Bovine placental extracellular vesicles carry the fusogenic syncytin BERV-K1.

Syncytins are endogenous retroviral envelope proteins which induce the fusion of membranes. A human representative of this group, endogenous retrovirus group W member 1 envelope (ERVW-1) or syncytin-1 is present in trophoblast-derived extracellular vesicles and supports the incorporation of these extracellular vesicles into recipient cells. During pregnancy, placenta-derived extracellular vesicles participate in feto-maternal communication. Bovine fetal binucleate trophoblast cells express the syncytin, bovine endogenous retroviral envelope protein K1 (BERV-K1). These cells release extracellular vesicles into the maternal stroma, but it is unclear whether BERV-K1 is included in these extracellular vesicles. Here, extracellular vesicles were isolated from bovine placental tissue using collagenase digestion, ultracentrifugation, and size exclusion chromatography. They were characterized with transmission electron microscopy, nanoparticle tracking analysis, immunoblotting and mass spectrometry. Immunohistochemistry and immunoelectron microscopy were used to localize BERV-K1 within the bovine placental tissue. The isolated extracellular vesicles range between 50 and 300 nm, carrying multiple extracellular vesicle biomarkers. Proteomic analysis and immunoelectron microscopy confirmed BERV-K1 presence on the isolated extracellular vesicles. Further, BERV-K1 was localized on intraluminal vesicles in secretory granules of binucleate trophoblast cells. The presence of BERV-K1 on bovine placental extracellular vesicles suggests their role in feto-maternal communication and potential involvement of BERV-K1 in uptake of extracellular vesicles by target cells.

The Prl3d1-Cre mouse line selectively induces the expression of Cre recombinase in parietal trophoblast giant cells.

The placenta plays a pivotal role in the maintenance of normal pregnancy, but how it forms, matures, and performs its function remains poorly understood. Here, we describe a novel mouse line (Prl3d1-iCre) that expresses iCre recombinase under the control of the endogenous prl3d1 promoter. Prl3d1 has been proposed as a marker for distinguishing trophoblast giant cells (TGCs) from other trophoblast cells in the placenta. The in vivo efficiency and specificity of the Cre line were analyzed by interbreeding Prl3d1-iCre mice with B6-G/R reporter mice. Through anatomical studies of the placenta and other tissues of Prl3d1-iCre/+; B6-G/R mouse mice, we found that the tdTomato signal was expressed in parietal trophoblast giant cells (P-TGCs). Thus, we report a mouse line with ectopic Cre expression in P-TGCs, which provides a valuable tool for studying human pathological pregnancies caused by implantation failure or abnormal trophoblast secretion due to aberrant gene regulation.

A possible function of Nik-related kinase in the labyrinth layer of delayed delivery mouse placentas.

In mice and humans, Nik-related protein kinase (Nrk) is an X-linked gene that encodes a serine/threonine kinase belonging to GCK group 4. Nrk knockout (Nrk KO) mice exhibit delayed delivery, possibly due to defective communication between the Nrk KO conceptus and its mother. However, the mechanism of delayed labor remains largely unknown. Here, we found that in pregnant mothers with the Nrk KO conceptus, the serum progesterone (P4) and placental lactogen (PL-2) concentrations in late pregnancy were higher than those in the wild type. Moreover, we demonstrated that Nrk is expressed in trophoblast giant cells (TGCs) and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is also expressed in Syn-T in villi. Both human Syn-T and mouse TGCs of the labyrinth layer are present within fetal tissues that are in direct contact with the maternal blood. The labyrinth layer of the Nrk KO conceptus was gigantic, with enlarged cytoplasm and Golgi bodies in the TGCs. To investigate the function of Nrk in the labyrinth layer, a differentially expressed gene (DEG) analysis was performed. The DEG analysis revealed that labor-promoting factors, such as prostaglandins, were decreased, and pregnancy-maintaining factors, such as the prolactin family and P4 receptor, were increased. These findings suggest that the Nrk KO mice exhibit delayed delivery owing to high P4 concentrations caused by the hypersecretion of pregnancy-maintaining factors, such as PL-2, from the placenta.

Padi2/3 Deficiency Alters the Epigenomic Landscape and Causes Premature Differentiation of Mouse Trophoblast Stem Cells.

