Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024:2818:65-80.
doi: 10.1007/978-1-0716-3906-1_4.

Efficient Enrichment of Synchronized Mouse Spermatocytes Suitable for Genome-Wide Analysis

Agustin Carbajal  1 , Irma Gryniuk  1   2 , Rodrigo O de Castro  1 , Roberto J Pezza  3   4
Affiliations

Efficient Enrichment of Synchronized Mouse Spermatocytes Suitable for Genome-Wide Analysis

Agustin Carbajal et al. Methods Mol Biol. 2024.

Abstract

Chromatin undergoes extensive remodeling during meiosis, leading to specific patterns of gene expression and chromosome organization, which ultimately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies we adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin-binding sites by ChIP-seq. High-quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin-binding sites.

Keywords: ChIP-seq; Genomic analysis; Mouse spermatocytes; Spermatocyte fraction enrichment.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.. Enrichment of synchronized mouse spermatocytes.
A. Schematic showing neonate mice treatment for synchronization of spermatocytes and collection of cells at different stages of prophase I. B. Diagram representing PFA-fixed spermatocyte centrifugation on a discontinuous Percoll gradient. C. Representative image showing zygotene and pachytene spermatocytes after Percoll gradient. D. Percentage of spermatocytes before Percoll gradient (mean ± standard deviation, 49.4 ± 13.1 %, n = 12), purified using a discontinuous Percoll gradient without cell fixation (“- PFA”, 63.7 ± 11.6 %, n = 3) or purified after fixing cells (“+PFA”, 81.3 ± 11.8%, n = 55). Tukey’s significance test, p < 0.05 (*) or < 0.001 (**).
Figure 2.
Figure 2.. Analysis of INO80 ChIP-seq obtained from synchronized zygotene spermatocytes.
A. Heatmap profiles corresponding to INO80 ChIP-seq signal of each replicate centered at INO80 peaks with IDR =< 0.05. Rep. #1 and Rep. #2 correspond to first and second replicate, respectively. B. Examples of INO80 ChIP-seq signals and their respective inputs at selected promoters visualized using IGV. Scales are shown between brackets at the top left corner of each sample. C. Ven diagram depicting the intersection of peaks of both INO80 ChIP-seq replicates. D. Genomic features occupied by INO80. Colors represent whether the number of peaks found at a given genomic feature is larger (overrepresented), equal (expected) or lower (underrepresented) than expected by chance.
Figure 3.
Figure 3.
Cross-correlation analysis corresponding to INO80 ChIP-seq peaks. Graphs correspond to replicate 1 and replicate 2.
See this image and copyright information in PMC

References

    1. Bellve AR et al. , Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. The Journal of cell biology 74, 68–85 (1977). - PMC - PubMed
    1. Bryant JM, Meyer-Ficca ML, Dang VM, Berger SL, Meyer RG, Separation of spermatogenic cell types using STA-PUT velocity sedimentation. J Vis Exp 10.3791/50648 (2013). - DOI - PMC - PubMed
    1. Barchi M, Geremia R, Magliozzi R, Bianchi E, Isolation and analyses of enriched populations of male mouse germ cells by sedimentation velocity: the centrifugal elutriation. Methods in molecular biology 558, 299–321 (2009). - PubMed
    1. Gaysinskaya V, Soh IY, van der Heijden GW, Bortvin A, Optimized flow cytometry isolation of murine spermatocytes. Cytometry A 85, 556–565 (2014). - PMC - PubMed
    1. Romer KA, de Rooij DG, Kojima ML, Page DC, Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting. Dev Biol 443, 19–34 (2018). - PubMed

LinkOut - more resources