Primordial Germ Cell Specification in Vertebrate Embryos: Phylogenetic Distribution and Conserved Molecular Features of Preformation and Induction

The differentiation of primordial germ cells (PGCs) occurs during early embryonic development and is critical for the survival and fitness of sexually reproducing species. Here, we review the two main mechanisms of PGC specification, induction, and preformation, in the context of four model vertebrate species: mouse, axolotl, Xenopus frogs, and zebrafish. We additionally discuss some notable molecular characteristics shared across PGC specification pathways, including the shared expression of products from three conserved germline gene families, DAZ (Deleted in Azoospermia) genes, nanos-related genes, and DEAD-box RNA helicases. Then, we summarize the current state of knowledge of the distribution of germ cell determination systems across kingdom Animalia, with particular attention to vertebrate species, but include several categories of invertebrates – ranging from the “proto-vertebrate” cephalochordates to arthropods, cnidarians, and ctenophores. We also briefly highlight ongoing investigations and potential lines of inquiry that aim to understand the evolutionary relationships between these modes of specification.

Identification and expression analysis of Dazl homologue in Cynops cyanurus

Summary The Dazl (deleted in azoospermia-like) gene encodes an RNA-binding protein containing an RNA recognition motif (RRM) and a DAZ motif. Dazl is essential for gametogenesis in vertebrates. In this study, we report the cloning of Dazl cDNA from Cynops cyanurus. Ccdazl mRNA showed a germline-specific expression pattern as expected. Ccdazl expression gradually decreased during oogenesis, suggesting that it may be involved in oocyte development. Phylogenetic analysis revealed that the Ccdazl protein shares conserved motifs/domains with Dazl proteins from other species. Cloning of Ccdazl provides a new tool to carry out comparative studies of germ cell development in amphibians.

DAZL regulates mRNA deadenylation independently of translation in germ cells

ABSTRACTAberrant gene expression during gametogenesis is one of the factors underlying infertility, which affects roughly 15% of couples worldwide. Deleted-in-Azoospermia-Like (DAZL), a member of the DAZ-gene family, encodes an mRNA-specific regulator of translation which is essential for gametogenesis in both sexes. In this study we show that DAZL controls gene expression in oocytes by regulating the length of the mRNA poly(A) tail, a major determinant of temporal and amplitudinal gene regulation in germ cells, in which gene expression is regulated entirely post-transcriptionally. We show that DAZL does not induce polyadenylation but that binding of DAZL efficiently inhibits mRNA deadenylation induced by oocyte maturation. We reveal that this activity depends on DAZL-mediated recruitment of poly(A)-binding protein, PABP, to the mRNA. Although DAZL also activates mRNA translation via PABP recruitment, mechanistic analysis revealed that neither translation nor translational activation are required for DAZL to stabilise the poly(A) tail, suggesting two mutually independent posttranscriptional roles for the DAZL-PABP complex. We show that recruited PABP must maintain its ability to bind RNA, leading to a model in which DAZL recruits PABP and/or stabilises PABP binding to poly(A) thereby preventing access of deadenylases. These results indicate that the role of DAZL in regulating germ-cell mRNA fate is more complex than previously thought and inform on the poorly understood links between mRNA translation and deadenylation, showing that they can be mechanistically separable.

Hypomethylation of the DAZ3 promoter in idiopathic asthenospermia: a screening tool for liquid biopsy

Given the role of the deleted in azoospermia gene in male infertility, whether the somatic deleted in azoospermia methylation status is associated with idiopathic asthenospermia should be determined. To investigate the methylation levels of the deleted in azoospermia promoter in peripheral white blood cells from idiopathic asthenospermia patients relative to those in normozoospermia controls, 61 ethylene diamine tetraacetic acid anticoagulant blood samples were drawn from all participants for DNA isolation. The deleted in azoospermia promoter methylation ratio was detected by MassARRAY-based methylation quantification and confirmed by quantitative methylation-specific polymerase chain reaction. A MassARRAY-based methylation analysis showed that the deleted in azoospermia 3 promoter (0 to − 2 kbp) was significantly hypomethylated in peripheral white blood cells from idiopathic asthenospermia males, specifically one CpG site (− 246 to − 247). Quantitative methylation-specific polymerase chain reaction data further confirmed that the methylation level of the deleted in azoospermia 3 promoter region in idiopathic asthenospermia patients was significantly lower than that in normozoospermia males. The area under the receiver operating characteristic curve determined by quantitative methylation-specific polymerase chain reaction was 0.737 (95% confidence interval: 0.552 to 0.924), with a sensitivity of 53.9% and a specificity of 88.2% at a cut-off level of 74.7%. Therefore, our results suggested that methylation ratio detection of the deleted in azoospermia 3 promoter region by real-time polymerase chain reaction assay is a promising and feasible tool for liquid biopsy in the clinical laboratories. The methylation status of other reported infertility-related genes should also be investigated in peripheral white blood cells.

