Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell's phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low‐ and high‐density sEV subsets from a human mast cell line (HMC‐1) and an erythroleukemic cell line (TF‐1) were separated using high‐resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV‐associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high‐density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole‐genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology.

The fundamental underlying paradigm of sexual reproduction is the production of male and female gametes of sufficient genetic difference and quality that, following syngamy, they result in embryos with genomic potential to allow for future adaptive change and the ability to respond to selective pressure. The fusion of dissimilar gametes resulting in the formation of a normal and viable embryo is known as anisogamy, and is concomitant with precise structural, physiological, and molecular control of gamete function for species survival. However, along the reproductive life cycle of all organisms, both male and female gametes can be exposed to an array of “stressors” that may adversely affect the composition and biological integrity of their proteins, lipids and nucleic acids, that may consequently compromise their capacity to produce normal embryos. The aim of this review is to highlight gamete genome organization, differences in the chronology of gamete production between the male and female, the inherent DNA protective mechanisms in these reproductive cells, the aetiology of DNA damage in germ cells, and the remarkable DNA repair mechanisms, pre- and post-syngamy, that function to maintain genome integrity.