Search

Implantation is the first direct encounter between the embryo and uterus during pregnancy, and Hbegf is the earliest known molecular signaling for embryo–uterine crosstalk during implantation. The downstream effectors of heparin-binding EGF (HB-EGF) in implantation remain elusive due to the complexity of EGF receptor family. This study shows that the formation of implantation chamber (crypt) triggered by HB-EGF is disrupted by uterine deletion of Vangl2, a key planar cell polarity component (PCP). We found that HB-EGF binds to ERBB2 and ERBB3 to recruit VANGL2 for tyrosine phosphorylation. Using in vivo models, we show that uterine VAGL2 tyrosine phosphorylation is suppressed in Erbb2/Erbb3 double conditional knockout mice. In this context, severe implantation defects in these mice lend support to the critical role of HB-EGF-ERBB2/3-VANGL2 in establishing a two-way dialogue between the blastocyst and uterus. In addition, the result addresses an outstanding question how VANGL2 is activated during implantation. Taken together, these observations reveal that HB-EGF regulates the implantation process by influencing uterine epithelial cell polarity comprising VANGL2.

Nanospray desorption electrospray mass spectrometry imaging (nano-DESI MSI) enables quantitative imaging of hundreds of molecules in biological samples with minimal sample pretreatment and a spatial resolution down to 10 µm. We have recently developed an integrated glass microfluidic probe (iMFP) for nano-DESI MSI, which simplifies the experimental setup and enables imaging with a spatial resolution of 25 µm. Herein, we describe an improved design of the iMFP for the high-throughput imaging of tissue sections. We increased the dimensions of the primary and spray channels and optimized the spray voltage and solvent flow rate to obtain a stable operation of the iMFP at both low (0.04 mm/s) and high (0.4 mm/s) scan rates. We demonstrate the performance of the high-throughput iMFP by imaging mouse uterine and brain tissue sections. We observe that the sensitivity, molecular coverage, and spatial resolution obtained using the iMFP do not change to a significant extent as the scan rate increases. Using a scan rate of 0.4 mm/s, we obtained high quality images of mouse uterine tissue sections (scan area: 3.2 mm×2.3 mm) in only 9.5 minutes and of mouse brain tissue (scan area: 7.0 mm×5.4 mm) in 21.7 minutes, which corresponds to a 10-15-fold improvement in the experimental throughput. We have also developed a quantitative metric for evaluating the quality of ion images obtained at different scan rates. Specifically, by defining regions of interest (ROI) in both a representative ion image and optical image of the tissue section, we calculated the spatial deviation between the two ROIs by counting the number of mismatched pixels. Using this metric, we demonstrate that the percent deviation increases slightly from 6.7% to 10.2 % with an increase in the scan rate from 0.02 to 0.4 mm/s. The maximum experimental throughput achieved in this study is limited by the acquisition rate of a mass spectrometer necessary to achieve the desired spatial resolution and may be further improved using a faster instrument. The ability to image biological tissues with high throughput using iMFP-based nano-DESI MSI will substantially speed up tissue mapping efforts.

Supplementary Figure 3 from Loss of Cannabinoid Receptor 1 Accelerates Intestinal Tumor Growth

Supplementary Figure 2 from Repression of 15-Hydroxyprostaglandin Dehydrogenase Involves Histone Deacetylase 2 and Snail in Colorectal Cancer

