Nutritional and metabolic control of germ cell fate through O-GlcNAc regulation.

Fate determination of primordial germ cells (PGCs) is regulated in a multi-layered manner, involving signaling pathways, epigenetic mechanisms, and transcriptional control. Chemical modification of macromolecules, including epigenetics, is expected to be closely related with metabolic mechanisms but the detailed molecular machinery linking these two layers remains poorly understood. Here, we show that the hexosamine biosynthetic pathway controls PGC fate determination via O-linked ß-N-acetylglucosamine (O-GlcNAc) modification. Consistent with this model, reduction of carbohydrate metabolism via a maternal ketogenic diet that decreases O-GlcNAcylation levels causes repression of PGC formation in vivo. Moreover, maternal ketogenic diet intake until mid-gestation affects the number of ovarian germ cells in newborn pups. Taken together, we show that nutritional and metabolic mechanisms play a previously unappreciated role in PGC fate determination.

SETDB1 is essential for mouse primordial germ cell fate determination by ensuring BMP signaling.

In mouse embryos, primordial germ cells (PGCs) are fate-determined from epiblast cells. Signaling pathways involved in PGC formation have been identified, but their epigenetic mechanisms remain poorly understood. Here, we show that the histone methyltransferase SETDB1 is an epigenetic regulator of PGC fate determination. Setdb1-deficient embryos exhibit drastic reduction of nascent PGCs. Dppa2, Otx2 and Utf1 are de-repressed whereas mesoderm development-related genes, including BMP4 signaling-related genes, are downregulated by Setdb1 knockdown during PGC-like cell (PGCLC) induction. In addition, binding of SETDB1 is observed at the flanking regions of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs, and trimethylation of lysine 9 of histone H3 is reduced by Setdb1 knockdown at those regions. Furthermore, DPPA2, OTX2 and UTF1 binding is increased in genes encoding BMP4 signaling-related proteins, including SMAD1. Finally, overexpression of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs results in the repression of BMP4 signaling-related genes and PGC determinant genes. We propose that the localization of SETDB1 to Dppa2, Otx2 and Utf1, and subsequent repression of their expression, are crucial for PGC determination by ensuring BMP4 signaling.

Derivation of pluripotent stem cells from nascent undifferentiated teratoma.

Teratomas are tumors consisting of components of the three germ layers that differentiate from pluripotent stem cells derived from germ cells. In the normal mouse testis, teratomas rarely form, but a deficiency in Dead-end1 (Dnd1) in mice with a 129/Sv genetic background greatly enhances teratoma formation. Thus, DND1 is crucial for suppression of teratoma development from germ cells. In the Dnd1 mutant testis, nascent teratoma cells emerge at E15.5. To understand the nature of early teratoma cells, we established cell lines in the presence of serum and leukemia inhibitory factor (LIF) from teratoma-forming cells in neonatal Dnd1 mutant testis. These cells, which we designated cultured Dnd1 mutant germ cells (CDGCs), were morphologically similar to embryonic stem cells (ESCs) and could be maintained in the naïve pluripotent condition. In addition, the cells expressed pluripotency genes including Oct4, Nanog, and Sox2; differentiated into cells of the three germ layers in culture; and contributed to chimeric mice. The expression levels of pluripotency genes and global transcriptomes in CDGCs as well as these cells' adaption to culture conditions for primed pluripotency suggested that their pluripotent status is intermediate between naïve and primed pluripotency. In addition, the teratoma-forming cells in the neonatal testis from which CDGCs were derived also showed gene expression profiles intermediate between naïve and primed pluripotency. The results suggested that germ cells in embryonic testes of Dnd1 mutants acquire the intermediate pluripotent status during the course of conversion into teratoma cells.

The serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells.

