Giant Trophoblast Research Articles

Anti‐mouse renin expression in the rat kidney

The causes of toxemia of pregnancy are not fully understood. This project examines rat kidneys to determine if a mouse anti‐renin antibody will cross‐react rat renin‐producing cells in the rat. This will allow further study female reproductive tissues in the rat to examine for renin production. Renin is known as hormone produced by the juxtaglomerular cells of the afferent arteriole of the kidney in response low blood pressure. Also, renin is an immediate downstream target of Class I Hox gene regulation, important in embryonic development. In mice, renin is expressed in embryonic renal, adrenal and gonadal arteries, the adrenal glands, and tissue associated with embryonic pronephric and mesonephric kidneys. Using a renin/GFP reporter in transgenic mice, renin is expressed in placental giant trophoblast cells and uterine/ovarian artery cells of the tunica adventitia. In RCS rats, renin antibody stain that used anti‐rat renin and peroxidase labeled placental giant trophoblast cells and cells of the tunica adventitia of the uterine/ovarian artery. This project examines rat kidney tissue labeled by indirect immuno‐fluorescence with anti‐mouse renin antibody and fluorescent conjugate. Cells in the juxtaglomerular region of the kidney were renin positive. This demonstrates that anti‐mouse renin antibody can dependably label regions in the rat kidney that are known for renin production. This will allow for further studies to determine renin location in rat female reproductive tissues. Dr. Craig Jones of Roswell Park Cancer Institute generously donated mouse renin antibody and lab space for stain procedures. D'Youville College Faculty Council and Department of Math & Natural Sciences supported work.

Down-regulation of the transcription factor snail in the placentas of patients with preeclampsia and in a rat model of preeclampsia

BackgroundPlacental malfunction in preeclampsia is believed to be a consequence of aberrant differentiation of trophoblast lineages and changes in utero-placental oxygenation. The transcription factor Snail, a master regulator molecule of epithelial-mesenchymal transition in embryonic development and in cancer, is shown to be involved in trophoblast differentiation as well. Moreover, Snail can be controlled by oxidative stress and hypoxia. Therefore, we examined the expression of Snail and its downstream target, e-cadherin, in human normal term, preterm and preeclamptic placentas, and in pregnant rats that developed preeclampsia-like symptoms in the response to a 20-fold increase in sodium intake.MethodsWestern blotting analysis was used for comparative expression of Snail and e- cadherin in total protein extracts. Placental cells expressing Snail and e-cadherin were identified by immunohistochemical double-labeling technique.ResultsThe levels of Snail protein were decreased in human preeclamptic placentas by 30% (p < 0.01) compared to normal term, and in the rat model by 40% (p < 0.001) compared to control placentas. In preterm placentas, the levels of Snail expression varied, yet there was a strong trend toward statistical significance between preterm and preeclamptic placentas. In humans, e-cadherin protein level was 30% higher in preeclamptic (p < 0.05) placentas and similarly, but not significantly (p = 0.1), high in the preterm placentas compared to normal term. In the rat model of preeclampsia, e-cadherin was increased by 60% (p < 0.01). Immunohistochemical examination of human placentas demonstrated Snail-positive staining in the nuclei of the villous trophoblasts and mesenchymal cells and in the invasive trophoblasts of the decidua. In the rat placenta, the majority of Snail positive cells were spongiotrophoblasts of the junctional zone, while in the labyrinth, Snail-positive sinusoidal giant trophoblasts cells were found in some focal areas located close to the junctional zone.ConclusionWe demonstrated that human preeclampsia and the salt-induced rat model of preeclampsia are associated with the reduced levels of Snail protein in placenta. Down-regulation of the transcription factor Snail in placental progenitor cell lineages, either by intrinsic defects and/or by extrinsic and maternal factors, may affect normal placenta development and function and thus contribute to the pathology of preeclampsia.

Maternal diabetes affects cell proliferation in developing rat placenta.

