Interleukin-18 (IL-18) mRNA expression and localization of IL-18 mRNA-expressing cells in the mouse uterus

Interleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon-gamma inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and alpha subunit of IL-18 receptor mRNA were detected in the uterus. In the uterus, IL-18 mRNA levels increased during sexual maturation. In situ hybridization analysis demonstrated IL-18 mRNA-expressing cells in the mouse uterus of different ages. At 21 days of age, IL-18 mRNA-expressing cells were detected in the luminal epithelial cells and stromal cells although the IL-18 mRNA signal was weak. At 42 days of age, IL-18 mRNA signal was mainly detected in the stromal cells located near the myometrium, and in some of the luminal and glandular epithelial cells. In the uterus of 63-day-old adult mice, a strong hybridization signal for IL-18 mRNA was detected at estrus, but was weak at diestrus. IL-18 mRNA was mainly detected in the glandular epithelial cells and stromal cells. The effect of estradiol-17beta (E(2)) on IL-18 mRNA-expressing cells in the uterus was examined in ovariectomized mice. In oil-treated mice IL-18 mRNA signal was localized in luminal epithelial cells and stromal cells, while in E(2)-treated mice IL-18 mRNA signal was localized in stromal cells alone. These results suggest that the mouse uterus has an IL-18 system, and IL-18 exerts a physiological role within the uterus in a paracrine manner, and that IL-18 gene expression is regulated by estrogen.     

Progesterone and DNA damage encourage uterine cell proliferation and decidualization through up-regulating ribonucleotide reductase 2 expression during early pregnancy in mice

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.     

Expression of Ly-6A/E in the mouse uterus during implantation period

To investigate the molecular mechanisms of implantation, we constructed a complementary DNA library of mouse uterus enriched with pregnancy-induced genes by subtractive hybridization and polymerase chain reaction. One of the isolated clones was a part of complementary DNA for the Ly-6A/E. Ly-6A/E is reported to be differentially expressed on hematopoietic stem cells and some lymphoid and nonlymphoid tissues, mediate cell-cell adhesion on lymphoid cells, and associate with cell proliferation and angiogenesis of tumor cells. Northern blot, in situ hybridization, and immunohistochemical analyses demonstrated that the Ly-6A/E mRNA and protein were expressed in the endometrial epithelial cells as well as myometrial cells and vascular endothelial cells in the uterus of nonpregnant mouse. The expression was downregulated in luminal epithelial cells during pregnancy days 1-5, while it was upregulated in decidualized stromal cells around the implanted embryo at the time of implantation. The signals were primarily localized in stromal cells at the mesometrial pole on day 9. The increased expression was also observed in stromal cells of the embryo-transferred uterus and artificially-induced deciduoma, indicating that the expression of Ly-6A/E in the endometrial cells is concurrent with decidualization. These findings suggest that Ly-6A/E plays a role in embryo implantation.     

On the source of uterine 'luminal fluid' proteins in the mouse

Examination by light-, transmission electron- and scanning electron-microscopy showed that flushing the lumen of the mouse uterus with small volumes of fluid damaged the endometrium by rupturing and removing luminal epithelial cells, splitting the epithelial basement membrane and connective tissue stroma, and rupturing and leaching stromal cells and blood vessels. The damage increased with increasing progestation of the uterus and between Days 4 and 5 of pregnancy. I conclude that many so-called 'luminal fluid' proteins originate from luminal and stromal cells, intercellular fluid and blood and that apparent changes in luminal fluid protein content during early pregnancy may largely reflect alterations in the extent and type of damage produced by flushing, as a consequence of changes in the physical state of the uterus induced by hormones and the presence of blastocysts.     

