Expression of NADPH Oxidase by Trophoblast Cells: Potential Implications for the Postimplanting Mouse Embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [ reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.

Interferon-gamma alters the phagocytic activity of the mouse trophoblast

Interferon-gamma (IFN-gamma) mediates diverse functions in bone marrow-derived phagocytes, including phagocytosis and microbe destruction. This cytokine has also been detected at implantation sites under both physiological and pathological conditions in many different species. At these particular sites, the outermost embryonic cell layer in close contact with the maternal tissues, the trophoblast exhibits intense phagocytic activity. To determine whether IFN-gamma affects phagocytosis of mouse-trophoblast cells, ectoplacental cone-derived trophoblast was cultured and evaluated for erythrophagocytosis. Phagocytic activity was monitored ultrastructurally and expressed as percentage of phagocytic trophoblast in total trophoblast cells. Conditioned medium from concanavalin-A-stimulated spleen cells significantly enhanced trophoblast phagocytosis. This effect was blocked by pre-incubation with an anti-IFN-gamma neutralizing antibody. Introduction of mouse recombinant IFN-gamma (mrIFN-gamma) to cultures did not increase cell death, but augmented the percentage of phagocytic cells in a dose-dependent manner. Ectoplacental cones from mice deficient for IFN-gamma receptor alpha-chain showed a significant decrease of the phagocytosis, even under mrIFN-gamma stimulation, suggesting that IFN-gamma-induced phagocytosis are receptor-mediated. Reverse transcriptase-PCR analyses confirmed the presence of mRNA for IFN-gamma receptor alpha and beta-chains in trophoblast cells and detected a significant increase in the mRNA levels of IFN-gamma receptor beta-chain, mainly, when cultured cells were exposed to IFN-gamma. Immunohistochemistry and Western blot analyses also revealed protein expression of the IFN-gamma receptor alpha-chain. These results suggest that IFN-gamma may participate in the phagocytic activation of the mouse trophoblast, albeit the exact mechanism was not hereby elucidated. Protective and/or nutritional fetal benefit may result from this physiological response. In addition, our data also shed some light on the understanding of trophoblast tolerance to inflammatory/immune cytokines during normal gestation.

Effects of Matrix Metalloproteinase (MMP)-2 Polymorphisms on Responsiveness to Antihypertensive Therapy of Women with Hypertensive Disorders of Pregnancy

Imbalanced matrix metalloproteinase (MMP) expression, including MMP-2, has been demonstrated in pre-eclampsia. However, little is known about the effect of polymorphisms in MMP-2 gene on hypertensive disorders of pregnancy. We examined whether two functional MMP-2 polymorphisms (g.-1306C>T and g.-735C>T) are associated with pre-eclampsia and/or gestational hypertension and whether these polymorphisms affect therapeutic responses in women with these conditions. We studied 216 healthy pregnant women (HP), 185 patients with gestational hypertension (GH) and 216 patients with pre-eclampsia (PE). They were stratified as responsive or non-responsive to antihypertensive therapy according to clinical and laboratorial parameters of therapeutic responsiveness. Genomic DNA was extracted from whole blood and genotypes for g-1306C>T and g.-735C>T polymorphisms were determined by real-time PCR using Taqman allele discrimination assays. Haplotype frequencies were inferred using the PHASE 2.1 program. The distributions of MMP-2 genotypes and haplotypes were similar in HP, GH and PE patients (p > 0.05). In addition, we found no significant differences in MMP-2 genotype or haplotype frequencies when GH or PE patients were classified as responsive or non-responsive to antihypertensive therapy (p > 0.05). Our results suggest that MMP-2 polymorphisms do not affect the susceptibility to hypertensive disorders of pregnancy. In parallel, MMP-2 polymorphisms apparently do not affect the responsiveness to antihypertensive therapy of women with these hypertensive disorders of pregnancy.