BACKGROUND: Serine proteases act through the proteolytic cleavage of the hemagglutinin (HA) of influenza viruses for the entry of influenza virus into cells, resulting in infection. However, the inhibitory effects of serine protease inhibitors on influenza virus infection of human airway epithelial cells, and on their production of inflammatory cytokines are unclear. METHODS: Primary cultures of human tracheal epithelial cells were treated with four types of serine protease inhibitors, including camostat, and infected with A/Sendai-H/108/2009/(H1N1) pdm09 or A/New York/55/2004(H3N2). RESULTS: Camostat reduced the amounts of influenza viruses in the supernatants and viral RNA in the cells. It reduced the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. Camostat also reduced the concentrations of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants. Gabexate and aprotinin reduced the viral titers and RNA levels in the cells, and aprotinin reduced the concentrations of TNF-α in the supernatants. The proteases transmembrane protease serine S1 member (TMPRSS) 2 and HAT (human trypsin-like protease: TMPRSS11D), which are known to cleave HA0 and to activate the virus, were detected at the cell membrane and in the cytoplasm. mRNA encoding TMPRSS2, TMPRSS4 and TMPRSS11D was detectable in the cells, and the expression levels were not affected by camostat. CONCLUSIONS: These findings suggest that human airway epithelial cells express these serine proteases and that serine protease inhibitors, especially camostat, may reduce influenza viral replication and the resultant production of inflammatory cytokines possibly through inhibition of activities of these proteases.

Histone deacetylase inhibitor restores surfactant protein-C expression in alveolar-epithelial type II cells and attenuates bleomycin-induced pulmonary fibrosis in vivo.

AIM: Surfactant protein-C (SP-C) of alveolar epithelial type II cells (ATII) plays a key role in maintaining alveolar integrity and repair. Mutations or decreased expression of SFTPC, the gene encoding SP-C, causes ATII injury and aberrant repair of the lung tissue to develop pulmonary fibrosis. Histone deacetylases (HDACs) epigenetically remove acetyl groups from acetylated histones and regulate transcription. HDAC inhibitors attenuated epithelial-to-mesenchymal transition (EMT) and fibrotic disorders. The aim of this study is to investigate whether Trichostatin A (TSA), a pan-HDAC inhibitor, epigenetically exerts a protective effect on ATII against fibrotic changes via the restoration of SFTPC expression. MATERIALS AND METHODS: We treated A549 cells with TGF-ß1 to induce EMT, followed by TSA treatment. We evaluated SFTPC mRNA, histone acetylation levels in the SFTPC gene promoter region, and pro-SP-C protein. C57BL6/J mice were treated with intratracheal bleomycin instillation followed by TSA administration. Histological changes and Sftpc mRNA expression in isolated ATII were evaluated. RESULTS: TGF-ß1 treatment decreased SFTPC mRNA in A549 cells. TSA restored SFTPC mRNA, and increased histone H4 acetylation in the SFTPC promoter region in vitro. The administration of TSA partially attenuated BLM-induced pulmonary fibrosis and increased the Sftpc mRNA expression in isolated ATII from bleomycin-treated lungs in vivo. CONCLUSIONS: Decreased expression of SFTPC by TGF-ß1 treatment was restored by TSA via hyperacetylation of histone H4 in the promoter region. TSA partially attenuated pulmonary fibrosis and increased Sftpc mRNA in ATII. Our findings suggest that the epigenetic restoration of SP-C would be a therapeutic target for pulmonary fibrosis.

Localization of Notch signaling molecules and their effect on cellular proliferation in adult rat pituitary.

Notch signaling is a cell-to-cell signaling system involved in the maintenance of precursor cells in many tissues. Although Notch signaling has been reported in the pituitary gland, the histological characteristics of Notch receptors and ligands in the gland are unknown. Here, we report the histological gene expression pattern of Notch receptors and ligands and the role of Notch signaling in cellular proliferation in adult rat pituitary gland. In situ hybridization detected transcripts of Notch1 and 2 and Jagged1 and 2. Double-staining with a combination of in situ hybridization and immunohistochemistry revealed that their mRNAs were localized in almost half of the S100-protein-positive cells, which are generally regarded as marginal layer cells and folliculo-stellate cells. In primary culture of anterior pituitary cells, proliferation of S100-protein-positive cells was modulated by Notch signaling inhibitor and solubilized Notch ligand. Furthermore, quantitative analysis revealed that the inhibition of Notch signaling led to the down-regulation of mRNA for the Notch target gene Hes1 and the up-regulation of p57 gene expression. These findings suggest that Notch signaling is involved in the proliferation of S100-protein-positive cells, presumably precursor cells, in adult rat pituitary gland.

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

BACKGROUND: The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. METHODS: Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-ß, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. RESULTS: The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition. CONCLUSIONS: Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.

Differences in the released endothelial microparticle subtypes between human pulmonary microvascular endothelial cells and aortic endothelial cells in vitro.