Histone citrullination is a relatively poorly studied epigenetic modification that involves the irreversible conversion of arginine residues into citrulline. It is conferred by a small family of enzymes known as protein arginine deiminases (PADIs). PADI function supports the pluripotent state of embryonic stem cells, but in other contexts, also promotes efficient cellular differentiation. In the current study, we sought to gain deeper insights into the possible roles of PADIs in mouse trophoblast stem cells (TSCs). We show that Padi2 and Padi3 are the most highly expressed PADI family members in TSCs and are rapidly down-regulated upon differentiation. Padi2/3 double knockout (DKO) TSCs express lower levels of stem cell transcription factors CDX2 and SOX2 and are prone to differentiate into extremely large trophoblast giant cells, an effect that may be mediated by centrosome duplication defects. Interestingly, Padi2/3 DKO TSCs display alterations to their epigenomic landscape, with fewer H3K9me3-marked chromocentric foci and globally reduced 5-methylcytosine levels. DNA methylation profiling identifies that this effect is specifically evident at CpG islands of critical trophoblast genes, such as Gata3, Peg3, Socs3 and Hand1. As a consequence of the hypomethylated state, these factors are up-regulated in Padi2/3 DKO TSCs, driving their premature differentiation. Our data uncover a critical epigenetic role for PADI2/3 in safeguarding the stem cell state of TSCs by modulating the DNA methylation landscape to restrict precocious trophoblast differentiation.

Role of autocrine bone morphogenetic protein signaling in trophoblast stem cells†.

The Bone Morphogenetic Protein (BMP) pathway is involved in numerous developmental processes, including cell growth, apoptosis, and differentiation. In mouse embryogenesis, BMP signaling is a well-known morphogen for both mesoderm induction and germ cell development. Recent evidence points to a potential role in development of the extraembryonic compartment, including trophectoderm-derived tissues. In this study, we investigated the effect of BMP signaling in both mouse and human trophoblast stem cells (TSC) in vitro, evaluating the expression and activation of the BMP signaling response machinery, and the effect of BMP signaling manipulation during TSC maintenance and differentiation. Both mouse trophoblast stem cells (mTSC) and human trophoblast stem cells (hTSC) expressed various BMP ligands and the receptors BMPR1A and BMPR2, necessary for BMP response, and displayed maximal active BMP signaling when undifferentiated. We also observed a conserved modulatory role of BMP signaling during trophoblast differentiation, whereby maintenance of active BMP signaling blunted differentiation of TSC in both species. Conversely, the effect of BMP signaling on the undifferentiated state of TSC appeared to be species-specific, with SMAD-independent signaling important in maintenance of mTSC, and a more subtle role for both SMAD-dependent and -independent BMP signaling in hTSC. Altogether, these data establish an autocrine role for the BMP pathway in the trophoblast compartment. As specification and correct differentiation of the extraembryonic compartment are fundamental for implantation and early placental development, insights on the role of the BMP signaling in early development might prove useful in the setting of in vitro fertilization as well as targeting trophoblast-associated placental dysfunction.

Effects of Pparγ1 deletion on late-stage murine embryogenesis and cells that undergo endocycle.

Peroxisome proliferator-activated receptor (PPAR) γ1, a nuclear receptor, is abundant in the murine placenta during the late stage of pregnancy (E15-E16), although its functional roles remain unclear. PPARγ1 is encoded by two splicing isoforms, namely Pparγ1canonical and Pparγ1sv, and its embryonic loss leads to early (E10) embryonic lethality. Thus, we generated knockout (KO) mice that carried only one of the isoforms to obtain a milder phenotype. Pparγ1sv-KO mice were viable and fertile, whereas Pparγ1canonical-KO mice failed to recover around the weaning age. Pparγ1canonical-KO embryos developed normally up to 15.5 dpc, followed by growth delays after that. The junctional zone of Pparγ1canonical-KO placentas severely infiltrated the labyrinth, and maternal blood sinuses were dilated. In the wild-type, PPARγ1 was highly expressed in sinusoidal trophoblast giant cells (S-TGCs), peaking at 15.5 dpc. Pparγ1canonical-KO abolished PPARγ1 expression in S-TGCs. Notably, the S-TGCs had unusually enlarged nuclei and often occupied maternal vascular spaces, disturbing the organization of the fine labyrinth structure. Gene expression analyses of Pparγ1canonical-KO placentas indicated enhanced S-phase cell cycle signatures. EdU-positive S-TGCs in Pparγ1canonical-KO placentas were greater in number than those in wild-type placentas, suggesting that the cells continued to endoreplicate in the mutant placentas. These results indicate that PPARγ1, a known cell cycle arrest mediator, is involved in the transition of TGCs undergoing endocycling to the terminal differentiation stage in the placentas. Therefore, PPARγ1 deficiency, induced through genetic manipulation, leads to placental insufficiency.

