Involvement of cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) in interleukin 11 stimulation of decidualization in mice

Interleukin 11 receptor alpha (Il11ra) null mice are infertile due to defective decidualization and abnormal trophoblast invasion. We have previously shown in these mice that downregulation of decidual proteinase inhibitors plays a role in uncontrolled trophoblast invasion. However, the decidua is abnormally smaller in pseudopregnant Il11ra null mice, where trophoblast invasion is not a factor. Here, we examined whether defective decidualization is due to dysregulation of key molecules involved in decidual cell growth and differentiation. We found a dramatic downregulation of cyclin D3 in Il11ra null mice. We also found that IL11 robustly stimulates the expression of cyclin D3 in cell culture. CDK4 and CDK6, known partners of cyclin D3, are not affected. Immunolocalization studies show absence of cyclin D3 in the mesometrial site and absence of differentiated polyploid cells in the antimesometrial site of Il11ra null mice. We also examined the expression of cell differentiation factors CDKN1A (p21) and CDKN1B (p27), and found that in both in vivo and cell culture the expression of CDKN1A (p21) but not CDKN1B (p27) is under the control of IL11. Another clear target of IL11 in the decidua is BIRC5 (Survivin), whose expression is repressed in the decidua of Il11ra null mice and stimulated by IL11 in cell culture. Taken together, these results provide, at least in part, an explanation for the defective small decidua of mice lacking the Il11ra gene, and reveal for the first time that cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) are targets of IL11 in the decidua.     

A uterine decidual cell cytokine ensures pregnancy-dependent adaptations to a physiological stressor

In the mouse, decidual cells differentiate from uterine stromal cells in response to steroid hormones and signals arising from the embryo. Decidual cells are crucially involved in creating the intrauterine environment conducive to embryonic development. Among their many functions is the production of cytokines related to prolactin (PRL), including decidual prolactin-related protein (DPRP). DPRP is a heparin-binding cytokine, which is abundantly expressed in uterine decidua. In this investigation, we have isolated the mouse Dprp gene, characterized its structure and evaluated its biological role. Dprp-null mice were made by replacing exons 2 to 6 of the Dprp gene with an in-frame enhanced green fluorescent protein (EGFP) gene and a neomycin (neo) resistance cassette. Heterozygous intercross breeding of the mutant mice yielded the expected mendelian ratio. Pregnant heterozygote females expressed EGFP within decidual tissue in locations identical to endogenous Dprp mRNA and protein expression. Homozygous Dprp-null mutant male and female mice were viable, exhibited normal postnatal growth rates, were fertile and produced normal litter sizes. A prominent phenotype was observed when pregnant Dprp-null mice were exposed to a physiological stressor. DPRP deficiency interfered with pregnancy-dependent adaptations to hypoxia resulting in pregnancy failure. Termination of pregnancy was associated with aberrations in mesometrial decidual cells, mesometrial vascular integrity, and disruptions in chorioallantoic placenta morphogenesis. The observations suggest that DPRP participates in pregnancy-dependent adaptations to a physiological stressor.     

Apoptosis and related proteins in placenta of intrauterine fetal death in prostaglandin f receptor-deficient mice

The present study investigated whether the increase of apoptosis in the placenta is associated with intrauterine fetal death in prostaglandin F receptor-deficient mice. Apoptosis was demonstrated within placental and decidual tissue by the TUNEL method. The majority of apoptosis was found in syncytiotrophoblast tissues. Enhanced TUNEL-positive staining in the syncytiotrophoblast layer was scattered in the placental tissues in clusters of apoptotic cells in the death group. Marked TUNEL-positive cells were identified in decidua of both groups. The rate of apoptosis in the placenta and decidua in the death group was higher than that in the survival group (P < 0.05). Immunohistochemical analysis showed that the level of active caspase-3 protein expression in the placenta in the death group was much higher than that in the survival group. The level of Bcl-2 protein expression in the placenta in the death group was much lower than that in the survival group. Western blot analysis demonstrated that increased expression of the active form of caspase-3 was detected in the placenta and decidua in the death group compared with that in the survival group. In contrast, a decrease in the expression of Bcl-2 was detected in the placenta and decidua in the death group compared with that in the survival group. Enhanced expression of Bax:Bcl-2 ratio was detected in placenta and decidua in the death group compared with that in the survival group. Thus, significantly increased apoptosis in the mouse placenta and decidua might be involved in the pathophysiologic mechanism of intrauterine fetal death.     

