Project description:In order to study the effect of protease treatment on dissected testes, we performed different strategies of cleaning and dissociation on Drosophila melanogaster w1118 larval testes. We collected testes without protease treatment with fatbody just attached around the gonad, fatbody alone dissected from around the testes, testes without fatbody and testes dissociated by either by Papain or Trypsin and Collagenase cocktail. We prepared poly A+ RNA-Seq libraries and performed transcriptional profiling to generate 50 bp stranded single end reads.
Project description:Testes and testes terminal epithelium/seminal vesicles were dissected and separated from 4-5 day old Oregon-R-modENCODE Drosophila melanogaster males. RNA-sequencing was then performed on these two distinct biological structures.
Project description:The expression of a very large number of genes changes as male germ cells pass through differentiation into spermatids and then sperm. Much of this transcriptional programme requires the activity of the meiotic arrest genes. We extracted RNA from wild type testes, as well as aly mutants and Nxt1 mutants, and used microarrays to compare gene expression profiles. We dissected testes from 0-1 day old Drosophila males, taking care to remove accessory glands and ejaculatory ducts. The control sample expressed a tin-RNAi construct, and had normal testes. The aly mutant were homozygous for aly[5]. The Nxt1 mutants were Nxt1[z2-0488]/Nxt1[DG05102].
Project description:Expression of dCoREST or dLSD1 was reduced by RNAi in Drosophila testes and changes to gene transcription were determined by RNA-seq.
Project description:To investigate the role of CPES in germ cell differentiation during spermatogenesis in Drosophila testis. We have generated cpes null mutants using ends-out homologus recombination and rescued with Bam-Gal4 and UAS-CPES in cpes mutant background. We then dissected 200 pairs of testes for each of the 3 replicates from wild type, cpes mutant and Rescue (Bam-Gal4 and UAS-CPES) Drosophila males and total RNA was extracted using Trizol and RNA-Clean and concentrater column. About 1ug of RNA in 20ul was submitted for bulk RNAseq using Illumina machine (HWI-ST1276).
Project description:Whole genome expression analyses reveal little evidence for X chromosome dosage compensation or meiotic inactivation in Drosophila testes, whereas testes-specific transgene reporters suggest a novel form of X chromosome-specific regulation. Gene expression was measured in RNA extracted from adult male and female dissected thorax, and in cells dissected from the apical tip of 2-4 day old adult male testes. Each tissue was assayed on four replicate arrays.
Project description:Spermiogenesis in Drosophila melanogaster is a highly conserved process and essential for male fertility. In this haploid phase of spermatogenesis, motile sperm are assembled from round cells, flagella are assembled, and needle-shaped nuclei with highly compacted genomes are formed. We aimed at identifying proteins relevant for the maturation phase from spermatids to sperm. As transcription takes place mainly in spermatocytes, and transcripts with relevance for post-meiotic sperm development are translationally repressed for days, we comparatively analysed the prote-ome of larval testes (stages before meiotic divisions), of testes of 1–2-day-old pupae (meiotic and early spermatid stages) and adult flies (late spermatids and sperm). We identified 6677 pro-teins, with 422 solely detected in larval testes, 623 in pupal testes and 634 in adult testes. We analysed a few so far uncharacterized proteins with repect to stage specific expression and im-portance for male fertility. For example, Mst84B (gene CG1988), a very basic cysteine- and lysine-rich nuclear protein, was present in the phase of transition from a histone-based to a pro-tamine-based chromatin structure. CG6332 encodes d-Theg, which is related to the mouse tHEG and human THEG proteins. Mutants of d-Theg lacked sperm in the seminal vesicles and were sterile. The identification of numerous predicted proteins underscores the high potential of pro-teome analysis for future analyses of spermatogenesis.
Project description:DEAD-box RNA helicases DDX3 are important developmental regulators of multiple aspects of RNA metabolism of eukaryotes. belle, a single DDX3 ortholog in Drosophila, is essential for fly viability, fertility, and germline stem cell maintenance. Here we showed that RNAi belle knockdown in testis cyst cells caused a disruption of adhesion between germ cells and cyst cells and a generation of tumor-like clusters of stem-like germ cells. Ectopic expression of β-integrin in cyst cells rescued early stages of spermatogenesis in the belle knockdown testes, indicating that integrin adhesion complexes are required for interaction between somatic and germ cells in cyst. To address in details Belle functions in spermatogenesis we performed CLIP-seq analysis and identified multiple mRNAs which interacted with Belle in the testes. A set of Belle targets includes mRNAs of factors that are essential for preventing tumor-like cluster formation of early germ cells and ensuring of sustained gametogenesis.