Expression of Eph A molecules during swine embryo implantation

Eph–Ephrin system can induce repulsive forces in cell migration and adhesion during embryonic development in various mammals. In this study, the attachment sites of swine endometrium during pregnancy were used and the physiological role of this system in the step of mammalian embryo implantation was estimated to investigate the involvement of the Eph–Ephrin system in swine embryo implantation. Real-time quantitative PCR indicated that mRNA expression of Eph A1 on endometrium increased extremely significantly around the implantation period (P < 0.01), while expression of Eph A2 and A4 decreased significantly during this period (P < 0.05). Immunostaining showed that protein expression of Eph A1, A2 and A4 in the endometrial stroma underlying the luminal epithelium was higher during mid-implantation compared with early or post-implantation. Western blotting examination demonstrated that protein expression of Eph A1, A2 and A4 at the attachment sites of swine endometrium increased from pregnancy day 13 to 18 (P < 0.01), and then decreased from pregnancy day 18 to 24 (P < 0.01). These findings suggest that the Eph–Ephrin A system might play an important role in regulating the swine contact between blastocysts and endometrium during embryo implantation.     

A Novel Role of IGFBP7 in Mouse Uterus: Regulating Uterine Receptivity through Th1/Th2 Lymphocyte Balance and Decidualization

Previously we have screened out Insulin-like Growth Factor Binding Protein 7 (IGFBP7) as a differentially expressed gene in post-implantation uterus versus pre-implantation uterus by suppressive subtractive hybridation. However its function in uterus was not clearly identified. In this research, the expression and function of IGFBP7 during post-implantation were studied. We found that IGFBP7 was mainly located in the glandular epithelium and the stroma, and was upregulated after embryo implantation. The vector pCR3.1-IGFBP7-t expressing partial IGFBP7 was constructed. Inhibition of IGFBP7 by specific DNA immunization induced significant reduction of implanted embryos and pregnancy rate. The number of implanted embryos (5.68±0.46) was significantly reduced after immunization with pCR3.1-IGFBP7-t, as compared with that of the mice immunized with the control vector (12.29±0.36) or saline (14.58±0.40) (p<0.01). After specific inhibition of IGFBP7, the T helper type 1 (Th1) cytokine IFNγ, was significantly elevated (p<0.05) and the Th2 cytokines IL-4 and IL-10, were reduced in uteri (p<0.05). The increase of Tbet and the decrease of Gata3 were found in mice peripheral lymphocytes by flow cytometry. The expression of decidualization marker IGFBP1 and angiogenesis regulator VEGF were declined in uteri (p<0.05). The expression of apoptosis-associated proteins, caspase3 and Bcl-2, were also declined (p<0.05). These results showed that inhibition of IGFBP7 induced pregnancy failure by shifting uterine cytokines to Th1 type dominance and repressing uterine decidualization.     

Alkaline Phosphatase Protects Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women.     

Alteration in some pro and anti-inflammatory cytokines associated with complete and incomplete gestation cycle of cows

The maternal immune response during pregnancy is regulated by a complex array of pro and anti-inflammatory cytokines which provide optimum conditions for the embryo implantation and survival. The possible role of pro and anti-inflammatory cytokines during the complete gestation cycle of ruminants and the difference in their circadian rhythmicity between successful and unsuccessful pregnancies is still unknown. To study this, blood samples were collected from three groups of cows, pregnant (P), non-pregnant (NP) and aborted (ABORT) cows starting from the day of Artificial Insemination (AI) till calving in P cows and till stages of non pregnancy in other cows. Various pro and anti-inflammatory cytokines were estimated by bovine-specific ELISA test and compared. Successful pregnancies had lower levels of pro-inflammatory cytokines (IL-2, IL-6 and IL-8) but higher anti-inflammatory cytokine (IL-10) mainly during implantation. Significant (p < 0.05) increase in pro-inflammatory cytokines and low IL-10 levels was noticed at abortion and at calving. This study indicates that temporal and spatial aspects of reducing the release of various pro-inflammatory cytokines and maintaining high anti-inflammatory cytokines during pregnancy are essentially required for maintenance of pregnancy. Any disturbance in the cytokine equilibrium may lead to persistent inflammation and pregnancy failure.     