HGF/c-Met signaling regulates early differentiation of placental trophoblast cells.

Depletion of hepatocyte growth factor (HGF) or mesenchymal-epithelial transition factor (c-Met) in mice leads to fetal lethality and placental maldevelopment. However, the dynamic change pattern of HGF/c-Met signaling during placental development and its involvement in the early differentiation of trophoblasts remain to be elucidated. In this study, using in situ hybridization assay, we elaborately demonstrated the spatial-temporal expression of Hgf and c-Met in mouse placenta from E5.5, the very early stage after embryonic implantation, to E12.5, when the placental structure is well developed. The concentration of the soluble form of c-Met (sMet) in maternal circulation peaked at E10.5. By utilizing the induced differentiation model of mouse trophoblast stem cells (mTSCs), we found that HGF significantly promoted mTSC differentiation into syncytiotrophoblasts (STBs) and invasive parietal trophoblast giant cells (PTGCs). Interestingly, sMet efficiently reversed the effect of HGF on mTSC differentiation. These findings indicate that HGF/c-Met signaling participates in regulating placental trophoblast cell fate at the early differentiation stage and that sMet acts as an endogenous antagonist in this aspect.

Maternal oxycodone treatment causes pathophysiological changes in the mouse placenta.

INTRODUCTION: Pregnant women are increasingly being prescribed and abusing opioid drugs. As the primary communication organ between mother and conceptus, the placenta may be vulnerable to opioid effects but also holds the key to better understanding how these drugs affect long-term offspring health. We hypothesized that maternal treatment with oxycodone (OXY), the primary opioid at the center of the current crisis, deleteriously affects placental structure and gene expression patterns. METHODS: Female mice were treated daily with 5 mg OXY/kg or saline solution (Control, CTL) for two weeks prior to breeding and until placenta were collected at embryonic age 12.5. A portion of the placenta was fixed for histology, and the remainder was frozen for RNA isolation followed by RNAseq. RESULTS: Maternal OXY treatment reduced parietal trophoblast giant cell (pTGC) area and decreased the maternal blood vessel area within the labyrinth region. OXY exposure affected placental gene expression profiles in a sex dependent manner with female placenta showing up-regulation of many placental enriched genes, including Ceacam11, Ceacam14, Ceacam12, Ceacam13, Prl7b1, Prl2b1, Ctsq, and Tpbpa. In contrast, placenta of OXY exposed males had alteration of many ribosomal proteins. Weighted correlation network analysis revealed that in OXY female vs. CTL female comparison, select modules correlated with OXY-induced placental histological changes. Such associations were lacking in the male OXY vs. CTL male comparison. DISCUSSION: Results suggest OXY exposure alters placental histology. In response to OXY exposure, female placenta responds by upregulating placental enriched transcripts that are either unchanged or downregulated in male placenta. Such changes may shield female offspring from developmental origins of health and disease-based diseases.

Transcriptomic analysis reveals differential gene expression, alternative splicing, and novel exons during mouse trophoblast stem cell differentiation.

