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Supplemental Digital Content is available in the text., One driving factor for developing preeclampsia—a pregnancy disorder, often associated with poor spiral artery (SpA)-remodeling and fetal growth restriction—is the anti-angiogenic sFLT1 (soluble fms-like tyrosine kinase-1), which is found to be highly upregulated in preeclampsia patients. The sFLT1-mediated endothelial dysfunction is a common theory for the manifestation of maternal preeclampsia symptoms. However, the influence of sFLT1 on SpA-remodeling and the link between placental and maternal preeclampsia symptoms is less understood. To dissect the hsFLT1 (human sFLT1) effects on maternal and/or fetoplacental physiology in preeclampsia, sFLT1-transgenic mice with systemic hsFLT1 overexpression from midgestation onwards were used. SpA-remodeling was analyzed on histological and molecular level in placental/mesometrial triangle tissues. Maternal kidney and aorta morphology was investigated, combined with blood pressure measurements via telemetry. hsFLT1 overexpression resulted in maternal hypertension, aortic wall thickening, and elastin breakdown. Furthermore, maternal kidneys showed glomerular endotheliosis, podocyte damage, and proteinuria. preeclampsia symptoms were combined with fetal growth restriction already at the end of the second trimester and SpA-remodeling was strongly impaired as shown by persisted vascular smooth muscle cells. This phenotype was associated with shallow trophoblast invasion, delayed presence of uterine natural killer cells, and altered lymphatic angiogenesis. Overall, this study showed that circulating maternal hsFLT1 is sufficient to induce typical maternal preeclampsia-like symptoms in mice and impair the SpA-remodeling independent from the fetoplacental compartment, revealing new insights into the interaction between the placental and maternal contribution of preeclampsia.

Background: Cardioprotection by preventing or repairing mitochondrial damage is an unmet therapeutic need. To understand the role of cardiomyocyte mitochondria in physiopathology, the reliable characterization of the mitochondrial morphology and compartment is pivotal. Previous studies mostly relied on two-dimensional (2D) routine transmission electron microscopy (TEM), thereby neglecting the real three-dimensional (3D) mitochondrial organization. This study aimed to determine whether classical 2D TEM analysis of the cardiomyocyte ultrastructure is sufficient to comprehensively describe the mitochondrial compartment and to reflect mitochondrial number, size, dispersion, distribution, and morphology. Methods: Spatial distribution of the complex mitochondrial network and morphology, number, and size heterogeneity of cardiac mitochondria in isolated adult mouse cardiomyocytes and adult wild-type left ventricular tissues (C57BL/6) were assessed using a comparative 3D imaging system based on focused ion beam-scanning electron microscopy (FIB-SEM) nanotomography. For comparison of 2D vs. 3D data sets, analytical strategies and mathematical comparative approaches were performed. To confirm the value of 3D data for mitochondrial changes, we compared the obtained values for number, coverage area, size heterogeneity, and complexity of wild-type cardiomyocyte mitochondria with data sets from mice lacking the cytosolic and mitochondrial protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3; Bnip3−/−) using FIB-SEM. Mitochondrial respiration was assessed on isolated mitochondria using the Seahorse XF analyser. A cardiac biopsy was obtained from a male patient (48 years) suffering from myocarditis. Results: The FIB-SEM nanotomographic analysis revealed that no linear relationship exists for mitochondrial number (r = 0.02; P = 0.9511), dispersion (r = −0.03; P = 0.9188), and shape (roundness: r = 0.15, P = 0.6397; elongation: r = −0.09, P = 0.7804) between 3D and 2D results. Cumulative frequency distribution analysis showed a diverse abundance of mitochondria with different sizes in 3D and 2D. Qualitatively, 2D data could not reflect mitochondrial distribution and dynamics existing in 3D tissue. 3D analyses enabled the discovery that BNIP3 deletion resulted in more smaller, less complex cardiomyocyte mitochondria (number: P

Background B7-H4, a checkpoint molecule of the B7 family, regulates a broad spectrum such as T-cell activation, cytokine secretion, tumour progression and invasion capacities. Our previous data revealed that soluble B7-H4 (sB7-H4) blood serum levels are elevated in women at high risk for the hypertensive pregnancy disorder preeclampsia (PE) in the first trimester, as well as in patients with confirmed early/ late-onset PE. Aim We here aim to investigate the expression pattern of B7-H4 in placental tissues of PE and HELLP Syndrome versus control group. Methods B7-H4 protein expression and localization were investigated by immunoblotting as well as co-immunohistochemistry in placental chorionic villous and decidual basalis tissues. Results B7-H4 protein was prominently expressed at the cell membrane, in the cytoplasm of the syncytiotrophoblast (STB) and interstitial extravillous trophoblast (EVT). B7-H4 protein levels in placental chorionic villous tissue was significantly higher in women with early-onset/late-onset PE and HELLP, while it was decreased in decidual basalis tissues of early-onset PE and HELLP compared to controls. Conclusion B7-H4 was inversely expressed in placental chorionic villous and decidual basalis tissues of PE and HELLP patients. The increase of B7-H4 in the STB in PE and HELLP may lead to excessive apical expression and release of soluble B7-H4 in the maternal circulation. In contrast, the decrease of B7-H4 in decidual basalis tissues could be related to the decrease in invasion ability of the EVT in PE. Thus, the current results strongly suggest that B7-H4 is involved in the pathogenesis of PE and HELLP.