Progesterone and the immunology of pregnancy

The foetal–placental unit is a semi-allograft and the immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. This recognition of pregnancy results in an upregulation of progesterone receptors on activated lymphocytes amongst placental cells and decidual CD56+ cells. In the presence of sufficient progesterone, these cells synthesise progesterone induced blocking factor (PIBF), a mediator that exerts substantial anti-abortive activities. PIBF affects B cells and induces an increased production of asymmetric, non-cytotoxic antibodies. It also alters the profile of cytokine secretion by activated lymphocytes resulting in an increase in the production of non-inflammatory, non-cytotoxic interleukins (IL) (e.g. IL-3, IL-4 and IL-10) and a reduction in the production of inflammatory, cytotoxic cytokines (e.g. interferon (IFN)-δ, tumour necrosis factor (TNF)- and IL-2). PIBF also inhibits the cytotoxity of natural killer (NK) cells by blocking their degranulation and perforin release, as well as inhibiting IFN-δ, TNF- and IL-2-mediated transformation of NK cells into detrimental lymphokine activated killer (LAK) cells.     

The progesterone derivative dydrogesterone abrogates murine stress-triggered abortion by inducing a Th2 biased local immune response

Stress is known to induce abortions in mice and humans, putatively via increased levels of abortogenic Th1 cytokines and a decrease of progesterone. Adequate levels of progesterone exert an antiabortive response through binding to the progesterone-receptor, which induces the release of progesterone-induced blocking factor (PIBF) from lymphocytes. PIBF is highly pregnancy-protective by induction of a Th2 biased immune activity. The aim of this study was to investigate the effect of the progesterone derivative dydrogesterone (6-dehydro-retroprogesterone) in stress-triggered murine abortion. DBA/2J-mated CBA/J female mice were randomized in different groups: two groups were treated with different dydrogesterone dosages in a single injection before exposure to sound stress on Day 5 of pregnancy, one group was exposed to stress without dydrogesterone treatment, the fourth group received no stress and no dydrogesterone. On gestation Day 13, a highly elevated abortion rate was detected in stressed mice compared to control mice. Stressed animals presented lower levels of progesterone and PIBF in plasma and a reduced staining intensity of progesterone receptor at the feto-maternal interface. Injection of dydrogesterone abrogated the effect of stress on the abortion rate. Further, dydrogesterone increased levels of plasma PIBF in stressed mice, but did not affect progesterone levels. Interestingly, dydrogesterone dramatically increased the percentage of IL-4 positive decidual immune cells in stressed mice. Our data suggest that dydrogesterone abrogates stress-triggered abortion by inducing a Th2 biased local immune response.     

Cytokine production in Linomide-treated nod mice and the potential role of a Th (1)/Th(2) shift on autoimmune and anti-inflammatory processes

Linomide prevents the development of autoimmune insulitis and insulin-deficient diabetes mellitus in female NOD mice. Linomide prevents development of autoimmune manifestations in other experimentally induced and spontaneous autoimmune diseases as well, but the mechanism of action is unknown. The present report summarizes our investigations on the effect of Linomide on different functional T cell subsets in NOD mice analyzed according to their cytokine profile. Supernatants from cultured splenocytes and peritoneal cells taken from Linomide-treated mice contained lower levels of TNFalpha, IL-1 beta, IFN gamma and IL-12 versus higher levels of IL-4, IL-6 and IL-10 in comparison with supernatants from cultures of untreated mice. Our results suggest that regulation of autoimmunity following oral Linomide administration in NOD mice induces a shift from Th(1) to Th(2) phenotype response, thereby preventing the development of diabetes by active cytokine-induced immunoregulation of T cell subsets, including downregulation of Th(1) and upregulation of Th(2).     

Loss of cyclooxygenase-2 retards decidual growth but does not inhibit embryo implantation or development to term

Previous reports have described that female mice deficient in cyclooxygenase-2 (COX2) are largely infertile because of failure to ovulate, poor fertilization, and defective implantation and decidualization. In the present study, we reinvestigated reproduction in these mice and found they do show a reduction in the numbers of ovulated and fertilized eggs. However, we did not observe any substantial effect on embryo implantation frequencies or an inability of COX2-deficient females to support embryo development to weaning. Pseudopregnant COX2-null recipients do not show any alteration in the timing of implantation following blastocyst transfer, but they do show a delay in the initial rate of decidual growth after implantation that lags by approximately 24 h compared to that in heterozygous or wild-type recipients. These results support previous findings that COX2 has a role in mediating the initial uterine decidual response but is not essential to sustaining decidual growth and embryo development throughout the remainder of pregnancy.     

