Identification and Characterization of RBM44 as a Novel Intercellular Bridge Protein

Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14

Selective terminal α2–3 and α2–6 sialylation of glycosphingolipids with lacto-series type 1 and 2 chains in human meconium

AbstractHuman meconium was found to contain two kinds of gangliosides with the same carbohydrate sequences belonging to the lacto-series. They were detected by TLC-immunostaining with monoclonal antibodies directed to the NeuAcα2–Gal and Lc4Cer structures. One of these two gangliosides, a major one, which migrated on TLC to a position below that of standard IV3NeuAcnLc4Cer from human erythrocytes, reacted with the antibody to NeuAcα2–6Gal. The other minor one, which migrated on TLC to a position corresponding to standard IV3NeuAcnLc4Cer, was detected with the antibody to Lc4Cer only when the plate, on which the individual gangliosides were separated, was subjected to prior treatment with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation analysis and enzyme treatment after isolation with antibody monitoring, were shown to be IV6NeuAcnLc4Cer for the former and IV3NeuAcLc4Cer for the latter, indicating that the lacto-series type 2 (nLc4Cer) and 1 (Lc4Cer) chains are sialylated at different linkages, α2–6 and α2–3, respectively. IV6NeuAcLc4Cer and IV3NeuAcnLc4Cer were not detected, even in trace amounts, on TLC-immunostaining with the monoclonal antibodies. The concentrations of IV6NeuAcnLc4Cer and IV3NeuAcLc4Cer were 448 and 18 nmol/g dry wt of human meconium

Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells

Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/− spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term

Cholesterol and oxysterol sulfates:Pathophysiological roles and analytical challenges

Cholesterol and oxysterol sulfates are important regulators of lipid metabolism, inflammation, cell apoptosis, and cell survival. Among the sulfate-based lipids, cholesterol sulfate (CS) is the most studied lipid both quantitatively and functionally. Despite the importance, very few studies have analysed and linked the actions of oxysterol sulfates to their physiological and pathophysiological roles. Overexpression of sulfotransferases confirmed the formation of a range of oxysterol sulfates and their antagonistic effects on liver X receptors (LXRs) prompting further investigations how are the changes to oxysterol/oxysterol sulfate homeostasis can contribute to LXR activity in the physiological milieu. Here, we aim to bring together for novel roles of oxysterol sulfates, the available techniques and the challenges associated with their analysis. Understanding the oxysterol/oxysterol sulfate levels and their pathophysiological mechanisms could lead to new therapeutic targets for metabolic diseases

Cyanobacterial Cell Lineage Analysis of the Spatiotemporal hetR Expression Profile during Heterocyst Pattern Formation in Anabaena sp. PCC 7120

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation

Widespread Gene Conversion of Alpha-2-Fucosyltransferase Genes in Mammals

The alpha-2-fucosyltransferases (α2FTs) are enzymes involved in the biosynthesis of α2fucosylated glycan structures. In mammalian genomes, there are three α2FT genes located in tandem—FUT1, FUT2, and Sec1—each contained within a single exon. It has been suggested that these genes originated from two successive duplications, with FUT1 being generated first and FUT2 and Sec1 second. Despite gene conversion being considered the main mechanism of concerted evolution in gene families, previous studies of primates α2FTs failed to detect it, although the occurrence of gene conversion between FUT2 and Sec1 was recently reported in a human allele. The primary aim of our work was to initiate a broader study on the molecular evolution of mammalian α2FTs. Sequence comparison leads us to confirm that the three genes appeared by two rounds of duplication. In addition, we were able to detect multiple gene-conversion events at the base of primates and within several nonprimate species involving FUT2 and Sec1. Gene conversion involving FUT1 and either FUT2 or Sec1 was also detected in rabbit. The extent of gene conversion between the α2FTs genes appears to be species-specific, possibly related to functional differentiation of these genes. With the exception of rabbits, gene conversion was not observed in the region coding the C-terminal part of the catalytic domain. In this region, the number of amino acids that are identical between FUT1 and FUT2, but different in Sec1, is higher than in other parts of the protein. The biologic meaning of this observation may be related to functional constraints

HP1a Targets the Drosophila KDM4A Demethylase to a Subset of Heterochromatic Genes to Regulate H3K36me3 Levels

The KDM4 subfamily of JmjC domain-containing demethylases mediates demethylation of histone H3K36me3/me2 and H3K9me3/me2. Several studies have shown that human and yeast KDM4 proteins bind to specific gene promoters and regulate gene expression. However, the genome-wide distribution of KDM4 proteins and the mechanism of genomic-targeting remain elusive. We have previously identified Drosophila KDM4A (dKDM4A) as a histone H3K36me3 demethylase that directly interacts with HP1a. Here, we performed H3K36me3 ChIP-chip analysis in wild type and dkdm4a mutant embryos to identify genes regulated by dKDM4A demethylase activity in vivo. A subset of heterochromatic genes that show increased H3K36me3 levels in dkdm4a mutant embryos overlap with HP1a target genes. More importantly, binding to HP1a is required for dKDM4A-mediated H3K36me3 demethylation at a subset of heterochromatic genes. Collectively, these results show that HP1a functions to target the H3K36 demethylase dKDM4A to heterochromatic genes in Drosophila

Identification of Inappropriately Reprogrammed Genes by Large-Scale Transcriptome Analysis of Individual Cloned Mouse Blastocysts

Although cloned embryos generated by somatic/embryonic stem cell nuclear transfer (SECNT) certainly give rise to viable individuals, they can often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. In an effort to gain further insights into reprogramming and the properties of SECNT embryos, we performed a large-scale gene expression profiling of 87 single blastocysts using GeneChip microarrays. Sertoli cells, cumulus cells, and embryonic stem cells were used as donor cells. The gene expression profiles of 87 blastocysts were subjected to microarray analysis. Using principal component analysis and hierarchical clustering, the gene expression profiles were clearly classified into 3 clusters corresponding to the type of donor cell. The results revealed that each type of SECNT embryo had a unique gene expression profile that was strictly dependent upon the type of donor cells, although there was considerable variation among the individual profiles within each group. This suggests that the reprogramming process is distinct for embryos cloned from different types of donor cells. Furthermore, on the basis of the results of comparison analysis, we identified 35 genes that were inappropriately reprogrammed in most of the SECNT embryos; our findings demonstrated that some of these genes, such as Asz1, Xlr3a and App, were appropriately reprogrammed only in the embryos with a transcriptional profile that was the closest to that of the controls. Our findings provide a framework to further understand the reprogramming in SECNT embryos