Epithelial-mesenchymal interactions in uterus and vagina alter the expression of the cell surface proteoglycan, syndecan

The cell surface proteoglycan, syndecan, exhibits molecular and histological dimorphism in the mouse uterus and vagina. In the mature vagina, syndecan is localized at the surfaces of the basal and intermediate cells of the stratified epithelium and has a modal molecular mass of ca. 92 kDa. The uterus expresses a larger form of syndecan (ca. 110 kDa) which is detected at the basolateral surfaces of the simple columnar epithelial cells. We have investigated whether epithelial-mesenchymal interactions influence the expression of syndecan in these organs by analyzing tissue recombinants composed of mouse epithelium and rat mesenchyme or vice versa with monoclonal antibody 281-2, which recognizes mouse syndecan. In tissue recombinants composed of newborn mouse uterine epithelium and rat vaginal stroma, the uterine epithelium was induced to form a stratified vaginal epithelium which expressed syndecan in same the pattern and mass typical of vaginal epithelium. Likewise, rat uterine stroma induced newborn mouse vaginal epithelium to undergo uterine development, and this epithelium exhibited a uterine pattern of syndecan expression. Although stromal cells normally express little syndecan in most adult organs, analysis of recombinants composed of mouse stroma and rat epithelium revealed that both uterine and vaginal mouse stromata synthesized syndecan that was larger (ca. 170-190 kDa) than the epithelial syndecans. A quantitative increase in the amount of stromal syndecan was evident when stroma was grown in association with epithelium in comparison to stroma grown by itself. These data suggest that epithelial-mesenchymal interactions influence the amount, localization, and mass of both epithelial and stromal syndecan.     

Angiopoietin-like gene expression in the mouse uterus during implantation and in response to steroids

The purpose of this work was to determine if and where Angiopoietin-like genes are expressed in the mouse uterus during the implantation period of pregnancy and to determine if uterine expression of such genes is controlled by estrogen or progesterone. We found that all six known murine angiopoietin-like genes were expressed in the mouse uterus during implantation. The expression of four genes was controlled by either estrogen or progesterone. Only the levels of angiopoietin-like 4 (Angptl4) mRNA dramatically increased in implantation segments of the uterus during decidualization and was conceptus-independent. Due to this increased expression and the fact that angiopoietin-like 4 protein plays a role in lipid metabolism and angiogenesis in other tissues, only the expression of Angptl4 was further examined in the uterus and developing placenta. Angptl4 mRNA was localized to subpopulations of the endometrial stromal fibroblast and endothelial cell populations during decidualization. It was also localized to the ectoplacental cone, trophoblast giant cells and parietal endoderm of the conceptus at this time. By mid-pregnancy, Angptl4 mRNA was localized mainly to the mesometrial lymphoid aggregate region plus mesometrial endothelial cells of the uterus, as well as in various cell types of the conceptus. Additional work showed that Angptl4 expression increases in mouse endometrial stromal cells as they undergo decidualization in vitro. As in other cell types, the expression of Angptl4 in endometrial stromal cells was increased in response to an agonist of the peroxisome proliferator activated receptors. Taken together, the results of this work support the hypothesis that locally expressed Angptl4 might play a role in local uterine/placental lipid metabolism and vascular changes during implantation and thus provide a basis for future research.     

Cell-type-specific expression of transforming growth factor-alpha in the mouse uterus during the peri-implantation period.

Immunohistochemistry as well as in situ and Northern blot hybridization were employed to determine temporal and cell-type-specific expression of transforming growth factor-alpha (TGF-alpha) in the mouse uterus during the peri-implantation period. The co-localization of TGF-alpha (by immunohistochemistry) with its mRNA (by in situ hybridization) in the luminal and glandular epithelia on Days 1-4 of pregnancy (Day 1 = vaginal plug) and also in many stromal cells on Days 3 and 4 indicates that these cells are the primary sites of TGF-alpha synthesis during the preimplantation period. The higher levels of TGF-alpha mRNA in total uterine RNA on Day 4, as shown by Northern blotting, is consistent with the recruitment of stromal cells expressing this gene. During the post-implantation period (Days 5-8), the co-localization of the mRNA and protein in the decidua at the implantation sites suggests that the decidualizing stromal cells synthesize TGF-alpha. Although in situ hybridization showed the presence of mRNA in embryos on Days 5-8, immunostaining was noted in the embryo only on Days 5 and 6. These results suggest that uterine and embryonic expression of TGF-alpha during the peri-implantation period could be involved in embryonic development, preparation of the uterus for implantation, and decidualization.     

Osteopontin Is Expressed in the Mouse Uterus during Early Pregnancy and Promotes Mouse Blastocyst Attachment and Invasion In Vitro

Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.     

