By: Ewart Kuijk, recipient of the De Snoo award 2017
Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands
First of all, I am highly grateful for having received the De Snoo award in 2017. In addition to having been able to perform the study explained below, the De Snoo award has propelled my research further resulting in additional funding to extend my studies on instability of the embryonic genome, thereby helping me to establish my own independent research line.
Sperm DNA damage affects post zygotic genome function and interferes with proper embryo development, thereby impacting on miscarriage rates, fertility and postnatal pathologies. Nevertheless, the direct consequences of sperm DNA damage on the embryonic genome are largely unknown, because this requires single cell and low input sequencing techniques that have only recently been developed. The aim of this study was to elucidate how sperm DNA damage affects embryonic genomes. By absence of homologous templates we hypothesized that damaged sperm will be repaired through the inaccurate process of non-homologous end-joining which will result in the formation of Structural Variants (SVs) such as copy number variants (CNVs), translocations and inversions. Bovine embryos were produced with sperm subjected to different doses of γ-radiation (experimental group) and with untreated sperm (control group). Increasing doses of γ-radiation resulted in decreasing blastocyst rates. Moreover, embryos produced with γ-radiated sperm contained more micronuclei and other nuclear aberrations than control embryos. In follow-up experiments, the resulting zygotes were cultured until the 2-cell stage after which both individual blastomeres were collected and subjected to single-cell sequencing (control group n = 36 embryos, 2.5 Gray group n =42 embryos, 10 Gray group n = 18 embryos). This revealed that embryos produced with damaged sperm contain significantly more chromosomal segmental gains and deletions than control embryos (fig. 1). Additionally, experimental embryos contained more aneuploidies than control embryos, indicating that sperm DNA damage can induce mis-segregations (fig. 1). Strikingly, for all the observed gain, a reciprocal loss was observed in the sister cell (fig. 1, 2). If the 2-cell stage embryo would have been sequenced “in bulk” instead of both blastomeres separately, the genome would have appeared copy neutral and all variants would have been missed.
Figure 1: (Left) Number of copy number changes per cell for control embryos and embryos produced with 2 different doses of γ -radiated sperm, (Right) Number of aneuploidies per cell for control embryos and embryos produced with 2 different doses of γ -radiated sperm.
Figure 2; Single cell sequencing data for individual blastomeres. (Left) Circos plot showing the copy number state for all of the chromosomes for both cells of a 2-cell stage embryo from the control group. (Right) Circos plot showing the copy number state for all of the chromosomes for both cells of a 2-cell stage embryo from the 10 Gray group. Dark green = copy state 2 on the Crick strand, dark purple = copy state 1 on the Crick strand, light green = copy state 2 on the Watson strand, light purple = copy state 1 on the Watson strand. The total copy number state of Watson and Crick strands is indicated by the color in the inner ring for each cell: yellow = copy number state 2 (normal), blue = copy number state 3 (triploid), red = copy number state 1 (monozygous). From this picture, it is clear that the same chromosomes and chromosomal fragments that have a copy number state of 1 in one cell have a copy number state 3 in the sister cell, resulting in an average copy number state of 2 for both cells. Note: the data is derived through Strand-seq, a single cell sequencing technique that sequences the DNA strand that was used as a template in the previous cell division. When the Watson strand has been used as template for both the maternal and the paternal chromosome, the sequencing reads will map to the Watson side of the chromosome, when the Crick strand has been used as template for both the maternal and the paternal chromosome, the sequencing reads will map to the Crick side of the chromosome, if both the Watson and the Crick strands have been used as templates, the sequencing reads will map to both sides of the chromosome.
Single-cell sequencing of all blastomeres of 8-cell stage embryos produced with damaged sperm indicates that the reciprocal gains and losses observed at the 2-cell stage are preserved within their separate cellular lineages. I am currently sequencing entire blastocysts derived from damaged sperm to gain more insight in the effects of sperm DNA damage on the embryonic genome at later stages of development. Together, these results demonstrate that sperm DNA damage leads to reduced developmental competence, probably caused by the observed genomic aberrations. Embryos that succeed to develop further, are at risk to carry de novo structural variation. These findings may be of relevance for fertility treatments of males with high levels of sperm DNA damage. This study delivers the required proof of principle to be extended to study the effects of sperm DNA damage on human embryonic genomes, as was originally proposed. The manuscript for this study is currently in preparation.
This study was performed in collaboration with: S.H.A. Middelkamp1, D.C.J. Spierings2, H.T. A. van Tol3, V. Guryev2, P.M. Lansdorp2,4, B.A.J. Roelen3, E.P.J. Cuppen1
1 Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands
2 European Research Institute for the Biology of Ageing (ERIBA), University of Groningen, UMC Groningen, Groningen, The Netherlands.
3 Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
4 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada; Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada