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.     

Hormone-induced preimplantation Lyt 2+ murine uterine suppressor cells persist after implantation and may reduce the spontaneous abortion rate in CBA/J mice

Immunoregulatory cells in the maternal uterine endometrium and decidua are thought to play an important role in ensuring the success of the semiallogeneic conceptus. Two phases of suppression have been described in pregnant mice. Prior to implantation, the hormonal changes triggered by mating activate or recruit a population of nonspecific Lyt 2+ suppressor cells that inhibit cytotoxic T lymphocyte generation: this suppression appears to wane at the time or implantation and 4-5 days after implantation, a non-T suppressor cell population activated or recruited by fetal trophoblast cells develops. In this paper we confirm the non-major histocompatibility complex specificity of the hormone-regulated preimplantation suppressor cell. We show that this activity persists in the uterus during the early postimplantation period where its suppressive activity is masked by an Fc-receptor-positive cell population recruited by the implanting embryo. The potential importance of the persisting suppressor cells is suggested by an increase in the rate of spontaneous abortion of DBA2-mated CBA/J mice following injection of monoclonal anti-Lyt 2+ antibody in the early postimplantation period.     

Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy.

Uterine NK (uNK) cells are abundant in human and murine uteri during decidualization. It is unclear whether precursors of uNK (pre-uNK) cells self-renew or are recruited from other sites. To assess self-renewal of pre-uNK cells, uterine segments from NK cell-competent mice were grafted orthotopically into NK/uNK cell-deficient or wild-type mice. Only in wild-type recipients did decidualized grafts contain uNK cells, indicating that pre-uNK cells do not self-renew in uterus. To identify pre-uNK cell sources, thymus, bone marrow, lymph node, or spleen cells were grafted from virgin or pregnant NK cell-competent donors into mated NK/uNK cell-deficient recipients. Cells from secondary lymphoid tissues of pregnant donors gave high level uNK cell reconstitution, which was independent of chemokine receptors CCR2 or CCR5. Pregnancy-induced changes to lymphocyte-endothelial cell interactions were documented using adhesion of human lymphocytes to frozen mouse tissue sections under shear. A dynamic increase was observed in L-selectin- and alpha(4) integrin-dependent adhesion of CD56(bright) NK cells to decidualizing uterus and in human PBL adhesion to lymph node endothelium. These data support a model that attributes the dramatic increases in human and murine uNK cells during decidualization to precursor cell recruitment.     

A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation

The cellular actions of steroid hormone progesterone (P) are mediated via its nuclear receptors, which regulate the expression of specific target genes. The identity of gene networks that are regulated by the P receptors (PRs) in the uterus at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. In this study, we have used oligonucleotide microarrays to identify mRNAs whose expression in the pregnant mouse uterus is modulated by RU486, a well-characterized PR antagonist, which is also an effective inhibitor of implantation. We found that, in response to RU486, expression of mRNAs corresponding to 78 known genes was down-regulated at least 2-fold in the preimplantation mouse uterus. The PR regulation of several of these genes was ascertained by administering P to ovariectomized wild-type and PR knockout (PRKO) mice. Detailed spatio-temporal analysis of these genes in the pregnant uterus indicated that their expression in the epithelium and stroma could be correlated with the expression of PR in those cell types. Furthermore, time-course studies suggested that many of these genes are likely primary targets of PR regulation. We also identified 70 known genes that were up-regulated at least 2-fold in the pregnant uterus in response to RU486. Interestingly, initial examination of a number of RU486-inducible genes reveals that their uterine expression is also regulated by estrogen. The identification of several novel PR-regulated gene pathways in the reproductive tract is an important step toward understanding how P regulates the physiological events leading to implantation.     

Expression of hypoxia-inducible factors in the peri-implantation mouse uterus is regulated in a cell-specific and ovarian steroid hormone-dependent manner. Evidence for differential function of HIFs during early pregnancy

Increased uterine vascular permeability and angiogenesis are hallmarks of implantation and placentation. These events are profoundly influenced by vascular endothelial growth factor (VEGF). We previously showed that VEGF isoforms and VEGF receptors are expressed in the uterus, suggesting the role of VEGF in uterine vascular permeability and angiogenesis required for implantation and decidualization. We have recently shown that estrogen promotes uterine vascular permeability but inhibits angiogenesis, whereas progesterone stimulates angiogenesis with little effect on vascular permeability. However, the mechanism of differential steroid hormonal regulation of uterine angiogenesis remains unresolved. Oxygen homeostasis is essential for cell survival and is primarily mediated by hypoxia-inducible factors (HIFs). These factors are intimately associated with vascular events and induce VEGF expression by binding to the hypoxia response element in the VEGF promoter. HIFalpha isoforms function by forming heterodimers with the aryl hydrocarbon nuclear translocator (ARNT) (HIF-beta) family members. There is very limited information on the relationship among HIFs, ARNTs, and VEGF in the uterus during early pregnancy, although the role of HIFs in regulating VEGF and angiogenesis in cancers is well documented. Using molecular and physiological approaches, we here show that uterine expression of HIFs and ARNTs does not correlate with VEGF expression during the preimplantation period (days 1-4) in mice. In contrast, their expression follows the localization of uterine VEGF expression with increasing angiogenesis during the postimplantation period (days 5-8). This disparate pattern of uterine HIFs, ARNTs, and VEGF expression on days 1-4 of pregnancy suggests HIFs have multiple roles in addition to the regulation of angiogenesis during the peri-implantation period. Using pharmacological, molecular, and genetic approaches, we also observed that although progesterone primarily up-regulates uterine HIF-1alpha expression, estrogen transiently stimulates that of HIF-2alpha.     

Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus

Populations of macrophages and neutrophils in the uterus are under the control of the female sex steroids estrogen and progesterone (P4). Their influx is induced by estrogen, while P4 can both stimulate and inhibit leukocyte influx depending on the timing of P4 with respect to estrogen. Regulation of leukocytes has been implicated in changes in uterine immune responses during the estrous cycle, pregnancy, and implantation. This work demonstrates that P4 given concurrently with estrogen to ovariectomized mice for 4 days antagonizes the ability of estrogen to recruit macrophages and neutrophils into the mouse uterus. Using progesterone receptor knockout (PRKO) mice, we show that this effect is dependent on progesterone receptors (PR). In the absence of PR, neutrophils recruited by estrogen were found to be degranulated, partially explaining the edema that is observed with long-term treatment of PRKO mice with estrogen and P4. Populations of B lymphocyte cells were shown to be unchanged by estrogen and P4 treatment in both wild-type and PRKO mice. The neutrophil chemotactic chemokine MIP-2 was examined for down-regulation by P4 but was found to be unaffected by hormonal treatment. Together, these observations demonstrate that PR has a strong anti-inflammatory role in the mouse uterus when estrogen and P4 are present together.     

Down-regulation of anandamide hydrolase in mouse uterus by sex hormones.

Endocannabinoids are an emerging class of lipid mediators, which mimic several effects of cannabinoids. Anandamide (arachidonoylethanolamide) is a major endocannabinoid, which has been shown to impair pregnancy and embryo development. The activity of anandamide is controlled by cellular uptake through a specific transporter and intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). We characterized FAAH in mouse uterus by radiochromatographic and immunochemical techniques, showing that the enzyme is confined to the epithelium and its activity decreases appreciably during pregnancy or pseudopregnancy because of lower gene expression at the translational level. Ovariectomy prevented the decrease in FAAH, and both progesterone and estrogen further reduced its basal levels, suggesting hormonal control of the enzyme. Anandamide was shown to induce programmed cell death in mouse blastocysts, through a pathway independent of type-1 cannabinoid receptor. Blastocysts, however, have a specific anandamide transporter and FAAH, which scavenge this lipid. Taken together, these results provide evidence of an interplay between endocannabinoids and sex hormones in pregnancy. These findings may also be relevant for human fertility, as epithelial cells from healthy human uterus showed FAAH activity and expression, which in adenocarcinoma cells was increased fivefold.     

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.     

Molecular mechanisms involved in progesterone receptor regulation of uterine function

The ovarian steroid hormone progesterone is a major regulator of uterine function. The actions of this hormone is mediated through its cognate receptor, the progesterone receptor, Pgr. Ablation of the Pgr has shown that this receptor is critical for all female reproductive functions including the ability of the uterus to support and maintain the development of the implanting mouse embryo. High density DNA microarray analysis has identified direct and indirect targets of Pgr action. One of the targets of Pgr action is a member of the Hedgehog morphogen Indian Hedgehog, Ihh. Ihh and members of the Hh signaling cascade show a coordinate expression pattern in the mouse uterus during the preimplantation period of pregnancy. The expression of Ihh and its receptor Patched-1, Ptc1, as well as, down stream targets of Ihh-Ptch1 signaling, such as the orphan nuclear receptor COUP-TF II show that this morphogen pathway mediates communication between the uterine epithelial and stromal compartments. The members of the Ihh signaling axis may function to coordinate the proliferation, vascularization and differentiation of the uterine stroma during pregnancy. This analysis demonstrates that progesterone regulates uterine function in the mouse by coordinating the signals from the uterine epithelium to stroma in the preimplantation mouse uterus.     

Differential PGE2 and cAMP production by allogeneic and syngeneic pregnant mice uteri.

Prostaglandin E2 (PGE2) and cAMP production in allogeneic and syngeneic pregnant mice uteri, were measured in relation to the ratio of plasma estrogen/progesterone levels. PGE2 generation by allopregnant uteri varied with the days of pregnancy. The increment of the prostanoid coincided with the increase in plasma estrogen concentration, whereas the decrement of its production was in parallel with the increment of plasma progesterone. The syngeneic pregnant uterus was unable to increase the release of PGE2 above basal values during the whole pregnancy. The rise of PGE2 production by the allogeneic pregnant uterus was correlated with an increase in cAMP levels. It is proposed that the pregnant mouse uterus increases its ability to release PGE2 in response to an ovarian steroids.     

Receptors to Dolichos biflorus Agglutinin

Dolichos biflorus agglutinin (DBA), a lectin specific to N-acetylgalactosamine residue, identifies cell surface markers on teratocarcinoma cells. These receptors are found in very limited types of adult tissues. In the present investigation, mouse embryos collected on days 1 (2-cell) to 3 (early blastocyst) were shown to be stained with FITC-conjugated DBA. Embryos homozygous for t ^w32 were also positively stained. These results suggest that receptors for DBA on preimplantation embryos include components distinct from Forssman, F9, and SSEA-1 antigens.