Modulation of Maternal Immune System During Pregnancy in the Cow

There is a molecular crosstalk between the trophoblast and maternal immune cells of bovine endometrium. The uterine cells are able to secrete cytokine/chemokines to either induce a suppressive environment for establishment of the pregnancy or to recruit immune cells to the endometrium to fight infections. Despite morphological differences between women and cows, mechanisms for immune tolerance during pregnancy seem to be conserved. Mechanisms for uterine immunesuppression in the cow include: reduced expression of major histocompatability proteins by the trophoblast; recruitment of macrophages to the pregnant endometrium; and modulation of immune-related genes in response to the presence of the conceptus. Recently, an eGFP transgenic cloned embryo model developed by our group showed that there is modulation of foetal proteins expressed at the site of syncytium formation, suggesting that foetal cell can regulate not only by the secretion of specific factors such as interferon-tau, but also by regulating their own protein expression to avoid excessive maternal recognition by the local immune system. Furthermore, foetal DNA can be detected in the maternal circulation; this may reflect the occurrence of an invasion of trophoblast cells and/or their fragment beyond the uterine basement membrane in the cow. In fact, the newly description of exosome release by the trophoblast cell suggests that could be a new fashion of maternal-foetal communication at the placental barrier. Additionally, recent global transcriptome studies on bovine endometrium suggested that the immune system is aware, from an immunological point of view, of the presence of the foetus in the cow during early pregnancy.

Chorioallantoic and yolk sac placentation in Thrichomys laurentinus (Echimyidae) and the evolution of hystricognath rodents

The evolutionary history of Hystricognathi is associated with major transformations in their placental system. Data so far indicate that key characters are independent from size dimensions in medium to very large species. To better understand the situation in smaller species, we analyzed placental development in a spiny rat, Thrichomys laurentinus. Fourteen individuals ranging from early implantation to near term were investigated by histology, immunohistochemistry, proliferation activity and electron microscopy. Placentation in Thrichomys revealed major parallels to the guinea pig and other hystricognath rodents with respect to the early and invasive implantation, the process of trophoblast invasion, the internal organization of the labyrinth and the trophospongium as well as the establishment of the complete inverted yolk sac placenta. In contrast to systematically related small-sized species, the placental regionalization in Thrichomys was characterized by a remarkable lobulated structure and associated growing processes. Reverse to former perspectives, these conditions represented ancient character states of hystricognaths. The subplacenta was temporarily supplied by both the maternal and fetal blood systems, a rare condition among hystricognaths. The extraplacental trophoblast originating from the subplacenta was partly proliferative in mid gestation. In conclusion, the presented results indicated that only minor variations occurred in small-sized hystricognath species, independent of their systematic interrelationships. Previous views were supported that placentation in hystricognaths followed an extraordinary stable pattern, although the group had distinct habitats in South America and Africa that were separated 30-40 million years ago. J. Exp. Zool. (Mol. Dev. Evol.) 318:13-25, 2012. (C) 2011 Wiley Periodicals, Inc.

Placentation in the anteaters Myrmecophaga tridactyla and Tamandua tetradactyla (Eutheria, Xenarthra)

Background: Since Xenarthra are serious candidates for being basal to Eutheria, their characteristics, e.g. the placental system, influence perceptions of evolution. However, in the subgroup containing the anteaters, data are very limited. The present study aims to elucidate the nature of the feto-maternal interface in the anteater placenta and to interpret these data within an evolutionary context. Methods: Placentas of two species were investigated with histology, immunohistochemistry and transmission electron microscopy. Results: Remnants of the maternal vessel endothelium were absent, resulting in a fully haemochorial barrier throughout the placenta. Two structurally different parts, the villous and trabecular areas were complex and intermingled. In particular, the trabeculae which consisted of cellular, proliferative trophoblast, associated with connective tissue, were attached to the decidua. The villi contained fetal capillaries and hypertrophied mesenchymal cells that occured near the surface near the end of gestation. The surface of the villi consisted of flat, syncytial trophoblast, interspersed with proliferative trophoblast cells. Conclusions: Based on fundamental differences between anteaters and armadillos, we inferred that placental evolution was more complex than previously thought. The haemochorial pattern of anteaters was likely an ancient condition of xenarthrans. Consequently, villous placentation may be attributed, at least in part, by convergent evolution, but was also characterized by some features that were widespread among xenarthrans.

