Abstract
Germline mutations underlie genetic diversity and species evolution. Previous studies have assessed the theoretical mutation rates and spectra in germ cells mostly by analyzing genetic markers and reporter genes in populations and pedigrees. This study reported the direct measurement of germline mutations by whole-genome sequencing of cultured spermatogonial stem cells in mice, namely germline stem (GS) cells, together with multipotent GS (mGS) cells that spontaneously dedifferentiated from GS cells. GS cells produce functional sperm that can generate offspring by transplantation into seminiferous tubules, whereas mGS cells contribute to germline chimeras by microinjection into blastocysts in a manner similar to embryonic stem cells. The estimated mutation rate of GS and mGS cells was approximately 0.22 × 10−9 and 1.0 × 10−9 per base per cell population doubling, respectively, indicating that GS cells have a lower mutation rate compared to mGS cells. GS and mGS cells also showed distinct mutation patterns, with C-to-T transition as the most frequent in GS cells and C-to-A transversion as the most predominant in mGS cells. By karyotype analysis, GS cells showed recurrent trisomy of chromosomes 15 and 16, whereas mGS cells frequently exhibited chromosomes 1, 6, 8, and 11 amplifications, suggesting that distinct chromosomal abnormalities confer a selective growth advantage for each cell type in vitro. These data provide the basis for studying germline mutations and a foundation for the future utilization of GS cells for reproductive technology and clinical applications.
Highlights
Germline mutations underlie genetic diversity and species evolution
To assess the genome stability of mouse germline stem (GS) and multipotent GS (mGS) cells, de novo mutations that accumulate in clonally derived cell populations of each cell type during a defined culture period were analyzed
Clonal cultures from single cells were first carried out, and each clone of GS and mGS cells was expanded for 100 population doublings (Fig. 1)
Summary
Germline mutations underlie genetic diversity and species evolution. Previous studies have assessed the theoretical mutation rates and spectra in germ cells mostly by analyzing genetic markers and reporter genes in populations and pedigrees. The addition of glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), both self-renewal factors for SSCs, stimulated the proliferation of spermatogonia in vitro and induced the formation of grape-like clusters of germ cells These cells, designated as germline stem (GS) cells, can proliferate for more than 2 years without significant loss of SSC activity to recolonize seminiferous tubules[9]. Androgenetic DNA methylation patterns were maintained after 5 years of culture, one of the two lines showed a partial deletion of chromosome 1711 They still continued to proliferate despite significantly shortened telomeres. The lower mutation rate in ES cells and its mechanism have been well c haracterized[16], little is known about the impact of cell culture on the genome integrity of GS cells
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