Expression of Fas/Fas ligand by decidual leukocytes in hydatidiform mole

Complete hydatidiform moles are entirely paternally derived and, therefore, represent a complete intrauterine allograft that might be expected to provoke an altered maternal immune response compared with that of normal pregnancy. Uterine decidua contains a large leukocyte population, of which 10%-20% are T lymphocytes. Fas ligand (FasL) expression by placental trophoblast may induce apoptosis of Fas+ lymphocytes, thereby facilitating immune tolerance and survival of the molar trophoblast. Our previous studies have shown an increase in activated CD4+ decidual T cells in molar pregnancy compared with normal pregnancy. This study was designed to characterize and quantitate Fas/FasL expression by decidual leukocytes in complete and partial hydatidiform mole compared with that in normal early pregnancy using single and double immunohistochemical labeling (i.e., avidin-biotin-peroxidase and avidin-biotin-alkaline phosphatase). A significant increase was found in Fas and FasL expression by decidual CD4+ T cells in complete (Fas+, P = 0.0106; FasL+, P = 0.0081) and partial (Fas+, P = 0.0131; FasL+, P = 0.0051) hydatidiform moles, as was a significant decrease in Fas expression by decidual CD8+ T cells in complete (P = 0.0137) and partial (P = 0.0202) hydatidiform mole compared with normal early pregnancy. The implications of altered Fas/FasL status of decidual T-cell subsets in hydatidiform mole are also discussed.     

Expression and function of PDCD1 at the human maternal-fetal interface

The failure to reject the semiallogenic fetus by maternal T lymphocytes suggests that potent mechanisms regulate these cells. PDCD1 is a CD28 family receptor expressed by T cells, and its ligand CD274 is strongly expressed by trophoblast cells of the human placenta. In this study, we examined whether human maternal T cells express PDCD1. Immunofluorescence examination of uterine tissues revealed PDCD1 expression on CD3+ cells was low in nonpregnant endometrium but increased in first-trimester decidua and remained elevated in term decidua (P < 0.05). In addition, higher relative proportions of term decidual CD8 bright, CD4+, and regulatory T cells expressed PDCD1 in comparison to autologous peripheral blood (P < 0.05). Term decidual T cells also expressed full-length and soluble PDCD1 mRNA isoforms more abundantly than their peripheral blood counterparts (P 相似文献   

Induction of cytotoxic T-lymphocyte antigen-2beta, a cysteine protease inhibitor in decidua: a potential regulator of embryo implantation

During early pregnancy, the steroid hormone progesterone induces differentiation of uterine stroma to decidual cells, which regulate embryo-uterine interactions. The progesterone-induced signaling molecules that participate in the formation and function of decidua remain poorly understood. We recently utilized high-density oligonucleotide microarrays to identify several genes whose expression is markedly altered in pregnant uterus in response to RU486, a well characterized antagonist of the progesterone receptor (PR). Our study revealed that the gene encoding cytotoxic T-lymphocyte antigen-2beta (CTLA-2beta), a cysteine protease inhibitor, is expressed during PR-induced decidualization. The spatio-temporal expression of CTLA-2beta mRNA precisely overlapped with the decidual phase of pregnancy. Interestingly, administration of progesterone to estrogen-primed ovariectomized mice failed to induce CTLA-2beta expression. A concomitant artificial decidual stimulation was necessary to trigger this expression. Uteri of PR knockout mice failed to express this mRNA, even after a combined administration of steroid hormones and artificial stimulation. The uterine expression of CTLA-2beta was, therefore, dependent on PR as well as other unknown factor(s) associated with decidual response. To identify the molecular target(s) of CTLA-2beta,we analyzed its interaction with proteins present in soluble extracts prepared from day 7 pregnant uteri containing implanted embryos. A protein affinity strategy employing recombinant CTLA-2beta helped us to determine that cathepsin L, a cysteine protease, is one of its targets in the pregnant uterus. Consistent with this finding, expression of cathepsin L was detected in the giant trophoblast cells of the ectoplacental cone on day 7 of pregnancy. Collectively, our results support the hypothesis that expression of CTLA-2beta in the decidua may regulate implantation of the embryo by neutralizing the activities of one or more proteases generated by the proliferating trophoblast.     