Placentation starts with the formation of a spheroidal trophoblastic shell surrounding the embryo, thus facilitating both implantation into the uterine stroma and contact with maternal blood. Although it is known that diabetes increases the placental size and weight, the mechanisms responsible for this alteration are still poorly understood. In mammals, cellular proliferation occurs in parallel to placental development and it is possible that diabetes induces abnormal uncontrolled cell proliferation in the placenta similar to that seen in other organs (e.g. retina). To test this hypothesis, the objective of this work was to determine cell proliferation in different regions of the placenta during its development in a diabetic rat model. Accordingly, diabetes was induced on day 2 of pregnancy in Wistar rats by a single injection of alloxan (40 mg/kg i.v.). Placentas were collected on days 14, 17, and 20 postcoitum. Immunoperoxidase was used to identify Ki67 nuclear antigen in placental sections. The number of proliferating cells was determined in the total placental area as well as in the labyrinth, spongiotrophoblast and giant trophoblast cell regions. During the course of pregnancy, the number of Ki67 positive cells decreased in both control and diabetic rat placentas. However, starting from day 17 of pregnancy, the number of Ki67 positive cells in the labyrinth and spongiotrophoblast regions was higher in diabetic rat placentas as compared to control. The present results demonstrate that placentas from the diabetic rat model have a significantly higher number of proliferating cells in specific regions of the placenta and at defined developmental stages. It is possible that this increased cell proliferation promotes thickness of the placental barrier consequently affecting the normal maternal-fetal exchanges.

Endopolyploid and proliferating trophoblast cells express different patterns of intracellular cytokeratin and glycogen localization in the rat placenta

The presence of keratin intermediate filaments is a characteristic of trophoblast differentiation. Meantime, their intracellular localization in the functionally different subtypes of placental trophoblast is poorly investigated in rodent, whereas their placentae are being broadly investigated in recent years as a model of the feto-maternal interaction. The purpose was to study the intracellular distribution of cytokeratin filaments in correlation with glycogen deposits, both being important constituents of the trophoblast cells in rat placenta. Different rat trophoblast cell populations exhibited different patterns of cytokeratin immunolocalization. The most intensive immunostaining was observed in the highly endopolyploid SGTCs (secondary giant trophoblast cells) at the border with decidua basalis. The most prominent cytokeratin-positive threads were found at the periphery of cytoplasm and in the extensive system of cytoplasmic sprouts by which the SGTC connect each other. Similar cytokeratin intensity and distribution was detected in the TSC (trabecular spongiotrophoblast cells) of the junctional zone of placenta that line the lacunae with the maternal blood. Clusters of highly proliferative pre-glycogen as well as glycogen cells showed some weaker cytokeratin signals mostly in the perinuclear and peripheral zones of cytoplasm. At the 11.5th to the 13.5th day of gestation, the interstitial and endovascular invasive endopolyploid TGTCs (tertiary giant trophoblast cells) prove the intensive cytokeratin staining throughout the cytoplasm and its sprouts. Meantime, the TGTCs were glycogen negative. By contrast, glycogen was heavily accumulated in the glycogen cells that belong both to the junctional zone of placenta and the cuff of the central arterial channel underlying the monolayer of endovascularly invading TGTCs. Thus, the TGTCs that are first to penetrate into the depth of the uterine wall do not contain glycogen but are accompanied by the glycogen-rich cells. The SGTC also contained the prominent deposits of glycogen at the periphery of cytoplasm and in the cytoplasmic sprouts. At the 16th day of gestation, an extensive interstitial invasion of the cytokeratin-positive glycogen trophoblast cells from the junctional zone was observed. The patterns of cytokeratin and glycogen intracellular localization are specific for each subtype of the rat trophoblast; that is, most probably, accounted for by the functional diversity of different trophoblast populations, i.e. patterns of invasion/phagocytosis and their involvement in a barrier at the feto-maternal interface.

Regulation of proteinases during mouse peri-implantation development: urokinase-type plasminogen activator expression and cross talk with matrix metalloproteinase 9

