Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of &lt;em&gt;Drosophila&lt;/em&gt; Testes

Sitaram, Poojitha; Hainline, Sarah G.; Lee, Laura Anne

doi:10.3791/51058

Cited by 27 publications

(27 citation statements)

References 42 publications

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“…Spermatocyte squashes were performed as previously described 64 , 65 . In brief, tubules from 10 days or 8–10 week old mice (unless stated otherwise) were placed directly in fixative solution (2% formaldehyde, 0.05% Triton-X 100 in PBS) for 10 minutes.…”

Section: Methodsmentioning

confidence: 99%

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination

et al. 2015

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CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63 deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63 deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination.

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Section: Methodsmentioning

confidence: 99%

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination

et al. 2015

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“…Testes from 1-d-old male flies were dissected in PBS and prepared for microscopy as described ( Sitaram et al. , 2014 ).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis

Williams

Krug

McMillan

et al. 2016

MBoC

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“…For immunostaining of actin-cone proteins, ring canals, and acetylated αtubulin, testes were squashed on slides and frozen in liquid N 2 before fixation and staining, as described by Sitaram et al (2014).…”

Section: Fluorescence Staining and Imagingmentioning

confidence: 99%

Combover interacts with the axonemal component Rsp3 and is required for sperm individualization

et al. 2019

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Gamete formation is key to survival of higher organisms. In male animals, spermatogenesis gives rise to interconnected spermatids that differentiate and individualize into mature sperm, each tightly enclosed by a plasma membrane. In Drosophila melanogaster, individualization of sister spermatids requires the formation of specialized actin cones that synchronously move along the sperm tails, removing inter-spermatid bridges and most of the cytoplasm. Here, we show that Combover (Cmb), originally identified as an effector of planar cell polarity (PCP) under control of Rho kinase, is essential for sperm individualization. cmb mutants are male sterile, with actin cones that fail to move in a synchronized manner along the flagella, despite being correctly formed and polarized initially. These defects are germline autonomous, independent of PCP genes, and can be rescued by wild-type Cmb, but not by a version of Cmb in which known Rho kinase phosphorylation sites are mutated. Furthermore, Cmb binds to the axonemal component Radial spoke protein 3, knockdown of which causes similar individualization defects, suggesting that Cmb coordinates the individualization machinery with the microtubular axonemes.

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

Cited by 27 publications

References 42 publications

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination

Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis

Combover interacts with the axonemal component Rsp3 and is required for sperm individualization

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