RNA-binding proteins (RBPs) have been described for cancer cell progression and differentiation, although there is still much to learn about their mechanisms. Here, using in vivo decidualization as a model, we describe the role of RBP cold shock domain containing C2 (CSDC2) in the endometrium. Csdc2 messenger RNA expression was differentially regulated depending on time and areas of decidua development, with the most variation in antimesometrium (AM) and, to a lesser degree, in the junctional zone (JZ). Immunohistochemistry of CSDC2 showed a preferentially cytoplasmic localization at AM and JZ, and nuclear localization in underneath myometrium and mesometrium (M). Cytoplasmic localization coincided with differentiated, DESMIN-marked areas, while nuclear localization coincides with proliferative zones. Uterine suppression of CSDC2 through intrauterine-injected-specific small interfering RNA (siRNA) led to abnormal decidualization in early pregnancy, with more extended antimesometrial area and with poor M development if compared with control siRNA-injected animals. These results suggest that CSDC2 could be a regulator during decidua development. Fil: Vallejo, Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Mestre Citrinovitz, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Winterhager, Elke. Universitat Essen; Alemania Fil: Saragueta, Patricia Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Química Biologica; Argentina

Cardiomyocytes consist of longitudinally oriented myofibril bundles with a misaligned composition caused by the uneven contours of the intercalated discs. The cytoplasmic space harbors the organelles, including mitochondria. This study investigated whether cardiomyocytes contain spatially and ultrastructurally discrete pools of mitochondria that can be separated for structurally and functionally appraisal in (patho)physiology. Transmission electron microscopy disclosed continuous transitions of mitochondria without attributable characteristics from beneath the sarcolemma directly into the barrier-free cytoplasmic space between myofibrils. The various shapes and sizes of mitochondria are formed by myofibril positioning and the space available independent of their localization within the cardiomyocytes. Furthermore, the established enzymatic isolation procedure including proteinase treatment resulted in loss of mitochondrial proteins, as evidenced by immunogold labeling of Connexin43 in situ, a postulated marker for distinguishing mitochondrial subpopulations. Moreover, mitochondrial ATP produced in those mitochondria was not different. These findings preclude a spatial and ultrastructural grading of cardiac mitochondria and their distinct separation and classification in subsarcolemmal and interfibrillar subpopulations.

Since it is known that placental overexpression of the human anti-angiogenic molecule sFlt-1, the main candidate in the progression of preeclampsia, lead to intrauterine growth restriction (IUGR) in mice by lentiviral transduction of mouse blastocysts, we hypothesize that sFlt-1 influence placental morphology and physiology resulting in fetal IUGR. We therefore examined the effect of sFlt-1 on placental morphology and physiology at embryonic day 18.5 with histologic and morphometric analyses, transcript analyses, immunoblotting, and methylation studies. Interestingly, placental overexpression of sFlt-1 leads to IUGR in the fetus and results in lower placental weights. Moreover, we observed altered trophoblast differentiation with reduced expression of IGF2, resulting in a smaller placenta, a smaller labyrinth, and the loss of glycogen cells in the junctional zone. Changes in IGF2 are accompanied by small changes in its DNA methylation, whereas overall DNA methylation is unaffected. In addition, the expression of placental nutrient transporters, such as the glucose diffusion channel Cx26, is decreased. In contrast, the expression of the fatty acid transporter CD36 and the cholesterol transporter ABCA1 is significantly increased. In conclusion, placental sFlt-1 overexpression resulted in a reduction in the differentiation of the spongiotrophoblast into glycogen cells. These findings of a reduced exchange area of the labyrinth and glycogen stores, as well as decreased expression of glucose transporter, could contribute to the intrauterine growth restriction phenotype. All of these factors change the intrauterine availability of nutrients. Thus, we speculate that the alterations triggered by increased anti-angiogenesis strongly affect fetal outcome and programming. J. Cell. Biochem. 118: 1316-1329, 2017. (C) 2016 Wiley Periodicals, Inc.

Several gap junction connexins have been shown to be essential for appropriate placental development and function. It is known that the expression and distribution of connexins change in response to environmental oxygen levels. The placenta develops under various oxygen levels, beginning at a low oxygen tension of approximately 2% and increasing to a tension of 8% after the onset of the uteroplacental circulation. Moreover, it has been shown that during preeclampsia (PE) placentas are subjected to chronic hypoxia. Therefore, we investigated oxygen sensitivity of placental connexins 43 and 46. Using the trophoblast cell line Jar, we demonstrated that the expression of connexin43 increased during acute hypoxia but decreased during chronic hypoxia. Chronic hypoxia resulted in the translocation of connexin43 from the membrane to the cytoplasm and in a reduction in its communication properties. In contrast, the expression of connexin46 was down-regulated during chronic hypoxia and was translocated from perinuclear areas to the cell membrane. Hypoxia-inducible factor (HIF) knockdown showed that the translocation of connexin43 but not that of connexin46 was HIF-2α dependent and was mediated by phosphoinositide 3-kinase. The up-regulation of connexin43 in combination with the down-regulation of connexin46 was confirmed in placental explants cultivated under low oxygen and in placentas with early-onset PE. Taken together, in Jar cells, placental connexins 43 and 46 are regulated during periods of low oxygen in opposite manners. The oxygen sensing of connexins in the trophoblast may play a role in physiological and pathophysiological oxygen conditions and thus may contribute to PE.