BACKGROUND: Differentiation of mouse trophoblast stem cells (TSCs) to trophoblast giant cells (TGCs) has been widely used as a model system to study placental development and function. While several differentially expressed genes, including regulators of TSC differentiation, have been identified, a comprehensive analysis of the global expression of genes and splice variants in the two cell types has not been reported. RESULTS: Here, we report ~ 7800 differentially expressed genes in TGCs compared to TSCs which include regulators of the cell cycle, apoptosis, cytoskeleton, cell mobility, embryo implantation, metabolism, and various signaling pathways. We show that several mitotic proteins, including Aurora A kinase, were downregulated in TGCs and that the activity of Aurora A kinase is required for the maintenance of TSCs. We also identify hitherto undiscovered, cell-type specific alternative splicing events in 31 genes in the two cell types. Finally, we also report 19 novel exons in 12 genes which are expressed in both TSCs and TGCs. CONCLUSIONS: Overall, our results uncover several potential regulators of TSC differentiation and TGC function, thereby providing a valuable resource for developmental and molecular biologists interested in the study of stem cell differentiation and embryonic development.

Morphological Characterization of Basally Located Uninucleate Trophoblast Cells as Precursors of Bovine Binucleate Trophoblast Giant Cells.

Binucleate trophoblast giant cells (TGCs) are one characteristic feature of the ruminant placenta. In cows, the frequency of TGCs remains constant for most of the duration of pregnancy. As TGCs are depleted by their fusion with uterine epithelial cells, they need to be constantly formed. It is still unclear whether they develop from stem cells within the trophectoderm or whether they can arise from any uninucleate trophoblast cell (UTC). Within the latter, generally accepted theory, a basally located uninucleate cell (BUC) without contact to the feto-maternal interface would represent a transient cell between a UTC and a TGC. So far, no evidence for the existence of such transient cells or for the presence of stem cells has been shown. The aim of the present study is to morphologically characterize the early stages of TGC development. Placentomal tissue of 6 pregnant cows from different gestational stages (gestational days 51-214) was examined for BUCs, UTCs, and TGCs either in serial sections (light and transmission electron microscopy, TEM, n = 3), in single sections (TEM, n = 2), or by serial block face-scanning electron microscopy (n = 1). These investigations revealed the occurrence of BUCs, as well as young TGCs showing contact with the basement membrane (BM), but without apical contact to the feto-maternal interface. The study morphologically defines these 2 cell types as early stages of TGC development and shows that binucleation of TGCs can precede detachment from the BM.

Advanced maternal age impairs spatial learning capacity in young adult mouse offspring.

Effects of maternal aging on the offspring cognitive function remain controversial in population-based investigations, and information available in animal studies is very limited. We investigated the impact of a delayed first natural pregnancy on pregnancy outcomes in the mouse model. Spatial learning capacity in young adult mouse offspring was observed by step-down passive avoidance task and Morris water maze (MWM). Maternal serum α-klotho was measured by ELISA. Morphological characteristics of fetoplacental unit and offspring brain were identified by H&E and immunohistochemistry. Klotho, VDR and other related genes expression were quantified by real-time-RT-PCR and western blot. We found delayed pregnancy reduced fertility in female mice by three-fold (Young vs. Old: 5.0% vs. 20.7%), and increased adverse pregnant outcomes by eight-fold (Young vs. Old: 3.0% vs. 27.5%). Mice born to old mothers exhibited shorter retention trial latency in passive avoidance task and longer latency to find the platform in MWM, suggesting worse performance on the tests that measure learning and memory. Serum α-klotho level was lower in old female mice before pregnancy, whereas became comparable after pregnancy. Vitamin D receptor (VDR) expression, both in mRNA and protein, markedly decreased during the early stage of fetoplacental unit in old mice, especially in trophoblast giant cells when compared with that of young mice. Importantly, consistent with fetoplacental unit, VDR expression also declined in hippocampus from offspring born to old mice. These results suggest that young adult offspring from aged mothers exhibited worse cognitive function and the reduced VDR expression during fetoplacental development might play an important role.

A Critical Role of TET1/2 Proteins in Cell-Cycle Progression of Trophoblast Stem Cells.

The ten-eleven translocation (TET) proteins are well known for their role in maintaining naive pluripotency of embryonic stem cells. Here, we demonstrate that, jointly, TET1 and TET2 also safeguard the self-renewal potential of trophoblast stem cells (TSCs) and have partially redundant roles in maintaining the epithelial integrity of TSCs. For the more abundantly expressed TET1, we show that this is achieved by binding to critical epithelial genes, notably E-cadherin, which becomes hyper-methylated and downregulated in the absence of TET1. The epithelial-to-mesenchymal transition phenotype of mutant TSCs is accompanied by centrosome duplication and separation defects. Moreover, we identify a role of TET1 in maintaining cyclin B1 stability, thereby acting as facilitator of mitotic cell-cycle progression. As a result, Tet1/2 mutant TSCs are prone to undergo endoreduplicative cell cycles leading to the formation of polyploid trophoblast giant cells. Taken together, our data reveal essential functions of TET proteins in the trophoblast lineage.

Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells.

Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate "BNC". Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering.

Differential patterning of genes involved in serotonin metabolism and transport in extra-embryonic tissues of the mouse.

INTRODUCTION: Serotonin (5-HT) is an important neuromodulator, but recently has been shown to be involved in neurodevelopment. Although previous studies have demonstrated that the placenta is a major source of forebrain 5-HT during early forebrain development, the processes of how 5-HT production, metabolism, and transport from placenta to fetus are regulated are unknown. As an initial step in determining the mechanisms involved, we investigated the expression patterns of genes critical for 5-HT system function in mouse extraembryonic tissues. METHODS: Mid-through late gestation expression of 5-HT system-related enzymes, Tph1, Ddc, Maoa, and 5-HT transporters, Sert/Slc6a4, Oct3/Slc22a3, Vmat2/Slc18a2, and 5-HT in placenta and yolk sac were examined, with cell type-specific resolution, using multiplex fluorescent in situ hybridization to co-localize transcripts and immunocytochemistry to co-localize the corresponding proteins and neurotransmitter. RESULTS: Tph1 and Ddc are found in the syncytiotrophoblast I (SynT-I) and sinusoidal trophoblast giant cells (S-TGC), whereas Maoa is expressed in SynT-I, syncytiotrophoblast II (SynT-II) and S-TGC. Oct3 expression is observed in the SynT-II only, while Vmat2 is mainly expressed in S-TGC. Surprisingly, there were comparatively high expression of Tph1, Ddc, and Maoa in the yolk sac visceral endoderm. DISCUSSION: In addition to trophoblast cells, visceral endoderm cells in the yolk sac may contribute to fetal 5-HT production. The findings raise the possibility of a more complex regulation of 5-HT access to the fetus through the differential roles of trophoblasts that surround maternal and fetal blood space and of yolk sac endoderm prior to normal degeneration.

Reduced Gene Dosage of Tfap2c Impairs Trophoblast Lineage Differentiation and Alters Maternal Blood Spaces in the Mouse Placenta.

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

The dual roles of geminin during trophoblast proliferation and differentiation.

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.

Developmental effects of oral exposure to diethylstilbestrol on mouse placenta.

Placental growth and function are of biological significance in that placental tissue promotes prenatal life and the maintenance of pregnancy. Exposure to synthetic estrogens causes embryonic mortality and placental growth restriction in mice. The aim of the present study was to examine the effects of diethylstilbestrol (DES) on placenta in mice. DES at 1, 5, 10 or 15 µg kg(-1) day(-1) , or 17ß-estradiol (E2 ) at 50 µg kg(-1) day(-1) , was administered orally to ICR mice on days 4 through to 8 of gestation. Expression of ERα, ERß, ERRß or ERRγ mRNA in the junctional or labyrinth zone of the placentas on day 13 was assessed using RT-PCR, as well as the embrynic mortality, embryonic and placental weight, histological changes of labyrinth and ultrastructural changes of the trophoblast giant cells (TGCs). Embryo mortalities in the DES 10 and 15 µg kg(-1) day(-1) groups were markedly increased. No significant changes in embryonic and placental weight were observed in any DES- or E2 -exposed groups. Expression of ERα mRNA in the junctional zone with male embryos in the 5 µg kg(-1) day(-1) group was significantly higher than that in the control, whereas expression was not determined in the 15 µg kg(-1) day(-1) group. Histological observation revealed that the placentas exposed to DES at 10 µg kg(-1) day(-1) lacked the developing labyrinth. Ultrastructural observation of the TGCs showed poor rough-surfaced endoplasmic reticulum in the DES 10 µg kg(-1) day(-1) group. The present data suggest that developmental changes induced by DES may be related to interference with the nutrition and oxygen exchange between mother and embryo or decreased protein synthesis, resulting in a high frequency of embryo mortality.