Microvascularization on Collared Peccary Placenta: a Microvascular Cast Atudy in Late Pregnancy

The microvascularization of the collared peccary (Tayassu tajacu) placenta was studied by vascular casts and immunolocalization of alpha-smooth muscle actin and vimentin, to identify the three-dimensional organization and vascular flow interrelation in the microvasculature between the maternal and fetal compartments of the placentae. The immunolocalization of vimentin in the vascular endothelium and in the smooth muscle cells of blood vessels showed indented capillaries along the uterine epithelium and the trophoblast at the sides of complementary maternal and fetal microfolds, or rugae. This confers the three-dimensional structure observed in vascular casts. On the maternal side, casts demonstrated uterine folds coated by with primary and secondary ridges, and by areolae dispersed between these ridges. The arteriole runs through the center/middle of ridges, branching at the top into a microvascular network wall in a basket-like fashion. At the base of these baskets venules were formed. On the fetal side, arterioles branched centrally in the fetal rugae into a capillary network in a bulbous form, complementary to the opposite maternal depressions forming the baskets. At the base of the bulbous protrusions, the fetal venules arise. The blood vessel orientation in the materno-fetal interface of the placentae of collared peccaries suggests a blood flow pattern of the type countercurrent to crosscurrent. The same pattern has been reported in domestic swine demonstrating that, even after 38 million years, the Tayassuidae and Suidae families exhibit similar placental morphology, which is here characterized at the microvascular level.

Toll-like receptor 9 activation: a novel mechanism linking placenta-derived mitochondrial DNA and vascular dysfunction in pre-eclampsia

Emerging evidence suggests that in addition to being the 'power houses' of our cells, mitochondria facilitate effector responses of the immune system. Cell death and injury result in the release of mtDNA (mitochondrial DNA) that acts via TLR9 (Toll-like receptor 9), a pattern recognition receptor of the immune system which detects bacterial and viral DNA but not vertebrate DNA. The ability of mtDNA to activate TLR9 in a similar fashion to bacterial DNA stems from evolutionarily conserved similarities between bacteria and mitochondria. mtDNA may be the trigger of systemic inflammation in pathologies associated with abnormal cell death. PE (pre-eclampsia) is a hypertensive disorder of pregnancy with devastating maternal and fetal consequences. The aetiology of PE is unknown and removal of the placenta is the only effective cure. Placentas from women with PE show exaggerated necrosis of trophoblast cells, and circulating levels of mtDNA are higher in pregnancies with PE. Accordingly, we propose the hypothesis that exaggerated necrosis of trophoblast cells results in the release of mtDNA, which stimulates TLR9 to mount an immune response and to produce systemic maternal inflammation and vascular dysfunction that lead to hypertension and IUGR (intra-uterine growth restriction). The proposed hypothesis implicates mtDNA in the development of PE via activation of the immune system and may have important preventative and therapeutic implications, because circulating mtDNA may be potential markers of early detection of PE, and anti-TLR9 treatments may be promising in the management of the disease.

Histomorphometrical and proliferative aspects of placenta and uterus of the collared peccary (Tayassu tajacu)

The histomorphometric and proliferative characteristics of the collared peccary (Tayassu tajacu) placenta and uterus were analyzed. The material was examined by standard histological techniques and histochemistry (PAS, Perls and Alcian Blue pH 0.5 and 2.5%) and the cellular proliferation by AgNORs and flow cytometry. All the analyzed morphometric variables differed between pregnant and non-pregnant uteri in the luteal phase using the Dunnet test. Height and gland diameter of uterine glands increased linearly during pregnancy, with an intense positive PAS and Perls reaction in all stages. The cells with more than seven AgNORs per nuclei and the cells in the G2M cell cycle phase in the maternal tissue also increased after 70 days of pregnancy. The uteroplacental ridges had a linear increase in size with two distinct areas, base and top, with uterine epithelium and trophoblastic cells changing their morphology following the placental ridge development. Flow cytometry analysis showed the percentage of cells in each cell cycle phase with a quadratic behavior for stages G2/M in the maternal tissue, suggesting an increase in proliferative capacity of maternal tissue after 65 days of pregnancy. The same quadratic effect was observed in the G0/G1 phase in both maternal and fetal tissues. Cells in apoptosis showed cubic behavior in both tissues. The morphometric and cellular dynamic aspects observed in this study have not been previously described and they extend our knowledge of functions relating to maternal-fetal dynamics in this species.

