From two to one: spatiotemporal mapping of chromatin remodelling in the protamine-lacking early zygote of zebrafish.

BACKGROUND: At fertilization, two highly specialized gametic genomes must rapidly reprogram into a single totipotent nucleus. In mammals, this transformation is marked by a dramatic protamine-to-histone exchange and pronounced asymmetry between the parental pronuclei. Zebrafish offer a contrasting vertebrate model in which sperm chromatin is already organized in nucleosomes and carries both active and repressive histone modifications, eliminating the need for protamine replacement. This histone-based configuration provides a unique opportunity to examine how parental chromatin transitions are initiated and coordinated directly in vivo. Although zebrafish is a well-established model organism in developmental biology, the detailed cellular and molecular steps of its fertilization process, particularly the timing and coordination of parental chromatin remodelling, remain largely unknown.
RESULTS: Using immunofluorescence coupled with spinning-disk confocal microscopy, we mapped histone variants and post-translational modifications with temporal sampling at minute-level intervals across 3,549 zebrafish embryos from fertilization to the first cleavage. This analysis resolved the main steps of fertilization, from sperm entry and chromatin decondensation to the formation, expansion, and apposition of the parental pronuclei. Throughout these stages, maternal and paternal genomes were remodelled almost synchronously. Activation-associated marks (H3K4me1/2/3, H3K9ac, H4K12ac) appeared shortly after fertilization, spread to both pronuclei by apposition, and disappeared before mitosis. The histone modification H3K9me3, commonly associated with compact or heterochromatin-like chromatin states, was present from the earliest time point examined and became more prominent during pronuclear maturation, whereas H3K27me3 was not detectable at any stage. H2A.Z showed strong paternal enrichment after sperm entry and was later detected in both pronuclei during apposition with weaker maternal signal. Polar bodies did not show evidence of undergoing the extensive chromatin remodelling observed in the zygotic pronuclei. In gametes, sperm contained core histones and methylation marks but lacked acetylation, indicating a compact, semi-active chromatin state, while unfertilized eggs displayed H3K9me3 and H3S10ph but no activating marks, consistent with meiotic arrest.
CONCLUSIONS: Zebrafish fertilization involves direct, histone-based chromatin remodelling that occurs without protamine replacement and proceeds almost simultaneously in both parental pronuclei. The sperm genome enters fertilization already in a semi-active, histone-bound state, enabling rapid and coordinated remodelling of both parental chromatin. This process results in a transient change in chromatin configuration that is more symmetrical between the parental genomes than that reported in mammalian systems.