Implantation-associated gene-1 (Iag-1): a novel gene involved in the early process of embryonic implantation in rat.

BACKGROUND: In order to study the novel genes related to rat embryonic implantation, a novel implantation-associated gene, Iag-1, was identified and characterized from rat uterus of early pregnancy. Iag-1 was initially derived from suppressive subtracted hybridization of a cDNA library of rat uterus, which was used to analyse differentially expressed genes between the preimplantation and implantation period. METHODS: The full-length cDNA sequence of Iag-1 was cloned from rat uterus on D5.5 of pregnancy by 5'- and 3'-RACE. The expression of Iag-1 in the uterus of early pregnancy, pseudopregnancy, artificial decidualization and activation of delayed implantation was detected by northern blotting, in situ hybridization, western blotting and immunofluorescence. Endometrial stromal cells (ESCs) were isolated from rat uterus. The effect of Iag-1 on ESCs proliferation and apoptosis were determined by MTT assay, TUNEL and Hoechst staining. Apoptosis-related proteins in ESCs were detected by western blotting. RESULTS: Differential patterns of Iag-1 expression were detected in rat embryo and in the uterus during the peri-implantation period. Iag-1 was specifically localized in glandular epithelium and luminal epithelium. In contrast, the expression of Iag-1 was not significantly altered in uterus of pseudopregnancy and artificial decidualization, but was significantly increased in the uterus after activation of delayed implantation. Stable expression of introduced Iag-1 inhibited the proliferation of in vitro-cultured ESCs. Significant apoptosis was also detected in the ESCs overexpressing Iag-1, along with the enhancement of p53 and Bax protein expression. CONCLUSIONS: Overexpression of Iag-1 can inhibit ESCs proliferation and induce ESCs apoptosis, and p53 and Bax may play an important role in the process of Iag-1-induced apoptosis.

IFN-gamma promotes apoptosis of the uterus and placenta in pregnant rat and human cytotrophoblast cells.

Growth and development of placentas in all pregnancy periods and that of fetuses in late pregnancy were inhibited after administration of interferon-gamma (IFN-gamma). Apoptosis can be detected by TUNEL at the maternal-fetal interface during normal rat pregnancy. Apoptosis locations at the maternal-fetal interface changed according to the period of pregnancy. The results of immunohistochemistry and the DNA ladder assay showed that IFN-gamma could promote the apoptosis levels during the entire pregnancy, but it did not change the apoptosis locations. IFN regulatory factor-1 (IRF-1), FasL, and p53 expressions were modulated by IFN-gamma during the entire pregnancy. In vitro cell proliferation assay indicated that IFN-gamma could inhibit proliferation of human cytotrophoblast cells, and flow assay showed that this effect was mainly due to apoptosis induction. TUNEL and Hoechst staining also showed that IFN-gamma could induce apoptosis of human cytotrophoblast cells. Expression of IRF-1 was induced and expression of active caspase-3 was promoted by IFN-gamma treatment, but IFN-gamma did not affect the expression of IFNGR and p53.

Effect of interferon-gamma treatment on the expression of interleukin-1beta at the maternal-fetal interface of pregnant rats.

In the present study, the possible mechanisms by which interferon (IFN)-gamma affects pregnancy were investigated using the cytokine network model. The IFN-gamma-induced expression of interleukin (IL)-1beta was examined using western blotting, immunohistochemistry and immunofluorescence. The results showed that IFN-gamma treatment significantly decreased the expression of uterine IL-1beta protein during the preimplantation, post-implantation and mid-gestation periods. The expression of IL-1beta protein was increased after IFN-gamma treatment compared with the control group in late pregnancy. In the placenta, IL-1beta protein levels were significantly increased after IFN-gamma treatment in early and mid-pregnancy. In late pregnancy, IFN-gamma treatment significantly decreased placental IL-1beta protein levels. IL-1beta was mainly expressed in the myometrium, uterine arteries, decidua basalis, trophospongium of the junctional layer and trophoblastic epithelium of the labyrinthine layers. IL-1beta was mainly located in the cytoplasm of in vitro cultured endometrial stromal cells (ESCs). IFN-gamma treatment did not affect the distribution of IL-1beta, only the expression of IL-1beta. The effects of IFN-gamma on the proliferation of ESCs were determined using an MTS (a novel tetrazolium compound) assay. IFN-gamma treatment inhibited the proliferation of ESCs and decreased the weight of the fetus and placenta. These results indicate that exogenous IFN-gamma affects the expression of IL-1beta and inhibits ESC proliferation.

Effects of IL-1 beta on RT1-A/RT1-DM at the maternal-fetal interface during pregnancy in rats.

Interleukin-1 (IL-1beta) beta and major histocompatibility complex (MHC) play an important role during pregnancy. Expression of non-classical class MHC II RT1-DM antigen and classical class MHC I RT1-A antigen induced by IL-1beta was examined by Northern blotting, Western blotting and immunohistochemistry. IL-1beta treatment significantly increased the expression of RT1-A and RT1-DM in early and mid pregnancy. In late pregnancy, expression of RT1-DM significantly increased in uteri and decreased in placenta. Immunohistochemical studies indicated that, in early pregnancy, RT1-DM protein mainly localized to luminal and glandular uterine epithelium, and RT1-A was present in deciduas basalis, outer layer of luminal epithelium and glandular epithelium. During mid and late pregnancy, RT1-DM was present in maternal blood vessels and syncytiotrophoblast of labyrinthine zone, and RT1-A was present in maternal blood vessels and trophoblastic epithelium of the labyrinthine layers. These findings show that exogenous IL-1beta affects expression of RT1-DM and RT1-A and does not affect the localization of corresponding molecules during pregnancy.

IFNgamma pretreatment sensitizes human choriocarcinoma cells to etoposide-induced apoptosis.

Choriocarcinoma is a malignant trophoblast-derived tumour, which can arise in any type of gestation. Cell proliferation assays showed that interferon gamma (IFNgamma) alone significantly inhibited proliferation of choriocarcinoma JAR and JEG-3 cells. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assays and Hoechst staining indicated that IFNgamma alone could not induce apoptosis of JAR and JEG-3 cells, but IFNgamma could enhance the sensitivity of JAR cells to etoposide-induced apoptosis. RT-PCR and western blotting were performed to detect expression of apoptosis-related molecules IFNgammaR, interferon regulatory factor-1 (IRF-1), p53 and pro-caspase 3. In JAR cells, etoposide increased expression of the proteins including IFNgammaR, p53 and pro-caspase 3 as well as IRF-1 mRNA and IFNgamma-pretreatment apparently promoted up-regulation of these molecules expression. In addition, the responses of IRF-1, p53 and pro-caspase 3 expression to IFNgamma pretreatment were dose dependent. IRF-1 knock down assays demonstrated that IRF-1 directly mediated IFNgamma pretreatment enhanced sensitivity of JAR cells to etoposide-induced apoptosis and that pro-caspase 3 was one of the target genes of IRF-1.

The expression of membrane protein augments the specific responses induced by SARS-CoV nucleocapsid DNA immunization.

Nucleocapsid protein plays a critical role in SARS-CoV pathogenesis, and high-level anti-nucleocapsid antibodies are detected in the patients infected by severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Several studies have shown that there exists an interaction between nucleocapsid (N) and membrane (M) protein. In this paper, we investigate whether the expression of membrane protein can affect the immune responses induced by nucleocapsid DNA immunization. Two recombinant plasmids containing M and N coding sequence were constructed. Moreover, in order to get the antigen for ELISA and in vitro stimulation assay, N protein were expressed and purified from E. coli bacteria. Injection of 20mug of the mixture of pVAX1-M and pVAX1-N into the Balb/c mice could elicit the humoral and cellular responses. The ELISA analysis using the N antigen or inactivated SARS-CoV particles as capture antigen showed that co-injection of SARS-M could enhance N-induced antibody production, especially IgG2a subclass. After lymphocytes were stimulated with 10mug/ml purified N antigen, The CD4+ and CD8+ T cells of N and M plus N group were increased compared with those of control groups, and the M protein could augment the activation of lymphocytes induced by N DNA vaccine. Cytokine ELISA analysis revealed that co-injection of M could enhance the levels of IFN-gamma, IL-2 release induced by N antigen. Further experiments in field mouse also support the claim that membrane protein can augment the N-specific immune responses. Virus challenge test was conducted in BSL3 bio safety laboratory with Brandt's vole SARS-CoV model, and the results indicated that co-immunization of M and N antigens could reduce the mortality and pathological changes in lung from the virus infection.