Cyclooxygenases-1 and -2 (Cox-1 and Cox-2) are two distinct isoforms that catalyze the conversion of arachidonic acid to prostaglandins. The role of Cox-2 in a variety of cancers is well recognized, but the contribution of Cox-1 remains much less explored. We have previously shown that human epithelial ovarian tumors have increased levels of Cox-1, but not Cox-2. We also observed that Cox-1 is highly expressed in a mouse model of epithelial ovarian cancer (EOC), which lacks p53 but overexpresses c-myc and K-ras or c-myc and Akt. More importantly, a Cox-1-selective inhibitor, SC-560, attenuates EOC growth. In the present investigation, we used various genetically engineered mouse models of EOC to determine whether Cox-1 overexpression is unique to specific genetic and oncogenic alterations or is widespread. These models include: (a) deletion of both p53 and Rb, (b) induction of the transforming region of SV40 under the control of Mullerian inhibitory substance type II receptor, or (c) activation of K-Ras in the absence of Pten locally in the ovarian surface epithelium. We found that these three models, which produce spontaneous EOC, also show up-regulated expression of Cox-1, but not Cox-2. The results provide further evidence that Cox-1 overexpression is common in various models of EOC. Thus, Cox-1 serves as a potential marker of EOC and is a possible target for the prevention and/or treatment of this deadly disease. (Cancer Res 2006; 66(5): 2527-31)

Supplementary Table 1 from Repression of 15-Hydroxyprostaglandin Dehydrogenase Involves Histone Deacetylase 2 and Snail in Colorectal Cancer

Supplementary Table 1 from Loss of Cannabinoid Receptor 1 Accelerates Intestinal Tumor Growth

Supplementary Figure 3 from Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice

Supplementary Figure 2 from Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice

Supplementary Figure 2 from Cyclooxygenase-1 Is Overexpressed in Multiple Genetically Engineered Mouse Models of Epithelial Ovarian Cancer

Supplementary Figure 4 from Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice

Supplementary Figure 5 from Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice

Supplementary Figure 1 from Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice

Supplementary Figure 6 from Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice

Supplementary Figure Legends 1-2 from Repression of 15-Hydroxyprostaglandin Dehydrogenase Involves Histone Deacetylase 2 and Snail in Colorectal Cancer

Supplementary Figure 1 from Cyclooxygenase-1 Is Overexpressed in Multiple Genetically Engineered Mouse Models of Epithelial Ovarian Cancer

Supplementary Figures 1-2 from Extracellular Signal-Regulated Kinase Is a Target of Cyclooxygenase-1-Peroxisome Proliferator-Activated Receptor-δ Signaling in Epithelial Ovarian Cancer

Supplementary Figure 2 from Loss of Cannabinoid Receptor 1 Accelerates Intestinal Tumor Growth

The underlying causes of epithelial ovarian cancer (EOC) are unclear, and treatment options for patients with advanced disease are limited. There is evidence that the use of nonsteroidal anti-inflammatory drugs is associated with decreased risk of developing EOC. Nonsteroidal anti-inflammatory drugs inhibit cyclooxygenase (COX)-1 and COX-2, which catalyze prostaglandin biosynthesis. We previously showed that mouse and human EOCs have increased levels of COX-1, but not COX-2, and a COX-1–selective inhibitor, SC-560, attenuates prostaglandin production and tumor growth. However, the downstream targets of COX-1 signaling in EOC are not yet known. To address this question, we evaluated peroxisome proliferator-activated receptor δ (PPARδ) expression and function in EOC. We found that EOC cells express high levels of PPARδ, and neutralizing PPARδ function reduces tumor growth in vivo. More interestingly, aspirin, a nonsteroidal anti-inflammatory drug that preferentially inhibits COX-1, compromises PPARδ function and cell growth by inhibiting extracellular signal-regulated kinases 1/2, members of the mitogen-activated protein kinase family. Our study, for the first time, shows that whereas PPARδ can be a target of COX-1, extracellular signal-regulated kinase is a potential target of PPARδ. The ability of aspirin to inhibit EOC growth in vivo is an exciting finding because of its low cost, lack of cardiovascular side effects, and availability. [Cancer Res 2007;67(11):5285–92]

Supplementary Figure 1 from Repression of 15-Hydroxyprostaglandin Dehydrogenase Involves Histone Deacetylase 2 and Snail in Colorectal Cancer

