Being a conservative marker of germ cells across metazoan species, DEAD box RNA helicase Vasa (DDX4) remains the subject of worldwide investigations thanks to its multiple functional manifestations. Vasa takes part in the preformation of primordial germ cells in a group of organisms and contributes to the maintenance of germline stem cells. Vasa is an essential player in the piRNA-mediated silencing of harmful genomic elements and in the translational regulation of selected mRNAs. Vasa is the top hierarchical protein of germ granules, liquid droplet organelles that compartmentalize RNA processing factors. Here, we survey current advances and problems in the understanding of the multifaceted functions of Vasa proteins in the gametogenesis of different eukaryotic organisms, from nematodes to humans.

The Y chromosome is one of the sex chromosomes found in males of animals of different taxa, including insects and mammals. Among all chromosomes, the Y chromosome is characterized by a unique chromatin landscape undergoing dynamic evolutionary change. Being entirely heterochromatic, the Y chromosome as a rule preserves few functional genes, but is enriched in tandem repeats and transposons. Due to difficulties in the assembly of the highly repetitive Y chromosome sequence, deep analyses of Y chromosome evolution, structure, and functions are limited to a few species, one of them being Drosophila melanogaster. Despite Y chromosomes exhibiting high structural divergence between even closely related species, Y-linked genes have evolved convergently and are mainly associated with spermatogenesis-related activities. This indicates that male-specific selection is a dominant force shaping evolution of Y chromosomes across species. This review presents our analysis of current knowledge concerning Y chromosome functions, focusing on recent findings in Drosophila. Here we dissect the experimental and bioinformatics data about the Y chromosome accumulated to date in Drosophila species, providing comparative analysis with mammals, and discussing the relevance of our analysis to a wide range of eukaryotic organisms, including humans.

Species of the genus Drosophila have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are under-investigated. Here, we performed an analysis of hybrid female sterility by crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis and molecular, cellular, and genetic approaches, we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Because of the differential expression of genes encoding piRNA pathway components, rhino and deadlock, the functional RDC^mel complex in hybrid ovaries was not assembled. However, the activity of the RDC^sim complex was maintained in hybrids independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, we found no evidence that their activity can be considered the main cause of hybrid sterility. We revealed a complicated pattern of Vasa protein expression in the hybrid germline, including partial AT-chX piRNA targeting of the vasa^sim allele and a significant zygotic delay in vasa^mel expression. We arrived at the conclusion that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species.

The tight interaction between somatic and germline cells is conserved in animal spermatogenesis. The testes of Drosophila melanogaster are the model of choice to identify processes responsible for mature gamete production. However, processes of differentiation and soma-germline interactions occurring in somatic cyst cells are currently understudied. Here we focused on the comparison of transcriptome expression patterns of early and mature somatic cyst cells to find out the developmental changes taking place in them. We employed a FACS-based approach for the isolation of early and mature somatic cyst cells from fly testes, subsequent preparation of RNA-Seq libraries, and analysis of gene differential expression in the sorted cells. We found increased expression of genes involved in cell cycle-related processes in early cyst cells, which is necessary for the proliferation and self-renewal of a crucial population of early cyst cells, cyst stem cells. Genes proposedly required for lamellipodium-like projection organization for proper cyst formation were also detected among the upregulated ones in early cyst cells. Gene Ontology and interactome analyses of upregulated genes in mature cyst cells revealed a striking over-representation of gene categories responsible for metabolic and catabolic cellular processes, as well as genes supporting the energetic state of the cells provided by oxidative phosphorylation that is carried out in mitochondria. Our comparative analyses of differentially expressed genes revealed major peculiarities in early and mature cyst cells and provide novel insight into their regulation, which is important for male fertility.

DDX3 subfamily DEAD-box RNA helicases are essential developmental regulators of RNA metabolism in eukaryotes. belle, the single DDX3 ortholog in Drosophila, is required for fly viability, fertility, and germline stem cell maintenance. Belle is involved both in translational activation and repression of target mRNAs in different tissues; however, direct targets of Belle in the testes are essentially unknown. Here we showed that belle RNAi knockdown in testis cyst cells caused a disruption of adhesion between germ and cyst cells and generation of tumor-like clusters of stem-like germ cells. Ectopic expression of β-integrin in cyst cells rescued early stages of spermatogenesis in belle knockdown testes, indicating that integrin adhesion complexes are required for the interaction between somatic and germ cells in a cyst. To address Belle functions in spermatogenesis in detail we performed cross-linking immunoprecipitation and sequencing (CLIP-seq) analysis and identified multiple mRNAs that interacted with Belle in the testes. The set of Belle targets includes transcripts of proteins that are essential for preventing the tumor-like clusters of germ cells and for sustaining spermatogenesis. By our hypothesis, failures in the translation of a number of mRNA targets additively contribute to developmental defects observed in the testes with belle knockdowns both in cyst cells and in the germline.

Species of the genus Drosophila have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are underinvestigated. Here we performed the analysis of hybrid female sterility by crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis, molecular, cellular, and genetic approaches, we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Due to the differential expression of genes encoding piRNA pathway components, rhino and deadlock, the functional RDCmel complex in hybrid ovaries was not assembled. However, the activity of the RDCsim complex was maintained in hybrids, independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, we found no evidence that their activity can be considered the main cause of hybrid sterility. We revealed a complicated pattern of Vasa protein expression in the hybrid germline, including partial AT-chX piRNA targeting of the vasasim allele and a significant zygotic delay in vasamel expression. We arrived at the conclusion that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species.