Trophoblast cells express urokinase-type plasminogen activator (PLAU) and may depend on its activity for endometrial invasion and tissue remodeling during peri-implantation development. However, the developmental regulation, tissue distribution, and function of PLAU are not completely understood. In this study, the expression of PLAU and its regulation by extracellular matrix proteins was examined by RT-PCR, immunocytochemistry, and plasminogen-casein zymography in cultured mouse embryos. There was a progressive increase in Plau mRNA expression in blastocysts cultured on gestation days 4-8. Tissue-type plasminogen activator (55 kDa) and PLAU (a triplet of 40, 37, and 31 kDa) were present in conditioned medium and embryo lysates, and were adsorbed to the culture plate surface. The temporal expression pattern of PLAU, according to semi-quantitative gel zymography, was similar in non-adhering embryos and embryos cultured on fibronectin, laminin, or type IV collagen, although type IV collagen and laminin upregulated Plau mRNA expression. Immunofluorescence revealed PLAU on the surface of the mural trophectoderm and in non-spreading giant trophoblast cells. Exogenous human plasminogen was transformed to plasmin by cultured embryos and activated endogenous matrix metalloproteinase 9 (MMP9). Indeed, the developmental expression profile of MMP9 was similar to that of PLAU. Our data suggest that the intrinsic developmental program predominantly regulates PLAU expression during implantation, and that PLAU could be responsible for activation of MMP9, leading to localized matrix proteolysis as trophoblast invasion commences.

Renin Immunofluorescence in the metestrus rat uterus

Renin, produced by juxtaglomerular cells of the kidney to activate the renin‐angiotensin‐aldosterone pathway in response to low blood volume and/or pressure, also is a hox gene product that is important in the development of abdominal structures. When linked to green fluorescent protein, renin is expressed by adrenal, renal, and gonadal vessels in the mouse embryo. The ovarian/uterine artery continues to express renin after birth, although the testicular artery does not, and the quantity of expression varies with stage of estrus cycle and during pregnancy. Renin is also expressed in the giant trophoblast cells of the placenta. In the rat, pericytes of the ovarian/uterine artery are immunofluorescent for renin. This project specifically examines the rat uterus in the metestrus stage of the estrus cycle. Using anti‐mouse renin antibody stain, renin is localized to the tunica media of spiral arteries in the uterus and a larger vessel coursing through the serosa. With anti‐rat renin antibody stain, renin also localized to the tunica media of the same vessels. These results help define a baseline in the amount of renin expression in the uterus with which to compare other stages of the estrus cycle and pregnancy to possibly link renin expression with human conditions of premenstrual syndrome and toxemia of pregnancy. Support by NSF grants #0132823 &amp; 0726185, and D'Youville faculty council research fund

FITC localization of renin in rat mesometrial tissue

Renin, a hormone produced primarily by the kidneys in response to low blood pressure, is an immediate downstream gene product of Class I HOX gene regulation in the embryo. Examination of transgenic mice in which renin was linked to gfp reveals renin in renal, adrenal, and gonadal arteries in the embryo, giant trophoblast cells of the placenta, kidney and submandibular gland in both male and female, and uterine artery. In rat, giant trophoblast cells, other cells in fetal and maternal regions of the placenta, and cells of the tunica adventita of uterine artery as well as in the wall of the afferent arteriole of the kidney were labeled with antibody stain. This project examines pregnant and nonpregnant rat mesometrial tissues to identify cells that localize FITC labeled sheep anti‐rat renin antibody. Fluorescent granules were seen in cells resembling fibroblasts throughout the mesometrium and along the walls of small vessels within the mesometrium. Afferent arterioles were labeled and control tissues were not. These results demonstrate that the mesometrium is another site within rat female reproductive tissues in which renin is located, adding support to the hypothesis that extrarenal renin production may play a role in preecclampsia. Work is supported by NSF grant #0132823 and 0726185, and D’Youville College faculty council research funding.

Targeting of the mouse Slc39a2 (Zip2) gene reveals highly cell‐specific patterns of expression, and unique functions in zinc, iron, and calcium homeostasis

Fourteen members of the Slc39a superfamily of metal ion uptake transporters have been identified in mice and humans, but the physiological functions of most remain obscure. Herein, we created mice with Zip2 (Slc39a2) genes in which the open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP), to study temporal and spatial patterns of Zip2 gene expression and examine the physiological roles of this transporter. Expression of this gene was remarkably cell-type specific and developmentally regulated in pericentral hepatocytes, developing keratinocytes, and a subset of immature dendritic cells in the immune system. In addition, the Zip2 gene was transiently expressed in giant trophoblast cells in the placenta. Although the Zip2 gene was not essential under conditions of normal dietary zinc, it played an important role in adapting to dietary zinc deficiency during pregnancy, and in the homeostasis of iron in the liver as well as iron and calcium in developing embryos. These studies suggest that active expression of the Zip2 gene in these few specific cell types, aforementioned, plays a particularly important role during zinc deficiency. These studies further reveal novel interactions between zinc transporter function and the homeostasis of other essential metals.

