Allison M. Felker

Uterine natural killer cells: supervisors of vasculature construction in early decidua basalis

Mammalian pregnancy involves tremendous de novo maternal vascular construction to adequately support conceptus development. In early mouse decidua basalis (DB), maternal uterine natural killer (uNK) cells oversee this process directing various aspects during the formation of supportive vascular networks. The uNK cells recruited to early implantation site DB secrete numerous factors that act in the construction of early decidual vessels (neoangiogenesis) as well as in the alteration of the structural components of newly developing and existing vessels (pruning and remodeling). Although decidual and placental development sufficient to support live births occur in the absence of normally functioning uNK cells, development and structure of implantation site are optimized through the presence of normally activated uNK cells. Human NK cells are also recruited to early decidua. Gestational complications including recurrent spontaneous abortion, fetal growth restriction, preeclampsia, and preterm labor are linked with the absence of human NK cell activation via paternally inherited conceptus transplantation antigens. This review summarizes the roles that mouse uNK cells normally play in decidual neoangiogenesis and spiral artery remodeling in mouse pregnancy and briefly discusses changes in early developmental angiogenesis due to placental growth factor deficiency.

The Transcription Factor NFIL3 Is Essential for Normal Placental and Embryonic Development but Not for Uterine Natural Killer (UNK) Cell Differentiation in Mice

ABSTRACT Mice ablated for the gene encoding the transcription factor Nfil3 lack peripheral natural killer (NK) cells but retain tissue-resident NK cells, particularly in mucosal sites, including virgin uterus. We undertook a time course histological study of implantation sites from syngeneically (Nfil3−/−) and allogeneically (BALB/c) mated Nfil3−/− females. We also examined implantation sites from Rag2−/−Il2rg−/− females preconditioned by adoptive transfer of Nfil3−/− marrow or uterine cell suspensions to identify the Nfil3−/− pregnancy aberrations that could be attributed to nonlymphoid cells. Uterine NKs (UNKs) reactive and nonreactive with the lectin Dolichos biflorus agglutinin (DBA) differentiate, localize, and mature within Nfil3−/− implantation sites, although at reduced abundance. The DBA nonreactive UNK cells were enriched following Nfil3−/− marrow transplantation. Uterine lumen closure, early embryonic development, and differentiation of antimesometrial decidua were delayed in Nfil3−/− implantation sites. Major disturbances to the decidual-trophoblast interface that did not lead to fetal death were attributed to NFIL3 deficiency in trophoblast. At midgestation, vessels of the placental labyrinth were enlarged, suggestive of reduced branching morphogenesis. A major term complication in most Nfil3−/− × Nfil3−/− pregnancies but not Nfil3−/− × Nfil3+/− pregnancies was dystocia. These studies highlight the differentiation potential and functions of Nfil3−/− UNK cell progenitors and illustrate that much of the implantation site histopathology associated with this strain is due to Nfil3 deletion in nonlymphoid cell lineages.

Uterine natural killer cell partnerships in early mouse decidua basalis

The decidua basalis of developing mouse implantation sites is highly enriched in CD45+ leukocytes. In intact, syngeneically mated C57BL/6 decidua basalis examined at gestation day 8.5 by whole‐mount in situ immunohistochemistry, leukocyte, but not trophoblast, conjugations were reported. Nothing is known regarding time course, frequency, composition, or importance of physiologic decidual CD45+ cell pairing. In this study, we confirmed the presence of anti‐CD54+/anti‐CD11a+ immune synapses in CD45+ decidual cell conjugates and characterized their cellular heterogeneity. Conjugated cell pairs were virtually absent before implantation (virgin and gestation days 3.5 and 4.5), were infrequent at gestation day 5.5, but involved 19% of all CD45+ cells by gestation day 8.5, then declined. By gestation day 8.5, almost all CD45+ cells coexpressed CD31, and 2 CD45+CD31+ cells composed most conjugates. Conjugation partners were defined for 2 nonoverlapping uterine natural killer cell subsets (Ly49C/I +/Dolichos biflorus agglutinin lectin− and Ly49C/I−/Dolichos biflorus agglutinin lectin+). Ly49C/I+ uterine natural killer cells were the major subset from before mating up to gestation day 6.5. At gestation day 5.5/6.5, uterine natural killer cell conjugates involving Ly49C/I + cells were more abundant. By gestation day 8.5/9.5, Dolichos biflorus agglutinin lectin+ uterine natural killer cells were the dominant subset with Dolichos biflorus agglutinin lectin+/Dolichos biflorus agglutinin lectin+ homologous conjugates and Dolichos biflorus agglutinin lectin+/Dolichos biflorus agglutinin lectin− heterologous conjugates dominating uterine natural killer cell pairings. At gestation day 6.5, both Ly49C/I+/CD45+ and Dolichos biflorus agglutinin lectin+/CD45+ heterologous conjugate pairs strongly engaged antigen‐presenting cells (CD11c+, CD68+, or major histocompatibility complex class II+). By gestation day 8.5, dominant partners of Ly49C/I+/CD45+ and Dolichos biflorus agglutinin lectin+/CD45+ heterologous conjugates are T cells (CD8+ >CD4+). Heterologous conjugates that did not involve uterine natural killer cells occurred but did not suggest antigen presentation to T cells. These data identify gestation day 6.5–8.5 in the pregnant mouse as a critical window for leukocyte interactions that may establish immune regulation within implantation sites.

Whole Mount In Situ Immunohistochemistry

Chapter Summary Whole mount in situ immunohistochemistry is a simple technique that can be customized very easily to the investigator’s interests. It permits acquisition of multidimensional information on cellular relationships in intact, live tissue. By varying the antibodies used and strains of pregnant mouse and their manipulations, biologically accurate data concerning the maternal–fetal interface are obtained.

Changes in Structures and Relationships of Trophoblasts, Leukocytes, and Maternal Vessels in Implantation Sites to Midpregnancy

Chapter Summary Change is the only “true” constant during mouse implantation site development. Changes occur to the uterine stroma (including its vasculature), leukocytes are recruited, the conceptus enlarges during development and growth, trophoblast cells invade, and the placenta forms. These complex changes are most accurately found in intact tissue and are described through serial time course analyses. Whole mount in situ immunohistochemistry of gd4.5–9.5 implant sites is a simple imaging technique applicable to almost any mouse pregnancy model and research question. Fluorochrome-tagged antibodies to cell surface molecules are applied to intact, viable implant sites. Specimens are then studied using fluorescence photomicroscopy. Combined with the use of genetic reporter molecules expressing fluorescent gene-tracking tags and embryo or adoptive cell transfers, applications of this technique in mouse pregnancy research are almost limitless. Baseline findings in early implant sites for genetically normal mice are described in this chapter, as are example studies that address the roles of lymphocytes and their activation in genetically modified mice.

Ly49 knockdown in mice results in aberrant uterine crypt formation and impaired blastocyst implantation

Genetic knockdown (KD) of the mouse Ly49 receptor family is reported to result in infertility despite the presence of zona-enclosed blastocysts in the uterus. Ly49 receptors regulate leukocyte functions particularly Natural Killer (NK) cell functions and are analogous to human killer immunoglobulin-like receptors (KIRs). Histological analyses of gd3.5-4.5 B6.Ly49(KD) uteri identified hatched but retarded blastocysts with pyknotic nuclei, aberrant endometrial crypt formation and impaired uterine lumen closure accompanied by a lack of primary decidualization These data support peri-implantation roles for leukocytes expressing the Ly49 receptor repertoire and may give insight into KIR-based regulation of human infertility.