The fetomaternal interface in the placenta of three species of armadillos (Eutheria, Xenarthra, Dasypodidae)

Background: Placental characters vary among Xenarthra, one of four supraordinal clades of Eutheria. Armadillos are known for villous, haemochorial placentas similar to humans. Only the nine-banded armadillo has been well studied so far. Methods: Placentas of three species of armadillos were investigated by means of histology, immunohistochemistry including proliferation marker, and transmission and scanning electron microscopy. Results: The gross anatomy differed: Euphractus sexcinctus and Chaetophractus villosus had extended, zonary placentas, whereas Chaetophractus vellerosus had a disk. All taxa had complex villous areas within the maternal blood sinuses of the endometrium. Immunohistochemistry indicated the validity of former interpretations that the endothelium of the sinuses was largely intact. Tips of the villi and the columns entering the maternal tissue possessed trophoblast cell clusters with proliferation activity. Elsewhere, the feto-maternal barrier was syncytial haemochorial with fetal vessels near the surface. Conclusions: Differences among armadillos occurred in regard to the extension of the placenta, whereas the fine structure was similar. Parallels to the human suggest that armadillos are likely to be useful animal models for human placentation.

Expression of angiogenic factors in placenta of stressed rats

The aim of the present study was to analyse the influence of stress on pregnant rats, particularly in terms of maternal, placental and fetal weight, placental morphology and placental gene expression of the angiogenic factors Vegfa and Pgf and their receptors. The parameters were evaluated on gestation Day 20. Maternal, fetal and placental weights were statistically lower in stressed animals than controls, suggesting abnormalities in gestational physiology. Morphologically the placentas of rats subjected to stress were reduced in size and weight, with few glycogen cells and a significant increase in the number of apoptotic cells. Stress caused an increase in placental gene expression of Vegfa (P < 0.05) and a reduction in Pgf, Flt1 and Kdr expression (P < 0.05). It has been suggested that increased VEGF is associated with vasodilatation and hypotension, but in this model persistent hypertension was present. This study suggests that the limited hypotensive Vegfa response to stress-induced hypertension could result from reduced expression of Flt1/Kdr disrupting specific VEGF pathways. These findings may elucidate one of the multiple possible factors underlying how stress modulates placental physiology, and could aid the understanding of stress-induced gestational disorders.

Association between matrix metalloproteinase (MMP)-2 polymorphisms and MMP-2 levels in hypertensive disorders of pregnancy

We examined whether two functional polymorphisms (g.-1306 C> T and g.-735 C>T) in matrix metalloproteinase (MMP)-2 gene are associated with preeclampsia (PE) or gestational hypertension (GH), and whether they modify MMP-2 or tissue inhibitor of metalloproteinase (TIMP)-2 plasma concentrations in these hypertensive disorders of pregnancy. We studied 130 healthy pregnant (HP), 130 pregnant with GH, and 133 pregnant with PE. Genomic DNA was extracted from whole blood and genotypes for g.-1306 C>T and g.-735 C>T polymorphisms were determined by Real Time-PCR, using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by ELISA. The main findings were that pregnant with PE have higher plasma MMP-2 and TIMP-2 concentrations than HP (P<0.05), although the MMP-2/TIMP-2 ratios were similar (P>0.05). Moreover, pregnant with GH have elevated plasma MMP-2 levels and MMP-2/TIMP-2 ratios compared to HP (P<0.05). While MMP-2 genotypes and haplotypes are not linked with hypertensive disorders of pregnancy, MMP-2 genotypes and haplotypes are associated with significant alterations in plasma MMP-2 and TIMP-2 concentrations in preeclampsia (P<0.05). Our findings may help to understand the relevance of MMP-2 and its genetic polymorphisms to the pathophysiology of hypertensive disorders of pregnancy. It is possible that patients with PE and the MMP-2 haplotype combining the C and T alleles for the g.-1306 C>T and g.-735 C>T polymorphisms may benefit from the use of MMPs inhibitors such as doxycycline. However, this possibility remains to be determined. (C) 2012 Elsevier Inc. All rights reserved.