Circulating endothelial microparticles (EMPs) are membrane vesicles that are shed into the blood stream from activated or apoptotic endothelial cells. We previously reported that circulating EMP numbers significantly increased in stable chronic obstructive pulmonary disease (COPD) patients and during exacerbation compared with healthy control subjects. However, different types of circulating EMPs with distinct time profiles were detectable during exacerbations. We hypothesized that the released EMP subtypes correlated with differences in the inflammatory stimuli and the endothelial cell type. We compared the EMP subtypes from human aortic endothelial cells (Aortic ECs) and human lung microvascular endothelial cells (Pulmonary microvascular ECs) released in response to various stimuli, including proinflammatory cytokines (TNFα), oxidative stress (H2O2), and cigarette smoke extracts (CSE) in vitro. We defined circulating EMPs by the expression of endothelial antigens: CD144(+) MPs (VE-cadherin EMPs), CD31(+)/CD41(-) MPs (PECAM EMPs), CD62E(+) MPs (E-selectin EMPs), and CD146(+) MPs (MCAM EMPs). E-selectin EMPs were released from both pulmonary microvascular and aortic ECs in response to TNFα but not to H2O2 or CSE stimulation. The amount of MCAM EMPs released from pulmonary microvascular ECs differed significantly between the cells stimulated with H2O2 and those stimulated with CSE. VE-cadherin EMPs were only released from aortic ECs, whereas PECAM EMPs were released exclusively from pulmonary microvascular ECs. The EMP subtypes released differ in vitro among TNFα, H2O2, and CSE stimulation as well as between pulmonary microvascular and aortic ECs. The differences in circulating EMP subtypes may reflect a condition or site of endothelial injury and may serve as markers for endothelial damage in COPD patients.

Inheritance of environment-induced phenotypic changes through epigenetic mechanisms.

Growing evidence suggests that epigenetic changes through various parental environmental factors alter the phenotypes of descendants in various organisms. Environmental factors, including exposure to chemicals, stress and abnormal nutrition, affect the epigenome in parental germ cells by different epigenetic mechanisms, such as DNA methylation, histone modification as well as small RNAs via metabolites. Some current remaining questions are the causal relationship between environment-induced epigenetic changes in germ cells and altered phenotypes of descendants, and the molecular basis of how the abnormal epigenetic changes escape reprogramming in germ cells. In this review, we introduce representative examples of intergenerational and transgenerational inheritance of phenotypic changes through parental environmental factors and the accompanied epigenetic and metabolic changes, with a focus on animal species. We also discuss the molecular mechanisms of epigenomic inheritance and their possible biological significance.

Identification of spermatogenesis-associated changes in DNA methylation induced by maternal exposure to chemicals in male germ cells.

It is now recognized that maternal environmental factors, including chemical exposure and nutritional conditions, alter DNA methylation patterns in fetal germ cells, subsequently affecting germ cell development as well as offspring phenotypes. Here, we describe steps for detecting DNA methylation changes in mouse germ cells isolated from both embryonic and spermatogenic stages after maternal exposure to a chemical compound. For complete details on the use and execution of this protocol, please refer to Tando et al. (2021).1.

Immunohistochemical localization of anterior pituitary hormones in S-100 protein-positive cells in the rat pituitary gland.

In the anterior and intermediate lobes of the rat pituitary gland, non-hormone-producing cells that express S-100 protein coexist with various types of hormone-producing cells and are believed to function as phagocytes, supporting and paracrine-controlling cells of hormone-producing cells and stem cells, among other functions; however, their cytological characteristics are not yet fully understood. Using a transgenic rat that expresses green fluorescent protein under the promoter of the S100ß protein gene, we immunohistochemically detected expression of the luteinizing hormone, thyroid-stimulating hormone, prolactin, growth hormone and proopiomelanocortin by S-100 protein-positive cells located between clusters of hormone-producing cells in the intermediate lobe. These findings lend support to the hypothesis that S-100 protein-positive cells are capable of differentiating into hormone-producing cells in the adult rat pituitary gland.

Live staining and isolation of specific hormone-producing cells from rat anterior pituitary by cytochemistry with lectins and cholera toxin B subunit.