The gap junction channel forming connexins (Cx) Cx31 (Gjb3) and Cx31.1 (Gjb5) are co-expressed in the mouse trophoblast lineage. Inactivation of either gene results in partial embryonic loss at mid gestation (60% and 30%, respectively, between embryonic days E10.5and E13.5) caused by placental phenotypes. Cx31 deficiency results in loss of stem cell potential and enhanced trophoblast giant cell (TGC) differentiation, whereas the molecular role of the co-expressed Cx31.1 remained unclear. It was assumed that both isoforms have overlapping functions and can compete for each loss in placentation as both knockout mice show similar survival rates, reduced placental weights, and growth restricted embryos. Instead, here we show that Cx31.1 has opposed functions in regulating trophoblast differentiation. Cx31.1 deficiency causes a shift in placental subpopulations, reduced area of fetal blood spaces, and a reduced number of secondary TGC in the junctional zone, as shown by stereology at E10.5. Cx31.1 is critical for terminal differentiation of trophoblast cells during placentation resulting in a delayed induction of marker genes Tpbpa, Prl3b1/Pl-2, and Ctsq in Cx31.1-deficient placentas. Derivation and analysis of Cx31.1-deficient trophoblast stem lines clearly indicates a delayed trophoblast differentiation manifested by repression of marker genes for placental subpopulations and continued expression of stem cell marker genes Id2 and Ascl2, which is correlated to enhanced proliferation capacity of differentiating stem cells These findings clarify the disparate actions of Cx31.1 and Cx31 that act in opposition to balance the fate of trophoblast cells during differentiation, with Cx31.1 promoting, and Cx31 delaying terminal differentiation.

Purpose To investigate the effect of daily exposure in utero to static magnetic fields during prenatal development on germ cell development and fertility of exposed offspring in adulthood. Materials and Methods Mice were exposed daily in utero to different static magnetic field strengths at the bore entrance or in the isocenter of 1.5 T and 7 T MRI systems during the entire course of prenatal development. Results In utero-exposed male mice revealed no effect of magnetic field strength on weight of testes and epididymis or on sperm count, sperm morphology, or fertility. Exposed pregnant female mice showed no reduced fertility in terms of pregnancy rates and litter size, pointing to a normal ovarian function. However, a reduced placental weight of offspring of intrauterine exposed female mice was observed that correlated with a decrease in embryonic weight in those animals exposed at the strongest magnetic field. This effect seemed to be parent-dependent, since it was not observed in those embryos fathered by in utero-exposed male mice. Conclusion Repetitive in utero exposure to strong static magnetic fields does not impair fertility but may have a parental-dependent effect on fetal programming with regard to placental development and fetal growth. J. Magn. Reson. Imaging 2014;39:683–690. © 2013 Wiley Periodicals, Inc.

Purpose To evaluate possible risks of strong static magnetic fields for embryo implantation, gestation, organogenesis, and embryonic development. Materials and Methods Pregnant mice were exposed for 75 minutes daily during the entire course of pregnancy at the bore entrance, representing the position of medical staff, and at the isocenter, representing the position of patients, of a 1.5 T and a 7 T human MRI scanner. Results No effect of static magnetic field strength was observed with regard to pregnancy rate, duration of pregnancy, litter size, still births, malformations, sex distribution, or postpartum death of offspring. During the first 8 weeks postnatal, mice exposed in utero to a magnetic field strength of 1.5 T or stronger showed a slight delay in weight gain and in time to eye opening compared to controls. Conclusion Daily exposure to strong magnetic fields during pregnancy had no deleterious effect on offspring; however, a developmental retardation could be observed postnatally with regard to weight gain and eye opening. J. Magn. Reson. Imaging 2014;39:691–699. © 2013 Wiley Periodicals, Inc.

