The stem cell niche: lessons from theDrosophilatestis

Cuevas, Margaret de; Matunis, Erika

doi:10.1242/dev.056242

Cited by 211 publications

(243 citation statements)

References 87 publications

(122 reference statements)

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“…Drosophila testes are an ideal model system for the study of many biological processes including the regulation of stem cells, meiosis, and sperm development [13][14][15][16][17][18] . The spermatocytes and their meiotic spindles are large and hence convenient for cytological analysis, and relaxed cell cycle checkpoints during spermatogenesis facilitate the study of mutations in cell cycle genes.…”

Section: Introductionmentioning

confidence: 99%

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Sitaram

Hainline

Lee

2014

JoVE

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Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries and testes [3][4][5][6][7][8][9][10][11][12] . Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins. Video LinkThe video component of this article can be found at

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Section: Introductionmentioning

confidence: 99%

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Sitaram

Hainline

Lee

2014

JoVE

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“…It is characterized by a series of crucial events that culminates in the formation of morphologically and epigenetically highly specialized spermatozoa. In Drosophila melanogaster, germ-line stem cells at the tip of the testis divide asymmetrically to give rise to a new stem cell and the spermatogonium, i.e., the daughter cell that is committed to differentiation (see Figure 1 for an overview) 1,2 . The spermatogonium undergoes four rounds of mitotic divisions and, with the onset of meiosis, a phase of impressive growth and transcriptional activity called the spermatocyte stage.…”

Section: Introductionmentioning

confidence: 99%

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Gärtner

Rathke

Renkawitz‐Pohl

et al. 2014

JoVE

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During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin-the histone-toprotamine switch.Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre-and post-meiotic spermatogenesis.

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“…Spermatogenesis is one of the most conserved biological processes from Drosophila to humans (7,8). For example, mutations in boule cause sterility in Drosophila, and mutants of the human homologous gene, DAZ, are associated with azoospermia.…”

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confidence: 99%

Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans

Sun

Wen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Processing of pre-mRNA into mRNA is an important regulatory mechanism in eukaryotes that is mediated by the spliceosome, a huge and dynamic ribonucleoprotein complex. Splicing defects are implicated in a spectrum of human disease, but the underlying mechanistic links remain largely unresolved. Using a genome-wide association approach, we have recently identified single nucleotide polymorphisms in humans that associate with nonobstructive azoospermia (NOA), a common cause of male infertility. Here, using genetic manipulation of corresponding candidate loci in Drosophila, we show that the spliceosome component SNRPA1/U2A is essential for male fertility. Loss of U2A in germ cells of the Drosophila testis does not affect germline stem cells, but does result in the accumulation of mitotic spermatogonia that fail to differentiate into spermatocytes and mature sperm. Lack of U2A causes insufficient splicing of mRNAs required for the transition of germ cells from proliferation to differentiation. We show that germ cellspecific disruption of other components of the major spliceosome manifests with the same phenotype, demonstrating that mRNA processing is required for the differentiation of spermatogonia. This requirement is conserved, and expression of human SNRPA1 fully restores spermatogenesis in U2A mutant flies. We further report that several missense mutations in human SNRPA1 that inhibit the assembly of the major spliceosome dominantly disrupt spermatogonial differentiation in Drosophila. Collectively, our findings uncover a conserved and specific requirement for the major spliceosome during the transition from spermatogonial proliferation to differentiation in the male testis, suggesting that spliceosome defects affecting the differentiation of human spermatogonia contribute to NOA.

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The stem cell niche: lessons from theDrosophilatestis

Cited by 211 publications

References 87 publications

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of <em>Drosophila</em> Testes

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of <em>Drosophila</em> Testes

<em>Ex vivo</em> Culture of <em>Drosophila</em> Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans

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