The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen

The activation function-1 (AF-1) domain of the estrogen receptor alpha (ERalpha) in stromal cells has been shown to be required for epithelial responses to estrogen in the mouse uterus. To investigate the role of the stroma in estrogenic responses of human uterine epithelium (hUtE), human/mouse chimeric uteri composed of human epithelium and mouse stroma were prepared as tissue recombinants (TR) that were grown in vivo under the renal capsule of female nude mouse hosts. In association with mouse uterine stroma (mUtS), hUtE formed normal glands surrounded by mouse endometrial stroma and the human epithelium influenced the differentiation of stroma into myometrium, such that a histologically normal appearing uterine tissue was formed. The hUtE showed a similar proliferative response and increase in progesterone receptors (PR) in response to 17beta-estradiol (E2) in association with either human or mUtS, as TRs. However, under identical endocrine and micro-environmental conditions, hUtE required 5-7 days exposure to E2 rather than 1 day, as shown for mouse uterine epithelium, to obtain a maximal proliferative response. Moreover, this extended length of E2 exposure inhibited mouse epithelial proliferation in the presence of mouse stroma. In addition, unlike the mouse epithelium, which does not proliferate or show regulation of PR expression in response to E2 in association with uterine stroma derived from mice that are null for the AF-1 domain of ERalpha, hUtE proliferates and PR are up-regulated in response to E2 in association genetically identical ERalpha knock-out mouse stromal cells. These results clearly demonstrate fundamental differences between mouse and human uterine epithelia with respect to the mechanisms that regulate estrogen-induced proliferation and expression of PR. Moreover, we show that genetically engineered mouse models could potentially aid in dissecting molecular pathways of stromal epithelial interactions in the human uterus.     

Bone morphogenetic protein 2 functions via a conserved signaling pathway involving Wnt4 to regulate uterine decidualization in the mouse and the human

A critical role of progesterone (P) during early pregnancy is to induce differentiation of the endometrial stromal cells into specialized decidual cells that support the development of the implanting embryo. The P-induced signaling pathways that participate in the formation and function of the decidual cells remain poorly understood. We report here that the expression of the bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFbeta superfamily, is induced downstream of P action in the mouse uterine stroma during decidualization. To determine the function of BMP2 during this differentiation process, we employed a primary culture system in which undifferentiated stromal cells isolated from pregnant mouse uterus undergo decidualization. When recombinant BMP2 was added to these stromal cultures, it markedly advanced the differentiation program. We also found that siRNA-mediated silencing of BMP2 expression in these cells efficiently blocked the differentiation process. Gene expression profiling experiments identified Wnt4 as a downstream target of BMP2 regulation in stromal cells undergoing decidualization. Attenuation of Wnt4 expression by siRNAs greatly reduced stromal differentiation in vitro, indicating that it is a key mediator of BMP2-induced decidualization. We also observed a remarkable induction in the expression of BMP2 in human endometrial stromal cells during decidualization in vitro in response to steroids and cAMP. Addition of BMP2 to these cultures led to a robust enhancement of Wnt4 expression and stimulated the differentiation process. Collectively, our studies uncovered a unique conserved pathway involving BMP2 and Wnt4 that mediates P-induced stromal decidualization in the mouse and the human.     

Effects of estrogen on the uterus of mice of different strains]

The strain differences in the response of uterus to the administration of exogenous oestrogen was studied. The uterus of mouse strain CBA appeared to be more susceptible than in strains C57B16 and C3HA, this manifested itself in a lower threshold for development of pseudoestrus and a more pronounced increase of uterine weight.     