Synergistic induction of apoptosis in primary rat decidual cells by INF-gamma and TNF

In the rat, in response to blastocyst implantation, stromal cells of the endometrium proliferate and differentiate into decidual cells, forming the decidua. After reaching its maximum development, the decidua undergoes regression. This phenomenon appears to be due to an active process involving apoptosis. As there is sparse knowledge concerning the mechanisms of induction of decidual cell death, the potential role of cytokines present in the uterine environment during pregnancy, such as tumor necrosis factor (TNF) and interferon-gamma (INF-gamma) was explored in primary cultures of rat decidual cells. The effects of these factors upon cellular viability, nuclear morphologic alterations, expression, and enzymatic activities of the effector caspases-3/7 were evaluated. The results obtained demonstrated that in contrast to TNF, which did not induce any alteration, INF-gamma and in association with TNF caused a decrease in cell viability and an increase in the appearance of apoptotic bodies in a time-dependent manner that was augmented in the co-presence of TNF. An increase in caspase-3/7 activities after 12 hr of TNF/INF-gamma treatment was also observed. These findings suggest that INF-gamma expressed in the uterine environment may play an important role in regulating apoptosis through potential synergistic mechanisms with TNF and thereby modulate decidual stability and regression during pregnancy.     

Maternal Smad3 deficiency compromises decidualization in mice

Transforming growth factor (TGF)‐β and activin, members of TGF‐β superfamily, are abundantly expressed in the endometrium and regulate decidualization of endometrial stroma. Smad2 and Smad3 are receptor‐regulated Smads (R‐Smads) that transduce extracellular TGF‐β/activin/Nodal signaling. In situ hybridization results showed that Smad3 was highly expressed in the decidual zone during the peri‐implantation period in mice. By using artificial decidualization, we found that Smad3 null mice showed partially compromised decidualization. We therefore hypothesized that Smad2 might compensate for the function of Smad3 during the process of decidualization. Smad2 was also highly expressed in the decidual zone and phosphorylated Smad2 was much more abundantly increased in the deciduoma of Smad3 null mice than for wild‐type (WT) mice. We further employed an in vitro uterine stromal cell decidualization model, and found that decidual prolactin‐related protein (dPRP) and cyclin D3, which are well‐known markers for decidual cells, were significantly down‐regulated in Smad3 null decidual cells, and were much more significantly reduced when the expression of Smad2 was simultaneously silenced by its siRNA (P < 0.05). However, the expression levels of dPRP and cyclin D3 remained the same when Smad2 was silenced in WT decidual cells. Collectively, these findings provide evidence for an important role of Smad3 in decidualization and suggest that Smad2 and Smad3 may have redundant roles in decidualization. J. Cell. Biochem. 113: 3266–3275, 2012. © 2012 Wiley Periodicals, Inc.     

Prostaglandin-mediated inactivation of natural killer cells in the murine decidua

Previous studies from this laboratory have demonstrated a large influx of null lymphocytes into the murine decidua during pregnancy. We had also shown that trophoblast cells of the murine placenta bear target structures recognized by NK cells. Since NK lineage cells belong to the null category of lymphocytes, we examined whether cells of this lineage appear in the murine decidua, and if so, whether their activity is locally regulated by NK suppressor cells. We further investigated the identity of the suppressor cells as well as their suppressor products. NK lineage cells, irrespective of their activation status, were identified morphologically in radioautographic preparations as the non-T, non-B (null) lymphocytes capable of binding YAC-1 lymphoma targets. NK activity of nucleated cells was measured with a 4-hr 51Cr-release assay against labeled YAC-1 targets. Studies with outbred CD1 mice, and to a smaller extent, inbred CBA mice revealed that the incidence of NK lineage cells remained fairly constant within the decidua throughout pregnancy, but their activity decreased steadily to negligible levels by Day 12-14 of gestation. This was found to result from an inactivation caused by NK-suppressor cells in the decidua. A mixing of Ficoll-Paque-separated nucleated cells of the decidua with normal splenic effector cells (at 1:1 ratio) led to a suppression of their NK activity tested immediately or after a 20-hr coculture. This suppression was MHC unrestricted. Suppressor cells were identified both in plastic nonadherent fraction highly enriched for typical decidual cells as well as in the plastic adherent fraction containing decidual cells and macrophages. Addition of indomethacin (10(-5) M), an inhibitor of prostaglandin synthesis, or anti PGE2 antibody, revived the NK activity in the mixed population, as well as in the decidua, suggesting a PGE2-mediated suppression. High levels of PGE2 were detectable in decidual cell supernatants with a sensitive radioimmunoassay. Addition of pure PGE2 (10(-7)-10(-6) M) but not PGF2 alpha (10(-6) M) during the NK assay or to the effector cells for a 20-hr period prior to the assay led to an inhibition of NK activity. These results reveal that NK cells appearing in the murine decidua are progressively inactivated by PGE2 produced by decidual cells and decidual macrophages.     