梅山猪胚胎附植期EphB2的组织表达及RNA-seq分析

猪产仔数是一个重要的经济和繁殖性状, 而胚胎附植是影响猪产仔数的重要因素。为了研究促红细胞生成素产生肝细胞受体B2 (EphB2)对猪胚胎附植过程中子宫内膜迁移和粘附活动的影响, 文章以太湖流域梅山猪为研究对象, 利用实时荧光定量PCR (qRT-PCR)和蛋白免疫印迹(Western blot)方法, 检测EphB2基因在梅山猪胚胎附植前、中、后期子宫内膜附植点/非附植点和卵巢组织的mRNA和蛋白表达谱, 并用转录组测序(RNA-seq)方法分析了不同附植时期子宫内膜附植点和卵巢组织的差异表达基因。qRT-PCR和Western blot结果表明, EphB2在胚胎附植前、中、后期子宫内膜附植点和非附植点的mRNA和蛋白均呈现先升高后降低的表达趋势, 且附植中期的表达量显著高于前期和后期(P<0.01);EphB2在附植前、中、后期卵巢中的mRNA和蛋白表达趋势则相反, 为先降低后升高, 且不同时期间表达差异显著(P<0.05)。RNA-seq结果表明, EphB2在子宫内膜附植点的mRNA表达, 附植中期极显著高于附植前期(P<0.01);EphB2在卵巢的mRNA表达, 附植中期显著高于附植后期(P<0.05)。综上所述, EphB2很可能在猪胚胎附植过程中发挥着重要的调控作用, 为潜在的猪产仔数性状候选基因。     

Embryonic resorption in context to intragestational corpus luteum regression: A longitudinal ultrasonographic study in the European brown hare (Lepus europaeus PALLAS, 1778)

Embryonic resorption is frequently observed in polytocous mammals. Often it occurs as partial litter resorption affecting only single conceptuses of a whole litter. The aim of the study was to describe the incidence and morphology of embryonic resorption in the European brown hare (Lepus europaeus). In 154 pregnancies viable conceptuses, conceptuses undergoing resorption and CL of pregnancy were ultrasonographically monitored during the entire gestation period. Resorptions were classified into (1) “pre-implantation resorptions,” (2) “peri-implantation resorptions,” and (3) “post-implantation resorptions.” The incidence of resorption in the pre-implantation period was 9%, in the peri-implantation period 9%, and in the post-implantation period 24%. Post-implantation resorptions were found up to late pregnancy stages when fetal development was already in progress. The highest daily incidence of resorption was on Day 8 of the 42-day pregnancy. In 91% of the cases, the regression of one CL was observed, while an embryo was undergoing resorption at the same time. The number of resorptions did not significantly differ from the number of CL in regression during gestation, suggesting an interesting one-resorption-to-one-regression relationship. The ultrasonographic appearance of the luteal regression during pregnancy was similar to the morphology characteristic for postpartal luteolysis.     

Autotaxin–lysophosphatidic acid–LPA3 signaling at the embryo‐epithelial boundary controls decidualization pathways

During pregnancy, up‐regulation of heparin‐binding (HB‐) EGF and cyclooxygenase‐2 (COX‐2) in the uterine epithelium contributes to decidualization, a series of uterine morphological changes required for placental formation and fetal development. Here, we report a key role for the lipid mediator lysophosphatidic acid (LPA) in decidualization, acting through its G‐protein‐coupled receptor LPA₃ in the uterine epithelium. Knockout of Lpar3 or inhibition of the LPA‐producing enzyme autotaxin (ATX) in pregnant mice leads to HB‐EGF and COX‐2 down‐regulation near embryos and attenuates decidual reactions. Conversely, selective pharmacological activation of LPA₃ induces decidualization via up‐regulation of HB‐EGF and COX‐2. ATX and its substrate lysophosphatidylcholine can be detected in the uterine epithelium and in pre‐implantation‐stage embryos, respectively. Our results indicate that ATX–LPA–LPA₃ signaling at the embryo‐epithelial boundary induces decidualization via the canonical HB‐EGF and COX‐2 pathways.     

Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.     