Supplementary Figure 1 from Loss of Cannabinoid Receptor 1 Accelerates Intestinal Tumor Growth

Supplementary Table 1 from Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice

Unraveling the complexity of the lipidome requires the development of novel approaches for the structural characterization of lipid species with isomer-level discrimination. Herein, we introduce an online photochemical approach for lipid isomer identification through selective derivatization of double bonds by reaction with singlet oxygen. Lipid hydroperoxide products are generated promptly after laser irradiation. Fragmentation of these species in a mass spectrometer produces diagnostic fragments, which reveal the C=C locations in the unreacted lipids. This approach uses an inexpensive light source and photosensitizer making it easy to incorporate into any lipidomics workflow. We demonstrate the utility of this approach for the shotgun profiling of C=C locations in different lipid classes present in tissue extracts using electrospray ionization (ESI) and ambient imaging of lipid species differing only by the location of C=C bonds using nanospray desorption electrospray ionization (nano-DESI).

Embryonic diapause is a reproductive strategy in which embryo development and growth is temporarily arrested within the uterus to ensure the survival of neonates and mothers during unfavorable conditions. Pregnancy is reinitiated when conditions become favorable for neonatal survival. The mechanism of how the uterus enters diapause in various species remains unclear. Mice with uterine depletion of Foxa2, a transcription factor, are infertile. In this study, we show that dormant blastocysts are recovered from these mice on day 8 of pregnancy with persistent expression of uterine Msx1, a gene critical to maintaining the uterine quiescent state, suggesting that these mice enter embryonic diapause. Leukemia inhibitory factor (LIF) can resume implantation in these mice. Although estrogen is critical for implantation in progesterone-primed uterus, our current model reveals that FOXA2-independent estrogenic effects are detrimental to sustaining uterine quiescence. Interestingly, progesterone and anti-estrogen can prolong uterine quiescence in the absence of FOXA2. Although we find that Msx1 expression persists in the uterus deficient in Foxa2, the complex relationship of FOXA2 with Msx genes and estrogen receptors remains to be explored.

Simultaneous spatial localization and structural characterization of molecules in complex biological samples currently represents an analytical challenge for mass spectrometry imaging (MSI) techniques. In this study, we describe a novel experimental platform, which substantially expands the capabilities and enhances the depth of chemical information obtained in high spatial resolution MSI experiments performed using nanospray desorption electrospray ionization (nano-DESI). Specifically, we designed and constructed a portable nano-DESI MSI platform and coupled it with a drift tube ion mobility (IM) spectrometer-mass spectrometer. We demonstrate imaging of drift time-separated ions with a high spatial resolution of better than ∼25 μm using uterine tissues on day 4 of pregnancy in mice. Collision cross-section measurements provide unique molecular descriptors of molecules observed in nano-DESI-IM-MSI necessary for their unambiguous identification by comparison with databases. Meanwhile, isomer-specific imaging reveals variations in the isomeric composition across the tissue. Furthermore, IM separation efficiently eliminates isobaric and isomeric interferences originating from solvent peaks, overlapping isotopic peaks of endogenous molecules extracted from the tissue, and products of in-source fragmentation, which is critical to obtaining accurate concentration gradients in the sample using MSI. The structural information provided by the IM separation substantially expands the molecular specificity of high-resolution MSI necessary for unraveling the complexity of biological systems.

