ESX1 gene as a potential candidate responsible for male infertility in nonobstructive azoospermia.

Infertility is a problem that affects approximately 15% of couples, and male infertility is responsible for 40-50% of these cases. The cause of male infertility is still poorly diagnosed and treated. One of the prominent causes of male infertility is disturbed spermatogenesis, which can lead to nonobstructive azoospermia (NOA). Whole-genome sequencing (WGS) allows us to identify novel rare variants in potentially NOA-associated genes, among others, in the ESX1 gene. The aim of this study was to activate the ESX1 gene using CRISPRa technology in human germ cells (testicular seminoma cells-TCam-2). Successful activation of the ESX1 gene in TCam-2 cells using the CRISPRa system was achieved, and the expression level of the ESX1 gene was significantly higher in modified TCam-2 cells than in WT cells or the negative control with nontargeted gRNA (p < 0.01). Using RNA-seq, a network of over 50 genes potentially regulated by the ESX1 gene was determined. Finally, 6 genes, NANOG, CXCR4, RPS6KA5, CCND1, PDE1C, and LINC00662, participating in cell proliferation and differentiation were verified in azoospermic patients with and without a mutation in the ESX1 gene as well as in men with normal spermatogenesis, where inverse correlations in the expression levels of the observed genes were noted.

NME3 binds to phosphatidic acid and mediates PLD6-induced mitochondrial tethering.

Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.

TAGAP instructs Th17 differentiation by bridging Dectin activation to EPHB2 signaling in innate antifungal response.

The TAGAP gene locus has been linked to several infectious diseases or autoimmune diseases, including candidemia and multiple sclerosis. While previous studies have described a role of TAGAP in T cells, much less is known about its function in other cell types. Here we report that TAGAP is required for Dectin-induced anti-fungal signaling and proinflammatory cytokine production in myeloid cells. Following stimulation with Dectin ligands, TAGAP is phosphorylated by EPHB2 at tyrosine 310, which bridges proximal Dectin-induced EPHB2 activity to downstream CARD9-mediated signaling pathways. During Candida albicans infection, mice lacking TAGAP mount defective immune responses, impaired Th17 cell differentiation, and higher fungal burden. Similarly, in experimental autoimmune encephalomyelitis model of multiple sclerosis, TAGAP deficient mice develop significantly attenuated disease. In summary, we report that TAGAP plays an important role in linking Dectin-induced signaling to the promotion of effective T helper cell immune responses, during both anti-fungal host defense and autoimmunity.

Two isoforms of the RAC-specific guanine nucleotide exchange factor TIAM2 act oppositely on transmission ratio distortion by the mouse t-haplotype.

Transmission ratio distortion (TRD) by the mouse t-haplotype, a variant region on chromosome 17, is a well-studied model of non-Mendelian inheritance. It is characterized by the high transmission ratio (up to 99%) of the t-haplotype from t/+ males to their offspring. TRD is achieved by the exquisite ability of the responder (Tcr) to trigger non-Mendelian inheritance of homologous chromosomes. Several distorters (Tcd1-Tcd4), which act cumulatively, together promote the high transmission ratio of Tcr and the t-haplotype. Molecularly, TRD is brought about by deregulation of Rho signaling pathways via the distorter products, which impair sperm motility, and the t-sperm specific rescue of sperm motility by the responder. The t-sperm thus can reach the egg cells faster than +-sperm and fertilize them. Previously we have shown that the responder function is accomplished by a dominant negative form of sperm motility kinase (SMOKTCR), while the distorter functions are accomplished by the Rho G protein regulators TAGAP, FGD2 and NME3 proposed to function in two oppositely acting pathways. Here we identify the RAC1-specific guanine nucleotide exchange factor TIAM2 as modifier of t-haplotype TRD. Tiam2 is expressed in two isoforms, the full-length (Tiam2l) and a short transcript (Tiam2s). Tiam2s expression from the t-allele is strongly increased compared to the wild-type allele. By transgenic approaches we show that Tiam2s enhances t-haplotype transmission, while Tiam2l has the opposite effect. Our data show that a single modifier locus can encode different gene products exerting opposite effects on a trait. They also suggest that the expression ratio of the isoforms determines if the outcome is an enhancing or a suppressive effect on the trait.