Uterine NK cells mediate inflammation-induced fetal demise in IL-10-null mice

Specialized NK cells are recruited in high numbers to the mammalian embryo implantation sites, yet remain pregnancy compatible. It is not well understood whether uterine NK (uNK) cells become adversely activated and mediate fetal demise, a common complication of early pregnancy. In this study we show that mating of IL-10(-/-) mice resulted in fetal resorption or intrauterine growth restriction in response to very low doses of LPS. Pregnancy in congenic wild-type mice was normal even at 10-fold higher LPS doses. Fetal resorption in IL-10(-/-) mice was associated with a significant increase in uNK cell cytotoxic activation and invasion into the placenta. Depletion of uNK cells, TNF-alpha neutralization, or IL-10 administration rescued pregnancy in LPS-treated IL-10(-/-) animals. Our results identify an immune mechanism of fetal demise involving IL-10 deficiency, NK cells, and inflammation. These results may provide insight into adverse pregnancy outcomes in humans.     

Expression and regulation of integrin receptors in human trophoblast cells: role of estradiol and cytokines

Embryo implantation and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-cell communication amongst the implanting blastocyst and the receptive endometrium through integrins, a large family of proteins involved in the attachment, migration, invasion and control of cellular functions. Integrins display dynamic temporal and spatial patterns of expression by the trophoblast cells during early pregnancy in humans. However, the precise mechanism of embryo implantation and the modulation of the integrin receptors during blastocyst attachment and further implantation remain elusive in the humans. The present study elucidates the expression and hormonal modulation of fibronectin, vitronectin and laminin integrin receptors by estradiol and IL-1alpha in human trophoblast cells. Human first trimester trophoblast cells showed the induction of the classical estrogen receptor (ER)-alpha by its own ligand, estradiol. Treatment with either estradiol or IL-1alpha induced the expressions of alpha4, alpha5, alpha6 and alpha(v) integrin receptor subunits at both the mRNA and protein levels, while expression of beta1 remained unaltered. Furthermore, estradiol upregulated the expression of IL-1alpha, thereby suggesting the possibility that estrogen may either directly or via the proinflammatory cytokine induces the expression of the cell surface integrin receptors. The findings delineate the role of hormones and the cytokines in modulating the adhesiveness and attachment of the trophoblast cells. This may reflect the in vivo scenario where the implanting embryo is surrounded by a hormone-cytokine rich uterine microenvironment that may precisely regulate the expression of integrins and thereby facilitate implantation.     

Transforming growth factor-β and Th17 responses in resistance to primary murine schistosomiasis mansoni

Discovery of the T-helper (Th) 17 cell lineage and functions in immune responses of mouse and man prompted us to investigate the role of transforming growth factor-beta (TGF-β) and interleukin (IL)-17 in innate resistance to murine schistosomiasis mansoni. Schistosoma mansoni-infected BALB/c and C57BL/6 mice were administered with recombinant TGF-β or mouse monoclonal antibody to TGF-β to evaluate the impact of this cytokine on host immune responses against lung-stage schistosomula, and subsequent effects on adult worm parameters. Developing schistosomula elicited increase in peripheral blood mononuclear cells (PBMC) mRNA expression and/or plasma levels of IL-4, IL-17, and interferon-gamma (IFN-γ), cytokines known to antagonize each other, resulting in impaired Th1/Th2, and Th17 immune responses and parasite evasion. Mice treated with TGF-β showed elevated PBMC mRNA expression of IL-6, IL-17, TGF-β, and TNF-α mRNA and increased IL-23 and IL-17 or TGF-β plasma levels, associated with significantly (P < 0.02–<0.0001) lower S. mansoni adult worm burden compared to controls in both mouse strains, thus suggesting that TGF-β led to heightened Th17 responses that mediated resistance to the infection. Mice treated with antibody to TGF-β showed increase in PBMC mRNA expression and plasma levels of IL-4, IL-12p70, and IFN-γ, and significantly (P < 0.02 and <0.0001) reduced worm burden and liver worm egg counts than untreated mice, indicating that Th1/Th2 immune responses were potentiated, resulting in significant innate resistance to schistosomiasis. The implications of these observations for schistosome immune evasion and vaccination were discussed.     