Prostaglandins are important lipids involved in mediating many physiological processes, such as allergic responses, inflammation, and pregnancy. However, technical limitations of in-situ prostaglandin detection in tissue have led researchers to infer prostaglandin tissue distributions from localization of regulatory synthases, such as COX1 and COX2. Herein, we apply a novel mass spectrometry imaging method for direct in situ tissue localization of prostaglandins, and combine it with techniques for protein expression and RNA localization. We report that prostaglandin D2, its precursors, and downstream synthases co-localize with the highest expression of COX1, and not COX2. Further, we study tissue with a conditional deletion of transformation-related protein 53 where pregnancy success is low and confirm that PG levels are altered, although localization is conserved. Our studies reveal that the abundance of COX and prostaglandin D2 synthases in cellular regions does not mirror the regional abundance of prostaglandins. Thus, we deduce that prostaglandins tissue localization and abundance may not be inferred by COX or prostaglandin synthases in uterine tissue, and must be resolved by an in situ prostaglandin imaging., Duncan et al. use a mass spectrometry imaging method to assess the localization and concentration of prostaglandins (PGs) in mouse tissues during pregnancy. This study brings new biological insights into the spatial evaluation of PGs in tissues, which could reveal the functional significance of each PGs during different stages of embryo development/pregnancy.

International audience; Directed trophoblast migration toward the maternal mesometrial pole is critical for placentation and pregnancy success. Trophoblasts replace maternal arterial endothelial cells to increase blood supply to the placenta. Inferior trophoblast invasion results in pregnancy complications including preeclampsia, intrauterine growth restriction, miscarriage, and preterm delivery. The maternal chemotactic factors that direct trophoblast migration and the mechanism by which trophoblasts respond to these factors are not clearly understood. Here, we show that invasive trophoblasts deficient in Vangl2, a core planar cell polarity (PCP) component, fail to invade in maternal decidua, and this deficiency results in middle-gestational fetal demise. Previously, we have shown that tightly regulated endocannabinoids via G protein-coupled cannabinoid receptor CB1 are critical to the invasion of trophoblasts called spiral artery trophoblast giant cells (SpA-TGCs). We find that CB1 directly interacts with VANGL2. Trophoblast stem cells devoid of Cnr1 and/or Vangl2 show compromised cell migration. To study roles of VANGL2 and CB1 in trophoblast invasion in vivo, we conditionally deleted Cnr1 (coding CB1) and Vangl2 in progenitors of SpA-TGCs using trophoblast-specific protein alpha (Tpbpa)-Cre. We observed that signaling mediated by VANGL2 and CB1 restrains trophoblasts from random migration by keeping small GTPases quiescent. Our results show that organized PCP in trophoblasts is indispensable for their directed movement and that CB1 exerts its function by direct interaction with membrane proteins other than its canonical G protein-coupled receptor role.

Biological tissues exhibit complex spatial heterogeneity that directs the functions of multicellular organisms. Quantifying protein expression is essential for elucidating processes within complex biological assemblies. Imaging mass spectrometry (IMS) is a powerful emerging tool for mapping the spatial distribution of metabolites and lipids across tissue surfaces, but technical challenges have limited the application of IMS to the analysis of proteomes. Methods for probing the spatial distribution of the proteome have generally relied on the use of labels and/or antibodies, which limits multiplexing and requires a priori knowledge of protein targets. Past efforts to make spatially resolved proteome measurements across tissues have had limited spatial resolution and proteome coverage and have relied on manual workflows. Here, we demonstrate an automated approach to imaging that utilizes label-free nanoproteomics to analyze tissue voxels, generating quantitative cell-type-specific images for >2000 proteins with 100-µm spatial resolution across mouse uterine tissue sections preparing for blastocyst implantation., Imaging mass spectrometry is a powerful emerging tool for mapping the spatial distribution of biomolecules across tissue surfaces. Here the authors showcase an automated technology for deep proteome imaging that utilizes ultrasensitive microfluidics and a mass spectrometry workflow to analyze tissue voxels, generating quantitative cell-type-specific images.

