface in favor of conceptus attachment. The co-expression of MUC-1 and PAGs in trophoblast TGCs on D26 could suggest a synergistic role of these proteins during TGC migration, invasion, and remodeling of the maternal interface. Further investigation is warranted to elucidate if other mucins and matrix remodeling proteases contribute to early placentation events in cows.