Estrogen and progesterone regulate the IL-6 signal transduction pathway in antibody secreting cells

Regulation of the immune response is necessary to allow successful pregnancy. Asymmetric IgG antibodies are involved in fetal maintenance. We have previously demonstrated that estrogen (E2) and progesterone (P4) modulate the synthesis of asymmetric antibodies but the underlying mechanisms remain unclear. Since IL-6 and a progesterone-induced blocking factor (PIBF) were shown to regulate asymmetric antibody synthesis, in this work we analyzed whether E2 and P4 were able to modulate IL-6 signal transduction pathways and the ability of P4 to induce PIBF synthesis, in hybridoma B cells was also evaluated. We found that the IL-6 treatment induced an increase in the expression of gp130 and JAK1 by the hybridoma. E2 and P4 diminished the IL-6-induced gp130 expression in a dose-dependent manner, whereas the expression of JAK1 was not significantly affected. At 10(-6)M concentration, the steroids inhibited the phosphorylation of gp130 and diminished the IL-6-induced STAT3 phosphorylation and traslocation to the nucleus. Maximal PIBF expression was observed when the hybridoma was cultured with 10(-10)M P4, compared to the control (p<0.05). Results demonstrate two molecular mechanisms, the modulation of the IL-6R signal transduction pathway and PIBF induction, which could be involved in the immunoregulatory role of sexual steroids during pregnancy.     

Development of a Novel Model for the Assessment of Dead-Space Management in Soft Tissue

Following extensive surgical debridement in the treatment of infection, a “dead space” can result following surgical closure that can fill with hematoma, an environment conducive to bacterial growth. The eradication of dead space is essential in order to prevent recurrent infection. This study describes a novel small animal model to investigate dead-space management in muscle tissue. Two absorbable test materials were implanted in each animal; beads of calcium sulfate alone, and beads loaded with vancomycin and tobramycin. In-life blood samples and radiographs were taken from each animal following implantation. Animals were sacrificed at 1, 7, 21, 42, and 63 days post-operatively (n = 4), and implant sites were analysed by micro-computed tomography, histology and immunohistochemistry. Complete resorption was confirmed radiographically at 3 weeks post-implantation. Histologically, the host tissue response to both materials was identical, and subsequent healing at the implant sites was observed with no dead space remaining. Vancomycin was not detected in blood serum. However, peak tobramycin levels were detected in all animals at 6 hours post-implantation with no detectable levels in any animals at 72 hours post implantation. Serological inflammatory cytokine expression for IL-6, TNF-α and IL-1β indicated no unusual inflammatory response to the implanted materials or surgical procedure. The model was found to be convenient and effective for the assessment of implant materials for management of dead space in muscle tissue. The two materials tested were effective in resolving the surgically created dead space, and did not elicit any unexpected adverse host response.     

Matrix metalloproteinase-9 deficiency leads to prolonged foreign body response in the brain associated with increased IL-1β levels and leakage of the blood-brain barrier

Matrix metalloproteinases (MMPs) are enzymes with specificity towards extracellular matrix (ECM) components. MMPs, especially MMP-9, have been shown to degrade components of the basal lamina and disrupt the blood-brain barrier (BBB) and thus, contribute to neuroinflammation. In the present study we examined the role of MMP-9 in the foreign body response in the brain. Millipore filters of mixed cellulose ester were implanted into the brain cortex of wild type and MMP-9-null mice for a period of 2 d to 8 wks and the response was analyzed by histology and immunohistochemistry. We observed enhanced and prolonged neuroinflammation in MMP-9-null mice, evidenced by persistence of neutrophils, macrophages/microglia, and reactive astrocytes up to 8 wks post-implantation. In addition, blood vessel density around implants was increased in MMP-9-null mice and detection of mouse serum albumin (MSA) indicated that vessels were leaky. Immunohistochemical and western blot analyses indicated that this defect was associated with the absence of tight junction proteins zonula occludens-1 (ZO-1) and ZO-2 from vessels in proximity to implants. Analysis of brain sections and brain protein extracts revealed that the levels of the pro-inflammatory cytokine interleukin-1β (IL-1β), which is a substrate for MMP-9, were significantly higher in MMP-9-null mice at 8wks post-implantation. Collectively, our studies suggest that increased levels of IL-1β and the delayed repair of BBB are associated with prolongation of the FBR in MMP-9-null mice.     

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice,and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross‐talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G‐protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G‐protein activation, plays an important role in embryo implantation by interfering in the cross‐talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17β‐estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17β‐estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17β‐estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.