Mass spectrometry imaging (MSI) enables label-free spatial mapping of hundreds of biomolecules in tissue sections. This capability provides valuable information on tissue heterogeneity that is difficult to obtain using population-averaged assays. Despite substantial developments in both instrumentation and methodology, MSI of tissue samples at single-cell resolution remains challenging. Herein, we describe a protocol for robust imaging of tissue sections with a high (better than 10-μm) spatial resolution using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry, an ambient ionization technique that does not require sample pretreatment before analysis. In this protocol, mouse uterine tissue is used as a model system to illustrate both the workflow and data obtained in these experiments. We provide a detailed description of the nano-DESI MSI platform, fabrication of the nano-DESI and shear force probes, shear force microscopy experiments, spectral acquisition, and data processing. A properly trained researcher (e.g., technician, graduate student, or postdoc) can complete all the steps from probe fabrication to data acquisition and processing within a single day. We also describe a new strategy for acquiring both positive- and negative-mode imaging data in the same experiment. This is achieved by alternating between positive and negative data acquisition modes during consecutive line scans. Using our imaging approach, hundreds of high-quality ion images were obtained from a single uterine section. This protocol enables sensitive and quantitative imaging of lipids and metabolites in heterogeneous tissue sections with high spatial resolution, which is critical to understanding biochemical processes occurring in biological tissues.

Scribble (Scrib) is a scaffold protein with multifunctional roles in PCP, tight junction and Hippo signaling. This study shows that Scrib is expressed in stromal cells around the implantation chamber following implantation. Stromal cells transform into epithelial-like cells to form the avascular primary decidual zone (PDZ) around the implantation chamber (crypt). The PDZ creates a permeability barrier around the crypt restricting immune cells and harmful agents from maternal circulation to protect embryonic health. The mechanism underlying PDZ formation is not yet known. We found that uterine deletion of Scrib by a Pgr-Cre driver leads to defective PDZ formation and implantation chamber (crypt) formation, compromising pregnancy success. Interestingly, epithelial-specific Scrib deletion by a lactoferrin-Cre (Ltf-Cre) driver does not adversely affect PDZ formation and pregnancy success. These findings provide evidence for a previously unknown function of stromal Scrib in PDZ formation, potentially involving ZO-1 and Hippo signaling., The scaffold protein Scribble (Scrib) has roles in PCP and tight junction formation. Here the authors show that during embryo implantation in mouse, Scrib is needed to form the avascular primary decidual zone by transforming stromal cells into an epithelial cell-like barrier around the crypt, protecting the embryo from harmful infiltrations.

Cannabinoid/endocannabinoid signaling is primarily mediated by cannabinoid receptor type 1 (CB1; encoded by Cnr1) and/or type 2 (CB2; encoded by Cnr2). Here, we show that Cnr1(−/−)Cnr2(−/−) mice are subfertile as a result of compromised implantation. Upon implantation, the epithelium is smooth and adhered to the blastocyst trophectoderm within the implantation chamber (crypt) in wild-type mice, whereas the epithelium in Cnr1(−/−)Cnr2(−/−) mice is ruffled, which compromises appropriate blastocyst-uterine interactions. The suboptimal implantation leads to higher incidence of pregnancy failure in Cnr1(−/−)Cnr2(−/−) mice. Histological analysis revealed heightened edema around the implantation chamber in these deleted females. With the use of a reporter mouse line, we observed that CB2 is present on endothelial cells of uterine blood vessels, and its absence leads to blood vessel leakage during implantation. These results suggest that appropriately regulated uterine edema is important to optimal implantation.

Unraveling the complexity of the lipidome requires the development of novel approaches for the structural characterization of lipid species with isomeric resolution. Herein, we introduce an online photochemical approach for lipid isomer identification through selective derivatization of double bonds by reaction with singlet oxygen. Lipid hydroperoxide products are generated promptly after laser irradiation. Fragmentation of these species in a mass spectrometer produces diagnostic fragments, which reveal the C=C locations in the unreacted lipids. This approach uses an inexpensive light source and photosensitizer making it easy to incorporate into any lipidomics workflow. We demonstrate the utility of this approach for the shotgun profiling of C=C locations in different lipid classes present in tissue extracts using electrospray ionization (ESI) and for spatially-resolved analysis of lipids in tissue sections using nanospray desorption electrospray ionization (nano-DESI). These results provide a path for both rapid profiling and ambient imaging of positional isomers in biological samples.

