Advertisement

Chromosoma

pp 1–14 | Cite as

Copy-number variation introduced by long transgenes compromises mouse male fertility independently of pachytene checkpoints

  • Ondrej MiholaEmail author
  • Tatyana Kobets
  • Klara Krivankova
  • Eliska Linhartova
  • Srdjan Gasic
  • John C. Schimenti
  • Zdenek Trachtulec
Original Article

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.

Keywords

Fertility Transgene Interspecific hybrid Spermatogenesis Proteasome 

Notes

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).

Compliance with ethical standards

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).

Supplementary material

412_2019_730_MOESM1_ESM.pdf (243 kb)
ESM 1 (PDF 242 kb)

References

  1. Anderson LK, Reeves A, Webb LM, Ashley T (1999) Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein. Genetics 151:1569–1579PubMedPubMedCentralGoogle Scholar
  2. Baudat F, Buard J, Grey C, Fledel-Alon A, Ober C, Przeworski M, Coop G, de Massy B (2010) PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice. Science 327:836–840.  https://doi.org/10.1126/science.1183439 CrossRefPubMedGoogle Scholar
  3. Bellve AR, Cavicchia JC, Millette CF, O'Brien DA, Bhatnagar YM, Dym M (1977) Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol 74:68–85.  https://doi.org/10.1083/jcb.74.1.68 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bernardini F, Kriezis A, Galizi R, Nolan T, Crisanti A (2019) Introgression of a synthetic sex ratio distortion system from Anopheles gambiae into Anopheles arabiensis. Sci Rep 9:5158.  https://doi.org/10.1038/s41598-019-41646-8 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bhattacharyya T, Gregorova S, Mihola O, Anger M, Sebestova J, Denny P, Simecek P, Forejt J (2013) Mechanistic basis of infertility of mouse intersubspecific hybrids. Proc Natl Acad Sci U S A 110:E468–E477.  https://doi.org/10.1073/pnas.1219126110 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Carballar-Lejarazu R, James AA (2017) Population modification of Anopheline species to control malaria transmission. Pathog Glob Health 111:424–435.  https://doi.org/10.1080/20477724.2018.1427192 CrossRefPubMedGoogle Scholar
  7. Davisson MT, Akeson EC (1987) An improved method for preparing G-banded chromosomes from mouse peripheral blood. Cytogenet Cell Genet 45:70–74.  https://doi.org/10.1159/000132432 CrossRefPubMedGoogle Scholar
  8. Di Siena S, Gimmelli R, Nori SL, Barbagallo F, Campolo F, Dolci S, Rossi P, Venneri MA, Giannetta E, Gianfrilli D, Feigenbaum L, Lenzi A, Naro F, Cianflone E, Mancuso T, Torella D, Isidori AM, Pellegrini M (2016) Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury. Cell Death Dis 7:e2317.  https://doi.org/10.1038/cddis.2016.205 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Faisal I, Kauppi L (2016) Sex chromosome recombination failure, apoptosis, and fertility in male mice. Chromosoma 125:227–235.  https://doi.org/10.1007/s00412-015-0542-9 CrossRefPubMedGoogle Scholar
  10. Fischer K, Kraner-Scheiber S, Petersen B, Rieblinger B, Buermann A, Flisikowska T, Flisikowski K, Christan S, Edlinger M, Baars W, Kurome M, Zakhartchenko V, Kessler B, Plotzki E, Szczerbal I, Switonski M, Denner J, Wolf E, Schwinzer R, Niemann H, Kind A, Schnieke A (2016) Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing. Sci Rep 6:29081.  https://doi.org/10.1038/srep29081 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Flachs P, Mihola O, Simecek P, Gregorova S, Schimenti JC, Matsui Y, Baudat F, de Massy B, Pialek J, Forejt J, Trachtulec Z (2012) Interallelic and intergenic incompatibilities of the Prdm9 (Hst1) gene in mouse hybrid sterility. PLoS Genet 8:e1003044.  https://doi.org/10.1371/journal.pgen.1003044 CrossRefGoogle Scholar