Modifications of nuclear envelope during differentiation and depolyploidization of rat trophoblast cells

An increased activity of membranes of the nuclear envelope (NE) was observed electron microscopically in the trophoblast cells of the rat placenta. The activity of the membranes was manifested as formation of various NE derivatives, such as the annulate lamellae (AL), the intranuclear tubules, and the concentric membranous structures. At the period of terminal differentiation of the secondary giant trophoblast cells (SGTC) the NE derivatives play active role in subdivision of the initial highly polyploid nuclei into the numerous low-ploidy fragments. 3H-thymidine labeling showed that attenuation of the DNA replication precedes the nuclear fragmentation. In the course of the nuclear fragmentation the narrow deep NE invaginations subdivide the nucleus into the separate lobes that subsequently are detached from the initial nucleus. By the beginning of the fragmentation, the accumulated membranous structures, i.e. the intranuclear AL, tubules, clusters of pore complexes, etc., seem to be the source of the reserve material that is necessary for formation of the great amount of the NE membranes of the newly formed nuclear fragments. Thus, the intranuclear membranous structures that seem to increase the active surface of the growing endopolyploid nucleus at the earlier stage of differentiation then take part in genome isolation that results in formation of a multinucleate cell with diploid and low-polyploid nuclei. The outer NE membrane of the initial nucleus plays an active role in compartmentalization of cytoplasmic areas around the nuclear fragments within the giant polykaryocyte. Apart from the membranous structures the bundles of intermediate filaments (IF) located in the cytoplasm perinuclear zones seem to participate in the nuclear fragmentation. These processes are likely to provide formation of the giant polykaryocytes incapable for further proliferation.

Global gene expression analysis and regulation of the principal genes expressed in bovine placenta in relation to the transcription factor AP-2 family

BackgroundCell-cell communication is an important factor in feto-maternal units during placentogenesis. The placenta produces pivotal hormones and cytokines for communication between cotyledonary villi and the maternal caruncle. Gene expression in bovine placenta throughout pregnancy was comprehensively screened by a cDNA microarray, and we searched for a common transcription factor in a gene cluster that showed increasing expression throughout gestation in cotyledonary villi and caruncle.MethodsPlacentomal tissues (villi and caruncle) were collected from Day 25 to Day 250 of gestation for microarray analysis. Global gene expression profiles were analyzed using the k-means clustering method. A consensus sequence cis-element that may control up-regulated genes in a characteristic cluster was examined in silico. The quantitative expression and localization of a specific transcription factor were investigated in each tissue using quantitative real-time RT-PCR and in situ hybridization.ResultsThe microarray expression profiles were classified into ten clusters. The genes with most markedly increased expression became concentrated in cluster 2 as gestation proceeded. Cluster 2 included placental lactogen (CSH1), pregnancy-associated glycoprotein-1 (PAG1), and sulfotransferase family 1E estrogen-preferring member 1 (SULT1E1), which were mainly detected in giant trophoblast binucleate cells (BNC). Consensus sequence analysis identified transcription factor AP-2 binding sites in some genes in this cluster. Quantitative real-time RT-PCR analysis confirmed that high level expression of transcription factor AP-2 alpha (TFAP2A) was common to cluster 2 genes during gestation. In contrast, the expression level of another AP-2 family gene, transcription factor AP-2 beta (TFAP2B), was extremely low over the same period. Another gene of the family, transcription factor AP-2 gamma (TFAP2C), was expressed at medium level compared with TFAP2A and TFAP2B. In situ hybridization showed that TFAP2A, TFAP2B and TFAP2C mRNAs were localized in trophoblast cells but were expressed by different cells. TFAP2A was expressed in cotyledonary epithelial cells including BNC, TFAP2B was specifically expressed in BNC, and TFAP2C in mononucleate cells.ConclusionWe detected gestational-stage-specific gene expression profiles in bovine placentomes using a combination of microarray and in silico analysis. In silico analysis indicated that the AP-2 family may be a consensus regulator for the gene cluster that characteristically appears in bovine placenta as gestation progresses. In particular, TFAP2A and TFAP2B may be involved in regulating binucleate cell-specific genes such as CSH1, some PAG or SULT1E1. These results suggest that the AP-2 family is a specific transcription factor for clusters of crucial placental genes. This is the first evidence that TFAP2A may regulate the differentiation and specific functions of BNC in bovine placenta.