Effect on expression of RT1-A and RT1-DM molecules of treatment with interferon-gamma at the maternal--fetal interface of pregnant rats.

BACKGROUND: Recent studies suggest a role for interferon-gamma (IFN-gamma) in the pregnancy process. METHODS: The expression of non-classical class II major histocompatibility complex (RT1-DM) antigens and classical class I major histocompatibility complex (RT1-A) antigens induced by IFN-gamma was examined by reverse transcription-PCR, western blotting and immunohistochemistry. RESULTS: IFN-gamma treatment increased expression of RT1-DM and RT1-A during early pregnancy and decreased them during mid pregnancy at the maternal-fetal interface. In late pregnancy, expression of RT1-A decreased in placenta and increased in uterus, and RT1-DM increased in both placenta and uterus with IFN-gamma treatment compared with untreated controls. Immunohistochemical studies suggested that in early pregnancy, RT1-DM protein mainly localized to uterine luminal epithelium and glandular epithelium, and RT1-A mainly localized to decidual blood vessels and decidua basalis. During mid and late pregnancy, RT1-A mainly localized in decidual blood vessels and spongiotrophoblast cells of the junction zone. RT1-DM mainly localized in blood vessels and the labyrinthine zone during mid and late gestation. CONCLUSIONS: RT1-A and RT1-DM can both be expressed at the maternal-fetal interface during normal pregnancy. Their localizaion changed according to the period of pregnancy. IFN-gamma can modulate the expression of these two molecules during the whole pregnancy.

[Research progress on regulation of class II MHC expression].

Class II MHC antigens play a critical role in the induction of immune responses through presentation of processed antigen to CD4+ T lymphocytes. The absence of MHC II normal expression results in severe primary immunodeficiency diseases, such as the bare lymphocyte syndrome (BLS). Four different MHC II regulatory genes have been identified. These genes encode RFXANK, RFX5, RFXAP and CIITA. The first three are subunits of RFX, a ubiquitously expressed factor that binds to the promoters of all MHC II genes. CIITA is the master control factor for MHC II expression. The highly regulated expression pattern of CIITA ultimately dictates the cell type specificity, induction and level of MHC II expression. This review focuses on research progress on regulation of class II MHC expression in recent years.

Effect of IFNgamma on caspase-3, Bcl-2 and Bax expression, and apoptosis in rabbit placenta.

The purpose of this study was to determine whether apoptosis in placenta was affected by IFNgamma, which can induce abortion, and whether the effect of IFNgamma on apoptosis resulted from an intrinsic program of apoptosis, which was regulated by Bcl-2 and Bax. DNA fragmentation analysis indicated that cleavage of DNA into 180 bp and its polymers were recognized in placenta in control and IFNgamma treated groups. Quantitative analysis of low molecular weight fragments of DNA revealed a significant increase in cases of 100,000 IU IFNgamma treatment compared with those in normal pregnancy (P<0.05). An analysis in situ revealed that apoptosis occurred predominantly in syncytiotrophoblast. Expression of Bcl-2 and Bax in placenta was evaluated by immunoblot analysis and immunohistochemistry study. Bcl-2 was expressed predominantly in syncytiotrophoblast, and was not expressed in cytotrophoblast of all cases. Whereas Bax was expressed in cytotrophoblast, syncytiotrophoblasts were found to be negative for Bax protein expression in all cases. Both Bcl-2 and Bax expression was decreased 0.44 fold and 0.46 fold by 50,000 IU IFNgamma and 0.41 fold and 0.03 fold by 100,000 IU IFNgamma. This resulted in change of a 0.07 fold increase in the Bax:Bcl-2 ratio in 50,000 IU IFNgamma treated groups and 0.41 fold increase in 100,000 IU IFNgamma treated groups as compared with those in control groups. The difference in Bax to Bcl-2 ratio between control and 100,000 IU IFNgamma treated groups was significant (P<0.05). The localization of caspase-3, the executioner of apoptosis, was detected in some cytotrophoblast and syncytiotrophoblast and increased 0.03 fold and 0.68 fold in 50,000 IU IFNgamma and 100,000 IU IFNgamma treated groups, respectively. There was significant difference between control and 100,000 IU IFNgamma treated groups (P<0.05). The results showed that high dose of IFNgamma administration increased the extent of apoptosis in placenta, the Bax to Bcl-2 ratio, and the activated caspase-3.