Patterning and gastrulation defects caused by the tw18 lethal are due to loss of Ppp2r1a.

The mouse t haplotype, a variant 20 cM genomic region on Chromosome 17, harbors 16 embryonic control genes identified by recessive lethal mutations isolated from wild mouse populations. Due to technical constraints so far only one of these, the tw5 lethal, has been cloned and molecularly characterized. Here we report the molecular isolation of the tw18 lethal. Embryos carrying the tw18 lethal die from major gastrulation defects commencing with primitive streak formation at E6.5. We have used transcriptome and marker gene analyses to describe the molecular etiology of the tw18 phenotype. We show that both WNT and Nodal signal transduction are impaired in the mutant epiblast, causing embryonic patterning defects and failure of primitive streak and mesoderm formation. By using a candidate gene approach, gene knockout by homologous recombination and genetic rescue, we have identified the gene causing the tw18 phenotype as Ppp2r1a, encoding the PP2A scaffolding subunit PR65alpha. Our work highlights the importance of phosphatase 2A in embryonic patterning, primitive streak formation, gastrulation, and mesoderm formation downstream of WNT and Nodal signaling.

An image-based genetic assay identifies genes in T1D susceptibility loci controlling cellular antiviral immunity in mouse.

The pathogenesis of complex diseases, such as type 1 diabetes (T1D), derives from interactions between host genetics and environmental factors. Previous studies have suggested that viral infection plays a significant role in initiation of T1D in genetically predisposed individuals. T1D susceptibility loci may therefore be enriched in previously uncharacterized genes functioning in antiviral defense pathways. To identify genes involved in antiviral immunity, we performed an image-based high-throughput genetic screen using short hairpin RNAs (shRNAs) against 161 genes within T1D susceptibility loci. RAW 264.7 cells transduced with shRNAs were infected with GFP-expressing herpes simplex virus type 1 (HSV-1) and fluorescent microscopy was performed to assess the viral infectivity by fluorescence reporter activity. Of the 14 candidates identified with high confidence, two candidates were selected for further investigation, Il27 and Tagap. Administration of recombinant IL-27 during viral infection was found to act synergistically with interferon gamma (IFN-γ) to activate expression of type I IFNs and proinflammatory cytokines, and to enhance the activities of interferon regulatory factor 3 (IRF3). Consistent with a role in antiviral immunity, Tagap-deficient macrophages demonstrated increased viral replication, reduced expression of proinflammatory chemokines and cytokines, and decreased production of IFN-ß. Taken together, our unbiased loss-of-function genetic screen identifies genes that play a role in host antiviral immunity and delineates roles for IL-27 and Tagap in the production of antiviral cytokines.

The nucleoside diphosphate kinase gene Nme3 acts as quantitative trait locus promoting non-Mendelian inheritance.

The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95%) from heterozygous (t/+) males to their offspring. This phenotype is termed transmission ratio distortion (TRD) and is caused by the interaction of the t-complex responder (Tcr) with several quantitative trait loci (QTL), the t-complex distorters (Tcd1 to Tcd4), all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+)-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S) that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr), a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and spermatozoa.

Retention of gene products in syncytial spermatids promotes non-Mendelian inheritance as revealed by the t complex responder.

The t complex responder (Tcr) encoded by the mouse t haplotype is able to cause phenotypic differences between t and + sperm derived from t/+ males, leading to non-Mendelian inheritance. This capability of Tcr contradicts the concept of phenotypic equivalence proposed for sperm cells, which develop in a syncytium and actively share gene products. By analyzing a Tcr minigene in hemizygous transgenic mice, we show that Tcr gene products are post-meiotically expressed and are retained in the haploid sperm cells. The wild-type allele of Tcr, sperm motility kinase-1 (Smok1), behaves in the same manner, suggesting that Tcr/Smok reveal a common mechanism prone to evolve non-Mendelian inheritance in mammals.

