New phenotypic aspects of the decidual spiral artery wall during early post-implantation mouse pregnancy

During pregnancy the walls of decidual spiral arteries (SAs) undergo clinically important structural modifications crucial for embryo survival/growth and maternal health. However, the mechanisms of SA remodeling (SAR) are poorly understood. Although an important prerequisite to this understanding is knowledge about the phenotype of SA muscular wall prior to and during the beginning of mouse SAR, this remains largely unexplored and was the main aim of this work. Using histological and immunohistochemical techniques, this study shows for the first time that during early mouse gestation, from embryonic day 7.5 (E7.5) to E10.5, the decidual SA muscular coat is not a homogeneous structure, but consists of two concentric layers. The first is a largely one cell-thick sub-endothelial layer of contractile mural cells (positive for α-smooth muscle actin, calponin and SM22α) with pericyte characteristics (NG2 positive). The second layer is thicker, and evidence is presented that it may be of the synthetic/proliferative smooth muscle phenotype, based on absence (α-smooth muscle actin and calponin) or weak (SM22α) expression of contractile mural cell markers, and presence of synthetic smooth muscle characteristics (expression of non-muscle Myosin heavy chain-IIA and of the cell proliferation marker PCNA). Importantly, immunohistochemistry and morphometrics showed that the contractile mural cell layer although prominent at E7.5-E8.5, becomes drastically reduced by E10.5 and is undetectable by E12.5. In conclusion, this study reveals novel aspects of the decidual SA muscular coat phenotype prior to and during early SAR that may have important implications for understanding the mechanisms of SAR.     

Serglycin proteoglycan synthesis in the murine uterine decidua and early embryo

This study has explored the localization and synthesis of the serglycin proteoglycan in the murine embryo and uterine decidua during midgestation. Embryos in deciduae were subjected to in situ hybridization with cRNA probes and to immunohistochemical detection with a specific antibody against murine serglycin. Adherent decidual cell cultures were prepared from freshly isolated deciduae. Proteoglycan biosynthesis was investigated by labeling intact deciduae and decidual cultures with (35)S-sulfate. Serglycin mRNA was detected by in situ hybridization throughout the mesometrial portion and at the periphery of the antimesometrial portion of the decidua at Embryonic Day (E) 8.5, and in the parietal endoderm surrounding the embryo. Serglycin mRNA was detected in fetal liver at E11.5-E14.5. Serglycin was detected by immunohistochemistry in decidua and parietal endoderm at E8.5 and in liver at E13.5. Most of the proteoglycans synthesized by cultured intact deciduae (78%) and adherent decidual cultures (91%) were secreted into the medium. Serglycin proteoglycan may play an important role in uterine decidual function during early postimplantation development.     

The uterine NK cell population requires IL-15 but these cells are not required for pregnancy nor the resolution of a Listeria monocytogenes infection

During pregnancy in mice, uterine natural killer (uNK) cells abundantly accumulate on the mesometrial side of the placenta. In this study, we show that the presence of both mature and immature uNK cells requires IL-15. Bone marrow transplantation of NK cell-negative mice due to null mutations in the recombination-activating gene (Rag) 2/common cytokine receptor gamma-chain (Rag2(-/-)gamma(c)(-/-)) genes indicated that uNK cells originate from the bone marrow and require IL-15 to develop. NK cells are thought to be central players in the immune response to intracellular pathogens such as Listeria monocytogenes, a bacterium that also has a predilection for replication in the placenta. However, IL-15(-/-), NK cell-deficient mice were relatively protected from this infection compared with wild-type mice, and during pregnancy the absence of NK cells did not compromise the immune response at this site. The loss of uNK cells results in decidual abnormalities, including thickening of the arterial walls with luminal narrowing and a hypocellular decidua basalis. These defects were rescued by bone marrow transplantation of the Rag2(-/-)gamma(c)(-/-) mice that restored the uNK cell population. The decidual abnormalities in the IL-15(-/-) mice however did not result in infertility as gestation times and litter sizes were comparable to those of wild-type mice. Fetal weights were mildly compromised, consistent with the arterial pathologies. These results show that uNK cells are not required for successful pregnancy and that NK cells are not essential for an adequate immune response to L. monocytogenes in either pregnant or nonpregnant mice.     

Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration

Rat gestation sites were obtained on days 10 through 16 of normal pregnancy. Light and electron microscopic examination of day-10 sites revealed a consistent complex pattern of stromal cell morphologies. Six distinct regions were identified: an antimesometrial region of epithelioid decidual cells that form the gestation chamber containing the embryo and extraembryonic membranes; an abembryonic antimesometrial decidual region, the decidual crypt, where the cells are separated by large extracellular spaces; a mesometrial region with granule-containing cells and mesometrial decidual cells; a region of spiny cells that are lateral to the antimesometrial decidual cells and continuous with the mesometrial decidual cells; and a region of undifferentiated stromal cells adjacent to the myometrium. Between days 12 and 16, the antimesometrial decidua becomes thinner and is eventually sloughed into the newly formed uterine lumen. The role of the antimesometrial decidual cells is discussed with reference to trophoblast invasiveness, protein synthesis, and especially remodeling of the gestation chamber. Differences between decidua and deciduoma are considered.     

Effect of the conceptus on uterine natural killer cell numbers and function in the mouse uterus during decidualization

Uterine natural killer (uNK) cells are the most abundant lymphocytes in the uterus during early pregnancy and play a role in spiral arteriole modifications. In the present study, we investigated whether uNK cell populations differed between mouse decidua and deciduoma. Histochemical staining using the Dolichos biflorus agglutinin (DBA) lectin was used to identify uNK cells and classify their stages of maturation. We found differences in the pattern of localization and density of uNK cells between the decidua and deciduoma at Days 2-4 after the onset of decidualization. The cells were more distributed and the densities were significantly greater in the mesometrial region of the decidua than in the deciduoma. Using double-labeling for DBA lectin binding and bromodeoxyuridine incorporation, we found that the higher number of uNK cells in the decidua was not due to an increase in uNK cell proliferation. Western blot analyses revealed that the increase in uNK cell number was accompanied by significant increases in the levels of interferon gamma (IFNG) and prointerleukin 18 when a conceptus was present. Vascular morphometry revealed that modifications of the spiral arterioles occurred in the mesometrial decidua but not in the deciduoma, which could be attributed to the differences observed in uNK cell number and IFNG production. The present study demonstrates that differences exist in uNK cell populations between the decidua and deciduoma, providing evidence that the conceptus generates signals that regulate uNK cell number and function in the uterus during implantation.     

Pathways participating in activation of mouse uterine natural killer cells during pregnancy

Activated natural killer (NK) cells proliferate in large numbers in murine mesometrial endometrium from Day 6 to Day 12 of gestation (term = 19 gestation days) to become the most abundant uterine lymphocytes. Early human decidua contains analogous CD56+/CD16- cells. Murine uterine (u)NK cells localize to decidua basalis and mesometrial lymphoid aggregate of pregnancy (MLAp). Decidua and MLAp are transient, pregnancy-associated tissues traversed by maternal arteries to the placentas. Uterine NK cells sensitize these arteries, facilitating their structural changes into high-volume conduits by Gestation Day 10 through release of interleukin (IL)-18, interferon (IFN)-gamma, vascular endothelial growth factor (VEGF), and other molecules. Little information exists concerning where, when, or how murine or human uNK cells become activated. In murine lymphoid tissue, three NK cell adaptor-mediated activation pathways are known: FcRgamma/CD3zeta, DNAX-activating protein (DAP) 10, and DAP12 (genes Fcgr3/Cd3z, Hcst, and Tyrobp, respectively). Expression of ligands for these receptors was demonstrated in implantation sites of normal C57BL/6J mice. Then, histological and morphometric analyses of implantation sites in mice with genetic inactivation of each pathway were undertaken. Implantation sites in DAP10-/- (Hcst deleted) mice appeared normal, spiral artery modification occurred, and concentrations of IFN-gamma in MLAp and decidua basalis were similar to those in time-matched C57BL/6J. Implantation sites of FcRgamma-/-/CD3zeta-/- (Fcgr3/Cd3z double knockout), DAP12 (Tyrobp)-loss-of-function-mutant, and FcRgamma-/-/DAP12-/- (Fcgr3/Tyrobp double knockout) mice differentiated abundant but functionally impaired uNK cells that could not modify spiral arteries. These data reveal key importance of FcRgamma-/-/CD3zeta-/- and thus maternal IgG during activation of mouse uNK cells and assign DAP12 but not DAP10 signaling contributions.     