Immunohistochemical localization of renin in rat female reproductive tissues

This analysis is to determine if any relationship exists between renin expression in female tissues and toxemia of pregnancy. Renin, produced by juxtaglomerular cells of kidney afferent arterioles, catalyzes the rate-limiting step of the renin-angiotensin system to regulate blood pressure and electrolyte balance. The renin gene is an immediate downstream target of Class I Hox gene regulation. Expression was detected in embryonic transgenic mice in renal, adrenal and gonadal arteries, adrenal glands and tissue associated with the mesonephric kidney, and placental giant trophoblast cells with a renin/green fluorescent protein transgenic reporter construct. Renin expression in mouse ovarian/uterine artery revealed intensity differences between stages of the estrus cycle. In female rats, anti-rat renin antibody and HRP-labeled conjugate localized renin to giant trophoblast cells, and anti-mouse renin antibody and fluorescein conjugate located renin to afferent arterioles and cells of the uterus and placenta. In this study, anti-mouse renin antibody localized renin positive cells to the tunica externa of rat ovarian/uterine artery, with intensity differences between pregnant and nonpregnant rats, and cells in stages of the placenta. Continuing studies are looking to provide rationale to these differences, and determine the significance of the production and presence of this protein through pregnancy and placental development. Work supported by NSF MCB 0132823 and the Department of Math & Natural Sciences of D'Youville College.

Cell reproduction and genome multiplication in the proliferative and invasive trophoblast cell populations of mammalian placenta

Spatiotemporal "time-table" of ways of cell reproduction (mitosis, restitutional mitosis, endomitosis, endoreduplication) of trophoblast cell populations is described. The populations of mitotically active trophoblast cells (diploid and low-polyploid) are located mostly out of contact with maternal tissues. In rodent placenta they mainly switch from mitotic cycle to polyploidizing (restitutional) mitoses and reach 4c-8c. Thereafter they switch to endoreduplication and reach 16c-64c. Following a series of endoreduplication cycles a part of this cell population sets apart and penetrates deeply into the decidualized endometrium and myometrium, their capabilities for replication being lost progressively (in rodent--256c-1024c). The invasive trophoblast cells that reach 256c-1024c via endoreduplication simultaneously form a barrier between semiallogenic fetal and maternal tissues. Arrest of mitoses and complete repression of DNA replication after a series of endoreduplication cycles makes hardly probable the renewal of mitotic activity in the deeply invading tertiary giant trophoblast cells, thereby preventing the possibility of their ectopic expanding in the maternal tissues during the normal pregnancy.

Whole-genome chromosome distribution during nuclear fragmentation of giant trophoblast cells of Microtus rossiaemeridionalis studied with the use of gonosomal chromatin arrangement

Gonosomal chromatin bodies (GCBs), i.e. blocks of condensed chromatin consisting of heterochromatized region of the sex chromosomes of the field vole M. rossiaemeridionalis, were used as a natural interphase chromosome marker in order to clarify the regularities of GCB rearrangement during nuclear fragmentation of secondary giant trophoblast cells (SGTCs) at the end of their differentiation. Cytophotometrical measurements of DNA content in the nuclei, nuclear fragments and simultaneously in the GCBs were made in the secondary giant SGTCs of field vole M. rossiaemeridionalis. In most cases 1 to 2 GCBs get into the nuclear fragments at different ploidy levels. In the nuclear fragments, GCB DNA content decreased mostly proportionally to DNA content in the whole fragments corresponding to 2c, 4c and 8c. The data obtained demonstrate a regular whole-genome chromosome distribution into nuclear fragments. A possible mechanism of nuclear fragmentation that largely ensures a balanced genome in nuclear fragments is discussed.