Anterior pituitary glands contain five types of hormone-producing cells. Distinguishing and isolating specific types of living cells are essential for studying their function. Although many such attempts have been made, the results have been disappointing. In the present study, we labeled specific types of living hormone-producing cells by using potential differences in sugar chains on the cell surfaces. Cytochemical analysis with lectins and cholera toxin B subunit revealed that PNA, S-WGA, and cholera toxin B subunit recognized sugar chains specific to prolactin cells, ACTH cells, and GH cells, respectively, and that UEA-I recognized most of prolactin cells and GH cells. Next, fluorescence-activated cell sorting was used to isolate GH cells labeled by fluoresceinated cholera toxin B. The purity of the GH cell fraction estimated by immunocytochemistry and quantitative real-time PCR for cell type-specific genes was more than 98%, which was higher than that reported in earlier studies, including those using transgenic animals. We conclude that cytochemistry with lectins and cholera toxin B subunit is a straightforward, acceptable method of isolating specific types of anterior pituitary cells and that the cells isolated by this method can serve as useful materials in the study of anterior pituitary cells.

Epi-mutations for spermatogenic defects by maternal exposure to di(2-ethylhexyl) phthalate.

Exposure to environmental factors during fetal development may lead to epigenomic modifications in fetal germ cells, altering gene expression and promoting diseases in successive generations. In mouse, maternal exposure to di(2-ethylhexyl) phthalate (DEHP) is known to induce defects in spermatogenesis in successive generations, but the mechanism(s) of impaired spermatogenesis are unclear. Here, we showed that maternal DEHP exposure results in DNA hypermethylation of promoters of spermatogenesis-related genes in fetal testicular germ cells in F1 mice, and hypermethylation of Hist1h2ba, Sycp1, and Taf7l, which are crucial for spermatogenesis, persisted from fetal testicular cells to adult spermatogonia, resulting in the downregulation of expression of these genes. Forced methylation of these gene promoters silenced expression of these loci in a reporter assay. These results suggested that maternal DEHP exposure-induced hypermethylation of Hist1h2ba, Sycp1, and Taf7l results in downregulation of these genes in spermatogonia and subsequent defects in spermatogenesis, at least in the F1 generation.

Functional characteristics of amphioxus troponin in regulation of muscle contraction.

Troponin regulates contraction of vertebrate striated muscle in a Ca(2+)-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca(2+) during contraction. In vertebrates, this regulatory mechanism is unlike that in some less highly derived taxa. Troponin in the smooth muscle of the protochordate ascidian species Halocynthia roretzi regulates actinmyosin contraction as an activator in the presence of Ca(2+), not as an inhibitor in the absence of Ca(2+) as is the case in vertebrates. In this study, contractile regulation of striated muscle from another protochordate, the amphioxus Branchiostoma belcheri, was analyzed using recombinant troponin components TnT, TnI, and TnC that were produced in an Escherichia coli expression system to further elucidate their roles in Ca(2+)-dependent regulation of the actin-myosin interaction. Combination of these troponin components in an actin-myosin ATPase activity assay showed that troponin in amphioxus striated muscle functions in a similar manner to troponin in vertebrate striated muscle, and differently from ascidian smooth muscle troponin. Thus, troponin function appears to have evolved differently in different protochordate muscles.

A homolog of the vertebrate thyrostimulin glycoprotein hormone alpha subunit (GPA2) is expressed in Amphioxus neurons.

The cystine-knot glycoprotein hormone alpha (GPA) family regulates gonadal and thyroid functions in vertebrates. Little is known concerning GPA family members in primitive chordates. A previous genomic analysis revealed the presence of two genes homologous to the thyrostimulin alpha subunit (GPA2) in an amphioxus (Branchiostoma florideae); however only one GPA2 homolog contained both the cystine-knot structure and N-glycosylation site characteristic of family members. Gene-specific PCR was used to obtain the cDNA and genomic sequences of the GPA2 homolog of the amphioxus Branchiostoma belcheri. Whole-mount in situ hybridization revealed GPA2 mRNA expression in the anterior part of the nerve cord and on the left side of the central canal. Because amphioxus possesses only one true GPA2 homolog, while vertebrates possess two glycoprotein hormone alpha subunits (thyrostimulin alpha, or GPA2, and the common alpha subunit of gonadal and thyroid glycoprotein hormones, GPA1), our results suggest that GPA1 was acquired later in the vertebrate lineage through gene duplication.

Expression of the gene for ancestral glycoprotein hormone beta subunit in the nerve cord of amphioxus.