Apoptosis in uterine epithelium and decidua in response to implantation: evidence for two different pathways

During the initial steps of implantation, the mouse uterine epithelium of the implantation chamber undergoes apoptosis in response to the interacting blastocyst. With progressing implantation, regression of the decidual cells allows a restricted and coordinated invasion of trophoblast cells into the maternal compartment. In order to investigate pathways of apoptosis in mouse uterine epithelium and decidua during early pregnancy (day 4.5–7.0 post coitum), we have investigated different proteins such as TNFalpha, TNF receptor1, Fas ligand, Fas receptor1, Bax and Bcl2 as well as caspase-9 and caspase-3 using immunohistochemistry. To detect cells undergoing apoptosis the Tunel assay was performed. Immunoreactivity for TNFalpha as well as for TNF receptor1 was observed exclusively in the epithelium of the implantation chamber and the adjacent luminal epithelium from day 4.5 post coitum onwards. In the developing decidua the Fas ligand, but not the Fas receptor, was expressed. Bax and Bcl2 revealed a complementary expression pattern with Bax in the primary and Bcl2 in the adjacent decidual zone. Strong immunolabelling for the initiator caspase-9 was restricted to the decidual compartment, whereas caspase-3 expression characterized the apoptotic uterine epithelium. Only some caspase-3 positive decidual cells were found around the embryo which correlated to the pattern of Tunel staining. Taken together, the apoptotic degeneration of the uterine epithelium seems to be mediated by TNF receptor1 followed by caspase-3, whereas the very moderate regression of the decidua did not show the investigated death receptor, but Bax and Blc2 instead and in addition caspase-9, which indicates a different regulation for epithelial versus decidual apoptosis.     

Apoptosis and related proteins during parturition in prostaglandin F receptor-deficient mice

This study investigated whether apoptosis and related proteins are involved in parturition by comparative observation of FP-deficient mice without labor and wild type mice with vaginal delivery. We examined the expression of apoptosis, Fas, FasL, active caspase-3 and bcl-2 proteins in the amnion, placenta and decidua. DNA laddering in the amnion, placenta and decidua tissue did not significantly differ between FP-deficient and wild type mice on day 18 of pregnancy. Similar TUNEL staining results were found in all tissues of FP-deficient mice compared with those of wild type mice. A higher intensity of apoptotic cells was found in the decidua basalis. The index of TUNEL-positive cells were not significantly different in the amnion, placenta and decidua of FP-deficient mice compared with that of wild type mice on day 18 of pregnancy. Specific bands for Fas were clearly observed in the amnion, placenta and decidua tissue. FasL specific bands were observed in the placenta and decidua, but a few in amnion tissue. A great number of active caspase-3 specific bands were detected in decidua, while a few such bands were detected in the placenta and few bands in the amniotic tissue. Bands for bcl-2 were detected in the amnion, placenta and decidua tissue. The weakest band was in decidual tissue. Fas, FasL, active caspase-3, and bcl-2 specific bands did not show any significant differences between the two groups. These findings demonstrate that apoptosis, Fas, FasL, caspase-3, and Bcl-2 occur in mouse term placenta that is not involved in parturition.     

