Susanne Tranguch

MicroRNA regulation of cyclooxygenase-2 during embryo implantation

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 miRNA-mediated 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.

Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice

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(+/-) mice are currently being used as a model to study EMC. These females develop atypical endometrial hyperplasia of which approximately 20% 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 approximately 5 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 up-regulation. 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.

Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice

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(2alpha) pathway. Taken together, our observations underscore what we believe to be a new critical role of uterine p53 in parturition.

Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation

Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4–8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A2&agr; and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.

Inactivation of Nuclear Wnt-β-Catenin Signaling Limits Blastocyst Competency for Implantation

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-β-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-β-catenin signaling with attenuation of the non-canonical 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.

Cyclooxygenase-1 Is a Potential Target for Prevention and Treatment of Ovarian Epithelial Cancer

The precise genetic and molecular defects underlying epithelial ovarian cancer (EOC) remain largely unknown, and treatment options for patients with advanced disease are limited. Cyclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandins. Whereas overwhelming evidence suggests a role for COX-2 in a variety of cancers, the contribution of COX-1 remains much less explored. The expression status of COX isoforms in ovarian cancers also remains confusing. We have previously shown that human epithelial ovarian tumors have increased levels of COX-1 but not COX-2. To more carefully examine the role of COXs in ovarian cancer, we used a mouse model of EOC in which genetic and oncogenic modifications were experimentally engineered into ovarian surface epithelial cells (OSE) thought to be the cells of origin for human EOC. These OSE cells produce tumors when allografted into host mice. Using multiple approaches, we observed that OSE cells and the tumors comprised of these cells express high levels of COX-1 but not COX-2. Prostacyclin (PGI(2)) is the major prostaglandin generated downstream of COX-1 in these cells, and SC-560, a COX-1-selective inhibitor, dramatically inhibits PGI(2) production. More importantly, SC-560 reduced the growth of tumors when OSE cells were allografted in nude female mice. In contrast, the COX-2-selective inhibitor celecoxib had little effect on tumor growth. The growth inhibitory effects of SC-560 result from reduced cell proliferation and/or accelerated apoptosis. Our results imply COX-1 as a target for the prevention and/or treatment of EOC.

Conditional Deletion of MSX Homeobox Genes in the Uterus Inhibits Blastocyst Implantation by Altering Uterine Receptivity

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.

Maternal heparin-binding-EGF deficiency limits pregnancy success in mice

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation.

FKBP52 deficiency–conferred uterine progesterone resistance is genetic background and pregnancy stage specific

Immunophilin FKBP52 serves as a cochaperone to govern normal progesterone (P(4)) receptor (PR) function. Using Fkbp52(-/-) mice, we show intriguing aspects of uterine P(4)/PR signaling during pregnancy. Implantation failure is the major phenotype found in these null females, which is conserved on both C57BL6/129 and CD1 backgrounds. However, P(4) supplementation rescued implantation and subsequent decidualization in CD1, but not C57BL6/129, null females. Surprisingly, experimentally induced decidualization in the absence of blastocysts failed in Fkbp52(-/-) mice on either background even with P(4) supplementation, suggesting that embryonic signals complement uterine signaling for this event. Another interesting finding was that while P(4) at higher than normal pregnancy levels conferred PR signaling sufficient for implantation in CD1 null females, these levels were inefficient in maintaining pregnancy to full term. However, elevating P(4) levels further restored PR signaling to a level optimal for successful term pregnancy with normal litter size. Collectively, the results show that the indispensability of FKBP52 in uterine P(4)/PR signaling is a function of genetic disparity and is pregnancy stage specific. Since there is evidence for a correlation between P(4) supplementation and reduced risks of P(4)-resistant recurrent miscarriages and remission of endometriosis, these findings have clinical implications for genetically diverse populations of women.

Molecular complexity in establishing uterine receptivity and implantation

Abstract.Implantation is the process by which the blastocyst comes into intimate physical and physiological contact with the uterine endometrium. This process is governed by an intimate cross-talk between the activated blastocyst and the receptive uterus. An increased understanding of mammalian implantation has been gained through the use of the mouse model. This review highlights the more recently defined signaling cascades involved in this dialogue, focusing specifically on cyclooxygenase-2-derived prostaglandins, endocannabinoids, Wnt proteins, homeotic transcription factors, and immunophilins. Unraveling the nature of these signals and discovering additional molecular cascades may lead to strategies to correct implantation failure and improve pregnancy rates in women.