Sperm are remarkable cells specialized for the delivery of the paternal genome to the oocyte, while embryos consist of rapidly dividing, largely undifferentiated cells. Unlike embryos, sperm have jettisoned most cellular components, have highly compacted chromatin, and are thought to be largely transcriptionally inactive. Thus, sperm rely on substitution histones and post-translational modification (PTM) of existing proteins for development and function. In other organisms, replacement of S-phase histones by histone variants and protamines fundamentally changes chromosome architecture at the nucleosome level. How chromatin differs between these two tissue types has been largely unexplored. To investigate the nature of C. elegans sperm chromatin composition, we conducted a global analysis of sperm and embryo chromatin proteins using Multidimensional Protein Identification Technology (MudPIT) mass spectroscopy. One story that emerges from these data reveals that variant histone H2A proteins are incorporated to a higher degree in a terminally differentiated cell type compared to actively dividing cells. In embryos, spectral counts of peptides corresponding to the four histone H2A variants confirms S-phase histone H2A as the most abundant form, while HTZ-1 and HIS-35 (an alternative H2A that differs from S-phase H2A by one residue) are much less abundant. However, this profile differs in sperm. Here, we detect the sperm-specific incorporation of the HTAS-1 variant. HTAS-1 is required for optimal male fertility, is expressed only in sperm as detected by Western Blot and immunostaining, and intriguingly marks paternally-contributed chromatin after fertilization. Also, despite the high level of sequence identity, HIS-35 is employed in greater proportion in sperm. Interestingly, unlike S-phase H2A histone genes,
his-35 contains an intron. This feature may reflect a mechanism for regulating HIS-35 expression to fine-tune chromatin status during spermatogenesis. Our novel approach to determine chromatin composition between disparate cell types has identified over 1000 proteins from embryos and sperm each, laying the groundwork for analysis of other histone subtypes as well as protamine orthologs. In addition, our ongoing large-scale identification of histone PTMs is a valuable resource to examine how paternal epigenetic marks contribute to embryonic development.