The protamines of the spider <i>Steatoda</i> sp. provide an example of liquid–liquid phase separation chromatin transitions during spermiogenesis

Although there is extensive information about sperm nuclear basic proteins (SNBP) in vertebrates, there is, by comparison, very little information in Arthropoda. This study aims to contribute to filling this gap by analyzing these proteins in the sperm of the noble false widow spider Steatoda nobilis (order Araneae, family Theridiidae). To this end, we have developed a protein extraction method that allows the extraction of both cysteine-containing and non-cysteine-containing protamines that is suitable for the preparation and analysis of SNBPs from samples in which the amount of starting tissue material is limited. We carried out top-down mass spectrometry sequencing and molecular phylogenetic analyses to characterize the protamines of S. nobilis and other spiders. We also used electron microscopy to analyze the chromatin organization of the Steatoda sperm and we found it to exhibit liquid-liquid phase spinodal decomposition during the late stages of spermiogenesis. These experiments further our knowledge on the distribution of SNBPs within the animal kingdom and provide additional support for a proposed evolutionary origin of many protamines from a histone H1 (H5) replication-independent precursor.