Cell fate determination has always fascinated scientists predominantly embryologists and challenged them to decipher master genes or networks of transcriptional factors which together with cell signaling cascades act as key regulators for the cell lineage allocation. With the rediscovery of the pluripotent precursor cells in an adult body and the possibility to induce pluripotency in somatic cells (iPSCs), as sources for alternative therapeutic options in translational medicine, this field has attracted high interest in medical research and application (1). iPSC have several advantages over embryonic stem cells such as avoiding tumor formation and to circumvent ethical concerns. Meanwhile the understanding of pluripotency has broadened thanks to abundant studies on converting ESC and somatic cells into another cell fate using predominantly transfection of a bunch of lineage specific transcription factors. While many studies focused on the regulation of ESC conversion less is known about the hierarchically organized network of transcriptional factors which determine the trophoblast lineage for placenta development.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

As a prerequisite for successful embryo implantation in mammals, before implantation ovarian hormones regulate the transformation of the endometrium into the receptive phase. During the implantation process, gene expression in the receptive endometrium is additionally modulated by the presence of a blastocyst. During this complex differentiation process, in humans as well as in rodents, gap junction connexin 26 (Cx26) is suppressed in the uterine epithelium and Cx43 is suppressed in the endometrial stromal cells during the receptive phase. In rodents, a blastocyst-mediated induction of Cx26 takes place locally in the uterine epithelial cells of the implantation chamber surrounding the blastocyst, followed by an increase in Cx43 in the cells of the developing decidua. The Cx26 induction is dependent on the presence of a blastocyst and occurs even before adhesion and invasion of the trophoblast takes place. The signal cascades involved in this blastocyst-mediated connexin induction are still elusive. The process of implantation is considered as a proinflammatory response, and inflammatory factors have been shown to be involved in the implantation process. In fact, Cx26 expression can be induced in the receptive rat endometrium by mediators of the inflammatory cascade including prostaglandin-F2α and IL1β by an ER-independent pathway similar to the blastocyst-mediated connexin induction at the time of implantation. Thus, in the receptive endometrium induction of connexin expression may also be induced by mediators of the inflammatory signaling cascade, and the implantation-related induction of intercellular communication may in part be due to an inflammatory response.

Objective To analyze the effects of delayed ovulation on embryonic development in mice, because intrafollicular oocyte development may be delayed during assisted reproductive technology (ART) treatment in humans. Design Experimental mouse study. Setting University hospital. Animal(s) Female C57Bl/6 mice. Intervention(s) Cetrorelix is used as a GnRH-antagonist in ART treatments. To assess the effect of delayed ovulation on embryonic development, cetrorelix was applied concomitantly with follicle stimulation by pregnant mare serum gonadotropin. Ovulation was induced by hCG. Controls were stimulated with pregnant mare serum gonadotropin without delaying ovulation. Suppression of ovulation was assessed from the number of tertiary follicles, ruptured follicles, and corpora lutea in mouse ovaries after cetrorelix treatment. Number and weight of embryos and placentas, as well as number of resorption sites and dead embryos, was determined on day 17.5 of pregnancy. Main Outcome Measure(s) Inhibition of ovulation, embryonic development. Result(s) Cetrorelix inhibited ovulation in mice, as shown by an increase in number of tertiary follicles concomitant with a significant inhibition of follicle rupture and corpora lutea formation. Delayed ovulation caused by Cetrorelix treatment led to a significant increase in resorption sites and a significant decrease in embryonic weight of offspring. Conclusion(s) Preovulatory oocyte overripeness might have an effect on fertility and embryonic development during ART treatment.

Introduction The anti-angiogenic factor sFlt-1 is the main candidate in the progression of preeclampsia, a disease which causes placental dysfunctions often leading to IUGR. Mostly is known about the impairment of the endothelial cell function by sFlt-1, but how sFlt-1 leads to IUGR and affects the placenta and fetus is currently unknown as well as therapeutic agents against such diseases are missing. Objectives Therefore we have established two transgenic, inducible sFlt-1 mouse models: (1) maternal ubiquitous (sFlt-1/rtTA mice) and (2) placental (sFlt-1/tTA/TpbpaCre mice) overexpression of human sFlt-1. We hypothesize that sFlt-1 is involved in the development of IUGR by influencing placental development and function and is therefore a potential candidate for intervention strategies. Material and Methods We examined the effects of sFlt-1 on placental morphology and function at 18.5 dpc with morphometric and immunohistochemical analyses and transcript expression of placental marker genes, nutrient transporters and proteomic analyses. Results Ubiquitous overexpression of sFlt-1 led to IUGR of the fetuses as shown by reduced fetal weights and signs of retardation. In addition, we observed a severely impaired placental phenotype shown by enlarged maternal blood sinusoids, a reduced number of fetal vessels and an inadequate placental differentiation of the labyrinth. Glucose, fatty acid and amino acid transport seem to be negatively affected. Preliminary results of the placental overexpression of sFlt-1 revealed while expressing lower levels of sFlt-1 also IUGR. Conclusion We assume that sFlt-1 has an inhibitory effect on placental differentiation, especially on fetal vessel development. A possible reactive response could be an increase in maternal blood flow promoting dilatation of the maternal sinusoids to fulfil the nutrient requirements of the fetus. This ultimately resulted in an uteroplacental insufficiency leading to IUGR. Thus, we speculate that the alterations triggered by increased anti-angiogenesis upon sFlt-1 strongly may affect fetal outcome and programming.

