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Chromosomal positioning in spermatogenic cells is influenced by chromosomal factors associated with gene activity, bouquet formation and meiotic sex chromosome inactivation


Chromosome territoriality is not random along the cell cycle and it is mainly governed by intrinsic chromosome factors and gene expression patterns. Conversely, very few studies have explored the factors that determine chromosome territoriality and its influencing factors during meiosis. In this study, we analysed chromosome positioning in murine spermatogenic cells using three-dimensionally fluorescence in situ hybridization-based methodology, which allows the analysis of the entire karyotype. The main objective of the study was to decipher chromosome positioning in a radial axis (all analysed germ-cell nuclei) and longitudinal axis (only spermatozoa) and to identify the chromosomal factors that regulate such an arrangement. Results demonstrated that the radial positioning of chromosomes during spermatogenesis was cell-type specific and influenced by chromosomal factors associated to gene activity. Chromosomes with specific features that enhance transcription (high GC content, high gene density and high numbers of predicted expressed genes) were preferentially observed in the inner part of the nucleus in virtually all cell types. Moreover, the position of the sex chromosomes was influenced by their transcriptional status, from the periphery of the nucleus when its activity was repressed (pachytene) to a more internal position when it is partially activated (spermatid). At pachytene, chromosome positioning was also influenced by chromosome size due to the bouquet formation. Longitudinal chromosome positioning in the sperm nucleus was not random either, suggesting the importance of ordered longitudinal positioning for the release and activation of the paternal genome after fertilisation.

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We thank Dr. Ignasi Roig for his advice in optimising the technique of immunofluorescence and cell stage identification. We thank M.A Handel from Jackson Laboratories for providing us the H1T antibody.


This study was supported by CF-180034 (UAB), DPI2015-65286-R/SAF2016-77165-P/RTI2018-095209 (MINECO), 2017-SGR-1624 and CERCA (Generalitat de Catalunya). Mireia Solé is the recipient of a grant from UAB (PIF/2015). Debora Gil is a Serra Hunter Fellow.

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MS performed the scientific experiments, image analysis and interpretation of the results. MS wrote the manuscript with support of ZS and JB. ZS and JB conceived, designed and supervised the study. DG designed the customised developed scripts for Matlab analysis. OV conducted the statistical analyses. AP performed part of the spermatozoa analysis. BC optimised the cell type identification methodology step. EA and FV helped shape the research and critically revised the manuscript. GF and RF provided the Chromoprobe Multiprobe® OctoChrome Murine System kit. All authors reviewed and approved the final version of the manuscript.

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Correspondence to Z. Sarrate.

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The Ethics Committee on Animal and Human Experimentation (CEEAH) of the Autonomous University of Barcelona declares that according to the Spanish regulation RD53/2013, the use of biological material in the term used in this study does not require formal consent by the Committee.

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Solé, M., Blanco, J., Gil, D. et al. Chromosomal positioning in spermatogenic cells is influenced by chromosomal factors associated with gene activity, bouquet formation and meiotic sex chromosome inactivation . Chromosoma 130, 163–175 (2021).

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  • Chromosome territories
  • FISH
  • Spermatogenesis
  • Three-dimensional analysis