Conceptus trophectoderm and uterine luminal epithelial cells interact via endocrine, paracrine, and autocrine modulators to mediate pregnancy recognition and implantation. Pig conceptuses not only release estrogens for pregnancy recognition but also secrete interferons during implantation. Because interferon-stimulated genes are increased by interferons secreted for pregnancy recognition in ruminants, we asked whether the interferon-stimulated gene, STAT1, is up-regulated in pig endometrium by conceptus estrogens and/or interferons. STAT1 expression in response to day of pregnancy, estrogen injection, and intrauterine infusion of conceptus secretory proteins in pigs indicated 1) estrogen increases STAT1 in luminal epithelial cells, 2) conceptus secretory proteins that contain interferons increase STAT1 in stroma, 3) STAT1 increases in close proximity to the conceptus, and 4) early estrogen results in conceptus death and no STAT1 in stroma. The interactions of estrogen and interferons to regulate cell-type-specific expression of STAT1 highlight the complex interplay between endometrium and conceptus for pregnancy recognition and implantation.

Pig conceptuses secrete estrogen for pregnancy recognition, and they secrete interferons (IFNs) gamma and delta during the peri-implantation period. The uterine effects of pig IFNs are not known, although ruminant conceptuses secrete IFN tau for pregnancy recognition, and this increases the expression of IFN-stimulated genes (ISGs) in the endometrium. In sheep, the transcriptional repressor interferon-regulatory factor 2 (IRF2) is expressed in the endometrial luminal epithelium (LE) and appears to restrict IFN tau induction of most ISGs, including IRF1, to the stroma and glands. Interestingly, MX1, which is an ISG in sheep, is also expressed in the endometrial stroma of pregnant pigs. The objective of the present study was to determine if estrogen and/or conceptus secretory proteins (CSPs) that contain IFNs regulate IRF1 and IRF2 in pig endometria. The endometrial levels of IRF1 and IRF2 were low throughout the estrus cycle. After Day 12 of pregnancy, the levels of the classical ISGs, which include IRF1, STAT2, MIC, and B2M, increased in the overall endometrium, with expression of IRF1 and STAT2 being specifically localized to the stroma. IRF2 increased in the LE after Day 12. To determine the effects of estrogen, pigs were treated with 17 beta-estradiol benzoate (E2). To determine the CSP effects, pigs were treated with E2 and implanted with mini-osmotic pumps that delivered control serum proteins (CX) to one ligated uterine horn and CSP to the other horn. Estrogen increased the level of IRF2 in the endometrial LE. The administration of E2 and infusion of CSP increased the level of IRF1 in the stroma. These results suggest that conceptus estrogen induces IRF2 in the LE and limits the induction of IRF1 by conceptus IFNs to the stroma. The cell-specific expression of IRF1 and IRF2 in the pig endometrium highlights the complex and overlapping events that are associated with gene expression during the peri-implantation period, when pregnancy recognition signaling and uterine remodeling for implantation and placentation are necessary for successful pregnancy.

The integrity of the fetal-maternal interface is critical for proper fetal nourishment during pregnancy. Integrins are important adhesion molecules present at the interface during implantation; however, in vivo evidence for integrin activation and focal adhesion formation at the maternal-conceptus interface is limited. We hypothesized that focal adhesion assembly in uterine luminal epithelium (LE) and conceptus trophectoderm (Tr) results from integrin binding of extracellular matrix (ECM) at this interface to provide increased tensile forces and signaling to coordinate utero-placental development. An ovine model of unilateral pregnancy was used to evaluate mechanotransduction events leading to focal adhesion assembly at the maternal-conceptus interface and within the uterine wall. Animals were hysterectomized on days 40, 80, or 120 of pregnancy, and uteri immunostained for integrins (ITGAV, ITGA4, ITGA5, ITGB1, ITGB3, and ITGB5), ECM proteins (SPP1, LGALS15, fibronectin (FN), and vitronectin (VTN)), cytoskeletal molecules (ACTN and TLN1), and a signal generator (PTK2). Focal adhesion assembly in myometrium and stroma was also studied to provide a frame of reference for mechanical stretch of the uterine wall. Large focal adhesions containing aggregates of ITGAV, ITGA4, ITGA5, ITGB1, ITGB5, ACTN, and PTK2 were detected in interplacentomal uterine LE and Tr of gravid but not non-gravid uterine horns and increased during pregnancy. SPP1 and LGALS15, but not FN or VTN, were present along LE and Tr interfaces in both uterine horns. These data support the idea that focal adhesion assembly at the maternal-conceptus interface reflects adaptation to increasing forces caused by the growing fetus. Cooperative binding of multiple integrins to SPP1 deposited at the maternal-conceptus interface forms an adhesive mosaic to maintain a tight connection between uterine and placental surfaces along regions of epitheliochorial placentation in sheep.

MHC class I molecules and β2-microglobulin (β2m) are membrane glycoproteins that present peptide Ags to TCRs, and bind to inhibitory and activating receptors on NK cells and other leukocytes. They are involved in the discrimination of self from non-self. Modification of these molecules in the placenta benefits pregnancy, but little is known about their genes in the uterus. We examined the classical class I swine leukocyte Ags (SLA) genes SLA-1, SLA-2, and SLA-3, the nonclassical SLA-6, SLA-7, and SLA-8 genes, and the β2m gene in pig uterus during pregnancy. Uterine SLA and β2m increased in luminal epithelium between days 5 and 9, then decreased between days 15 and 20. By day 15 of pregnancy, SLA and β2m increased in stroma and remained detectable through day 40. To determine effects of estrogens, which are secreted by conceptuses to prevent corpus luteum regression, nonpregnant pigs were treated with estradiol benzoate, which did not affect the SLA or β2m genes. In contrast, progesterone, which is secreted by corpora lutea, increased SLA and β2m in luminal epithelium, whereas a progesterone receptor antagonist (ZK137,316) ablated this up-regulation. To determine effects of conceptus secretory proteins (CSP) containing IFN-δ and IFN-γ, nonpregnant pigs were implanted with mini-osmotic pumps that delivered CSP to uterine horns. CSP increased SLA and β2m in stroma. Cell-type specific regulation of SLA and β2m genes by progesterone and IFNs suggests that placental secretions control expression of immune regulatory molecules on uterine cells to provide an immunologically favorable environment for survival of the fetal-placental semiallograft.