Objectives: The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in the human placenta under normal physiological circumstances and in preeclampsia.Study design: We investigated the expression pattern of the LXRs and their target genes in the human placenta during normal pregnancy and in preeclampsia. Placental explants and cell lines were studied under different oxygen levels and pharmacological LXR agonists.Main outcome measures: Gene expressions (Taqman PCR) and protein levels (Western Blot) were combined with immunohistochemistry to analyze the expression of LXR and its target genes.Results: In the human placenta, LXRA and LXRB expression increased during normal pregnancy. This was paralleled by the expression of their prototypical target genes, e.g., the cholesterol transporter ABCA1. Interestingly, early-onset preeclamptic placentae revealed a significant upregulation of ABCA1. Culture of JAr trophoblast cells and human first trimester placental explants under low oxygen lead to increased expression of LXRA and ABCA1 which was further enhanced by the LXR agonist T0901317.Conclusions: LXRA together with ABCA1 are specifically expressed in the human placenta and can be regulated by hypoxia. Deregulation of this system in early preeclampsia might be the result of placental hypoxia and hence might have consequences for maternal-fetal cholesterol transport. (c) 2010 Elsevier Ltd. All rights reserved.

In mammals, cell lineage specification is established at the blastocyst stage. At this stage, transcription factor Cdx2 represses pluripotency genes, thus promoting extraembryonic trophoblast fate. Recently, transcription factor Gata3 was shown to act in a parallel pathway in promoting trophoblast cell fate, suggesting that there are more factors working in the trophoblast lineage. Here, we report that the transcription factor Tcfap2c is expressed at a high level in the trophectoderm and is able to induce trophoblast fate in embryonic stem cells. Trophoblast fate induced by Tcfap2c does not require Cdx2 and vice versa, suggesting that the molecules act in alternative pathways. However, both Tcfap2c and Cdx2 are required for the upregulation of Elf5, a marker of trophoblast stem cell maintenance, suggesting that both factors are required for stable trophoblast induction. Tcfap2c-induced trophoblast-like cells are stable in long-term culture, indicating that they are capable of self-renewal. Tcfap2c-controlled trophoblast maintenance involves the induction of Cdx2 and the repression of the pluripotency factor Nanog. Tcfap2c-induced trophoblast-like cells differentiate to trophoblast derivatives in vitro and contribute to the trophectoderm in blastocysts in vivo. Taken together, these observations suggest that Tcfap2c and Cdx2 cooperate to override the pluripotency program and establish the extraembryonic trophoblast maintenance program in murine embryos.

Here, we review the expression, localization and the possible role of the different connexin isoforms in placental function and development in mice and men. Connexin gene deletion in mice has shown that Cx26 is responsible for transplacental uptake of glucose in the labyrinth, and Cx31 as well as Cx31.1 for trophoblast cell lineage development. In the human placenta, it appears that Cx43 is required for the fusion process of cytotrophoblastic cells leading to the formation of the syncytiotrophoblast. Thus Cx26 and Cx43 serve different species-specific functions in the functionally analogous placental compartments, mouse labyrinth and human villous trophoblast. However, like Cx31 in the mouse, Cx40 plays a critical role in the switch from a proliferative to an invasive phenotype of the trophoblast cells invading the endometrium. Both connexin channels seem to have similar functions in analogous compartments of the placentas. Taken together, connexins are important in regulating trophoblast cell differentiation in both species. In mouse, connexin channels are specifically involved in passive transport of molecules across the placental barriers.

The pro-angiogenic factor cysteine-rich protein 61 (CYR61/CCN1) mediates different signals in tumorigenesis, angiogenesis and is involved in the pathogenesis of endometriosis. In this study we investigated the temporal and spatial expression pattern in human endometrium during the menstrual cycle and its possible regulation mechanisms in the premenstrual phase. CYR61 transcript expression showed two distinct periods of elevated levels in the proliferative phase and in menstrual effluents. Because the menstrual breakdown of the functionalis is triggered by cytokines, prostaglandins (PGs), as well as hypoxia, we used a benign endometrial cell line to investigate if CYR61 is regulated by these factors. Hypoxic conditions transiently induced CYR61 mRNA levels and enhanced the secretion of the CYR61 protein into the medium. The hypoxia-inducible factor (HIF) 1alpha mediated this effect on CYR61 as evidenced by dimethyloxalylglycine treatment and by HIF1alpha short interfering RNA. CYR61 mRNA expression was further regulated by IL-1, TNFalpha, PGE2, and PGF2alpha. In addition, TNFalpha and PGE2 elevated significantly CYR61 cellular protein levels in well-oxygenated cells but had only a slight effect on the quantity of secreted protein. Moreover, PGE2 combined with hypoxic conditions increased CYR61 mRNA and protein levels synergistically, whereas the combination with TNFalpha abolished the CYR61 levels induced by hypoxia. Together, the up-regulation of CYR61 by hypoxia via HIF1alpha, TNFalpha, and PGE2 could represent possible mechanisms for the CYR61 increase at the onset of menstruation. The opposite effect of TNFalpha combined with hypoxia on CYR61 up-regulation could contribute to a balanced expression level of this angiogenic factor in the endometrium.