With implantation, mouse stromal cells begin to transform into epithelial-like cells surrounding the implantation chamber forming an avascular zone called the primary decidual zone (PDZ). In the mouse, the PDZ forms a transient, size-dependent permeable barrier to protect the embryo from maternal circulating harmful agents. The process of decidualization is critical for pregnancy maintenance in mice and humans. Mice deficient in cannabinoid receptors, CB1 and CB2, show compromised PDZ with dysregulated angiogenic factors, resulting in the retention of blood vessels and macrophages. This phenotype is replicated inCnr1-/-but not inCnr2-/-mice. In vitro decidualization models suggest thatCnr1levels substantially increase in mouse and human decidualizing stromal cells, and that neutralization of CB1 signaling suppresses decidualization and misregulates angiogenic factors. Taken together, we propose that implantation quality depends on appropriate angiogenic events driven by the integration of CB2 in endothelial cells and CB1 in decidual cells.

An early event after implantation is stromal cell transformation to decidual cells (decidualization) that support embryo development. In mice, this process begins at the antimesometrial (AM) pole with differentiation of stromal cells into epithelial-like cells (epithelioid cells) surrounding the implantation chamber. This is an avascular zone called the primary decidual zone (PDZ), and considered to function as a transient, size-dependent permeable barrier to protect the embryo from maternal circulating harmful agents, including immunoglobulins, immune cells, microorganisms and other noxious agents. This zone forms on day 5 afternoon and becomes fully established on day 6 with the loss of the crypt epithelium. The PDZ gradually degenerates with the appearance of the secondary decidual zone (SDZ) around the PDZ that peaks on day 8 in mice. Decidualization is critical for early pregnancy in mice and humans. We show that cannabinoid/endocannabinoid signaling influences decidualization in early pregnancy. Mice deficient in two major cannabinoid receptors, CB1 and CB2, show compromised PDZ. We found that angiogenic factors are dysregulated in Cnr1-/-Cnr2-/- mice with defective PDZs, resulting in the abnormal presence of blood vessels and macrophages in this zone; disruption of the PDZ compromises pregnancy outcomes. Using an in vitro decidualization model, we found that Cnr1 levels increase in mouse stromal cells and human uterine fibroblast (Huf) cells undergoing decidualization and that limiting CB1 signaling in these cell types suppresses decidualization in vitro. Since endothelial cells express Cnr2 and decidual cells express Cnr1, we hypothesize that angiogenic events driven by CB2 are integrated with CB1 in decidual cells, leading to proper PDZ formation, a critical step for pregnancy success.

Simultaneous spatial localization and structural characterization of molecules in complex biological samples currently represents an analytical challenge for mass spectrometry imaging (MSI) techniques. In this study, we describe a novel experimental platform, which substantially expands the capabilities and enhances the depth of chemical information obtained in high spatial resolution MSI experiments performed using nanospray desorption electrospray ionization (nano-DESI). Specifically, we designed and constructed a portable nano-DESI MSI platform and coupled it with a drift tube ion mobility spectrometer-mass spectrometer (IM-MS). Separation of biomolecules observed in MSI experiments based on their drift times provides unique molecular descriptors necessary for their identification by comparison with databases. Furthermore, it enables isomer-specific imaging, which is particularly important for unraveling the complexity of biological systems. Imaging of day 4 pregnant mouse uterine sections using the newly developed nano-DESI-IM-MSI system demonstrates rapid isobaric and isomeric separation and reduced chemical noise in MSI experiments. A direct comparison of the performance of the new nano-DESI-MSI platform operated in the MS mode with the more established nano-DESI-Orbitrap platform indicates a comparable performance of these two systems. A spatial resolution of better than ~16 µm and similar molecular coverage was obtained using both platforms. The structural information provided by the ion mobility separation expands the molecular specificity of high-resolution MSI necessary for the detailed understanding of biological systems.

