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In view of controversy about the source of placental multinuclear giant cells, we have re-examined the literature which clearly shows they are derived from trophoblastic elements that have populated the decidua. Archival material for electron microscopy from 17 to 18 week placentae demonstrates they can be found connected via desmosomes to the outer extravillous cytotrophoblast cells of anchoring columns, thus identifying a primary source. We suggest their formation is a terminal differentiation step occurring at all stages of invasion from the cell column to the myometrium, progressively reducing the invasive population.

Introduction: Prolyl oligopeptidase (prolyl endopeptidase, Prep), a multifunctional protease hydrolyzing -Pro-X-peptide bonds, is highly expressed in the mouse placenta, but the function during development is not known. We explored the possibility of Prep's involvement in placental differentiation. Methods: We cultured trophoblast stem cells (TSCs) derived from the E6.5 mouse embryo and investigated the detailed expression pattern of Prep during their differentiation. Prep-specific inhibitors were added to the TSC culture, and the effect on the differentiation was assessed by microscopic observation and the expression of marker gene for each placental cell. Results: During TSC differentiation for 6 days, Prep was constantly detected at mRNA, protein, and activity levels, and the proteinwas found mainly in the cytoplasm. The addition of 30 mu M and 10 mu M SUAM-14746, a Prep-specific inhibitor, effectively inhibited the differentiation into spongiotrophoblasts (SpTs) and trophoblast giant cells (TGCs), while the TSC viability was not affected. 5 mu M SUAM-14746 impaired the differentiation into SpTs, and 1 mu M SUAM-14746 exhibited no effects. Another Prep-specific inhibitor, KYP-2047, did not affect the differentiation. We confirmed efficient inhibition of Prep enzymatic activity in TSCs by both inhibitors. Conclusion: The dose-dependent effect of SUAM-14746 on TSCs suggests that Prep plays an important role in the differentiation into SpTs and TGCs in the mouse placenta. (C) 2017 Elsevier Ltd. All rights reserved.

Introduction The labyrinthine zone of the placenta is where exchange of nutrients and waste occurs between maternal and fetal circulations. Proper development of the placental labyrinth is essential for successful growth of the developing fetus and abnormalities in placental development are associated with intrauterine growth restriction (IUGR), preeclampsia and fetal demise. Our previous studies demonstrate that Hectd1 is essential for development of the junctional and labyrinthine zones of the placenta. Here we further characterize labyrinthine zone defects in the Hectd1 mutant placenta. Methods The structure of the mutant placenta was compared to wildtype littermates using histological methods. The expression of cell type specific markers was examined by immunohistochemistry and in situ hybridization. Results Hectd1 is expressed in the labyrinthine zone throughout development and the protein is enriched in syncytiotrophoblast layer type I cells (SynT-I) and Sinusoidal Trophoblast Giant cells (S-TGCs) in the mature placenta. Mutation of Hectd1 results in pale placentas with frequent hemorrhages along with gross abnormalities in the structure of the labyrinthine zone including a smaller overall volume and a poorly elaborated fetal vasculature that contain fewer fetal blood cells. Examination of molecular markers of labyrinthine trophoblast cell types reveals increased Dlx3 positive cells and Syna positive SynT-I cells, along with decreased Hand1 and Ctsq positive sinusoidal trophoblast giant cells (S-TGCs). Discussion Together these defects indicate that Hectd1 is required for development of the labyrinthine zonethe mouse placenta.

Introduction A few days after implantation, the embryo grows intensely and trophoblast giant cells (TGC) undergo cell rearrangement, especially of their cytoskeleton. Although we previously showed vimentin in mouse antimesometrial TGC at embryonic days (E) 8.5–10.5, by immunostaining, we did not demonstrate what is the first embryonic day that TGC synthesize vimentin and whether mouse chorioallantoic placental TGC express vimentin. This is of particular interest because cytokeratin is a marker for TGC in the placenta. Methods We performed in situ hybridization and immunolocalization, combined with histological and stereological techniques, to study vimentin expression between E6.5 and E12.5 and we investigated Vegf and Flt1/Vegfr1 expression in TGC. Results Analyses of morphologic parameters of TGC showed that the highest expansion of nuclear and cytoplasmic volumes (p ≤ 0.05) occurred at E7.5. We detected vimentin expression in TGC from E7.5 onwards; vimentin disappeared as TGC degeneration advanced. Primary and secondary TGC showed intense positive immunostaining for vimentin, Vegf and Flt1/Vegfr1 at E7.5. In the chorioallantoic placenta, parietal TGC (zone of giant cells), spiral artery-associated TGC, maternal blood canal-associated TGC and TGC within the sinusoidal spaces of the labyrinth exhibited an intense immunopositive-reaction for vimentin. Discussion At E7.5 TGC acquire vimentin, Vegf and Flt1/Vegfr1; at the same time, blood begins to drain from maternal vessels. Vimentin synthesis initiates during a differentiation process of TGC and continues throughout the stage of vascular TGC. Conclusions We propose that vimentin is a characteristic factor of specialized (vascular) TGC, being a valuable tool for studying pathological pregnancies associated with defects in vascular trophoblasts in mice.

In cattle no biological role has been definitely identified for placental estrogens and progesterone. However, in the bovine trophoblast androgens may also be produced and have local effects. Thus, the aims of this study were to identify androgen receptor (AR) expressing cells and to monitor testosterone tissue concentrations in bovine placentomes throughout gestation.Placental AR expression was characterized at the mRNA and protein level applying conventional and real-time RT-qPCR, western blot and immunohistochemistry. Testosterone was measured by radioimmunoassay.AR-mRNA was qualitatively detected from day 50 of gestation until term. Mean relative gene expression levels were constant between day 100 and late gestation. A slight non-significant increase was observed in the prepartal period. With immunohistochemistry distinct nuclear signals were predominantly observed in invasive trophoblast giant cells (TGC) from day 80 until term. In mature TGC of the trophoblast, immature TGC and uninucleated trophoblast cells, stromal cells of the chorionic villi, caruncular epithelial and stromal cells immunoreactive score values were low at early and midgestation but increased significantly (p 0.01) during late gestation and remained high until parturition. With western blot in placentomal tissue a specific band of approximately 110 kDa was detected as it was the case in epididymis used as a positive control. Testosterone concentrations increased from 0.70 ± 0.29 pmol/g wet tissue between days 60-220 to 4.22 ± 1.29 pmol/g during late gestation (p 0.001).The results are consistent with androgens as active products of bovine placental steroidogenesis. The substantial up-regulation of AR expression during TGC differentiation suggests that androgens may be related to this process.