  12. Flachs P, Bhattacharyya T, Mihola O, Pialek J, Forejt J, Trachtulec Z (2014) Prdm9 incompatibility controls oligospermia and delayed fertility but no selfish transmission in mouse intersubspecific hybrids. PLoS One 9:e95806.  https://doi.org/10.1371/journal.pone.0095806 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Forster D, Arnold-Ammer I, Laurell E, Barker AJ, Fernandes AM, Finger-Baier K, Filosa A, Helmbrecht TO, Kolsch Y, Kuhn E, Robles E, Slanchev K, Thiele TR, Baier H, Kubo F (2017) Genetic targeting and anatomical registration of neuronal populations in the zebrafish brain with a new set of BAC transgenic tools. Sci Rep 7:5230.  https://doi.org/10.1038/s41598-017-04657-x CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N (2003) A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917–925.  https://doi.org/10.1038/nature02033 CrossRefPubMedGoogle Scholar
  15. Gregorova S, Forejt J (2000) PWD/Ph and PWK/Ph inbred mouse strains of Mus m. musculus subspecies—a valuable resource of phenotypic variations and genomic polymorphisms. Folia Biol (Praha) 46:31–41Google Scholar
  16. Gregorova S, Gergelits V, Chvatalova I, Bhattacharyya T, Valiskova B, Fotopulosova V, Jansa P, Wiatrowska D, Forejt J (2018) Modulation of Prdm9-controlled meiotic chromosome asynapsis overrides hybrid sterility in mice. Elife 7.  https://doi.org/10.7554/eLife.34282
  17. Grunwald HA, Gantz VM, Poplawski G, Xu XS, Bier E, Cooper KL (2019) Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline. Nature 566:105–109.  https://doi.org/10.1038/s41586-019-0875-2 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hayashi K, Yoshida K, Matsui Y (2005) A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature 438:374–378.  https://doi.org/10.1038/nature04112 CrossRefPubMedGoogle Scholar
  19. Howell GR, Munroe RJ, Schimenti JC (2005) Transgenic rescue of the mouse t complex haplolethal locus Thl1. Mamm Genom 16:838–846.  https://doi.org/10.1007/s00335-005-0045-8 CrossRefGoogle Scholar
  20. Irvin N, Hoddle MS, O'Brochta DA, Carey B, Atkinson PW (2004) Assessing fitness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes. Proc Natl Acad Sci U S A 101:891–896.  https://doi.org/10.1073/pnas.0305511101 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kauppi L, Barchi M, Baudat F, Romanienko PJ, Keeney S, Jasin M (2011) Distinct properties of the XY pseudoautosomal region crucial for male meiosis. Science 331:916–920.  https://doi.org/10.1126/science.1195774 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Khor B, Bredemeyer AL, Huang CY, Turnbull IR, Evans R, Maggi LB Jr, White JM, Walker LM, Carnes K, Hess RA, Sleckman BP (2006) Proteasome activator PA200 is required for normal spermatogenesis. Mol Cell Biol 26:2999–3007.  https://doi.org/10.1128/MCB.26.8.2999-3007.2006 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321–334.  https://doi.org/10.1038/nrg3686 CrossRefPubMedGoogle Scholar
  24. Marrelli MT, Moreira CK, Kelly D, Alphey L, Jacobs-Lorena M (2006) Mosquito transgenesis: what is the fitness cost? Trends Parasitol 22:197–202.  https://doi.org/10.1016/j.pt.2006.03.004 CrossRefPubMedGoogle Scholar
  25. Mihola O, Trachtulec Z (2017) A mutation of the Prdm9 mouse hybrid sterility gene carried by a transgene. Folia Biol (Praha) 63:27–30Google Scholar
  26. Mihola O, Trachtulec Z, Vlcek C, Schimenti JC, Forejt J (2009) A mouse speciation gene encodes a meiotic histone H3 methyltransferase. Science 323:373–375.  https://doi.org/10.1126/science.1163601 CrossRefPubMedGoogle Scholar
  27. Mihola O, Pratto F, Brick K, Linhartova E, Kobets T, Flachs P, Baker CL, Sedlacek R, Paigen K, Petkov PM, Camerini-Otero RD, Trachtulec Z (2019) Histone methyltransferase PRDM9 is not essential for meiosis in male mice. Genome Res 29:1078–1086.  https://doi.org/10.1101/gr.244426.118 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Morgan AP, Fu CP, Kao CY, Welsh CE, Didion JP, Yadgary L, Hyacinth L, Ferris MT, Bell TA, Miller DR, Giusti-Rodriguez P, Nonneman RJ, Cook KD, Whitmire JK, Gralinski LE, Keller M, Attie AD, Churchill GA, Petkov P, Sullivan PF, Brennan JR, McMillan L, Pardo-Manuel de Villena F (2015) The Mouse Universal Genotyping Array: from substrains to subspecies. G3 (Bethesda) 6:263–279.  https://doi.org/10.1534/g3.115.022087 CrossRefPubMedCentralGoogle Scholar
  29. Morita Y, Perez GI, Maravei DV, Tilly KI, Tilly JL (1999) Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents spontaneous and chemotherapy-induced oocyte apoptosis in vitro. Mol Endocrinol 13:841–850.  https://doi.org/10.1210/mend.13.6.0306 CrossRefPubMedGoogle Scholar