Objective. Data on trophoblast particles in peripheral maternal circulation are rare. Applying a new enrichment technique, Oncoquick®, we evaluated the number of trophoblast particles in maternal blood in preeclamptic and non-preeclamptic pregnancies. Methods. After isolation of trophoblast particles using Oncoquick®, trophoblast particles were detected by immunocytochemistry, applying a monoclonal pan cytokeratin (CK) antibody. Fetal origin of the detected particles was verified by XY- fish analysis in women delivering male infants. Results. Trophoblast particles were identified in 25/52 (48%) of preeclamptic and 1/20 (5%) of non-preeclamptic pregnancies. Conclusion. The number of trophoblast particles in peripheral maternal blood of preeclamptic pregnancies was significantly higher than in non-preeclamptic pregnancies.

During placental development, continuous invasion of trophoblasts into the maternal compartment depends on the support of proliferating extravillous trophoblasts (EVTs). Unlike tumor cells, EVTs escape from the cell cycle before invasion into the decidua and spiral arteries. This study focused on the regulation properties of glycosylated and non-glycosylated matricellular CCN1 and CCN3, primarily for proliferation control in the benign SGHPL-5 trophoblast cell line, which originates from the first-trimester placenta. Treating SGHPL-5 trophoblast cells with the glycosylated forms of recombinant CCN1 and CCN3 decreased cell proliferation by bringing about G0/G1 cell cycle arrest, which was accompanied by the upregulation of activated Notch-1 and its target gene p21. Interestingly, both CCN proteins increased senescence-associated β-galactosidase activity and the expression of the senescence marker p16. The migration capability of SGHPL-5 cells was mostly enhanced in response to CCN1 and CCN3, by the activation of FAK and Akt kinase but not by the activation of ERK1/2. In summary, both CCN proteins play a key role in regulating trophoblast cell differentiation by inducing senescence and enhancing migration properties. Reduced levels of CCN1 and CCN3, as found in early-onset preeclampsia, could contribute to a shift from invasive to proliferative EVTs and may explain their shallow invasion properties in this disease.

Loss of TFAP2C in mouse leads to developmental defects in the extra-embryonic compartment with lethality at embryonic day (E)7.5. To investigate the requirement of TFAP2C in later placental development, deletion of TFAP2C was induced throughout extra-embryonic ectoderm at E6.5, leading to severe placental abnormalities caused by reduced trophoblast population and resulting in embryonic retardation by E8.5. Deletion of TFAP2C in TPBPA(+) progenitors at E8.5 results in growth arrest of the junctional zone. TFAP2C regulates its target genes Cdkn1a (previously p21) and Dusp6, which are involved in repression of MAPK signaling. Loss of TFAP2C reduces activation of ERK1/2 in the placenta. Downregulation of Akt1 and reduced activation of phosphorylated AKT in the mutant placenta are accompanied by impaired glycogen synthesis. Loss of TFAP2C led to upregulation of imprinted gene H19 and downregulation of Slc38a4 and Ascl2. The placental insufficiency post E16.5 causes fetal growth restriction, with 19% lighter mutant pups. Knockdown of TFAP2C in human trophoblast choriocarcinoma JAr cells inhibited MAPK and AKT signaling. Thus, we present a model where TFAP2C in trophoblasts controls proliferation by repressing Cdkn1a and activating the MAPK pathway, further supporting differentiation of glycogen cells by activating the AKT pathway.

The gap junction gene Connexin31.1 has been reported to be expressed predominantly in the epidermis of murine skin. To study the function of this gene, we generated mice in which the coding DNA of the Connexin31.1 gene was replaced by lacZ reporter coding DNA. Using β-galactosidase staining, we have shown that lacZ/Connexin31.1 was expressed in the spinous and granular layers of the epidermis, in cells of olfactory epithelium and in the vomeronasal organ. During embryogenesis, Connexin31.1 was co-expressed with another isoform, Connexin31, in the post-implantation trophoblast cell lineage and, later in gestation, in placental glycogen cells. Although homozygous Connexin31.1-deficient mice were fertile and showed no morphological or functional defects in adult organs expressing this gene, 30% of the offspring expected according to Mendelian inheritance were lost between embryonic days 11.5 and 14.5 and surviving embryos were significantly reduced in weight near the end of pregnancy. Placentas of Connexin31.1-deficient embryos were reduced in weight and showed altered morphology of the spongiotrophoblast and labyrinth layer. The spongiotrophoblast formed a compact barrier at the decidual border that might restrict the maternal blood supply. We conclude that Connexin31.1 is critical for normal placental development but appears to be functionally compensated by other connexin isoforms in the embryo proper and adult mouse.

