Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Dia, Ferdusy; Strange, Tierra; Liang, Jing; Hamilton, Jacob T.; Berkowitz, Karen M.

doi:10.3791/55378

Cited by 23 publications

(16 citation statements)

References 15 publications

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“…To obtain meiotic chromosome spreads, de-capsuled mouse testes were incubated in hypotonic extraction buffer (30-60 min, on ice) and seminiferous tubules were chopped to release germ cells [62]. A drop of sucrose solution containing germ cells was placed on a glass slide coated with 1× PBS containing 1% PFA and 0.15% (v/v) Triton-X100 (pH 9.2) and spread by swirling.…”

Section: Meiotic Chromosome Spreadsmentioning

confidence: 99%

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA

2021

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piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.

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Section: Meiotic Chromosome Spreadsmentioning

confidence: 99%

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA

2021

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“…A commercial humidity chamber is also available (see Table of Materials). We envision that any polystyrene foam box fitted with ridges and grooves (e.g., using glass pipettes 13 ) can be used. Meiotic chromosomes stained for Sycp3 imaged by SIM using a 20x objective.…”

Section: Discussionmentioning

confidence: 99%

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Blokhina

Olaya

Burgess

2020

JoVE

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Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

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“…Mounting medium with DAPI was added to the slides for imaging. For surface nuclear spread analysis, spermatocytes from 2, 4 and 6 dpt testes as well as the control were used according to the method described elsewhere (Dia et al, 2017;Peters et al, 1997) with minor modifications.…”

Section: Targeted Inactivation Of Ythdc2mentioning

confidence: 99%

YTHDC2 Is Essential for Pachytene Progression and Prevents Aberrant Microtubule-Driven Telomere Clustering in Male Meiosis

Liu

Kasowitz

Homolka

et al. 2021

Preprint

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Mechanisms driving the prolonged meiotic prophase I are poorly understood. The RNA helicase YTHDC2 is critical for mitosis to meiosis transition, as YTHDC2-deficient mouse germ cells initiate meiosis but arrest with mixed characteristics of mitotic and meiotic cell types. However, YTHDC2 is also highly expressed in normal pachytene cells. Here we identify an essential role for YTHDC2 in meiotic progression. Specifically, we find that YTHDC2 deficiency causes microtubule-dependent telomere clustering and apoptosis at the pachytene stage of prophase I, and thus a failure to advance to the diplotene stage. Depletion of YTHDC2 results in a massively dysregulated transcriptome in pachytene cells, with a tendency toward upregulation of genes normally expressed in mitotic germ cells and downregulation of meiotic transcripts. Dysregulation does not correlate with the m6A status of RNAs and YTHDC2-bound mRNAs are enriched in genes upregulated in mutant germ cells, revealing that YTHDC2 primarily targets its substrate mRNAs for degradation. Finally, altered transcripts in YTHDC2-deficient pachytene cells encode microtubule network proteins and inhibition of microtubule polymerization disperses clustered telomeres. Together, our results demonstrate that YTHDC2 regulates the prolonged pachytene stage of prophase I by perpetuating a meiotic transcriptome and preventing changes in the microtubule network that could lead to aberrant telomere clustering.

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Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Cited by 23 publications

References 15 publications

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

YTHDC2 Is Essential for Pachytene Progression and Prevents Aberrant Microtubule-Driven Telomere Clustering in Male Meiosis

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