DNA polymerase activity in trophoblast giant cells in the mouse /

In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.

Possible role of bovine trophoblast giant cells in transplacental transmission of Neospora caninum in cattle

Neospora caninum is an aplicomplexan parasite that has brought several concerns to cattle raisers worldwide due to its relationship to fetal loss. However, the mechanism of the parasite's transplacental infection and induced abortions are not completely understood. Bovine trophoblastic binucleated cells (BNC) play a major role in the maternal-fetal interactions, migrating during the entire pregnancy from chorionic connections to uterine epithelium. This study aimed to investigate the possible role of BNC as phagocytic cells and its participation in the bovine transplacental infection of N. caninum. BNC was isolated by discontinuous Percoll gradient, and characterized by Hoeschst 33342 nucleus-specific staining. Isolated BNC were cultured in DMEM supplemented with 10% bovine fetal serum, and infected with 10(4) tachyzoites of N. caninum NC-1 strain. Parasite invasion was visualized by indirect immunofluorescence and Giemsa technique. Multiplication of parasites took place in 2-3 day cycles. Healthy cows' placenta and normal and infected cultured BNC was immunostained with monoclonal antibodies against CD-163, MAC-387 and NOS, demonstrating their phagocyte capacity. Thus, BNC was characterized as cells with macrophagic activity, which may host N. caninum in vitro. Therefore, we may conclude that BNC could potentially participate in the transplacental infection of bovine neosporosis.

Culture of mature trophoblastic giant cells from bovine placentomes

The mostly binucleate trophoblast giant cells (TGC) found in bovine placentomes, in addition to synthesizing and releasing hormones play an important role in fetal development and maternal adaptation to pregnancy. Placentomes from early gestation were collected, and for isolation of mature TGC, three cellular disaggregation methods, mechanical (MECH), enzymatic by trypsin (TRYP) or collagenase (COLL) were compared to each other. Further on, the cell survival in culture medium (DMEM) supplemented with either 10% fetal calf serum (FCS) or 10% serum replacement (SR) on culture plates free of any substrate was evaluated over a period of 90 days by trypan blue exclusion. The cells were further characterized by HOECHST 33342 nuclear staining, and immunocytochemical staining with monoclonal antibodies against vimentim and cytokeratin. A mean total rate of TGC survival of 82.56% was recorded. Statistical analysis showed significantly higher survival rates after enzymatic disaggregation with COLL (86.23%) than following MECH (80.38%) or TRYP (80.91%) treatment. Supplementation of DMEM with FCS resulted in significantly higher cellular survival rates (87.13%) when compared to the addition of SR (77.73%). Analysis of the influence of both, disaggregation method and medium supplementation on TGC survival revealed statistically significant differences between the following groups: MECH-SR (71.09%) was significantly lower than all other groups; TRYP-SR (78.03%) was significantly different from all other groups; TRYP-FCS (83.43%) and COLL-SR (84.08%) were significantly lower than MECH-FCS (89.98%) which together with COLL-FCS (88.25%) showed the highest cellular survival rate. In summary, our results show that TGC isolated from early gestation placentomes may be viable for more than 90 days of culture. However, whether these TGC produce placental lactogen throughout this period has yet to be determined. (c) 2006 Elsevier B.V. All rights reserved.

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.

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Elevated concentration of TNF-a induces trophoblast differentiation in mouse blastocyst outgrowths

Elevated expression of tumour necrosis factora (TNF-a) is associated with adverse pregnancy outcome. This study has examined the expression of TNF-a and its receptors (TNF-Rs) by mouse blastocysts and blastocyst outgrowths from day 4 to 9.5 of pregnancy and investigated the effects of elevated TNF-a on the inner cell mass (ICM) and trophoblast cells of blastocyst outgrowths. RTPCR demonstrated TNF-a mRNA expression from day 7.5 to 9.5, TNF-R1 from day 6.5 to 9.5 and TNF-R2 from day 5.5 to 7.5 of pregnancy, and in situ hybridisation revealed the trophoblast giant cells (TGCs) of the early placenta as the site of TNF-a expression. Day 4 blastocysts were cultured in a physiologically high concentration of TNF-a (100 ng/ml) for 72 h to the outgrowth stage and then compared to blastocysts cultured in media alone. TNF-a-treated blastocyst outgrowths exhibited a significant reduction in ICM cells (mean € SD 23.90€10.42 vs 9.37€7.45, t-test, P<0.0001) with no significant change in the numbers of trophoblast cells (19.97€8.14 vs 21.73€7.79, t-test, P=0.39). Within the trophoblast cell population, the TNF-a-treated outgrowths exhibited a significant increase in multinucleated cells (14.10€5.53 vs 6.37€5.80, t-test, P<0.0001) and a corresponding significant decrease in mononucleated cells (5.87€3.60 vs 15.37€5.87, t-test, P<0.0001). In summary, this study describes the expression of TNF-a and its receptors during the peri-implantation period in the mouse. It also reports that elevated TNF-a restricts ICM proliferation in the blastocyst and changes the ratio of mononucleated to multinucleated trophoblast cells. These findings suggest a mechanism by which increased