The t-complex-encoded guanine nucleotide exchange factor Fgd2 reveals that two opposing signaling pathways promote transmission ratio distortion in the mouse.

Transmission ratio distortion (TRD), the preferential inheritance of the t haplotype from t/+ males, is caused by the cooperative effect of four t-complex distorters (Tcd1-4) and the single t-complex responder (Tcr) on sperm motility. Here we show that Fgd2, encoding a Rho guanine nucleotide exchange factor, maps to the Tcd2 region. The t allele of Fgd2 is overexpressed in testis compared with wild type. A loss-of-function allele of Fgd2 generated by gene targeting reduces the transmission ratio of the t haplotype t(h49), directly demonstrating the role of Fgd2 as Distorter. Fgd2 identifies a second Rho G protein signaling pathway promoting TRD.

Identification of keratins and analysis of their expression in carp and goldfish: comparison with the zebrafish and trout keratin catalog.

With more than 50 genes in human, keratins make up a large gene family, but the evolutionary pressure leading to their diversity remains largely unclear. Nevertheless, this diversity offers a means to examine the evolutionary relationships among organisms that express keratins. Here, we report the analysis of keratins expressed in two cyprinid fishes, goldfish and carp, by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot binding assay, and immunoblotting. We further explore the expression of keratins by immunofluorescence microscopy. Comparison is made with the keratin expression and catalogs of zebrafish and rainbow trout. The keratins among these fishes exhibit a similar range of molecular weights and isoelectric points, with a similar overall pattern on two-dimensional gels. In addition, immunofluorescence microscopy studies of goldfish and carp tissues have revealed the expression of keratins in both epithelial and mesenchymally derived tissues, as reported previously for zebrafish and trout. We conclude that keratin expression is qualitatively similar among these fishes, with goldfish and carp patterns being more similar to each other than to zebrafish, and the cyprinid fishes being more similar to each other than to the salmonid trout. Because of the detected similarity of keratin expression among the cyprinid fishes, we propose that, for certain experiments, they are interchangeable. Although the zebrafish distinguishes itself as being a developmental and genetic/genomic model organism, we have found that the goldfish, in particular, is a more suitable model for both biochemical and histological studies of the cytoskeleton, especially since goldfish cytoskeletal preparations seem to be more resistant to degradation than those from carp or zebrafish.

The t complex-encoded GTPase-activating protein Tagap1 acts as a transmission ratio distorter in mice.

Transmission ratio distortion in the mouse is caused by several t-complex distorters (Tcds) acting in trans on the t-complex responder (Tcr). Tcds additively affect the flagellar movement of all spermatozoa derived from t/+ males; sperm carrying Tcr are rescued, resulting in an advantage for t sperm in fertilization. Here we show that Tagap1, a GTPase-activating protein, can act as a distorter. Tagap1 maps to the Tcd1 interval and has four t loci, which encode altered proteins including a C-terminally truncated form. Overexpression of wild-type Tagap1 in sperm cells phenocopied Tcd function, whereas a loss-of-function Tagap1 allele reduced the transmission rate of the t6 haplotype. The combined data strongly suggest that the t loci of Tagap1 produce Tcd1a. Our results unravel the molecular nature of a Tcd and demonstrate the importance of small G proteins in transmission ratio distortion in the mouse.

Maternally supplied Smad5 is required for ventral specification in zebrafish embryos prior to zygotic Bmp signaling.

We have previously shown that the maternal effect dorsalization of zebrafish embryos from sbn(dtc24) heterozygous mothers is caused by a dominant negative mutation in Smad5, a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbn(dtc24) mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it remained open to what extent Smad5 itself is required for dorsoventral patterning. Here, we report the identification of novelsmad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. We rescued m169 mutant embryos by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169(-/-) females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169(-/-) embryos) are even more strongly dorsalized thanbmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169(-/-) embryos display reducedbmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry.