Implantation in all mammals involves shedding of the zona pellucida, followed by orientation, apposition, attachment and adhesion of the blastocyst to the endometrium. Endometrial invasion does not occur in domestic ruminants; thus, definitive implantation is achieved by adhesion of the mononuclear trophoblast cells to the endometrial lumenal epithelium (LE) and formation of syncytia by the fusion of trophoblast binucleate cells with the LE. This review highlights new information on mechanisms regulating the implantation cascade in sheep. The embryo enters the uterus on day 4 at the morula stage of development and then develops into a blastocyst by day 6. The blastocyst sheds the zona pellucida (day 8), elongates to a filamentous form (days 11–16), and adheres to the endometrial LE (day 16). Between days 14 and 16, the binucleate cells begin to differentiate in the trophoblast and subsequently migrate and fuse with the endometrial LE to form syncytia. Continuous exposure of the endometrium to progesterone in early pregnancy downregulates the progesterone receptors in the epithelia, a process which is associated with loss of the cell-surface mucin MUC1 and induction of several secreted adhesion proteins. Recurrent early pregnancy loss in the uterine gland knockout ewe model indicates that secretions of the endometrial epithelia have a physiologic role in blastocyst elongation and implantation. A number of endometrial proteins have been identified as potential regulators of blastocyst development and implantation in sheep, including glycosylated cell adhesion molecule 1 (GlyCAM-1), galectin-15, integrins and osteopontin. The epithelial derived secreted adhesion proteins (GlyCAM-1, galectin-15 and osteopontin) are expressed in a dynamic temporal and spatial manner and regulated by progesterone and/or interferon tau, which is the pregnancy recognition signal produced by the trophoblast during blastocyst elongation. The noninvasive and protracted nature of implantation in domestic animals provides valuable opportunities to investigate fundamental processes of implantation that are shared among all mammals. Understanding of the cellular and molecular signals that regulate uterine receptivity and implantation can be used to diagnose and identify causes of recurrent pregnancy loss and to improve pregnancy outcome in domestic animals and humans.

Uterine receptivity to implantation of blastocysts in mammals includes hatching from zona pellucida, precontact with uterine luminal (LE) and superficial glandular (sGE) epithelia and orientation of blastocyst, apposition between trophectoderm and uterine LE and sGE, adhesion of trophectoderm to uterine LE/sGE, and, in some species, limited or extensive invasion into the endometrial stroma and induction of decidualization of stromal cells. These peri-implantation events are prerequisites for pregnancy recognition signaling, implantation, and placentation required for fetal–placental growth and development through the remainder of pregnancy. Although there is a range of strategies for implantation in mammals, a common feature is the requirement for progesterone (P₄) to downregulate expression of its receptors in uterine epithelia and P₄ prior to implantation events. P₄ then mediates its effects via growth factors expressed by stromal cells in most species; however, uterine luminal epithelium may express a growth factor in response to P₄ and/or estrogens in species with a true epitheliochorial placenta. There is also compelling evidence that uterine receptivity to implantation involves temporal and cell-specific expression of interferon (IFN)-stimulated genes that may be induced directly by an IFN or induced by P₄ and stimulated by an IFN. These genes have many roles including nutrient transport, cellular remodeling, angiogenesis and relaxation of vascular tissues, cell proliferation and migration, establishment of an antiviral state, and protection of conceptus tissues from challenges by the maternal immune cells.

In pigs, endometrial functions are regulated primarily by progesterone and placental factors including estrogen. Progesterone levels are high throughout pregnancy to stimulate and maintain secretion of histotroph from uterine epithelia necessary for growth, implantation, placentation, and development of the conceptus (embryo and its extra-embryonic membranes). This study determined effects of long-term progesterone on development and histoarchitecture of endometrial luminal epithelium (LE), glandular epithelium (GE), and vasculature in pigs. Pigs were ovariectomized during diestrus (day 12), and then received daily injections of either corn oil or progesterone for 28 days. Prolonged progesterone treatment resulted in increased weight and length of the uterine horns, and thickness of the endometrium and myometrium. Hyperplasia and hypertrophy of GE were not evident, but LE cell height increased, suggesting elevated secretory activity. Although GE development was deficient, progesterone supported increased endometrial angiogenesis comparable to that of pregnancy. Progesterone also supported alterations to the apical and basolateral domains of LE and GE. Dolichos biflorus agglutinin lectin binding and α_v integrin were downregulated at the apical surfaces of LE and GE. Claudin-4, α₂β₁ integrin, and vimentin were increased at basolateral surfaces, whereas occludins-1 and -2, claudin-3, and E-cadherin were unaffected by progesterone treatment indicating structurally competent trans-epithelial adhesion and tight junctional complexes. Collectively, the results suggest that progesterone affects LE, GE, and vascular development and histoarchitecture, but in the absence of ovarian or placental factors, it does not support development of GE comparable to pregnancy. Furthermore, LE and vascular development are highly responsive to the effects of progesterone.

Pigs experience significant conceptus loss near mid-gestation, correlating with increasing glandular epithelial (GE) development and secretory activity. Secreted phosphoprotein 1 (SPP1, osteopontin) increases in GE between days 30 and 40 of pregnancy and is expressed in the GE of day 90 pseudopregnant pigs, suggesting that progesterone (P₄) from corpora lutea is responsible for induction of SPP1 in GE. In this study, pigs were ovariectomized and treated daily with P₄ to assess effects of 40 days of P₄ exposure on SPP1, P₄ receptor (PGR), uteroferrin (ACP5), and fibroblast growth factor 7 (FGF7) expression in porcine endometria. PGR mRNA decreased in pigs injected with P₄ compared with pigs injected with corn oil (CO), and PGRs were downregulated in the luminal epithelium (LE) and GE. ACP5 mRNA increased in pigs injected with P₄ compared with pigs injected with CO, and ACP5 was induced in the GE of P₄-treated pigs. FGF7 mRNA increased in pigs injected with P₄ compared with pigs injected with CO, and FGF7 was induced in the LE and GE of P₄-treated pigs. SPP1 mRNA was not different between pigs injected with P₄ compared with pigs injected with CO, and SPP1 was not present in the GE of P₄-treated pigs. Therefore, long-term P₄, in the absence of ovarian and/or conceptus factors, does not induce SPP1 expression in GE. We hypothesize that a servomechanism involving sequential effects of multiple hormones and cytokines, similar to those for sheep and humans, is required for GE differentiation and function, including the synthesis and secretion of SPP1.

Angiogenesis is fundamental to the expansion of the placental vasculature during pregnancy. Integrins are associated with vascular formation; and osteopontin is a candidate ligand for integrins to promote angiogenesis. Endothelial progenitor cells (EPCs) are released from bone marrow into the blood and incorporate into newly vascularized tissue where they differentiate into mature endothelium. Results of studies in women suggest that EPCs may play an important role in maintaining placental vascular integrity during pregnancy, although little is known about how EPCs are recruited to these tissues. Our goal was to determine the αv integrin mediated effects of osteopontin on EPC adhesion and incorporation into angiogenic vascular networks. EPCs were isolated from 6 h old piglets. RT-PCR revealed that EPCs initially had a monocyte-like phenotype in culture that became more endothelial-like with cell passage. Immunofluorescence microscopy confirmed that the EPCs express platelet endothelial cell adhesion molecule, vascular endothelial cadherin, and von Willebrand factor. When EPCs were cultured on OPN-coated slides, the αv integrin subunit was observed in focal adhesions at the basal surface of EPCs. Silencing of αv integrin reduced EPC binding to OPN and focal adhesion assembly. In vitro siRNA knockdown in EPCs,demonstrated that OPN stimulates EPC incorporation into human umbilical vein endothelial cell (HUVEC) networks via αv-containing integrins. Finally, in situ hybridization and immunohistochemistry localized osteopontin near placental blood vessels. In summary, OPN binds the αv integrin subunit on EPCs to support EPC adhesion and increase EPC incorporation into angiogenic vascular networks.

Integrins and OPN are potential mediators of blastocyst attachment to the endometrium to initiate implantation. The goals were to examine the temporal/spatial pattern of expression of integrins at the endometrial–placental interface of sheep encompassing Days 9 through 80 of gestation and determine if OPN co-localizes with integrins. Results show the following: (1) αv, α4, β1, β3 and β5 integrins at the apical surface of endometrial luminal epithelium (LE) from Days 11 through 16 of pregnancy that indicate a role for these integrins during implantation; (2) large, intermittent aggregates of αv, α4, α5, β1 and β5 integrins at the endometrial–placental interface from Days 20 through 55, suggesting adaptation to a localized tissue remodeling stage of placentation; and (3) integrin adhesion complexes (IACs) containing αv, α4, α5, β1 and β5 integrins precisely distribute at the apical surfaces of apposed endometrial LE and chorion along expanses of the interplacentomal endometrial–placental interface between Days 60 and 80 of gestation, suggesting engagement of these integrins with the ECM to stabilize adhesion between endometrial LE and chorion in response to the increasing mechanical stress on this interface by the increasing size of the fetus and volumes of fetal fluids. An advancement is the clear co-localization of OPN and integrins at the endometrial–placental interface throughout gestation in sheep. The comprehensive nature of these results provide evidence that integrins potentially interact with OPN to play key roles in the mechanisms required for implantation and placentation throughout pregnancy in sheep and have implications concerning implantation and placentation in other species.

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 (α 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 α 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 α 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.

Secreted phosphoprotein 1 (SPP1, osteopontin) is the most highly upregulated extracellular matrix/adhesion molecule/cytokine in the receptive phase human uterus, and Spp1 null mice manifest decreased pregnancy rates during mid-gestation as compared with wild-type counterparts. We hypothesize that Spp1 is required for proliferation, migration, survival, adhesion, and remodeling of cells at the conceptus–maternal interface. Our objective was to define the temporal/spatial distribution and steroid regulation of Spp1 in mouse uterus during estrous cycle and early gestation. In situ hybridization localized Spp1 to luminal epithelium (LE) and immune cells. LE expression was prominent at proestrus, decreased by estrus, and was nearly undetectable at diestrus. During pregnancy, Spp1 mRNA was not detected in LE until day 4.5 (day 1 = vaginal plug). Spp1-expressing immune cells were scattered within the endometrial stroma throughout the estrous cycle and early pregnancy. Immunoreactive Spp1 was prominent at the apical LE surface by day 4.5 of pregnancy and Spp1 protein was also co-localized with subsets of CD45-positive (leukocytes) and F4/80-positive (macrophages) cells. In ovariectomized mice, estrogen, but not progesterone, induced Spp1 mRNA, whereas estrogen plus progesterone did not induce Spp1 in LE. These results establish that estrogen regulates Spp1 in mouse LE and are the first to identify macrophages that produce Spp1 within the peri-implantation endometrium of any species. We suggest that Spp1 at the apical surface of LE provides a mechanism to bridge conceptus to LE during implantation, and that Spp1-positive macrophages within the stroma may be involved in uterine remodeling for conceptus invasion.

Attachment of the conceptus trophoblast (Tr) to the uterine luminal epithelium (LE) is critical for successful implantation. This study determined whether alpha v (av) integrins (ITGAV) directly mediate porcine trophoblast cell adhesion to secreted phosphoprotein 1 (SPP1, also known as osteopontin (OPN)) and examined the temporal/spatial expression of ITGAV, beta 3 (b3, ITGB3) and beta 6 (b6, ITGB6) integrin subunits, and SPP1, at the uterine–placental interface of pigs. Knockdown of ITGAV in porcine Tr (pTr2) cells by siRNA reduced pTr2 attachment to SPP1. In situ hybridization confirmed the presence of ITGAV, ITGB3 and ITGB6 mRNAs in uterine LE and conceptus Tr between Days 9 and 60 of gestation, with no change in the magnitude of expression over the course of pregnancy. Exogenous E2 or P4 did not affect ITGAV, ITGB3 and ITGB6 mRNA expression in the uteri of ovariectomized gilts. Immunofluorescence identified ITGAV, ITGB3 and SPP1 proteins in large aggregates at the uterine LE-placental Tr/chorion interface on Day 25, but aggregates were no longer observed by Day 50 of gestation. These results are the first to directly demonstrate that pTr2 cells engage ITGAV-containing integrin receptors to adhere to SPP1 and suggest that mechanical forces generated by tethering elongating conceptuses to uterine LE leads to assembly of focal adhesions containing ITGAV and SPP1; however, as placentation progresses, subsequent folding/interdigitation at the uterine–placental interface disperses mechanical forces resulting in the loss of focal adhesions.

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.