Ectoplacental Cone Induces Resistance to Apoptosis in High Doses of Interferon (IFN)-gamma-Treated Decidual Cells

Problem In this study, we explored the relationship between decidual cells (DC) and interferon (IFN)-gamma, in the presence or absence of ectoplacental cone (EC) using a coculture system. Method of study Decidual cells and EC were isolated from pregnant mice on gestation day 7.5. DCs were cultured for 48 hr and then treated with fresh EC. After characterization, they were treated with IFN-gamma, and cell death was evaluated. Results Interferon-gamma drastically increased decidual apoptosis, which was partially reverted by the addition of EC to the IFN-gamma-treated decidual culture. Moreover, the addition of EC to non-treated DC cultures was also capable of attenuating death rates. Conclusion Resistance to apoptosis may be induced in DC by the EC. This suggests that EC may participate in the inhibition of IFN-gamma-dependent apoptosis and, therefore, play important role for DC survival in a cytokineenriched placental environment.

Placentation in the paca (Agouti paca L)

Background: The paca is a South American rodent with potential as a commercial food animal. We examined paca placenta as part of a wider effort to understand the reproductive biology of this species. Methods: Thirteen specimens between midgestation and term of pregnancy were studied by light and transmission electron microscopy. Results: The placenta is divided into several lobes separated by interlobular trophoblast. Maternal arterial channels and fetal veins are found at the centre of each lobe. In the labyrinth, maternal blood flows through trophoblast-lined lacunae in close proximity to the fetal capillaries. The interhaemal barrier is of the haemomonochorial type with a single layer of syncytiotrophoblast. Caveolae occur in the apical membrane of the syncytiotrophoblast and recesses in the basal membrane, but there is no evidence of transtrophoblastic channels. The interlobular areas consist of cords of syncytiotrophoblast defining maternal blood channels that drain the labyrinth. Yolk sac endoderm covers much of the fetal surface of the placenta. The subplacenta comprises cytotrophoblast and syncytiotrophoblast. There are dilated intercellular spaces between the cytotrophoblasts and lacunae lined by syncytiotrophoblast. In the junctional zone between subplacenta and decidua, there are nests of multinucleated giant cells with vacuolated cytoplasm. The entire placenta rests on a pedicle of maternal tissue. An inverted yolk sac placenta is also present. The presence of small vesicles and tubules in the apical membrane of the yolk sac endoderm and larger vesicles in the supranuclear region suggest that the yolk sac placenta participates in maternal-fetal transfer of protein. Conclusion: The paca placenta closely resembles that of other hystricomorph rodents. The lobulated structure allows for a larger exchange area and the development of precocial young

The subplacenta of the red-rumped agouti (Dasyprocta leporina L)

Background: Hystricognath rodents have a lobed placenta, comprising labyrinthine exchange areas and interlobular trophoblast. These correspond to the labyrinthine and spongy zones of other rodent placentae. Beneath them, however, is a structure unique to hystricognath rodents called the subplacenta. We here describe the subplacenta of the red-rumped agouti and examine the possible functional correlates of this structure. Methods: Placentae were collected from early in midgestation to near term of pregnancy and examined by standard histological techniques, immunohistochemistry and transmission electron microscopy. In addition, to study the microvasculature of the subplacenta, vessel casts were inspected by scanning electron microscopy. Results: In the subplacenta, lamellae of connective tissue support a layer of mononuclear cytotrophoblast cells. Beneath this is found syncytiotrophoblast. Clusters of multinuclear giant cells occur in the transition zone between the subplacenta and decidua. There are prominent intercellular spaces between the cytotrophoblast cells. The basal membrane of these cells is often close to fetal blood vessels. The syncytiotrophoblast surrounds an extensive system of lacunae. Microvilli project into these lacunae from the plasma membrane of the syncytiotrophoblast. The syncytial cytoplasm contains electron-dense granules. This is probably the amylase-resistant PAS-positive material identified by histochemistry. The subplacenta is supplied entirely from the fetal circulation. Within it the vessels pursue a tortuous course with sinusoidal dilatations and constrictions. Conclusion: The functions that have been attributed to the subplacenta include hormone production. Our findings are consistent with this interpretation, but suggest that hormone secretion is directed towards the fetal circulation rather than the maternal tissues.