Abstract
Male infertility is associated with several causes affecting the paternal nucleus such as DNA lesions (breaks, deletions, mutations, ...) or numerical chromosome anomalies. More recently, male infertility has also been associated with changes in the sperm epigenome, including modification in the topology of chromatin (Olszewska et al., Chromosome Research 16:875–890, 2008; Alladin et al., Syst Biol Reprod Med 59: 146–152, 2013) ref with number 1, 2. Indeed, the positioning of chromosomes in the sperm nucleus is nonrandom and defines chromosome territories (Champroux et al., Genes (Basel) 9:501, 2018) ref with number 3 whose optimal organization determines the success of embryonic development. In this context, the study of the spatial distribution of chromosomes in sperm cells could be relevant for clinical diagnosis. We describe here a in situ fluorescence hybridization (FISH) strategy coupled with a fluorescent immunocytochemistry approach followed by confocal analysis and reconstruction (2D/3D) as a powerful tool to analyze the location of chromosomes in the sperm nucleus using the mouse sperm as a model. Already, the two-dimensional (2D) analysis of FISH and immunofluorescence data reveal the location of chromosomes as well as the different markings on the spermatic nucleus. In addition, a good 3D rendering after Imaris software processing was obtained when Z-stacks of images were acquired over a defined volume (10 μm × 13 μm × 15 μm) with a sequential scanning mode to minimize bleed-through effects and avoid overlapping wavelengths.
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Acknowledgments
The authors would like to thank the CNRS, INSERM, and UCA for their financial support and the Auvergne Rhône Alpes Region for their contribution to this research. We would like to thank the Anipath platform (Damon-Soubeyrand C. and Bravard S.) for the technical assistance in immunofluorescence as well as the CLIC (confocal imaging facility; Vachias C.; Pouchin P. and Desset S.) of the GReD laboratory.
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Champroux, A., Goubely, C., Henry-Berger, J., Drevet, J.R., Kocer, A. (2021). Three-Dimensional Confocal Analysis of Chromosome Positioning Coupled with Immunofluorescence in Mouse Sperm Nuclei. In: Ruzov, A., Gering, M. (eds) DNA Modifications. Methods in Molecular Biology, vol 2198. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0876-0_20
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