Objective To investigate the regulation of estrogen-converting enzymes in human ectopic endometrial tissue. Design Animal study. Setting Academic medical center. Animal(s) Sixty female nude mice with implanted human endometrial tissue. Patient(s) Twenty-two premenopausal women undergoing endometrial biopsy or hysterectomy. Intervention(s) Human endometrial tissue was implanted into the peritoneal cavity of nude mice, and the effect of therapeutic drugs on transcription of steroid receptors and estrogen-converting enzymes was analyzed. Main Outcome Measure(s) Transcript levels of steroid hormone receptors, 17β-hydroxysteroid dehydrogenase type 1 and 2, aromatase, and steroid sulfatase as well as proliferation rate were analyzed in the human ectopic endometrial tissue. Result(s) Steroid receptors and estrogen-converting enzymes were expressed in the ectopic human endometrial fragments. Application of medroxyprogesterone acetate, dydrogesterone, danazol, and the aromatase inhibitor finrozole significantly inhibited aromatase transcription. In addition, danazol caused a significant decrease in transcription of steroid sulfatase, and finrozole, of 17β-hydroxysteroid dehydrogenase type 1 in parallel to a decrease in proliferation rate in the ectopic human endometrial tissue. Conclusion(s) Pharmacological regulation of transcription of estrogen-converting enzymes in human endometrium cultured in nude mice may help to develop new therapeutic concepts based on local regulation of estrogen metabolism in endometriosis.

Erythrokeratodermia variabilis (EKV) is a rare autosomal dominant human genodermatosis. Its clinical appearance varies from transient, fast moving erythemas to persistent brown hyperkeratoses. So far, several mutations in the Cx31 or Cx30.3 gene have been reported to cause EKV in humans. We have generated a conditional mouse mutant that carries the human F137L mutation in the Cx31 gene which was described to act in a transdominant negative manner. The phenylalanine residue at position 137 is highly conserved in several human and mouse connexin genes. Mouse embryonic stem (ES) cells expressing one allele of the Cx31F137L mutation were stable but showed approximately 30% decreased transfer of neurobiotin. This is probably due to dominant negative effects of the Cx31F137L protein on wild type Cx31 and Cx43 protein expressed in ES cells. Surprisingly, the healing process of tail incision wounds in Cx31(+/F137L) mice was shortened by 1 day, i.e. very similar as previously reported for mice with decreased expression of Cx43 in the epidermis. This suggests again that Cx31 and Cx43 proteins functionally interact, possibly by forming heteromeric channels in the epidermis. Heterozygous Cx31(+/F137L) mice are viable and fertile, in contrast to homozygous Cx31(F137L/F137L) mice that die around ED 7.5. In Cx31(+/F137L) mice, the epidermal expression pattern and level of Cx26, Cx30, Cx30.3 and Cx43 proteins were not altered compared with wild-type mice. No erythemas were detected in young C31(+/F137L) mice before 2 weeks of age. In contrast to human EKV patients, hyperproliferation of the stratum germinativum was found in only 5% of the analyzed skin area.

Preeclampsia is a major cause of maternal, fetal and neonatal mortality worldwide. The etiology of preeclampsia is still unclear. Currently, the inadequate transformation of the smooth muscle cells of spiral arteries in the placental bed caused by an insufficient endovascular invasion of the trophoblast is considered to be the major reason for the development of preeclampsia. This insufficient invasion seems to lead to altered placental angiogenesis which thus has causal importance for the development of preeclampsia. Vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) are the most important angiogenic factors with regulatory functions for placental vessel development. In preeclamptic pregnancies the expression of VEGF and PLGF was decreased. However, the expression of the corresponding receptor, the soluble fms-like tyrosine kinase receptor, which is considered to have anti-angiogenic properties, was elevated in preeclamptic pregnancies. CYR61 and NOV which belong to a group of proteins, the CCN-family, play also an important role in the regulation of placental angiogenesis. Recently, a decreased expression of both proteins in the placentae of preeclamptic women was detected. This review summarizes our current understanding of the role of angiogenic factors in the pathogenesis of preeclampsia.

Tfap2c is required for placental development and trophoblast stem cell maintenance. Deletion of Tfap2c results in early embryonic loss because of failure in placental development. We evaluated the effect of reduced Tfap2c expression on fetal outcome and placental development. Sixty percent of the heterozygous mice were lost directly after birth. Labyrinthine differentiation was impaired, as indicated by enhanced proliferation and inclusions of cobblestone-shaped cell clusters characterized by expression of Tfap2c and glycogen stores. Moreover, expression of marker genes such as Cdx2, Eomes, Gata3, and Ascl2 are decreased in the spongiotrophoblast and indicate a lowered stem cell potential. On Day 18.5 postcoitum, the labyrinth layer of Tfap2c(+/-) placentas exhibited massive hemorrhages in the maternal blood spaces; these hemorrhages might have contributed to the significantly reduced number of live-born pups. These morphological alterations were accompanied by a shift toward sinusoidal trophoblast giant cells as the cell subpopulation lining the maternal sinusoids and toward reduction in expression of the prolactin gene family member Prl2c2, a finding characteristic of the spiral arteries lining trophoblast cells. The trophoblast stem cells heterozygous for Tfap2c exhibited a reduction in the expression level of stem cell markers and in their proliferation and differentiation capacity but did not exhibit changes in marker genes of the trophoblast giant cell lineage. Taken together, these findings indicate that a reduction in the gene dosage of placental Tfap2c leads to morphological changes in the labyrinth at midgestation and in the maternal blood spaces during late pregnancy.