Deletion of porcine BOLL causes defective acrosomes and subfertility in Yorkshire boars

SummaryA recessively inherited sperm defect of Finnish Yorkshire boars was detected more than a decade ago. Affected boars produce ejaculates that contain many spermatozoa with defective acrosomes resulting in low fertility and small litters. The acrosome defect was mapped to porcine chromosome 15 but the causal mutation has not been identified. We re-analyzed microarray-derived genotypes of affected boars and performed a haplotype-based association study. Our results confirmed that the acrosome defect maps to a 12.24 Mb segment of porcine chromosome 15 (P=3.38 × 10−14). In order to detect the mutation causing defective acrosomes, we sequenced the genomes of two affected and three unaffected boars to an average coverage of 11-fold. Read-depth analysis revealed a 55 kb deletion that segregates with the acrosome defect. The deletion encompasses the BOLL gene encoding the boule homolog, RNA binding protein which is an evolutionarily highly conserved member of the DAZ (deleted in azoospermia) gene family. Lack of BOLL expression causes spermatogenic arrest and sperm maturation failure in many species. Our study reveals that absence of BOLL is associated with a sperm defect also in pigs. The acrosomes of boars that carry the deletion in the homozygous state are defective suggesting that lack of porcine BOLL compromises acrosome formation. Our findings warrant further research to investigate the precise function of BOLL during spermatogenesis and sperm maturation in pigs.

Expression of DAZL Gene in Selected Tissues and Association of Its Polymorphisms with Testicular Size in Hu Sheep

The deleted in azoospermia-like (DAZL) gene encoding an RNA binding protein is pivotal in gametogenesis in lots of species and also acts as a pre-meiosis marker. The current study was conducted to detect expression profiles and single nucleotide polymorphisms (SNPs) of DAZL in sheep using qPCR, DNA-pooled sequencing, improved multiplex ligase detection reaction (iMLDR®) and restriction fragment length polymorphism (RFLP) methods. The results confirmed that ovine DAZL showed the highest expression level at six-months of age across five developmental stage. At six-month stage, DAZL expressed primarily in testis across seven tissues analyzed. The abundance of DAZL in the large-testis group is higher than that in the small-testis group although it is not significant. In addition, six SNPs (SNP1-SNP6) were identified in DAZL. Of those, SNP1 (p < 0.05) and SNP6 (p < 0.01) were significantly correlated with the variation coefficient between left and right epididymis weight (VCTW). The current study implies DAZL may play important roles in testicular development and its SNPs are associated with testicular parameters, which supply important indicators for ram selection at early stage.

Homozygous mutations in DZIP1 can induce asthenoteratospermia with severe MMAF

BackgroundAsthenoteratospermia, one of the most common causes for male infertility, often presents with defective sperm heads and/or flagella. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the common clinical manifestations of asthenoteratospermia. Variants in several genes including DNAH1, CEP135, CATSPER2 and SUN5 are involved in the genetic pathogenesis of asthenoteratospermia. However, more than half of the asthenoteratospermia cases cannot be explained by the known pathogenic genes.Methods and resultsTwo asthenoteratospermia-affected men with severe MMAF (absent flagella in >90% spermatozoa) from consanguineous families were subjected to whole-exome sequencing. The first proband had a homozygous missense mutation c.188G>A (p.Arg63Gln) of DZIP1 and the second proband had a homozygous stop-gain mutation c.690T>G (p.Tyr230*). Both of the mutations were neither detected in the human population genome data (1000 Genomes Project, Exome Aggregation Consortium) nor in our own data of a cohort of 875 Han Chinese control populations. DZIP1 encodes a DAZ (a protein deleted in azoospermia) interacting protein, which was associated with centrosomes in mammalian cells. Immunofluorescence staining of the centriolar protein Centrin1 indicated that the spermatozoa of the proband presented with abnormal centrosomes, including no concentrated centriolar dot or more than two centriolar dots. HEK293T cells transfected with two DZIP1-mutated constructs showed reduced DZIP1 level or truncated DZIP1. The Dzip1-knockout mice, generated by the CRSIPR-Cas9, revealed consistent phenotypes of severe MMAF.ConclusionOur study strongly suggests that homozygous DZIP1 mutations can induce asthenoteratospermia with severe MMAF. The deficiency of DZIP1 induces sperm centrioles dysfunction and causes the absence of flagella.