The concept of a network within the family of adhesion/growth regulatory galectins implies distinct spatio-temporal expression profiles. To test this assumption immunohistochemically for bovine placenta, placentomal (P) and interplacentomal tissues (IP) were collected at a slaughterhouse from the three stages of pregnancy (early gestation = day 30–130; mid gestation = day 130–220; late gestation = day 220–275). The specimens were snap-frozen or fixed in Bouin’s solution, then embedded in paraffin. Gene expression for galectins-1, -3, -4 and -9 in P and IP of late gestational stages was monitored by RT-PCR. Galectin-type-specific antibodies were used for immunohistochemical localization. In IP, galectin-1 was present in stroma cells and early gestational trophoblast giant cells (TGC), whereas galectin-3 was confined to uterine epithelial cells. In contrast, both galectins were found in epithelia of P tissue. Uterine epithelial cells and blood vessel walls were positive for galectin-4, while galectin-9 was detected predominantly in uterine epithelial cells and late gestational TGC. Our study thus reveals individual profiles among the galectins tested, an indication for specific functions exerted by each protein in the bovine endometrium and placenta.

During their invasion of the placental bed, interstitial trophoblasts fuse to multinuclear giant cells which are thought to have lost their invasive properties. Trophoblast fusion is associated with downregulation of E-cadherin, and persistent E-cadherin expression has been linked to defective placentation in preeclampsia. Since a previous study suggested 'premature' giant cell formation in preeclampsia, we started with the working hypotheses that fusion is increased in hypertensive pregnancies, and that the intensity of fusion correlates with the severity of disease. Using double immunostaining for E-cadherin and cytokeratin 7/17, nuclei in interstitially invasive trophoblasts (IT) in the myometrial compartment of the placental bed from normotensive pregnancies (NT, n=8), gestational hypertension (GH, n=4), preeclampsia (PE, n=9), and HELLP syndrome (n=5) were categorised according to the E-cadherin staining of the cell and their occurrence in single, clustered or multinuclear cells. GH and PE patients showed a higher percentage of nuclei in clustered non-fused E-cadherin-positive cells (P

Placental trophoblasts are an important source of endocrine, paracrine and autocrine acting hormones. The aim of the present study was to establish and evaluate a tissue culture model for bovine trophoblasts to study regulation of key genes of steroid hormone synthesis. Trophoblast cells were isolated from cotyledons by collagenase disaggregation and subsequent percoll density gradient centrifugation. The cells were seeded on collagen coated dishes and incubated for up to seven days. The cells were characterized for the presence of mesenchymal vimentin and epithelial cytokeratin filaments and for Dolichos biflorus agglutinin (DBA) binding, a marker for differentiated trophoblast giant cells. Transcripts of Hsd3b , Cyp17 and Cyp19 encoding 3β-HSD, P450c17 and P450arom, the key enzymes of progesterone, androgen, and oestrogen biosynthesis, respectively, and of Csh1 encoding the trophoblast-specific hormone placental lactogen (PL) were measured by qPCR. Uninucleate cotyledonary epithelial cells and bi- and trinucleate trophoblast giant cells efficiently formed a dense cell layer on the collagen coated dishes within 24 h. Bi- and trinucleate cells showed DBA binding and weak or undetectable cytokeratin immunoreactivity. Vimentin-positive, fibroblast-like cells were found on top of this cell layer. Cyp19 transcripts were found in freshly dissociated but not in cultured cells. Cyp17 expression continuously increased, Hsd3b transcripts largely and rapidly increased during the first days in culture, followed by a decline after three days, whereas Csh1 decreased towards day seven. Serum free culture conditions significantly enhanced Cyp17 and Csh1 but not Hsd3b expression. The data indicate that collagen is a favourable substrate for cultured binucleate trophoblast giant cells. The cells represent an in vitro model to study the regulation of key genes of placental progesterone and androgen but not of oestrogen biosynthesis.

Extraembryonic development in rodents depends on the differentiation and function of trophoblast giant cells. Morphologically striking, giant cells exhibit many extraordinary characteristics adapted to ensure the success of pregnancy. This review summarizes some of the intriguing aspects of giant cell morphology and function. Giant cells are highly polyploid as a result of a switch from a mitotic to an endoreduplicative cell cycle. They further partition their genome content into various fragments which may represent a mechanism to maximize protein synthesis. Similar to metastatic tumour cells, they breach basement membranes and invade deeply into a foreign tissue, the maternal decidualized uterine stroma. Their angiogenic and vasodilatory properties, combined with the ability to remodel arterial walls, enable them to redirect maternal blood flow towards the implantation site. Recent advances have recognized that the giant cell population is more diverse than previously recognized and future studies will have to show how these subtypes differ functionally and how their differentiation is controlled.

The keratin cytoskeleton is present in all trophoblast cell subtypes of the mouse and human placenta and is required to maintain the structural integrity of these cells. Recently, various genetic mouse models have shown that a normal keratin network is necessary for placental development. Keratin-deficiency leads to trophoblast giant cell fragility, breaking the barrier between the conceptus and the maternal blood circulation. Alternatively, keratin aggregation prevents chorioallantoic attachment, a key developmental milestone required for the formation of the labyrinth within the mouse placenta. These models give us insight into cytokeratin function in human trophoblast cell subtypes and suggest that defects in the keratin cytoskeleton may result in intrauterine growth restriction or miscarriage.

A specialized subtype of trophoblast giant cells (TGCs) line the torturous sinusoids of the murine placental labyrinth, and can be distinguished from most other TGCs by the expression of Ctsq. We generated a transgenic mouse line expressing Cre recombinase from the Ctsq promoter. Crosses with Cre-inducible tdTomato reporter mice indicated Cre activity was restricted to the sinusoidal TGCs of the labyrinth, as well as the recently characterized channel TGCs. When crossed with Cre-inducible DTA transgenic mice, ablation of sinusoidal TGCs was achieved in double transgenic embryos, resulting in fetal growth restriction by E16.5, and embryonic lethality by term.

The extracellular matrix protein fibulin-1 (FBLN1) is an important component of blood vessel walls, as shown by the lethality of mice with homozygous targeted deletion of the Fbln1 gene. Here, we show that a murine placental overgrowth phenotype is associated with elevated Fbln1 transcript levels, suggesting that the gene and its product have a functional role in placentation. Fbln1 exhibits a specific expression pattern in the mouse placenta. Transcripts could not be detected prior to day 12. In subsequent stages, Fbln1 was expressed strongly in the spongiotrophoblast. Other sites of expression were endothelia of large fetal blood vessels, a tissue type reported to not express this gene. In addition, a subset of giant cells expressed the gene. This giant cell specific expression was strongly increased in hyperplastic placentas. Analysis of the placentation in fibulin null mice did not show any abnormality. Attempts to rescue the placental phenotypes of a congenic model of interspecies hybrid placental dysplasia (IHPD) by normalizing expression of Fbln1 proved that Fbln1 alone is not the key cause of phenotypes in these models of placental hyperplasia.

Amino acid transport System A (SysA) activity is present within the rodent and human placentas. Inhibition of this transport system is associated with fetal growth retardation. Several cDNAs encoding SysA transport proteins have been discovered, and their presence documented within the human placenta. We have demonstrated the presence of mRNA encoding three of these transporters, SNAT1, 2, and 4 within the rat placenta over the final third of gestation. Abundance of these mRNA species increases from day 14 to day 20 of gestation. Immunohistochemistry demonstrates the presence of SNAT1 and 2 within the placental labyrinth at both days 14 and 20. Transport proteins are also present within marginal giant cells and, for SNAT1, within fetal endothelium. In conclusion, several proteins capable of SysA transport activity are present within the rodent placenta. mRNA expression increases over the final third of gestation, coincident with the period of greatest need for fetal amino acid delivery.

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.

Transferrin (TF), a 76-80 kDa glycoprotein, is responsible for the transport of iron to cells within both the fetal and maternal systems, but it does not cross the multiple cell layer barrier of the placenta. Recent findings that both rat and human placental cells produce TF indicated that placental TF may function in some manner to transport or regulate iron passage across this barrier. However, placental production of TF was brought into question because the cell preparations used to identify TF were obtained using dispersed tissue and may have contained non-placental contaminating elements. In this study, cultures of phenotypically distinct cell types containing only placental cells were used to firmly establish whether or not TF is expressed, and if so to begin to identify the cell(s) associated with its synthesis. Utilizing RT-PCR, in situ hybridization, and Western blot analysis, we identified TF mRNA and protein in three trophoblast cell types, HRP-1 (rat), Rcho-1 (rat), and BeWo (human) cells. Additionally, TF mRNA and protein were found in Giant cells, the differentiated form of Rcho-1 cells. When taken together, these results demonstrate clearly that TF is expressed by both differentiated and non-differentiated placental cells, and when viewed in light of previous findings, strengthen the possibility that placental TF may be central to the passage of iron from the mother to the fetus during development.

Trophoblast giant cells in the rodent placenta form the outermost layer of the extraembryonic compartment, establish direct contact with maternal cells, and produce a number of pregnancy-specific cytokine hormones. Giant cells differentiate from proliferative trophoblasts as they exit the cell cycle and enter a genome-amplifying endocycle, a process we show involves decreased expression of the G1 checkpoint proteins p53 and Rb. Although p53 mRNA levels are unchanged in proliferative compared to differentiated trophoblasts, p53 protein levels are markedly reduced in giant cells. Forced expression of wild type p53 in trophoblasts inhibits differentiation, and expression of a dominant negative p53 peptide stimulates differentiation. Consistent with the loss of p53 protein, differentiated trophoblasts become resistant to apoptosis-inducing agents. Decreased expression of Rb is also detected during differentiation, and overexpression of Rb in trophoblasts inhibits giant cell differentiation. Although an increase in E2F activity would be expected with the loss of Rb, what is observed is an overall decrease in E2F DNA-binding complexes, a shift to new complexes, and a decrease in E2F-dependent gene expression in differentiating trophoblasts. Overall, these results indicate that the combination of a decrease in p53 and Rb represents a functionally important part of the transition of trophoblasts from a proliferative cell cycle to an endocycle in the giant cell differentiation programme.

Introduction The tammar wallaby has a short gestation (26.5 days) and vascular modifications to expedite transport during that brief pregnancy. Here we examine trophoblast structural attributes that would facilitate materno-fetal exchange. Materials and methods Four specimens of Macropus eugenii between days 23 and 26 gestation were examined using electron microscopy and 24 lectins to characterise glycosylated secretions and their internalisation. Results Two trophoblast phenotypes were found, flattened cells generally in contact with the underlying uterine epithelium and giant cells associated with histiotrophe. The latter appeared to penetrate uterine clefts, occasionally detach and become necrotic. Lectin histochemistry and ultrastructure indicated the presence of many lysosomes and residual bodies especially in trophoblast giant cells; these contained glycans, mainly apically, which were also detected in secretions and cell debris. Trophoblast basal membranes bore extensive filopodia. Giant cells were less common in vascular trilaminar areas and here the trophoblast barrier became thinner near term. Discussion Loss of Maackia amurensis agglutinin binding suggested cleavage of terminal sialic acid residues as an early post-internalisation event in the trophoblast. Lectin staining indicated degradation occurred in an apical–basal direction, and the heavily glycosylated basal membrane appeared specialised for transport out of the cell. Conclusion Granules seen ultrastructurally and histochemically, particularly in giant trophoblast cells of the bilaminar area, suggest that internalised histiotrophe is broken down here and nutrients transferred to the embryo via the specialised basal plasma membrane. The trilaminar vascular area contained mostly flattened trophoblast cells, supporting the suggestion that gaseous exchange is its primary function.

The objective of this work was to determine if fluorescence-activated cell sorting (FACS) can be used to isolate trophoblast giant cells (TGCs) from bovine placentae. Cotyledons were harvested, minced and digested with dispase/pancreatin. Tissue homogenates were incubated with Vybrant Dye Cycle-Green. FACS was completed, and cells with DNA content 2- to 4-times greater than cells within the diploid peak contained between 65 and 84% TGCs. The relative abundance of CSH1 and PAG1 transcripts were greater (P < 0.05) in TGC-enriched fractions than cells within the diploid peak. These observations indicate that FACS can effectively enrich TGCs from bovine placentae.

Cytophotometric measurement of the DNA content of Feulgen-stained nuclei in touch preparations of bovine placentomes (n =5) revealed that 8C nuclei occurred in all, 16C nuclei in two, and 32C nuclei in one specimen. The determination of ploidy level by in situ hybridization with a Y-chromosome specific DNA probe showed that the majority of the fetal nuclei in touch preparations of placentomes from male fetuses (n =5) are tetraploid. Generally two tetraploid nuclei lie close together. These findings indicate that polyploidization is a normal feature in the development of the mostly binucleate trophoblast giant cells (TGCs). A new model for the development of these cells is proposed: a primary acytokinetic mitosis leads to a binucleate cell with two diploid nuclei. This cell enters a second acytokinetic mitosis during which the chromosomes of both nuclei form a common metaphase plate. The resulting cell with two tetraploid nuclei undergoes an additional S-phase but does not enter a renewed mitosis. The functional significance of this genome multiplication may be an increased synthetic capacity of bovine TGCs, caused by an increased number of gene copies available for transcription. Since genome multiplication is a property of invasive trophoblast cells of different species, it may be advantageous for trophoblast invasion.

To identify tumour necrosis factor (TNF)-alpha immunopositive cells, third trimester human placental bed biopsies were selected from nine normotensive control women, 16 severely pre-eclamptic patients and seven patients with pre-existing hypertension with superimposed pre-eclampsia. In addition, five first and early second trimester specimens were included in the study. Immunostaining was performed with a mouse IgG1 monoclonal antibody (J1D9) reactive specifically with human TNF-alpha (1:300 ascitic fluid), using a biotin-streptavidin-peroxidase technique. Variable staining of stromal cells was noted in all biopsies. Specimens of early pregnancy showed marked immunostaining for TNF-alpha on proliferating tips of anchoring villi, invasive interstitial cytotrophoblast (but not the multinuclear giant cells), and endovascular trophoblast invading the spiral arteries. At term, weak staining was found in trophoblast incorporated within spiral artery walls. In biopsies from pre-eclamptic patients, spiral arteries without physiological change showed very little staining except in atherotic vessels where the infiltrated lipophages often showed intense immunolabelling. The marked presence of TNF-alpha in extravillous cytotrophoblast of young specimens is suggestive of a role in early invasion. Immunostaining of foam cells in non-invaded spiral arteries in pre-eclampsia at or near-term indicates a potential role of this cytokine in the development of atherotic lesions.

Pregnancy-induced structural changes in spiral arteries seem to be a prerequisite for successful fetal outcome in humans. It is unknown whether these changes also occur in other preplacental vessels (radial and arcuate arteries) in normal pregnancies. Since the radial and arcuate arteries need to dilate in order to accommodate the increase in placental blood flow during pregnancy, it is expected that they are also invaded by trophoblast and respond with structural changes. The objective of the present study was to evaluate the extent of trophoblast invasion in the guinea-pig preplacental vasculature and its effect on the vascular structure of mesometrial, myometrial and arcade arteries. Under general anaesthesia the vascular system of non- ( n =4), mid- ( n =4) and late- ( n =8) pregnant guinea-pigs was fixed by immersion or perfusion. Cross-sections of immersion-fixed mesometrial and arcade arteries were stained with toluidine blue. Cross-sections of perfusion-fixed mesometrial, myometrial and arcade arteries were stained with haematoxylin-eosin, Elastica van Gieson staining and antibodies against α-smooth-muscle-actin (ASMA), cytokeratin and factor VIII, to detect vascular smooth muscle, trophoblastic, and endothelial cells, respectively. In addition, the external and internal vascular circumference of sections from perfusion-fixed tissue was determined. All cross-sections were evaluated by light microscopy. In the course of pregnancy, progressive endothelial swelling, disappearance of the elastic lamina interna and disarrangement of the tunica media were observed in the myometrial and throughout the mesometrial arteries up to the junction with the arcade arteries. These changes coincided the migration of keratin-positive staining giant cells. It is concluded that in normal guinea-pig pregnancy, structural changes occur in the entire mesometrial artery and at least a part of the myometrial artery, although such changes were not seen in the arcade artery.

Phorbol myristate acetate (PMA) and all-trans-retinal (retinal) were evaluated as possible phagocytic stimulators of cultured, post implantation, trophoblast cells. Ectoplacental cones dissected from 7.5 day-old mouse embryos provided the source of trophoblastic cells. Co-cultures were performed using stimulated and non-stimulated trophoblast cells and erythrocytes under standard conditions. Phagocytic activity was expressed as the total number of phagocytic cells per ectoplacental cone, and as phagosomic vacuoles per trophoblast giant cell, either in the presence or absence of the stimulators. Both chemical agents had similar effects; less than I2 h after stimulation, statistically significant numbers of erythrophagosomes appear in the trophoblast giant cells (TGC). These findings demonstrate that TGC, like neutrophils and macrophages, can be activated to phagocytosis by exogenous factors. This enhanced activity may result from the generation and release of reactive oxygen species. In conclusion, our data suggest that, because stimulation was provided, the remarkable in vivo phagocytic activity of the trophoblast can be maintained under in vitro conditions, allowing study of the pathways and regulatory steps involved in this process.

The ovaries and uteri were removed from four pregnant camels on days 14, 25, 35 and 56 after ovulation. The day 14 and 25 uteri were perfuse-fixed with 3 per cent glutaraldehyde: 3 per cent paraformaldehyde whereas the day 35 and 56 specimens were opened dorsally for photography before biopsies of allantochorion attached to endometrium were taken and fixed in either 3 per cent glutaraldehyde: 3 per cent paraformaldehyde or Bouin's fluid. Samples of each uterus were then processed and sectioned for light and transmission electron microscopy. By day 14 the majority of the trophoblast had become closely apposed to the luminal epithelium of the endometrium to form the start of an epitheliochorial placenta with microvillar interdigitation initiated in some places. By day 25 a well-developed microvillar junction had formed between the fetal and maternal tissues. The fetus was situated in the middle of the left uterine horn in the day 35 and 56 specimens and, histologically, large multinucleate giant trophoblast cells had developed at frequent but irregular intervals along the, otherwise single-cell, trophoblast. These giant cells were often situated over the mouth of an endometrial gland but their actual function in pregnancy is not yet known.

The outgrowth of the mouse blastocyst in culture represents an in vitro model of trophoblastic invasion. In the present study weanalysed trophoblast spreading by time lapse video microscopy. Trophoblast spreading consists of (1) the migration and (2) the giant cell transformation of trophoblast cells, (3) the proliferation of ectoplacental cone (EPC) cells and (4) the subsequent transformation of EPC cells into the secondary giant cells. During migration, ruffling of the trophoblast cell membrane is followed by the formation of lamellipodia. The mean surface areas of the spreading trophoblast, measured in more than 100 cultured blastocysts, increased linearly from 48 to 96 h of culture, while the linear migratory speed at the periphery of the outgrowth declined as the time of culture advanced. The EPC cells increased in size approximately eightfold during the giant cell transformation. The apparent nuclear: cytoplasmic ratios, i.e., ratios between the size of nucleus and that of the cytoplasm, measured as the surface areas on the photomicrographs, of EPC cells increased between 40–46 h of culture, but a sharp decline in the ratio occurred between 50 and 51 h of culture, reflecting either the sudden and tremendous increase in the cellular volume and/or spreading of the cytoplasm. The rates of trophoblast spreading varied considerably among the blastocysts of different genetic constitution examined (ICR, C57BL/6, C3H/He and (B6 × C3)F1. It was fastest in blastocysts obtained from matings of males and females of (B6 × C3)F1, and slowest in the C57BL/6 embryos. The differences in the rate of outgrowth observed may not simply be ascribed to difference in the developmental speed of the early embryos, because the rate of outgrowth reached a plateau at about 96–120 h and no ‘catch-up’ was observed by leaving the blastocysts in culture longer. Our results strongly suggest the possible presence of genetic regulatory mechanisms underlying trophoblast outgrowth; further analysis of the phenomenon may provide clues to understand the molecular mechanisms of trophoblastic invasion during the early phase of implantation, hopefully leading to improved success rates of in vitro fertilization-embryo transfer.

We performed studies to characterize the mechanisms responsible for development during gestation of a placental barrier to Theiler's murine encephalomyelitis virus (TMEV) in a murine model of gestational enterovirus infection. Electron microscopy of placentae infected in early gestation revealed TMEV-induced changes in the decidua, giant cell, spongiotrophoblast, and labyrinth layers; in contrast, placentae infected in middle and late pregnancy demonstrated degenerative changes in the decidua, giant cell, and spongiotrophoblast layers but not in the labyrinth. Immunohistochemistry and in situ hybridization of placentae infected in early or late gestation demonstrated accumulation of monocytes/macrophages in infected, histologically damaged labyrinths, but no infiltration of immune cells into infected but histologically normal placental regions. Silver staining of placentae from dams inoculated in late gestation with inert gold beads the size of TMEV virions revealed beads within the decidua, giant cell, and spongiotrophoblast layers, but restriction of beads from labyrinths, similar to the usual distribution of TMEV in placentae infected in late pregnancy. These experiments suggest that anatomical relationships, and not systemic immune response, appear to be a major contributor to the murine placental barrier to TMEV.

Normal placental development and function is essential for fetal growth of eutherian mammals. Mutational studies have shown that numerous growth factors are required for placental development and differentiation of placental lineages. Here, using a gene-trap mutant mouse line, Crim1(KST264), we show that Crim1 is essential for murine placental development. Crim1 is a developmentally expressed, trans-membrane regulator of growth factor activity. Crim1(KST264/KST264) mutant placentae displayed hypoplasia from 13.5 dpc, and altered structure from 15.5 dpc, including alterations in cell number in both the junctional and labyrinth zones. Using the reporter gene from the Crim1(KST264) allele, we found that Crim1 is expressed in multiple cell types of the placenta, including strong expression in the spongiotrophoblast cells of the junctional zone. In the junctional zone of Crim1(KST264/KST264) placentae, there was an increase in the glycogen trophoblast cells adjacent to the spongiotrophoblast cells. In the labyrinth zone, we found a decrease in the density of sinusoidal-trophoblast giant cells. Our findings show that Crim1 is required for placental development, and is necessary for the proper differentiation of sinusoidal-trophoblast giant cells and glycogen trophoblast cells.

Objectives: Reproduction in the plains viscacha is characterized by the polyovulation of hundreds of oocytes, the loss of implantation and the development of 1-3 offspring. Our goal was to determine whether placental development was affected by these specializations. Study design: Thirteen placentas from early pregnancy to near-term pregnancy were analyzed using histological, immunohistochemical and transmission electron microscopy. Results: An inverted, villous yolk sac was present. Placentas were formed by the trophospongium, labyrinth and subplacenta. A lobulated structure with a hemomonochorial barrier was established early in pregnancy. Proliferating trophoblast that was clustered at the outer border and inside the labyrinth was responsible for placental growth. Trophoblast invasion resulted from the cellular trophoblast and syncytial streamers derived from the subplacenta. Different from other caviomorphs, numerous giant cells were observed. Conclusions: The principle processes of placentation in caviomorphs follow an extraordinarily stable pattern that is independent of specializations, such as polyovulation., Facultad de Ciencias Veterinarias

Summary Theiler's murine encephalomyelitis virus (TMEV), a murine enterovirus, infects the majority of murine placentae and fetuses following inoculation in early gestation and infects most placentae but almost no fetuses in late gestation. The sequence of infection of TMEV following early gestation inoculation was studied. Mice were inoculated with TMEV on day 6 or 7 of pregnancy and sacrificed at intervals between 1 h and 4 days later. Culture of placenta-embryo units identified infection at 2, 3, and 4 days post-inoculation. In situ hybridization revealed TMEV RNA primarily in giant cells around the yolk cavity and in giant cells situated between the decidua and spongiotrophoblast layers of the placenta. Occasional decidual cells located near giant cells were also hybridization-positive. The giant cells were immunohistochemically identified as fetally derived trophoblast cells. Giant cells are the earliest predominant target of TMEV infection following early gestation inoculation and appear to be an integral part of the pathogenesis of gestational murine enterovirus infection.

The bovine placenta produces considerable amounts of pregnancy-associated estrogens, predominantly estrone sulfate, which does not interact with classical nuclear estrogen receptors. Thus, bovine placental estrogens may rather act as local regulatory factors than as hormones in a classical sense. To obtain information on the local availability of free estrogens in bovine placentomes, the expression pattern of the estrogen specific sulfotransferase SULT1E1 was characterized using antisera against the bovine and human enzyme, respectively. In western blot both antisera detected a band of the expected molecular weight (approx. 33 kDa) in placentomes and fetal liver. In immunohistochemistry the two antisera yielded virtually identical results. In placentomes distinct staining was restricted to the cytosol of uninucleated trophoblast cells (UTC). The staining pattern in UTC, immature and mature trophoblast giant cells (TGC) is consistent with a down-regulation of SULT1E1 during TGC differentiation. During early and midgestation staining intensity in UTC was higher in the trophoblast covering the chorionic plate and basal parts of stem villi compared to other regions of the villous tree, whereas during late gestation and at parturition an almost ubiquitous distinct staining of UTC was found. Correspondingly, relative SULT1E1-mRNA levels as measured by quantitative real-time RT-PCR increased significantly during late gestation (p = 0.0043). Comparative measurements showed that SULT1E1-mRNA levels in adult bovine organs were considerably lower compared to placentomes and fetal liver. The results suggest that the activities of free estrogens produced in bovine TGC are curtailed by SULT1E1 expressed in UTC and in fetal liver. Bovine pregnancy-associated estrogens produced in trophoblast giant cells are predominantly sulfonated in uninucleated trophoblast cells.