Mammary glands are comprised of ducts and terminal lobules that form tree-like structures. Luminal epithelial cells in these lobules undergo differentiation into alveolar cells in pregnancy to support milk production. This study reveals that Scribble (SCRIB), a scaffold protein expressed in progesterone receptor (PGR)-positive cells, plays a critical role in mammary gland alveologenesis in mice. We conditionally deleted Scrib using a Pgr-Cre driver. PGR is heterogeneously expressed throughout the luminal epithelium. Scrib loss in mammary glands by Pgr-Cre (Scribf/fPgrCre/+) shows inefficient alveologenesis and terminal end bud (TEB)-like morphology during pregnancy, resulting in poor milk production and subsequent death of pups after delivery. The differentiation of PGR-positive epithelial cells into Elf5-expressing alveolar cells is defective in Scribf/fPgrCre/+ mice. These changes are reflected in reduced activation of JAK2 and PAK1, resulting in downregulation of pSTAT5, a critical transcriptional factor for alveologenesis. These results provide evidence that SCRIB impacts PGR-positive cell lineage during alveologenesis, which impacts milk production and the health of offspring.

SUMMARY Protein kinase C (PKC) isozymes are commonly recognized as oncoproteins based on their activation by tumor-promoting phorbol esters. However, accumulating evidence indicates that PKCs can be inhibitory in some cancers, with recent findings propelling a shift in focus to understanding tumor suppressive functions of these enzymes. Here, we report that PKCα acts as a tumor suppressor in PI3K/AKT-driven endometrial cancer. Transcriptional suppression of PKCα is observed in human endometrial tumors in association with aggressive disease and poor prognosis. In murine models, loss of PKCα is rate limiting for endometrial tumor initiation. PKCα tumor suppression involves PP2A-family-dependent inactivation of AKT, which can occur even in the context of genetic hyperactivation of PI3K/AKT signaling by coincident mutations in PTEN, PIK3CA, and/or PIK3R1. Together, our data point to PKCα as a crucial tumor suppressor in the endometrium, with deregulation of a PKCα→PP2A/PP2A-like phosphatase signaling axis contributing to robust AKT activation and enhanced endometrial tumorigenesis., Graphical Abstract, In Brief Hsu et al. find that PKCα is frequently lost in human and murine endometrial tumors and that PKCα deficiency enhances PI3K/ AKT-driven endometrial neoplasia. PKCα suppresses aberrant AKT activity via a PP2A family phosphatase-dependent mechanism. Thus, PKCα loss appears to cooperate with PI3K/AKT perturbations to hyperactivate AKT and promote endometrial tumorigenesis.

The observation of pups born from recipient and donor mice after ovariectomy followed by ovarian transplant poses the interesting possibility of an extraovarian source of oocytes. However, whether mammalian adult oocytes reside in extragonadal tissues remains elusive. Using transgenic fluorescent reporter mice and transplantation surgeries, we demonstrate the presence of both donor- and recipient-derived corpora lutea and recovery of both donor- and recipient-derived offspring from ovariectomized mice after transplantation of donor ovaries. A potential region for extraovarian oocytes is the hilum, a ligament-like structure between the ovary and the reproductive tract. Immunofluorescent confocal microscopy of mouse ovaries and reproductive tracts revealed that a population of primordial follicles resides outside the ovary within the hilum. Ovariectomy-only controls confirmed that oocytes remain in the recipient hilum after surgery. These results provide evidence that the hilum is a reserve source of follicles, which likely return to the ovary for maturation and ovulation. By identifying a new follicle reservoir, our study addresses a long-standing question in reproductive biology and contributes to new conceptual knowledge about ovarian function and fertility.