Tissue inhibitor of matrix metalloproteinase-3 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation in mice, tissue inhibitor of matrix metalloproteinases-3 is believed to play a key role in inhibiting matrix metalloproteinase activity associated with embryo invasion and tissue remodeling. The first objective of this study was to quantitatively compare the steady-state mRNA levels of tissue inhibitors of matrix metalloproteinases between segments of the mouse uterus undergoing decidualization compared to those that are not during early pregnancy plus oil-induced decidualization. Steady-state tissue inhibitor of metalloproteinase-3 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 6 and 7 of pregnancy and in stimulated compared to nonstimulated uterine horns at 48 and 72 hr after artificial induction of decidualization. Steady-state tissue inhibitor of metalloproteinase-1 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 5-8 of pregnancy and in stimulated compared to nonstimulated uterine horns at 24, 48, and 72 hr after oil stimulation. Therefore, the steady-state mRNA levels of tissue inhibitors of metalloproteinase-1 and -3 increased in the uterus during decidualization. The second objective of this study was to determine if transforming growth factor-beta1 influences tissue inhibitors of metalloproteinase mRNA concentrations in mouse endometrial stromal cells. As determined by Northern blot analyses, transforming growth factor beta1 significantly increased tissue inhibitors of matrix metalloproteinases-1 and -3 mRNA levels in cultured mouse endometrial stromal cells isolated from uteri sensitized for decidualization. On the other hand, interleukin-1, epidermal growth factor, and leukemia inhibitory factor had no effect. The results of this study further characterize the tissue inhibitor of metalloproteinase expression in the uterus during implantation and artificially induced decidualization and the potential control of their expression in the stroma by transforming growth factor.     

Reversal of indomethacin-induced inhibition of implantation in the mouse by epidermal growth factor.

In the mouse, estriol (E3) can induce implantation as a phase I of estrogen action. E3-induced implantation was attenuated by indomethacin(INDO), an inhibitor of prostaglandin (PG) synthesis. The inhibitory effect of INDO was reversed by administration of epidermal growth factor (EGF), and this EGF effect was dose-dependent. These results suggest that one of the functions of estrogen could be to activate the EGF ligand-receptor signalling in the uterus in generating PGs required for initiation of implantation. This is consistent with the results of EGF stimulation of synthesis of PGs in uterine stromal cells in culture.     

MicroRNA expression and regulation in mouse uterus during embryo implantation

MicroRNAs (miRNAs) are 21-24-nucleotide non-coding RNAs found in diverse organisms. Although hundreds of miRNAs have been cloned or predicted, only very few miRNAs have been functionally characterized. Embryo implantation is a crucial step in mammalian reproduction. Many genes have been shown to be significantly changed in mouse uterus during embryo implantation. However, miRNA expression profiles in the mouse uterus between implantation sites and inter-implantation sites are still unknown. In this study, miRNA microarray was used to examine differential expression of miRNAs in the mouse uterus between implantation sites and inter-implantation sites. Compared with inter-implantation sites, there were 8 up-regulated miR-NAs at implantation sites, which were confirmed by both Northern blot and in situ hybridization. miR-21 was highly expressed in the subluminal stromal cells at implantation sites on day 5 of pregnancy. Because miR-21 was not detected in mouse uterus during pseudopregnancy and under delayed implantation, miR-21 expression at implantation sites was regulated by active blastocysts. Furthermore, we showed that Reck was the target gene of miR-21. Our data suggest that miR-21 may play a key role during embryo implantation.     

Apoptosis in endometrium of mouse during estrous cycle

The present study was carried out to evaluate apoptosis in endometrium and to correlate these changes with the circulating levels of estradiol and progesterone in the mouse. Apoptosis was observed in various compartments of mouse uterus i.e. stroma, glandular epithelium and luminal epithelium depending on the stage of cycle. Stromal cell apoptosis was observed during various stages of cyclicity except on estrus day. Luminal epithelial cells showed apoptotic changes during all stages of cyclicity except on diestrus day. During metestrus, apoptosis was observed in glandular and luminal epithelia as well as stromal cells. Steroid antagonists such as tamoxifen and onapristone altered the apoptotic changes in the uterus. The results suggest that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone.     

Adam12 plays a role during uterine decidualization in mice

In mouse, decidualization is characterized by the proliferation of stromal cells and their differentiation into specialized type of cells (decidual cells) with polyploidy, surrounding the implanting blastocyst. However, the mechanisms involved in these processes remain poorly understood. Using multiple approaches, we have examined the role of Adam12 in decidualization during early pregnancy in mice. Adam12 is spatiotemporally expressed in decidualizing stromal cells in intact pregnant females and in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Adam12 is upregulated after progesterone treatment, which is primarily mediated by nuclear progesterone receptor. In a stromal cell culture model, the expression of Adam12 gradually rises with the progression of stromal decidualization, whereas the attenuated expression of Adam12 after siRNA knockdown significantly blocks the progression of decidualization. Our study suggests that Adam12 is involved in promoting uterine decidualization during pregnancy.