Localization of Cathepsins D and B at the Maternal-Fetal Interface and the Invasiveness of the Trophoblast during the Postimplantation Period in the Mouse

A survey of existing data suggests that trophoblast cells produce factors involved in extracellular matrix degradation. In this study, we correlated the expression of cathepsins D and B in the murine ectoplacental cone with the ultrastructural progress of decidual invasion by trophoblast cells. Both proteases were immunolocalized at implantation sites in lysosome-endosome-like compartments of trophoblast giant cells. Cathepsin D, but not cathepsin B, was also detected ultrastructurally in extracellular compartments surrounded by processes of the invading trophoblast containing extracellular matrix components and endometrial cell debris. The expression of cathepsins D and B by trophoblast cells was confirmed by RT-PCR in ectoplacental cones isolated from implantation chambers at gestation day 7.5. Our data addressed a positive relationship between the expression and presence of cathepsin D at the extracellular compartment of the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period, suggesting a participation of invading trophoblast cells in the cathepsin D release. Such findings indicate that mouse trophoblast cells might exhibit a proteolytic ability to partake in the decidual invasion process at the maternal-fetal interface. Copyright (C) 2010 S. Karger AG, Basel

Caracterização das áreas hemófagas da placenta bovina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Do binucleate cardiomyocytes have a role in myocardial repair? Insights using isolated rodent myocytes and cell culture.

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape.Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made.All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a 'dormant' or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle.Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183).

Maternal nutrition modifies trophoblast giant cell phenotype and fetal growth in mice

Mammalian placentation is dependent upon the action of trophoblast cells at the time of implantation. Appropriate fetal growth, regulated by maternal nutrition and nutrient transport across the placenta, is a critical factor for adult offspring long-term health. We have demonstrated that a mouse maternal low-protein diet (LPD) fed exclusively during preimplantation development (Emb-LPD) increases offspring growth but programmes adult cardiovascular and metabolic disease. In this study, we investigate the impact of maternal nutrition on post-implantation trophoblast phenotype and fetal growth. Ectoplacental cone explants were isolated at day 8 of gestation from female mice fed either normal protein diet (NPD: 18% casein), LPD (9% casein) or Emb-LPD and cultured in vitro. We observed enhanced spreading and cell division within proliferative and secondary trophoblast giant cells (TGCs) emerging from explants isolated from LPD-fed females when compared with NPD and Emb-LPD explants after 24 and 48 h. Moreover, both LPD and Emb-LPD explants showed substantial expansion of TGC area during 24-48 h, not observed in NPD. No difference in invasive capacity was observed between treatments using Matrigel transwell migration assays. At day 17 of gestation, LPD- and Emb-LPD-fed conceptuses displayed smaller placentas and larger fetuses respectively, resulting in increased fetal:placental ratios in both groups compared with NPD conceptuses. Analysis of placental and yolk sac nutrient signalling within the mammalian target of rapamycin complex 1 pathway revealed similar levels of total and phosphorylated downstream targets across groups. These data demonstrate that early post-implantation embryos modify trophoblast phenotype to regulate fetal growth under conditions of poor maternal nutrition.

The expression and regulation of pregnancy-specific glycoproteins in the mouse

Pregnancy-Specific Glycoproteins (PSG) are the most abundant fetally expressed proteins in the maternal bloodstream at term. This multigene family are immunoglobulin superfamily members and are predominantly expressed in the syncytiotrophoblast of human placenta and in giant cells and spongiotrophoblast of rodent placenta. PSGs are encoded by seventeen genes in the mouse and ten genes in the human. Little is known about the function of this gene family, although they have been implicated in immune modulation and angiogenesis through the induction of cytokines such as IL-10 and TGFβ1 in monocytes, and more recently, have been shown to inhibit the platelet-fibrinogen interaction. I provide new information concerning the evolution of the murine Psg genomic locus structure and organisation, through the discovery of a recent gene inversion event of Psg22 within the major murine Psg cluster. In addition to this, I have performed an examination of the expression patterns of individual Psg genes in placental and non-placental tissues. This study centres on Psg22, which is the most abundant murine Psg transcript detected in the first half of pregnancy. A novel alternative splice variant transcript of Psg22 lacking the protein N1-domain was discovered, and similar to the full length isoform induces TGFβ1 in macrophage and monocytic cell lines. The identification of a bidirectional antisense long non-coding RNA transcript directly adjacent to Psg22 and its associated active local chromatin conformation, suggests an interesting epigenetic gene-specific regulatory mechanism that may be responsible for the high level of Psg22 expression relative to the other Psg family members upon trophoblast giant cell differentiation