  30. Myers S, Bowden R, Tumian A, Bontrop RE, Freeman C, MacFie TS, McVean G, Donnelly P (2010) Drive against hotspot motifs in primates implicates the PRDM9 gene in meiotic recombination. Science 327:876–879.  https://doi.org/10.1126/science.1182363 CrossRefPubMedGoogle Scholar
  31. Parvanov ED, Petkov PM, Paigen K (2010) Prdm9 controls activation of mammalian recombination hotspots. Science 327:835.  https://doi.org/10.1126/science.1181495 CrossRefPubMedGoogle Scholar
  32. Porter SN, Levine RM, Pruett-Miller SM (2019) A practical guide to genome editing using targeted nuclease technologies. Compr Physiol 9:665–714.  https://doi.org/10.1002/cphy.c180022 CrossRefPubMedGoogle Scholar
  33. Qian MX, Pang Y, Liu CH, Haratake K, Du BY, Ji DY, Wang GF, Zhu QQ, Song W, Yu Y, Zhang XX, Huang HT, Miao S, Chen LB, Zhang ZH, Liang YN, Liu S, Cha H, Yang D, Zhai Y, Komatsu T, Tsuruta F, Li H, Cao C, Li W, Li GH, Cheng Y, Chiba T, Wang L, Goldberg AL, Shen Y, Qiu XB (2013) Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis. Cell 153:1012–1024.  https://doi.org/10.1016/j.cell.2013.04.032 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Tengowski MW, Feng D, Sutovsky M, Sutovsky P (2007) Differential expression of genes encoding constitutive and inducible 20S proteasomal core subunits in the testis and epididymis of theophylline- or 1,3-dinitrobenzene-exposed rats. Biol Reprod 76:149–163.  https://doi.org/10.1095/biolreprod.106.053173 CrossRefPubMedGoogle Scholar
  35. Tilly JL (2003) Ovarian follicle counts—not as simple as 1, 2, 3. Reprod Biol Endocrinol 1:11.  https://doi.org/10.1186/1477-7827-1-11 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Torgovnick A, Heger JM, Liaki V, Isensee J, Schmitt A, Knittel G, Riabinska A, Beleggia F, Laurien L, Leeser U, Jungst C, Siedek F, Vogel W, Klumper N, Nolte H, Wittersheim M, Tharun L, Castiglione R, Kruger M, Schauss A, Perner S, Pasparakis M, Buttner R, Persigehl T, Hucho T, Herter-Sprie GS, Schumacher B, Reinhardt HC (2018) The Cdkn1aSUPER mouse as a tool to study p53-mediated tumor suppression. Cell Rep 25:1027–1039 e1026.  https://doi.org/10.1016/j.celrep.2018.09.079 CrossRefPubMedGoogle Scholar
  37. Trachtulec Z, Forejt J (1999) Transcription and RNA processing of mammalian genes in Saccharomyces cerevisiae. Nucleic Acids Res 27:526–531CrossRefGoogle Scholar
  38. Trachtulec Z, Vlcek C, Mihola O, Gregorova S, Fotopulosova V, Forejt J (2008) Fine haplotype structure of a chromosome 17 region in the laboratory and wild mouse. Genetics 178:1777–1784.  https://doi.org/10.1534/genetics.107.082404 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Vernet N, Mahadevaiah SK, Ojarikre OA, Longepied G, Prosser HM, Bradley A, Mitchell MJ, Burgoyne PS (2011) The Y-encoded gene Zfy2 acts to remove cells with unpaired chromosomes at the first meiotic metaphase in male mice. Curr Biol 21:787–793.  https://doi.org/10.1016/j.cub.2011.03.057 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Wang Z, Engler P, Longacre A, Storb U (2001) An efficient method for high-fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfection. Genome Res 11:137–142CrossRefGoogle Scholar
  41. Wang M, Sun Z, Yu T, Ding F, Li L, Wang X, Fu M, Wang H, Huang J, Li N, Dai Y (2017) Large-scale production of recombinant human lactoferrin from high-expression, marker-free transgenic cloned cows. Sci Rep 7:10733.  https://doi.org/10.1038/s41598-017-11462-z CrossRefPubMedPubMedCentralGoogle Scholar
  42. Winer J, Jung CK, Shackel I, Williams PM (1999) Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270:41–49.  https://doi.org/10.1006/abio.1999.4085 CrossRefPubMedGoogle Scholar
  43. Zhou CY, McInnes E, Copeman L, Langford G, Parsons N, Lancaster R, Richards A, Carrington C, Thompson S (2005) Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor. Xenotransplantation 12:142–148.  https://doi.org/10.1111/j.1399-3089.2005.00209.x CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Laboratory of Germ Cell Development, Division BIOCEVInstitute of Molecular Genetics of the Czech Academy of SciencesPragueCzech Republic
  2. 2.Department of Biomedical SciencesCornell UniversityIthacaUSA

Personalised recommendations