Mouse trophoblast stem (TS) cells represent a unique in vitro system that provides an unlimited supply of TS cells for the study of trophoblast differentiation and TS cell self-renewal. Although the mouse transcription factor Ets2 is required for TS cell self-renewal, its role in this and in TS cell differentiation has not been explored fully, partly due to the early lethality of Ets2 null mice. To address this, we developed a novel lentivirus-based system that resulted in efficient Ets2 knockdown in the overwhelming majority of TS cells. This system enables functional studies in TS cells, especially for genes required for TS cell self-renewal because TS cell derivation using gene-knockout embryos for such genes depends on TS cell self-renewal. Using morphological/morphometric criteria and gene expression analysis, we show that the requirement for Ets2 in self-renewal of TS cells cultured in 'stem cell medium' (SCM) involves maintenance of the expression of genes that inhibit TS cell differentiation in SCM, such as Cdx2 and Esrrb, and preservation of the undifferentiated TS cell morphology. During TS cell differentiation caused by Cdx2/Esrrb downregulation, due to either Ets2 knockdown in SCM or culture in differentiation medium (DM), Ets2 is also required for the promotion of trophoblast giant cell (TGC) and junctional zone trophoblast (JZT) differentiation. This TGC differentiation involves Ets2-dependent expression of Hand1, a gene required for the differentiation of all TGC types. This study uncovers new roles for Ets2 in TS cell self-renewal and differentiation and demonstrates the usefulness of this lentivirus system for gene function studies in TS cells. © 2010 Elsevier Ltd. All rights reserved. 31 630 640 Cited By :17

In the bovine placenta, multinucleate trophoblast giant cells (TGC), evolving from uninucleate trophoblast cells, are crucial for feto-maternal interaction as they show endocrine activity and the ability to migrate and fuse with caruncular epithelial cells. In contrast to caruncular epithelial cells, the isolation and culture of bovine trophoblast cells is complicated because they cease to express their specific products, like placental lactogen (PL), during prolonged culture. In the present study, we aimed to establish a bovine cotyledonary trophoblast cell line targeting our long term goal to develop an in vitro model for the bovine placenta. Therefore, the functional activity of important signalling pathways was tested. Primary trophoblast cells were isolated from a bovine cotyledon of a male fetus and successfully subcultured and cryopreserved. The obtained cell line, termed F3, showed epithelial morphology and characteristic binuclear giant cells in small numbers through all passages. The trophoblastic origin of F3 cells was verified by amplification of a Y-chromosome specific DNA-sequence and the presence of PL mRNA. Immunofluorescence demonstrated that F3 cells were continuously positive for zonula occludens-2 (ZO-2), cytokeratin and vimentin, whereas they expressed the TGC specific marker PL only in the first two passages. F3 cell growth was accelerated in medium supplied with epidermal growth factor (EGF). EGF-stimulated proliferation was mediated through activation of Ras and the phosphorylation of mitogen-activated protein kinase (MAPK) 42 and 44. In conclusion, the F3 cell line shows several in vivo characteristics of bovine cotyledonary trophoblast cells. The response to EGF stimulation indicates that EGF plays a role during bovine placentation, and illustrated that F3 cells may provide a valuable tool for further mechanistic studies elucidating the feto-maternal interplay.

Binucleate giant cells (BNC) decline in number and viability peripartum if the placenta is subsequently released normally. This suggests an involvement of BNC destruction in fetal membrane separation in the cow. Dexamethasone induction of parturition may result in fetal membrane retention through a stabilization oftheBNC (possibly the BNC plasma membranes). Prostaglandin F 2 a treatment in vitro induced a loss ofBNC when these were obtained from cows which subsequently retained the fetal membranes for a longer than normal interval. Overall, our results indicate an involvement of BNC loss and breakdown in placental separation and a possible involvement ofprostaglandins and glucocorticoids in modulating this process.

Neither parthenogenetic (PG) nor androgenetic (AG) mouse embryos survive after day 9.5 of pregnancy, owing to the inadequate growth of extraembryonic tissues, including the placenta. At day 9.5 of pregnancy, the placental structures are poorly developed in PG embryos, while trophoblast giant cells are abundant at the implantation site in AG embryos. These findings suggest that both parental genomes are required for placental development. To gain further insight into the trophoblast lineage in PG and AG embryos, we attempted to derive trophoblast stem (TS)-like cell lines from uniparental embryos. Furthermore, we sought to assess their ability to differentiate into cells of the trophoblast lineage by using gene expression analysis. Three cell lines that expressed marker genes for undifferentiated TS cells (Cdx2 and Errbeta) were derived from AG embryos. Under differentiation conditions, these cells expressed the trophoblast giant cell-specific genes, but did not express the spongiotrophoblast-specific genes. In contrast, none of the four cell lines from PG embryos expressed marker genes for undifferentiated TS cells, but they expressed Oct3/4, a marker gene for embryonic stem cells. Immunohistochemical analysis indicated that PG blastocysts expressed Oct3/4 and Cdx2 specifically in inner cell mass and the trophectoderm respectively. These results suggest that PG embryos do not possess TS cells, because of the lack of the developmental ability of trophoblast cells.

Summary Monoclonal antibody MA21 recognized a 44kDA plasma membrane protein on F9 teratocarcinoma cells, trophectoderm of mouse peri-implantation-stage blastocyst and ectoplacental cone cells of 5 day postcoitum implanted blastocyst (Vernon, Linnemeyer and Hamilton, 1989). We show here that this antigen is expressed by trophoblast cells of the maturing placenta. Immunohistochemical assays of early and mature placental tissue sections, indirect immunofluorescence labelling of placental cultures and blastocyst outgrowths in vitro, and immunoprecipitation of 35 S-labelled NP-40 extracts of placental cultures indicate the presence of a plasma membrane-associated antigen with the same characteristics as MA21 antigen of peri-implantation embryos and F9 teratocarcinoma cells. In sections of placentae, antigenpositive cells are always situated in a thin layer between trophoblastic giant cells and maternal tissue. In cultures of postimplantation stage embryos, attached trophoblast cells express MA21 antigen initially, but following transformation to the giant cell state, antigen is no longer expressed. These results indicate the presence of a plasma membrane protein antigen associated with a distinct population of cells believed to be, trophoblast. We believe that these cells are the foremost trophoblast cells opposing maternal decidua and that they may give rise to secondary trophoblastic giant cells.