Unique receptor repertoire in mouse uterine NK cells

Uterine NK (uNK) cells are a prominent feature of the uterine mucosa and regulate placentation. NK cell activity is regulated by a balance of activating and inhibitory receptors, however the receptor repertoire of mouse uNK cells is unknown. We describe herein two distinct subsets of CD3(-)CD122(+) NK cells in the mouse uterus (comprising decidua and mesometrial lymphoid aggregate of pregnancy) at mid-gestation: a small subset indistinguishable from peripheral NK cells, and a larger subset that expresses NKp46 and Ly49 receptors, but not NK1.1 or DX5. This larger subset reacts with Dolichus biflores agglutinin, a marker of uNK cells in the mouse, and is adjacent to the invading trophoblast. By multiparametric analysis we show that the phenotype of uNK cells is unique and unprecedented in terms of adhesion, activation, and MHC binding potential. Thus, the Ly49 repertoire and the expression of other differentiation markers strikingly distinguish uNK cells from peripheral NK cells, suggesting that a selection process shapes the receptor repertoire of mouse uNK cells.     

Role of uterine natural killer cells in angiogenesis of human decidua of the first-trimester pregnancy

Decidualization is accompanied by extensive angiogenesis, which is an essential step in the maturation of new blood vessels in mammalian pregnancy. The purpose of this study was to determine a distribution of uNK cells (CD56 uNK or CD56bright cells) in human decidua of the first-trimester pregnancy, and investigate whether uNK cells in human decidua could express vascular endothelial growth factor (VEGF-A) and/or angiopoietin2 (Ang2). Our immunohistochemical staining results demonstrated that a great amount of uNK (CD56 ) cells scattered throughout the decidual stroma and near endometrial gland and spiral vessels in human decidua. The protein expression of VEGF-A and Ang2 was detected in decidual stroma cells, capillary endothelial cells and glandular cells in tissue specimens. There was a positive correlation between microvessel density (MVD) and the number of the CD56-positive uNK cells in decidual stroma, and also between the number of the CD56-positive uNK cells and VEGF-A protein expression in the tissue. In addition, we found that uNK cells in human decidua could express VEGF-A mRNA, but not Ang2 mRNA, in isolated uNK cells in human decidua of the first-trimester gestation by combination of LCM and Nested-PCR. Our study indicated that uNK cells, through expressing VEGF-A, may play an important role in the angiogenic response at the time of human decidualization and early placenta development.     

Role of uterine natural killer cells in angiogenesis of human decidua of the first-trimester pregnancy

Decidualization is accompanied by extensive angiogenesis, which is an essential step in the maturation of new blood vessels in mammalian pregnancy. The purpose of this study was to determine a distribution of uNK cells (CD56⁺ uNK or CD56^bright cells) in human decidua of the first-trimester pregnancy, and investigate whether uNK cells in human decidua could express vascular endothelial growth factor (VEGF-A) and/or angiopoietin2 (Ang2). Our immunohistochemical staining results demonstrated that a great amount of uNK (CD56⁺) cells scattered throughout the decidual stroma and near endometrial gland and spiral vessels in human decidua. The protein expression of VEGF-A and Ang2 was detected in decidual stroma cells, capillary endothelial cells and glandular cells in tissue specimens. There was a positive correlation between microvessel density (MVD) and the number of the CD56-positive uNK cells in decidual stroma, and also between the number of the CD56-positive uNK cells and VEGF-A protein expression in the tissue. In addition, we found that uNK cells in human decidua could express VEGF-A mRNA, but not Ang2 mRNA, in isolated uNK cells in human decidua of the first-trimester gestation by combination of LCM and Nested-PCR. Our study indicated that uNK cells, through expressing VEGF-A, may play an important role in the angiogenic response at the time of human decidualization and early placenta development.     

Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy.

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High-to-moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8-9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9-12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.     

Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy

The identification of lytic cells in 6.5-day to 9.5-day murine decidua as NK cells has been extended. The cells with natural killer (NK) activity in early decidua were nonphagocytic and heterogeneous in size as assessed by velocity sedimentation at unit gravity. The numbers of lytic cells were reduced by treatment with anti-asialo GM1 in vivo and they were absent from the decidua of bg/bg mice. Thus, decidual NK cells were not distinct from NK cells in other tissues. The decline in the levels of decidual NK activity as pregnancy progressed was attributed to their regulation by other cells present in decidua by midgestation. The development of NK activity in decidua was dependent upon the presence of an embryo, however, decidual NK cells were not essential for successful pregnancy because viable offspring were obtained from mice lacking decidual NK activity. It was shown that NK cells from either spleen or decidua were unlikely to cause damage to embryos during the first half of pregnancy as freshly dissociated 9.5- and 11.5-day embryonic cells resisted NK lysis. Furthermore, blastocysts were not damaged by coincubation with splenic or decidual NK cells and were viable upon subsequent embryo transfer. These studies indicate that decidual NK cells are not essential for successful pregnancy and are not necessarily detrimental to early embryos. It is suggested that decidual NK cells may play other nonimmunological roles during embryonic development.     

Interferon-gamma contributes to the normalcy of murine pregnancy.

Uterine natural killer (uNK) cells are transient, large, heavily granulated, maternal lymphocytes present on the mesometrial side of the pregnant mouse uterus. These cells contribute to normal implantation site development. Cytokine production, particularly interferon (IFN)-gamma, is a major function of most NK cell subsets. In this study, uNK cells were assessed for IFN-gamma production. Local concentrations of IFN-gamma were measured in the mesometrial regions of murine implantation sites between Days 6 and 16 of gestation. IFN-gamma was detected by ELISA at all days studied in a random-bred (CD1) and an inbred (BALB/c) strain of immune-competent mouse and in two immune-deficient strains, SCID (NK(+), T(-), B(-)) and tgepsilon26 (NK(-), T(-), B(+)). Concentrations of IFN-gamma per implantation site peaked at Day 10 of gestation in NK(+) strains but were low and relatively constant in NK(-) mice. To evaluate the functions of IFN-gamma at murine implantation sites, pregnancy was studied in homozygously mated IFN-gamma(-/-) and IFN-gammaRalpha(-/-) mice and their congenic controls. Primiparous but not multiparous IFN-gamma(-/-) mice experienced significant fetal loss. Primiparous IFN-gammaRalpha(-/-) carried full litters to term. Implantation site pathology was demonstrated in both strains of gene-deleted mice by light microscopy and ultrastructurally. This included elevated numbers of uNK cells that contained fewer and smaller granules and, after Day 10 of gestation, progressive necrosis and loss of decidua. The presence of a fetus able to produce IFN-gamma did not modify the phenotype of pregnant IFN-gamma(-/-) mice. This study indicates that during murine pregnancy, uNK cells are the main source of IFN-gamma on the mesometrial side of the uterus and that IFN-gamma contributes to normal health of the midgestational decidua. Furthermore, evidence is presented that IFN-gamma-producing cells exist in mesometrial regions of implantation sites that are neither NK nor T cells.     

Assessment of requirements for IL-15 and IFN regulatory factors in uterine NK cell differentiation and function during pregnancy

In mouse and human, precursors of NK cell lineage home to decidualizing uteri. To assess the requirement for IL-15, an essential cytokine for NK differentiation in lymphoid tissue, on uterine NK (uNK) cell differentiation, implantation sites from IL-15(-/-) mice were analyzed histologically. IL-15(-/-) implantation sites had no uNK cells, no spiral-artery modification, and lacked the decidual integrity found in normal mice. IL-15(-/-) recipients of C57BL/6 marrow displayed similar pathology. However, implantation sites from recombination-activating gene-2(-/-)gamma(c)(-/-) (alymphoid) recipients of IL-15(-/-) marrow showed normal uNK cells, modified spiral arteries, and well-developed decidua basalis. Deletion of the IFN-regulatory factor (IRF)-1, but not IRF-2 (factors important in peripheral NK cell differentiation) limited but did not prevent uNK cell development. In situ hybridization localized IRF-1 largely to placental trophoblast cells. IRF-1(-/-) marrow transplanted into recombination-activating gene-2(-/-)gamma(c)(-/-) displayed competence for full uNK cell differentiation. IL-15 mRNA expression at implantation sites of IRF-1(-/-) and C57BL/6 was similar, suggesting that, unlike in bone marrow and spleen, IRF-1 does not regulate IL-15 in the pregnant uterus. Terminal differentiation of uNK cells was not promoted in pregnant IRF-1(-/-) mice by 5-day infusion of murine rIL-15, suggesting that IRF-1 deficiency rather than IL-15 deficiency limits uNK cell differentiation in these mice. Further, IRF-1 regulates placental growth, birth weight, and postnatal growth of offspring. These studies indicate that uNK cell development and maturation share some aspects with NK cell development in other tissues, but also display distinctive tissue-specific regulation.     

