AGARWAL, I. | Search bioRxiv

bioRxiv2026

Large disruptions to mammalian spermatogenesis downstream of genetic perturbations in meiotic double-strand break repair

Fertility in mammals relies on successful pairing of homologous chromosomes mediated by DNA double-strand breaks (DSBs). Here, we develop a system of mouse hybrids in which (a)symmetry of binding by the break-positioning protein PRDM9 to homologs varies over broad scales in the genome. Profiling transcription and chromatin in single nuclei, we trace how resulting delays in repair of variable subsets of meiotic DSBs propagate through spermatogenesis and drive large fertility differences in animals. We demonstrate that only asymmetry-generating mutations in PRDM9-binding motifs, not high average (~1%) divergence, disrupt chromosomal pairing. We observe substantial variation in animal-level sensitivity to asymmetry, and identify an interacting locus containing Dmc1 and Mei1 controlling (R2=0.64) this variation. Silencing of unpaired autosomes downstream of asynapsis and failure of normal sex chromosome silencing independently explain cell death in pachytene. Surprisingly, many cells with synaptic defects evade cell cycle arrest, and even those where physical division arrests still exhibit transcriptional progression to post-division states. Attrition of abnormal cells via arrest continues beyond the first division; nonetheless, cells that complete both meiotic divisions exhibit aneuploidy, especially of the sex chromosomes. This partly reflects de novo segregation errors explained by silencing of only chromosomes 16 and 19. Even "normal" euploid spermatids show crossovers redistributed at multi-megabase scales, indicating novel and potentially post-zygotic impacts of delays in meiotic DSB-repair. We thus elucidate cell-level and chromosome-specific impacts of regulatory variation in ~0.03% of the genome cascading through germline development, advancing our understanding of fertility and reproductive isolation.

genetics↗

bioRxiv2025

Sex differences in transcription-associated mutagenesis in the human germline

In humans, germline mutation rates are three- to four-fold higher in males than females, for largely unknown reasons. We investigated whether transcription, a well-documented source of both DNA damage and repair in somatic tissues, is associated with sex differences in germline mutations. To this end, we used expression data from male and female germline cells and phased de novo germline mutations from pedigrees. Focusing on protein-coding genes, we found no relationship between the male mutation rate and gene expression levels in the fetal germline or in adult testis tissue, despite evidence for transcriptional asymmetry. Individual stages of spermatogenesis differ in their contribution to mutation, however: expression levels in spermatogonial stem cells are significantly positively associated with paternal mutation rates, while those in primary spermatocytes are significantly negatively associated. Thus, transcription may have varying effects over male gametogenesis that are not readily detected from its cumulative effect on the total germline mutation rate. In females, by contrast, mutation rates increase significantly with transcription levels in the fetal germline, adult oocytes and adult ovary tissues, consistent with widespread transcription asymmetry. We confirm the difference between the sexes by analyzing phased mutations from three-generation pedigrees and the lack of an association in males by analyzing paternal mutations from seminiferous tubules and sperm. Thus, transcription has distinct effects on the mutation rate in the two sexes, leading to an increase in mutations in females but not males, in contrast to what one might expect from the overall paternal bias in germline mutations.

genetics↗