Abstract
Long transgenes are often used in mammalian genetics, e.g., to rescue mutations in large genes. In the course of experiments addressing the genetic basis of hybrid sterility caused by meiotic defects in mice bearing different alleles of Prdm9, we discovered that introduction of copy-number variation (CNV) via two independent insertions of long transgenes containing incomplete Prdm9 decreased testicular weight and epididymal sperm count. Transgenic animals displayed increased occurrence of seminiferous tubules with apoptotic cells at 18 days postpartum (dpp) corresponding to late meiotic prophase I, but not at 21 dpp. We hypothesized that long transgene insertions could cause asynapsis, but the immunocytochemical data revealed that the adult transgenic testes carried a similar percentage of asynaptic pachytene spermatocytes as the controls. These transgenic spermatocytes displayed less crossovers but similar numbers of unrepaired meiotic breaks. Despite slightly increased frequency of metaphase I spermatocytes with univalent chromosome(s) and reduced numbers of metaphase II spermatocytes, cytological studies did not reveal increased apoptosis in tubules containing the metaphase spermatocytes, but found an increased percentage of tubules carrying apoptotic spermatids. Sperm counts of subfertile animals inversely correlated with the transcription levels of the Psmb1 gene encoded within these two transgenes. The effect of the transgenes was dependent on sex and genetic background. Our results imply that the fertility of transgenic hybrid animals is not compromised by the impaired meiotic synapsis of homologous chromosomes, but can be negatively influenced by the increased expression of the introduced genes.
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Acknowledgments
We thank M. Fickerová, L. Šebestová, K. Třešňák, and P. Valtrová for technical assistance, employees of the animal facility of the Institute of Molecular Genetics of the Czech Academy of Sciences (IMG CAS) for mouse keeping, Dr. M. A. Handel for providing the anti-H1t antibody, and anonymous reviewers for comments. Some data were produced in the Microscopy Centre, IMG CAS. The authors were supported by CSF (16-19158S), by CAS (RVO 68378050), by MEYS (LQ1604, LM2015062, LM2015040), and by ERDF (CZ.1.05/1.1.00/02.0109 BIOCEV, CZ.1.05/2.1.00/19.0395).
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The European Community Council Directive 86/609/ EEC, Appendix A of the Council of Europe Convention ETS123, the Czech Republic Act 359/2012 Sb, and Decree 419/2012 of the Czech Ministry of Agriculture were followed during the mouse care and experiments. The study was approved by the Committee on the Ethics of Animal Experiments of the IMG (permit number 9/2016).
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Mihola, O., Kobets, T., Krivankova, K. et al. Copy-number variation introduced by long transgenes compromises mouse male fertility independently of pachytene checkpoints. Chromosoma 129, 69–82 (2020). https://doi.org/10.1007/s00412-019-00730-8
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DOI: https://doi.org/10.1007/s00412-019-00730-8