It has become apparent that astrocytes may be important contributors to inflammatory immune responses within the brain in response to microbial challenges. To date, the mechanisms that underlie activation of this major glial cell type by such challenges have not been investigated. In the present study, we present evidence for members of a recently discovered family of receptors for highly conserved microbial components, the Toll-like receptors (TLRs), in isolated cultures of primary murine astrocytes. We describe the low-level constitutive expression of messenger RNA-encoding TLR2, TLR4, TLR5, and TLR9 in resting cultures of these cells. Importantly, the level of expression of messenger RNA for each of these receptors is markedly elevated following exposure to specific bacteria-derived ligands for these receptors. The functional expression of these receptor proteins is further supported by the ability of known ligands for each TLR to induce both message expression and protein secretion of the proinflammatory cytokine, interleukin-6. In addition, the recent availability of antibodies to TLR2 and TLR4 has enabled us to demonstrate directly the presence of these receptors on astrocytes by Western blot and immunofluorescence analysis, respectively. Furthermore, we have confirmed the sensitivity of such receptor expression to ligand stimulation. The present demonstration of Toll-like microbial pattern-recognition receptors on primary astrocytes provides a mechanistic link between bacterial challenge and inflammatory immune responses that may be an important component of the pathologies of bacterially induced inflammatory CNS disorders.

An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs.

Many signaling pathways that contribute to tumorigenesis are also functional in pregnancy, although they are dysregulated in the former and tightly regulated in the latter. Transformation-related protein 53 (Trp53), which encodes p53, is a tumor suppressor gene whose mutation is strongly associated with cancer. However, its role in normal physiological processes, including female reproduction, is poorly understood. Mice that have a constitutive deletion of Trp53 exhibit widespread development of carcinogenesis at early reproductive ages, compromised spermatogenesis, and fetal exencephaly, rendering them less amenable to studying the role of p53 in reproduction. To overcome this obstacle, we generated mice that harbor a conditional deletion of uterine Trp53 and examined pregnancy outcome in females with this genotype. These mice had normal ovulation, fertilization, and implantation; however, postimplantation uterine decidual cells showed terminal differentiation and senescence-associated growth restriction with increased levels of phosphorylated Akt and p21, factors that are both known to participate in these processes in other systems. Strikingly, uterine deletion of Trp53 increased the incidence of preterm birth, a condition that was corrected by oral administration of the selective COX2 inhibitor celecoxib. We further generated evidence to suggest that deletion of uterine Trp53 induces preterm birth through a COX2/PGF synthase/PGF 2α pathway. Taken together, our observations underscore what we believe to be a new critical role of uterine p53 in parturition.

Etiology of endometrial cancer (EMC) is not fully understood. Animal models with rapidly and spontaneously developing EMC will help explore mechanisms of cancer initiation and progression. Pten +/À mice are currently being used as a model to study EMC. These females develop atypical endometrial hyperplasia of which f20% progresses to EMC. In addition, tumors develop in other organs, complicating the use of this model to specifically study EMC. Here, we show that conditional deletion of endometrial Pten results in EMC in all female mice as early as age 1 month with myometrial invasion occurring by 3 months. In contrast, conditional deletion of endometrial p53 had no phenotype within this time frame. Whereas mice with endometrial Pten deletion had a life span of f5 months, mice with combined deletion of endometrial Pten and p53 had a shorter life span with an exacerbated disease state. Such rapid development of EMC from homozygous loss of endometrial Pten suggests that this organ is very sensitive to this tumor suppressor gene for tumor development. All lesions at early stages exhibited elevated Cox-2 and phospho-Akt levels, hallmarks of solid tumors. More interestingly, levels of two microRNAs miR-199a * and miR-101a that posttranscriptionally inhibit Cox-2 expression were down-regulated in tumors in parallel with Cox-2 upregulation. This mouse model in which the loxP-Cre system has been used to delete endometrial Pten and/or p53 allows us to study in detail the initiation and progression of EMC. These mouse models have the added advantage because they mimic several features of human EMC.

The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. Because microRNAs (miRNAs) have major roles in regulating gene expression, we speculated that they participate in directing the highly regulated spatiotemporally expressed genetic network during implantation. Here, we show that two miRNAs, mmu-miR-101a and mmu-miR-199a*, are spatiotemporally expressed in the mouse uterus during implantation coincident with expression of cyclooxygenase-2, a gene critical for implantation. More interestingly, our in vitro gain-and loss-of-function experiments show that cyclooxygenase-2 expression is posttranscriptionally regulated by these two miRNAs. We report on miRNAmediated regulation of uterine gene expression in the context of implantation. We believe that many other critical genes related to this process are also regulated by miRNAs. Thus, elucidating the physiological roles of uterine miRNAs will help us better understand the genetic control of implantation, the gateway to a successful pregnancy.mouse ͉ uterus ͉ blastocyst ͉ decidua M icroRNAs (miRNAs) are a novel family of small (Ϸ19-22 nt) noncoding RNAs transcribed by genomes of most metazoa. They are diverse in sequence, evolutionary widespread, and involved in sequence-specific posttranscriptional gene regulation by affecting mRNA stability and/or translation (1-3). In mammals, mature miRNAs are generated via a unique biosynthetic cascade involving RNA polymerase II, nuclear and cytosolic RNase III endonucleases, and their dsRNA binding protein partners. miRNA-guided RNA silencing is executed by a RNA-induced silencing complex (RISC) that involves an interaction between a mature miRNA and its binding site located at the 3Ј