s / Placenta 36 (2015) 469e521 502 Methods: Histological and immunofluorescence analysis of Swiss Webster mouse placentas of 9 to 12 dpc, through serial sections and using different stains and antibodies to evaluate the presence, topography and origin of hematopoietic foci. Results: Only the labyrinth region seemed to have hematopoietic activity. Distinct erythroid cells were observed in fetal vessels consisting of larger acidophilic cells with more spherical shape than conventional erythrocytes probably seeded by yolk sac. These cells were often seen in cell division and attached to others and tomore immature cells in circulation. In some areas, they were surrounded by trophoblasts with no apparent vessels. Close to trophoblast giant cells (TGC), heterogeneous cellswere observed protruding toward fetal vessels, which were suggestive to be derived from trophoblast cells rather than fromtransendothelialmigration.Otherarrangements in the labyrinth showed erythroid cells with different levels of hemoglobinization forming agglomerates that resembled erythroid foci. Hematopoietic clusters similar to those of HSC formed by fetuses’ large vessels were also found near umbilical insertion. Immunostaining deepened the characterization of these arrangements, contributing to phenomena interpretation. Conclusions: The placenta is proposed to have hematopoietic potential producing blood cells by at least two different ways inside the labyrinth region. One might occurs near TGC and seems to be restricted to the erythroid lineage. Trophoblast cells are probably the precursors of these hematopoietic cells through migration into fetal circulation, where they proliferate and differentiate, and through their differentiation inside the trophoblast layer, forming erythropoietic foci. A possible second mechanism would occur near the umbilical cord and seems to be related to definitive hematopoiesis. Furthermore, it seems that the placenta not only generates hematopoietic cells but also promotes their expansion and/or differentiation before the fetal liver stage. PA.46. ENROFLOXACIN AND TOLTRAZURIL ARE ABLE TO CONTROL TOXOPLASMA GONDII INFECTION IN HUMAN TROPHOBLAST CELLS R.J. Silva , A.O. Gomes , J.R. Mineo , N.M. Silva , E.A.V. Ferro , B.F. Barbosa . 1 Laboratory of Reproduction Immunophysiology, Institute of Biomedical Science, Federal University of Uberlândia, Uberlândia, Brazil; 2 Laboratory of Immunoparasitology, Institute of Biomedical Science, Federal University of Uberlândia, Uberlândia, Brazil; 3 Laboratory of Immunopatology, Institute of Biomedical Science, Federal University of Uberlândia, Uberlândia

Extravillous trophoblast invasion of uterine stroma and spiral arteries (SA) is essential for normal pregnancy and is reduced in preeclampsia and late miscarriage. The control mechanisms are not understood, but transforming growth factor-beta (TGF-beta) may be a candidate. Placental and placental bed biopsies were obtained from early (8(+0)-12(+6) weeks) euploid miscarriages (n = 10), early aneuploid miscarriages (n = 10), late (13(+0)-19(+6) weeks) euploid miscarriages (n = 10) and controls of the same gestation (n = 20). Frozen sections were immunostained for TGF-beta1, 2 and 3. Immunoreactivity of trophoblast and uterine cell populations was assessed semi-quantitatively. TGF-beta1 immunolocalization was limited to extracellular matrix in cytotrophoblast islands and cytotrophoblast shell, perivascular fibrinoid and interstitial trophoblast and did not differ in miscarriage compared with controls. TGF-beta2 was expressed additionally in endovascular trophoblast and multinucleate trophoblast giant cells. There was no aberrant TGF-beta2 immunolocalization in late miscarriage, but TGF-beta2 immunoreactivity was increased in extracellular matrix in cytotrophoblast islands in early miscarriage. TGF-beta3 was absent from all cell populations. Stromal and extravillous trophoblast TGF-beta2 immunolocalization suggests a more important role in trophoblast invasion than TGF-beta1, but neither isoform was altered in miscarriage. Altered TGF-beta localization is therefore unlikely to play a role in abnormal trophoblast invasion and SA transformation in miscarriage.

Trafficking of cells between the fetus and its mother provides indirect clues to the underlying pathophysiology of pregnancy. Georg Schmorl first documented the presence of fetal cells in the maternal body and emphasized the importance of the placenta in eclampsia. Although his classic paper, written in 1893, is widely cited today, few investigators have actually read the paper, as it was published in German [Schmorl G., Pathologisch-anatomische Untersuchungen uber Puerperal-Eklampsie. Verlag FCW Vogel, Leipzig; 1893]. Our goal was to translate the paper into English and critically re-evaluate its conclusions from a 21st century perspective.Schmorl was remarkably astute in his assessment of the pathologic changes that were seen in the 17 women on whom he performed complete autopsies. He found similar severe changes in all of the women, implying a common pathogenesis. This was in direct contrast to the then current doctrine. He was the first to observe the presence of thrombi containing multinucleated syncytial giant cells in the lungs of the women and speculated that they were of placental origin. To support his hypothesis he performed animal experiments. He also recognized that feto-maternal trafficking occurred in normal gestations but was increased in pregnancies affected by eclampsia. Using sophisticated molecular techniques we can now precisely confirm what Schmorl so elegantly described. (c) 2006 Elsevier Ltd. All rights reserved.

Although the extravillous trophoblastic invasion has a critical role in human placental development, nothing is known about HERV-W expression in the extravillous phenotype. The aim of the present study was to localize in first trimester placenta the expression of HERV-W Env glycoprotein and its receptor all along the differentiation pathway of the extravillous phenotype. In addition using an in vitro model of extravillous cytotrophoblastic cell isolation and invasion we investigated the presence of HERV-W transcripts and envelope glycoprotein in cultured extravillous trophoblastic cells. Using monoclonal and polyclonal antibodies, the glycoprotein was immunolocalized in all the cell types of the extravillous phenotype lineage: cytotrophoblastic cells of the column, interstitial extravillous trophoblastic cells, multinucleated giant cells and endovascular trophoblast. Furthermore, using a polyclonal antibody, the D mammalian virus receptor was also localized in the various extravillous trophoblastic phenotypes. In addition, the presence of HERV-W transcripts and protein was demonstrated in cultured extravillous trophoblastic cells. HERV-W Env glycoprotein expressed in villous and extravillous trophoblast can be considered as a specific marker of the human trophoblast.