Murine T cell determination of pregnancy outcome.

At the fetomaternal interface, maternal effector cells come in intimate contact with fetal trophoblast cells which express paternal antigens. Failure of fetal trophoblast cells to activate maternal Th1 immune responses has been attributed in part to the absence of classical Class I and Class II major histocompatibilty complex (MHC) antigen expression and elaboration of factors which reduce TcR expression and shift any immune responses which may occur to Th2. Classical TcR alphabeta(+) T cells have not been found to be able to respond to trophoblasts. Recently, TcR gammadelta(+) T cells have been characterized in the low-abortion-rate pregnant C57Bl/10 mouse decidua, and the Vgamma1(+) subset may be able to respond to trophoblasts in a non-MHC-dependent manner. Trophoblast-recognizing T cells with Vgamma1 receptors are also present in the decidua of CBA/J mice pregnant by DBA/2, an abortion-prone mating combination. To test the role of the Vgamma1 subset of decidual gammadelta T cells in abortion-prone pregnancies, we altered this subset by injecting monoclonal anti-Vgamma1.1 antibody on gestation day 5.5, 1 day after implantation. This reduced detectability of a Vgammadelta subset producing TNF-alpha and reduced the abortion rate. Anti-Vgamma2, which reacts with a similar proportion of decidual gammadelta T cells as anti-Vgamma1.1, failed to prevent abortions. Vdelta6.3(+) cells are prominent at the fetomaternal interface, and anti-Vdelta6 antibody injected on day 5.5 prevented abortions. TGF-beta2(+) gammadelta cells first appear on day 8.5 of pregnancy; anti-Vgamma1.1 antibody injection on day 8.5 depleted these cells and boosted abortions; anti-Vdelta6.3 given on day 8.5 boosted abortions to the same level. These results suggest that two populations of Vgamma1.1(+)delta6.3(+) T cells may arise in the decidua: an early population that is Th1, abortogenic, and present during the time of implantation, and a Th2/3 cell subset that is present in the decidua later during pregnancy and which is pregnancy-protective.     

Chorioallantoic placenta formation in the rat: II. Angiogenesis and maternal blood circulation in the mesometrial region of the implantation chamber prior to placenta formation.

Rat gestation sites were examined on days 7 through 9 of pregnancy by light microscopy and transmission and scanning electron microscopy to determine the extent of vascular modifications in the vicinity of the mesometrial part of the implantation chamber (mesometrial chamber). At a later time, the mesometrial chamber is, in conjunction with the uterine lumen, the site of chorioallantoic placenta formation. On day 7, in the vicinity of the mesometrial chamber, vessels derived from a subepithelial capillary plexus and venules draining the plexus were dilating. By early day 8, this network of thin-walled dilated vessels (sinusoids) was further enlarged and consisted primarily of hypertrophied endothelial cells with indistinct basal laminas. Sinusoids were frequently close to the mesometrial chamber's luminal surface which was devoid of epithelial cells but was lined by decidual cell processes and extracellular matrix. By late day 8, cytoplasmic projections of endothelial cells extended between healthy-appearing decidual cells and out onto the mesometrial chamber's luminal surface, and endothelial cells were sometimes found on the luminal surface indicating that endothelial cells were migrating. The presence of maternal blood cells in the mesometrial chamber lumen suggested that there was continuity between the chamber and blood-vessel lumens. On day 9, the mesometrial chamber was completely lined with hypertrophied endothelial cells, and sinusoid lumens were clearly continuous with the lumen of the mesometrial chamber. Mesometrial sinusoids and possibly the mesometrial chamber lumen were continuous with vessels in vicinity of the uterine lumen that were fed by mesometrial arterial vessels. Clearing of the mesometrial chamber lumen during perfusion fixation via the maternal vasculature indicated the patency of this luminal space and its confluence with mesometrial arterial vessels and sinusoids. The conceptus occupied an antimesometrial position in the implantation chamber on days 7 through 9, and it was not in direct contact with uterine tissues in the vicinity of the mesometrial chamber. These observations suggest that angiogenesis, not trophoblast invasion or decidual cell death, plays a major role in the opening of maternal vessels into the mesometrial chamber lumen before the formation of the chorioallantoic placenta.