Uterine Notch2 facilitates pregnancy recognition and corpus luteum maintenance via upregulating decidual Prl8a2

The maternal recognition of pregnancy is a necessary prerequisite for gestation maintenance through prolonging the corpus luteum lifespan and ensuring progesterone production. In addition to pituitary prolactin and placental lactogens, decidual derived prolactin family members have been presumed to possess luteotropic effect. However, there was a lack of convincing evidence to support this hypothesis. Here, we unveiled an essential role of uterine Notch2 in pregnancy recognition and corpus luteum maintenance. Uterine-specific deletion of Notch2 did not affect female fertility. Nevertheless, the expression of decidual Prl8a2, a member of the prolactin family, was downregulated due to Notch2 ablation. Subsequently, we interrupted pituitary prolactin function to determine the luteotropic role of the decidua by employing the lipopolysaccharide-induced prolactin resistance model, or blocking the prolactin signaling by prolactin receptor-Fc fusion protein, or repressing pituitary prolactin release by dopamine receptor agonist bromocriptine, and found that Notch2-deficient females were more sensitive to these stresses and ended up in pregnancy loss resulting from abnormal corpus luteum function and insufficient serum progesterone level. Overexpression of Prl8a2 in Notch2 knockout mice rescued lipopolysaccharide-induced abortion, highlighting its luteotropic function. Further investigation adopting Rbpj knockout and DNMAML overexpression mouse models along with chromatin immunoprecipitation assay and luciferase analysis confirmed that Prl8a2 was regulated by the canonical Notch signaling. Collectively, our findings demonstrated that decidual prolactin members, under the control of uterine Notch signaling, assisted pituitary prolactin to sustain corpus luteum function and serum progesterone level during post-implantation phase, which was conducive to pregnancy recognition and maintenance.     

Decidual cells produce a heparin-binding prolactin family cytokine with putative intrauterine regulatory actions

Pregnancy in mice and rats is associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J-specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature.     

Expression of activins and TGF beta 1 and beta 2 RNAs in early postimplantation mouse embryos and uterine decidua.

The expression of the mesoderm inducing factors, activins and TGF beta s, was characterized in 5 1/2-9 1/2 day mouse embryos and implantation sites by in situ hybridization. Activin beta A RNA was not detected within the embryo, but is expressed in nearby decidual cells from 5 to 7 days. Thus activin A could play a role within the embyro during gastrulation. Activin beta A is also expressed in more mesometrially located decidual cells from 6 to 9 1/2 days. Activin beta B and inhibin alpha RNAs were not detected, while a control tissue was highly positive. TGF beta 1 is expressed in the secondary decidual zone and in developing endothelial cells in the decidua and embryo. TGF beta 2 is expressed in the mesometrial decidua at 6 1/2 days and in the midline of the cranial neural plate.     

Differential regulation of colony stimulating factor 1 and macrophage migration inhibitory factor expression by inflammatory cytokines in term human decidua: implications for macrophage trafficking at the fetal-maternal interface

Macrophages are a major component of the leukocyte population of human pregnant endometrium. Although several crucial functions have been ascribed to these cells, the mechanisms underlying macrophage trafficking in the placental bed are poorly understood. The aim of this study was to evaluate the in vivo expression of two potentially antagonistic macrophage-targeting chemokines, colony stimulating factor 1 (CSF1, also known as M-CSF) and macrophage migration inhibitory factor (MIF), in term decidua, and to examine the effects of the inflammatory cytokines tumor necrosis factor (TNF, also known as TNF alpha) and interleukin 1beta (IL1B) on CSF1 and MIF expression in cultured decidual cells. The expression of CSF1 and MIF in term decidua was evaluated by immunohistochemistry. Cultured decidual cells were primed with estradiol (E2) or with E2+medroxyprogesterone acetate (MPA), and then incubated with corresponding steroid(s) with or without TNF or IL1B. The levels of CSF1 and MIF protein and mRNA were assessed by ELISA and quantitative RT-PCR, respectively. Immunostaining for CSF1 and MIF was observed in term decidua. The levels of secreted CSF1 and MIF were similarly unchanged whether the decidual cells were incubated with E2 or with E2+MPA. The CSF1 levels significantly increased in cultures exposed to E2 or E2+MPA plus TNF or IL1B. In contrast, the MIF levels in TNF- and IL1B-treated cells were not changed significantly from the control cultures. The ELISA data were confirmed by quantitative RT-PCR analysis. These results indicate that CSF1 and MIF are involved in regulating macrophage trafficking at the fetal-maternal interface, and suggest a mechanism by which inflammatory cytokines influence pregnancy by regulating decidual macrophage infiltration.