Mammalian embryonic diapause is a reproductive phenomenon defined by the reversible arrest in blastocyst development and metabolic activity within the uterus which synchronously becomes quiescent to implantation. This natural strategy, evident in over 130 species across eight orders, can temporally uncouple conception from delivery until conditions are favorable for the survival of the mother and newborn. While the maternal endocrine milieu has been shown to be important for this process, the local molecular mechanisms by which the uterus and embryo achieve quiescence, maintain blastocyst survival and then resumes blastocyst activation with subsequent implantation in response to endocrine cues remains unclear. Here we review the first evidence that the proximal molecular control of embryonic diapause is conserved in three unrelated mammalian species which employ different endocrine programs to initiate diapause. In particular, uterine expression of muscle segment homeobox (Msx) genes Msx1 or Msx2 persists during diapause, followed by downregulation with blastocyst reactivation and implantation. Mice (Mus musculus) with conditional inactivation of Msx1 and Msx2 in the uterus fail to achieve diapause and reactivation. Remarkably, the mink (Neovison vison) and tammar wallaby (Macropus eugenii) share this pattern of MSX1 or MSX2 expression as in mice during delay - it persists during diapause and is rapidly downregulated upon implantation. Therefore, these findings were the first to provide evidence that there are common conserved molecular regulators in the uterus of unrelated mammals during embryonic diapause.

A reciprocal communication between the implantation-competent blastocyst and the receptive uterus is essential to successful implantation and pregnancy success. Progesterone (P(4)) signaling via nuclear progesterone receptor (PR) is absolutely critical for pregnancy initiation and its success in most eutherian mammals. Here we show that a nuclear protein high-mobility group box-1 (HMGB1) plays a critical role in implantation in mice by preserving P(4)-PR signaling. Conditional deletion of uterine Hmgb1 by a Pgr-Cre driver shows implantation defects accompanied by decreased stromal cell Hoxa10 expression and cell proliferation, two known signatures of inefficient responsiveness of stromal cells to PR signaling in implantation. These mice evoke inflammatory conditions with sustained macrophage accumulation in the stromal compartment on day 4 of pregnancy with elevated levels of macrophage attractants Csf1 and Ccl2. The results are consistent with the failure of exogenous P(4) administration to rescue implantation deficiency in the mutant females. These early defects are propagated throughout the course of pregnancy and ultimately result in substantial subfertility. Collectively, the present study provides evidence that nuclear HMGB1 contributes to successful blastocyst implantation by sustaining P(4)-PR signaling and restricting macrophage accumulation to attenuate harmful inflammatory responses.

A reciprocal communication between the implantation-competent blastocyst and the receptive uterus is essential to implantation. Blastocyst implantation is considered to be a regulated proinflammatory response in the uterus, however the underlining mechanism remains elusive. Here, we provide genetic evidence that High-mobility group protein Box-1 (HMGB1), expressed in uterine cell nuclei, restricts inflammatory responses during the periimplantation period. Conditional deletion of uterineHmgb1by using aPgr-Credriver (Pgrcre/+Hmgb1f/f) shows substantial infertility because of defective implantation and subsequent adverse ripple effects. These mice accumulate and retain an increased number of macrophages in the stroma on day 4 of pregnancy with a unique enrichment of macrophages in the stroma encircling the blastocyst on day 5, evoking inflammatory responses. These results are in contrast to previous findings that HMBG1 is an internal alarmin. In search for the mechanism, we found thatHmgb1-deleted stromal cells show reduced activation of PR and decreasedHoxa10expression, providing evidence that PR and Hoxa10 mediated regression of inflammation is mediated by HMGB1. In addition, levels of two macrophage attractants CSF1 and CCL2 are elevated in the stroma and in vitro studies show that CSF1 specifically attracts macrophages which is abrogated if challenged with a CSF1 receptor antagonist. The results suggest thatHmgb1contributes to successful blastocyst implantation by regulating macrophage trafficking in the stroma to prevent excessive inflammatory responses.