Extraembryonic expression of EPCR is essential for embryonic viability

AbstractThe endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex. Deletion of the EPCR gene (Procr) in mice leads to embryonic lethality before embryonic day 10 (E10.0). EPCR is detected in the giant trophoblast cells at the feto-maternal boundary from E7.5 and weakly in embryonic aortic endothelial cells from E13.5, suggesting that extraembryonic EPCR expression may be essential for embryonic viability. Using conditional knock-out strategies, we demonstrate that Procr-deficient embryos with EPCR expression on placenta giant trophoblasts can be carried to term and then develop normally. Conversely, EPCR expression in the embryo, without expression in the giant trophoblast cells, does not rescue the mice. In genetically modified mice with low tissue factor activity, Procr deficiency is not lethal to the embryo. As adults, Procr-deficient mice generate more thrombin and activate less protein C in response to procoagulant stimuli. Spontaneous thrombin formation in the deficient animals increases with age. These findings show that extraembryonic EPCR expression is critical for embryo development.

Expression of Golf in the rat placenta: Possible implication in olfactory receptor transduction

Olfactory receptors are G-protein coupled receptors and are encoded by an extremely large and diverse family of genes in mammals. There is increasing evidence that olfactory receptors are widely distributed in many organs, suggesting that olfactory receptors do not only recognize airborne odorants but also play important roles in chemotaxis or organ construction in embryo. In this study, we investigated whether olfactory receptors and their transduction molecule, Golf are expressed in the rat placenta. By RT-PCR, we identified 11 different olfactory receptor genes, which are all members of class II, in the rat placenta cDNAs, and our results suggested that particular members of the olfactory receptor gene family might be preferentially expressed in the placenta. By western blot analysis, we demonstrated that Golf protein is expressed in the placenta and its expression levels are developmentally regulated. We found that Golf immunoreactivity is exclusively localized to giant cell trophoblasts and spongiotrophoblast cells. These findings raised a possibility that a particular subset of olfactory receptors might be coupled with Golf and function in giant cell trophoblasts and spongiotrophoblast cells.

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.

274. Placental expression of secreted frizzled related protein (sFRP4) in the rat: association with β-catenin localization and regulation by glucocorticoids

Wnt genes regulate a diverse range of developmental processes including placental development. Activation of the wnt pathway results in nuclear translocation of β-catenin and activation of the TCF/Lef family of transcription factors. The secreted frizzled related proteins (sFRPs) modulate wnt signaling by binding to either the wnt ligand or its transmembrane frizzled receptor. The current study examined the spatial and temporal expression of one of these secreted molecules, sFRP4, in the rat placenta over the final third of pregnancy, and whether associated changes occurred in the expression and localization of β-catenin. The expression of sFRP4 and β-catenin was also analysed in a model of glucocorticoid-induced placental growth restriction. Analysis by quantitative RT-PCR over the final third of pregnancy demonstrated a dramatic increase in sFRP4 mRNA (14-fold, P &lt; 0.001) specifically within the basal zone of the placenta near term. In situ hybridization and immunohistochemistry localized sFRP4 expression primarily to giant trophoblasts of the basal zone. In addition, sFRP4 protein was notably upregulated in association with a restricted nuclear translocation of β-catenin. Maternal dexamethasone treatment (1 μg/mL in drinking water; day 13–22) further increased the expression of sFRP4 mRNA in both the basal (120%, P &lt; 0.05) and labyrinth (285%, P &lt; 0.01) zones of the placenta at day 22 (term = 23 days) compared to untreated controls. These data indicate that sFRP4 expression is increased in the basal zone of the rat placenta, the major site of apoptosis in late pregnancy, and is further stimulated by glucocorticoids. Moreover, the observed inhibition of β-catenin expression and its nuclear translocation suggest that sFRP4 inhibition of wnt signaling in the placenta may contribute to placental apoptosis and ultimately fetal growth restriction.

Increased matrix metalloproteinases 2 and 9 in placenta of diabetic rats at midgestation

Matrix metalloproteinases (MMPs) are involved in placental remodelling throughout pregnancy. Diabetes mellitus induces alterations in tissue production of NO, a regulator of MMPs activity. The present work evaluates placental and fetal MMPs and NO levels during midpregnancy in neonatal streptozotocin-induced diabetic rats. MMP-2 and MMP-9 immunolabelling was increased both in the labyrinth zone ( p < 0.001) and in the giant trophoblast cells of the junctional zone ( p < 0.001) from diabetic placenta, when compared with controls. Also MMP-2 ( p < 0.01) and MMP-9 ( p < 0.005) activities were increased in both maternal and fetal sides of diabetic placenta when related to controls. In both sides of the diabetic placenta, nitrate/nitrite concentrations (which indicate NO production) were higher than in controls ( p < 0.05). An intense immunostaining for nitrotyrosine, indicating peroxynitrite-induced damage, was found in both labyrinth ( p < 0.001) and junctional zones ( p < 0.001) of diabetic placenta. Enhanced MMP-2 activity ( p < 0.05) and NO production were also higher in the fetuses from diabetic rats when compared to controls ( p < 0.005). These findings demonstrate alterations in MMPs and NO in the feto-placental unit of diabetic rats, anomalies that are likely to be involved in the developmental alterations induced by maternal diabetes.

Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein.

The mammalian F-box protein Fbw7 and its Caenorhabditis elegans counterpart Sel-10 have been implicated in the ubiquitin-mediated turnover of cyclin E as well as the Notch/Lin-12 family of transcriptional activators. Both unregulated Notch and cyclin E promote tumorigenesis, and inactivating mutations in human Fbw7 suggest that it may be a tumor suppressor. To generate an in vivo system to assess the consequences of such unregulated signaling, we generated mice deficient for Fbw7. Fbw7-null mice die around 10.5 days post coitus because of a combination of deficiencies in hematopoietic and vascular development and heart chamber maturation. The absence of Fbw7 results in elevated levels of cyclin E, concurrent with inappropriate DNA replication in placental giant trophoblast cells. Moreover, the levels of both Notch 1 and Notch 4 intracellular domains were elevated, leading to stimulation of downstream transcriptional pathways involving Hes1, Herp1, and Herp2. These data suggest essential functions for Fbw7 in controlling cyclin E and Notch signaling pathways in the mouse.

154THE EFFECT OF FIBROBLAST GROWTH FACTOR-4 ON THE DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS

Fibroblast growth factor-4 (FGF-4) has been shown to be preferentially produced in the inner cell mass (ICM) of mouse blastocysts, to stimulate the proliferation of mouse trophectoderm (TE) cells and to repress their transformation and differentiation into giant trophoblasts. Recent studies have shown that nuclear transfer (NT) bovine embryos have aberrant allocations of ICM and TE cells (Koo et al., 2002 Biol. Report. 67, 487–492) and aberrant expression of FGF-4 gene (Daniels et al., 2000 Biol. Reprod.63, 1034–1040). In this study, we examined whether recombinant human FGF-4 (rhFGF-4) stimulates development of nuclear transfer bovine embryos. As donor cells for NT, bovine ear skin fibroblast cells of passage 5 to 8 were used. Oocytes were enucleated after in vitro maturation in TCM 199 supplemented with 10% FBS, 1μgmL−1 FSH and 1μgmL−1 estradiol-17β for 20h. Enucleated oocytes were fused with donor cells by a DC pulse of 25V/150μm for 10μs in Zimmerman cell fusion medium. For activation, fused oocytes were exposed to 10μM Ca-Ionophore for 5min, followed by 2mM 6-dimethylaminopurine for 3h. NT embryos were subsequently cultured in CR2 medium (containing 0.5% BSA) without or with rhFGF-4 (1, 10 and 100ngmL−1) at 39.0°C in 5% O2, 5% CO2 and 90% N2. After 7 days of culture, blastocyst formation was observed. Apoptotic cells in blastocysts were detected by a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay and total cell number was examined by propidium iodide (PI) counterstaining. Data were analyzed by chi-square test and Student’s t-test. Supplementation of serum-free medium with rhFGF-4 increased the proportion of embryos developing to the blastocyst stage (18.4, 29.4 and 23.5% for 0, 1 and 10ngmL−1 FGF-4, respectively) and total cell number of blastocysts (66.3±11.4, 75.9±25.5 and 74.4±22.4 for 0, 1 and 10ngmL−1 FGF-4). Particularly, 100ngmL−1 FGF-4 significantly (P&amp;lt;0.05) increased the proportion of blastocysts (40.4%) and total cell number of blastocysts (86.7±26.5) when compared with TCM 199 medium alone. FGF-4 also decreased the mean proportion of apoptotic cells in blastocysts (10.6±7.8, 7.4±5.3, 8.6±5.3 and 7.5±4.1% for 0, 1, 10 and 100ngmL−1 FGF-4). Our results suggest that FGF-4 may play a role in the early development of NT bovine embryos and might be a useful molecule for increasing development of NT bovine embryos in serum-free culture systems.