Amphioxus belongs to the subphylum cephalochordata, a clade of chordates phylogenetically placed at the most basal position. Despite many studies on the endocrine system of amphioxus, there were no confident lines of evidence on the presence of pituitary hormones, whereas recent amphioxus genome analysis reported that amphioxus has no pituitary hormone except for thyrostimulin, which is a glycoprotein hormone in the pituitary, brain, and other organs of vertebrates. In the present study, we cloned cDNA for one glycoprotein hormone beta subunit (GPB) from amphioxus, AmpGPB5, and phylogenetically indicated that AmpGPB5 is the ancestral molecule of glycoprotein hormone beta subunits of vertebrates including pituitary glycoprotein hormones. Synteny analyses showed conservation of chromosomal location of genes near GPB genes from amphioxus through human. The AmpGPB5 gene was expressed in a restricted region of the dorsal part of the nerve cord, glandular atrial cells of gills, and pre-vitellogenic oocytes in amphioxus. However, expression was not detected in the Hatschek's pit which is considered to be a primitive pituitary gland. On the basis of present results, we hypothesize that a portion of vertebrate pituitary hormones might be derived from an ancestral glycoprotein hormone of amphioxus that functions as a neuroendocrine hormone.

Evolution of cis-regulatory modules for the head organizer gene goosecoid in chordates: comparisons between Branchiostoma and Xenopus.

BACKGROUND: In cephalochordates (amphioxus), the notochord runs along the dorsal to the anterior tip of the body. In contrast, the vertebrate head is formed anterior to the notochord, as a result of head organizer formation in anterior mesoderm during early development. A key gene for the vertebrate head organizer, goosecoid (gsc), is broadly expressed in the dorsal mesoderm of amphioxus gastrula. Amphioxus gsc expression subsequently becomes restricted to the posterior notochord from the early neurula. This has prompted the hypothesis that a change in expression patterns of gsc led to development of the vertebrate head during chordate evolution. However, molecular mechanisms of head organizer evolution involving gsc have never been elucidated. RESULTS: To address this question, we compared cis-regulatory modules of vertebrate organizer genes between amphioxus, Branchiostoma japonicum, and frogs, Xenopus laevis and Xenopus tropicalis. Here we show conservation and diversification of gene regulatory mechanisms through cis-regulatory modules for gsc, lim1/lhx1, and chordin in Branchiostoma and Xenopus. Reporter analysis using Xenopus embryos demonstrates that activation of gsc by Nodal/FoxH1 signal through the 5' upstream region, that of lim1 by Nodal/FoxH1 signal through the first intron, and that of chordin by Lim1 through the second intron, are conserved between amphioxus and Xenopus. However, activation of gsc by Lim1 and Otx through the 5' upstream region in Xenopus are not conserved in amphioxus. Furthermore, the 5' region of amphioxus gsc recapitulated the amphioxus-like posterior mesoderm expression of the reporter gene in transgenic Xenopus embryos. CONCLUSIONS: On the basis of this study, we propose a model, in which the gsc gene acquired the cis-regulatory module bound with Lim1 and Otx at its 5' upstream region to be activated persistently in anterior mesoderm, in the vertebrate lineage. Because Gsc globally represses trunk (notochord) genes in the vertebrate head organizer, this cooption of gsc in vertebrates appears to have resulted in inhibition of trunk genes and acquisition of the head organizer and its derivative prechordal plate.

Improving the viability of tissue-resident stem cells using an organ-preservation solution.

Human clinical specimens are a valuable source of tissue-resident stem cells, but such cells need to be collected immediately after tissue collection. To extend the timescale for collection from fresh human samples, we developed a new extracellular fluid (ECF)-type preservation solution based on a high-sodium and low-potassium solution containing low-molecular-weight dextran and glucose, which is used for preservation of organs for transplantation. In this study, we compared the preservation of tissue-resident stem cells using our ECF solution with that using three other solutions: PBS, Dulbecco's modified Eagle's medium and Euro-Collins solution. These solutions represent a common buffer, a common culture medium and a benchmark organ-preservation solution, respectively. Lung tissues were removed from mice and preserved for 72 h under low-temperature conditions. Of the solutions tested, only preservation in the ECF-type solution could maintain the proliferation and differentiation capacity of mouse lung tissue-resident stem cells. In addition, the ECF solution could preserve the viability and proliferation of human alveolar epithelial progenitor cells when stored for more than 7 days at 4 °C. The mean viability of human alveolar type II cells at 2, 5, 8 and 14 days of low-temperature preservation was 90.9%, 84.8%, 85.7% and 66.3%, respectively, with no significant differences up to 8 days. Overall, our findings show that use of our ECF-type preservation solution may maintain the viability and function of tissue-resident stem cells. Use of this preservation solution may facilitate the investigation of currently unobtainable human tissue specimens for human stem cell biology.