Embryo implantation in the uterus is a critical step in mammalian reproduction, requiring preparation of the uterus receptive to blastocyst implantation. Uterine receptivity, also known as the window of implantation, lasts for a limited period, and it is during this period blastocysts normally implant. Ovarian steroid hormones estrogen and progesterone (P 4) are the primary regulators of this process. The immunophilin FKBP52 serves as a cochaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Here we show a critical role for FKBP52 in mouse implantation. This immunophilin has unique spatiotemporal expression in the uterus during implantation, and females missing the Fkbp52 gene have complete implantation failure due to lack of attainment of uterine receptivity. The overlapping uterine expression of FKBP52 with nuclear progesterone receptor (PR) in wild-type mice together with reduced P 4 binding to PR, attenuated PR transcriptional activity and down-regulation of several P4-regulated genes in uteri of Fkbp52 ؊/؊ mice, establishes this cochaperone as a critical regulator of uterine P4 function. Interestingly, ovulation, another P4-mediated event, remains normal. Collectively, the present investigation provides evidence for an in vivo role for this cochaperone in regulating tissue-specific hormone action and its critical role in uterine receptivity for implantation.mouse ͉ uterus ͉ ovulation ͉ blastocyst ͉ progesterone receptor P rogesterone (P 4 ) is essential for implantation and pregnancy maintenance in all mammalian species studied. In mice, P 4 priming of the uterus is obligatory for estrogen to prepare the uterus to the receptive state conducive to blastocyst implantation. P 4 acting through the nuclear P 4 receptor (PR) modulates uterine physiology and expression of various genes that are required for implantation (1, 2). Numerous defects in mice lacking the Pgr gene that encodes PR include failure in ovulation, mammary gland development, and sexual behavior along with uterine hyperplasia and inflammation, reflecting the critical role of P 4 in female reproduction (3). Appropriate functioning of nuclear steroid hormone receptors depends on interactions with the molecular chaperone machinery to maintain a functional state competent for hormone binding and subsequent transcriptional activation. Functionally mature steroid receptor complexes consist of a receptor monomer, a 90-kDa heat shock protein (Hsp90) dimer, the cochaperone p23, and one of four cochaperones that contain a tetratricopeptide repeat (TPR) domain. The TPR cochaperones include two members of the FK506 binding family of immunophilins, FKBP52͞FKBP4 and FKBP51͞FKBP5, a member of the cyclosporin-binding immunophilin cyclophilin 40 (CyP40) or the protein phosphatase PP5. FKBP52 and FKBP51 are similar to other FKBP family members in that both contain an active peptidylprolyl cis͞trans isomerase domain that catalyzes conformational changes in protein substrates (4, 5).Roles for Hsp90 and p23 in initia...

Early pregnancy failure often arises due to defects that occur before, during, or immediately after implantation. Even with in vitro fertilization and embryo-transfer techniques, implantation rates remain low in humans, most often as a result of embryos being transferred into nonreceptive uteri ( 1 ). Studying the molecular interactions that regulate implantation will provide a better understanding of these signaling pathways, eventually leading to new approaches to prevent implantation failure.There is evidence that phospholipid metabolism and signaling infl uences early pregnancy events ( 2 ). Phospholipids are vital structural and regulatory components of biological membranes and serve as precursors for many active biomolecules, such as eicosanoids and lysophospholipids ( 3, 4 ). Prostaglandins, one major group of eicosanoid lipid molecules, are produced from arachidonic acid (AA) that is released from membrane phospholipids by phospholipase A 2 . The released AA is acted upon by cyclooxygenases (COXs) to form prostaglandin H 2 , which is then converted to various prostaglandins by specifi c

The activation of the blastocyst, a process by which it gains competency to attach with the receptive uterus, is a prerequisite for successful implantation. However, the molecular basis of blastocyst activation remains largely unexplored. Combining molecular, pharmacological and physiological approaches, we show here that silencing of Wnt-␤-catenin signaling in mice does not adversely affect the development of preimplantation embryos to blastocysts and uterine preparation for receptivity, but, remarkably, blocks blastocyst competency to implantation. Using the physiologically relevant delayed implantation model and trophoblast stem cells in culture, we further demonstrate that a coordinated activation of canonical Wnt-␤-catenin signaling with attenuation of the noncanonical Wnt-RhoA signaling pathway ensures blastocyst competency to implantation. These findings constitute novel evidence that Wnt signaling is at least one pathway that determines blastocyst competency for implantation.