Loss of TFAP2C in mouse leads to developmental defects in the extra-embryonic compartment with lethality at embryonic day (E)7.5. To investigate the requirement of TFAP2C in later placental development, deletion of TFAP2C was induced throughout extra-embryonic ectoderm at E6.5, leading to severe placental abnormalities caused by reduced trophoblast population and resulting in embryonic retardation by E8.5. Deletion of TFAP2C in TPBPA(+) progenitors at E8.5 results in growth arrest of the junctional zone. TFAP2C regulates its target genes Cdkn1a (previously p21) and Dusp6, which are involved in repression of MAPK signaling. Loss of TFAP2C reduces activation of ERK1/2 in the placenta. Downregulation of Akt1 and reduced activation of phosphorylated AKT in the mutant placenta are accompanied by impaired glycogen synthesis. Loss of TFAP2C led to upregulation of imprinted gene H19 and downregulation of Slc38a4 and Ascl2. The placental insufficiency post E16.5 causes fetal growth restriction, with 19% lighter mutant pups. Knockdown of TFAP2C in human trophoblast choriocarcinoma JAr cells inhibited MAPK and AKT signaling. Thus, we present a model where TFAP2C in trophoblasts controls proliferation by repressing Cdkn1a and activating the MAPK pathway, further supporting differentiation of glycogen cells by activating the AKT pathway.

The keratitis–ichthyosis–deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome.

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS) 2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.

The connexin family of proteins are best known as oligomerizing to form intercellular membrane channels (gap junctions) that metabolically and ionically couple cells to allow for coordinated cellular function. Nowhere in the body is this role better illustrated than in the uterine smooth muscle during parturition, where gap junctions conduct the contraction wave throughout the tissue to deliver the baby. Parturition is followed by the onset of lactation with connexins contributing to both the dramatic reorganization of mammary gland tissue leading up to lactation and the smooth muscle contraction of the myoepithelial cells which extrudes the milk. This review summarizes what is known about the expression and roles of individual connexin family members in the uterus during labour and in the mammary glands during development and lactation. Connexin loss or malfunction in mammary glands and the uterus can have serious implications for the health of both the mother and the newborn baby.

Several gap junction connexins have been shown to be essential for appropriate placental development and function. It is known that the expression and distribution of connexins change in response to environmental oxygen levels. The placenta develops under various oxygen levels, beginning at a low oxygen tension of approximately 2% and increasing to a tension of 8% after the onset of the uteroplacental circulation. Moreover, it has been shown that during preeclampsia (PE) placentas are subjected to chronic hypoxia. Therefore, we investigated oxygen sensitivity of placental connexins 43 and 46. Using the trophoblast cell line Jar, we demonstrated that the expression of connexin43 increased during acute hypoxia but decreased during chronic hypoxia. Chronic hypoxia resulted in the translocation of connexin43 from the membrane to the cytoplasm and in a reduction in its communication properties. In contrast, the expression of connexin46 was down-regulated during chronic hypoxia and was translocated from perinuclear areas to the cell membrane. Hypoxia-inducible factor (HIF) knockdown showed that the translocation of connexin43 but not that of connexin46 was HIF-2α dependent and was mediated by phosphoinositide 3-kinase. The up-regulation of connexin43 in combination with the down-regulation of connexin46 was confirmed in placental explants cultivated under low oxygen and in placentas with early-onset PE. Taken together, in Jar cells, placental connexins 43 and 46 are regulated during periods of low oxygen in opposite manners. The oxygen sensing of connexins in the trophoblast may play a role in physiological and pathophysiological oxygen conditions and thus may contribute to PE.

The expression of human CYR61 (cysteine-rich, angiogenic inducer, 61; CCN1) mRNA has been previously shown to be deregulated in the endometrium of women with endometriosis. We have chosen the baboon model (Papio anubis) of induced endometriosis to clarify whether CYR61 mRNA upregulation is predisposed to an inappropriately differentiated endometrium or is deregulated as a response to the presence of ectopic lesions. In the baboon, endometrial CYR61 mRNA expression underwent moderate cyclical variation, with a significant 7.3-fold increase detected at Day 2 postmenses when compared to endometrium from the proliferative and secretory phases. The CYR61 transcript was extensively upregulated in the eutopic endometrium from all baboons with induced endometriosis, as early as 1 mo postinoculation of menstrual tissue into the peritoneal cavity. CYR61 mRNA expression then decreased throughout progression of the disease, but remained higher compared to control tissues. Ectopic endometriotic lesions showed a further increase in CYR61 mRNA, with highest expression found in red lesions. Moreover, the expression levels of CYR61 transcripts correlated significantly with those of VEGF. Immunohistochemistry revealed the presence of CYR61 protein in glandular and luminal epithelial cells as well as in blood vessels of eutopic and ectopic endometrium. As in humans, increased levels of CYR61 mRNA correlated with the development of endometriosis in baboons. The increase of CYR61 mRNA in eutopic endometrium of baboons following peritoneal inoculation with menstrual endometrium provides evidence for a feedback mechanism from resulting lesions to induce a shift in gene expression patterns in the eutopic endometrium. estradiol, female reproductive tract, menstrual cycle, uterus