High resolution ultrasound imaging of the mouse placenta during development revealed highly echogenic foci localized near the materno-placental interface in early gestation and, near term, in the placental labyrinth (the exchange region of the placenta). Echogenic foci and calcium deposits identified in histological sections using Alizarin red staining showed similar localization and changes with gestation. Calcium deposits caused the echogenic foci because incubating uteri in a decalcifying solution eliminated both the deposits and echogenic foci. Transmission electron microscopy, X-ray microanalysis, and electron diffraction were used to show that deposits were calcium hydroxyapatite crystals. Calcium deposits were extensive and densely packed at days 7.5-9.5 of gestation at the border between the maternal decidua and the fetal trophoblast giant cells of ectoplacental cone. After the formation of the chorio-allantoic placenta (approximately day 10.5), calcification deposits appeared larger and more rarefied but were still localized at the border between the maternal decidua and the fetal trophoblast giant cells of the placenta. Calcification deposits were not observed in the labyrinthine region of the mouse placenta until > or = day 15.5 (day 18.5 is full term). We conclude that deposits of calcium hydroxyapatite crystals in the mouse placenta are detectable by high resolution ultrasound imaging. These deposits provide an ultrasound detectable marker of the maternal-placental interface that is particularly prominent during the establishment of the chorio-allantoic placenta between days 7.5 and 9.5 of gestation.

The placenta of eutherian mammals is a remarkable biological structure. It is composed of both zygote-derived and maternal cells, and mediates the complex interactions between the mother and the fetus that are necessary for fetal growth and survival. While the genetic basis of human placental development and function is largely unknown, its understanding is of immense clinical importance because placentopathies of unknown genetic aetiology are thought to be the cause of many types of pregnancy complications including unexplained miscarriage and intrauterine growth retardation. The mouse is the best-studied mammalian experimental genetic model system and research is not restricted by the inherent ethical and practical limitations associated with the human. As a result, knowledge about the genetic control of mouse placental development has expanded greatly in recent years. In order for this to be of benefit to medical practice, extrapolations from murine to human placentation have to be made. However, comprehensive comparisons of the placentae of these two species are rare. This review therefore compares the developmental anatomy of the placenta between humans and mice with emphasis on structures and cell types that might be analogous between the two species. This could be of particular benefit to mouse developmental geneticists who study placental development and have an interest in the possible clinical implications of their work. © 2002 Harcourt Publishers Ltd. 23 3 19 Cited By :330

Lectin binding of rat trophoblast subpopulations was examined at days 10, 12 and 15 of gestation. Biotinylated lectins, specific for a range of carbohydrates, were applied to paraffin wax embedded tissues. At day 10 of pregnancy, a wide and similar range of lectins reacted with ectoplacental cone and giant cells: chorionic lamina was relatively unreactive except with wheat germ agglutinin. At days 12 and 15, the range of lectins reacting with the placental labyrinth was relatively restricted. Spongiotrophoblast at day 12 showed lectin reactivity similar to that of ectoplacental cone cells but at day 15 the reaction pattern indicated loss of N-acetylgalactosamine residues in the large, glycogen-rich cells. Trophoblastic giant cells also showed wide lectin reactivity: some reacted only with cytoplasmic components whilst others showed pericellular reactivity. Varying lectin reactivity of giant cells in different regions of the placenta suggests functional differences. There was reduced lectin reactivity of endovascular trophoblast at day 15, compared with day 12 of pregnancy, which may reflect altered invasive potential associated with loss of sialic acid and N-acetylgalactosamine residues. The pattern of lectin reactivity suggests that ectoplacental cells, as well as giving rise to giant cells, may also contribute to spongiotrophoblast and to endovascular trophoblast.

Phenotypically distinct rat trophoblast cell lines, the Rcho-1 and R8RP.3 cells, were compared for their responses to cytokines known to induce major histocompatibility (MHC) class I antigens, tumour necrosis factor (TNF), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma). Cell enzyme immunosorbent assays and flow cytometry experiments showed that only IFN-gamma could induce RT1 class I antigens on the Rcho-1 cells. Non-adherent cells were slightly less responsive than adherent, giant cell-like Rcho-1 cells. By contrast, RT1 class I antigens on the R8RP.3 cells were induced by both TGF-beta 1 and IFN-gamma. The cytokines also had different effects on mitochondrial enzyme activity in the two lines. TNF and TGF-beta 1 mRNAs were demonstrated in both lines by using Northern blot hybridization. Rcho-1 but not R8RP.3 cells contained two TNF messages (approximately 2.2, 1.9 kb). Steady state levels of transcripts from the TNF gene, and, to a lesser extent, the TGF-beta 1 gene, were increased in cultures of Rcho-1 cells that contained high proportions of giant cells. Thus, phenotypically distinct rat trophoblast cell lines do not respond identically to TNF, TGF-beta 1 or IFN-gamma, transcription of cytokine genes does not prevent the cells from responding to paracrine cytokine signals, and the cells contain novel TNF transcripts that might be important in cell maturation or differentiation.

Summary The neurotransmitter serotonin (5-HT) was localized in the ectoplacental cone (EPC) and placenta of the day 9–12 (E9–12) mouse embryo in vivo and in whole embryo cultures, using immunocytochemistry with a specific 5-HT antiserum. In uncultured conceptuses, 5-HT immunoreactivity (5-HT IR) was most intense in the EPC at E9 (2–7 somites), particularly in giant cells around the periphery. Nuclear staining was observed in lightly staining giant cells and in small cells in the core of the cone. By E10 (18–24 somites) 5-HT IR in the placenta was less intense and almost exclusively limited to giant cells, where it was localized to chromatin-like material in nuclei. The same pattern and level of 5-HT IR persisted through E12. In the placenta, 5-HT IR appeared to be most intense in giant cells located near aggregations of platelets in decidual blood vessels. 5-HT IR was enhanced in cultured conceptuses, and further increased when exogenous 5-HT was added to the culture medium. Immunoreactivity was greatly reduced by adding the 5-HT uptake inhibitor fluoxetine to the culture medium, or culturing conceptuses in medium containing 5-HT depleted rat serum. Thus, 5-HT was apparently taken up from the culture medium. In conceptuses exposed to exogenous 5-HT, immunoreactivity in the placenta appeared as a gradient from the giant cells to the inner layers, suggesting that these cells may transport 5-HT toward the embryo. No evidence of 5-HT synthesis by the EPC/placenta was found. These results suggest that 5-HT present in the EPC/placenta is due to uptake, not synthesis. Possible sources and functions of 5-HT in the developing placenta are discussed.