CENP-A is diluted during bovine spermatogenesis and is maintained at internally positioned centromere clusters in mature bull sperm

During spermatogenesis, chromatin structure is remodelled by the incorporation of distinct histone variants and associated posttranslational modifications, followed by the almost complete replacement of histones by protamines in sperm. However, the dynamics of the centromere-specific histone H3 variant CENP-A have not yet been elucidated during spermatogenesis in mammals. Here we investigate CENP-A localisation dynamics in cattle (Bos taurus). In bovine testis tissue sections, we quantify CENP-A intensity in key germ cell types; spermatogonia (pre-meiotic), primary spermatocytes (meiotic) and spermatids (post-meiotic). Our quantitation shows that spermatogonia harbour the highest amount of CENP-A compared to all other germ cell types. Spermatids have approximately one quarter the amount of CENP-A of spermatogonia indicating that overall, it is reduced and maintained through the two meiotic divisions. Yet, we also observed some unexpected dynamics. CENP-A is asymmetrically distributed such that undifferentiated spermatogonia harbour more CENP-A that differentiated spermatogonia that enter meiosis. We also noted an increase in CENP-A intensity in primary spermatocytes during meiotic prophase I, which is indicative of centromere assembly at this time. We also confirm the specific maintenance of CENP-A, and the absence of the centromeric DNA binding protein CENP-B, on mature bull sperm nuclei that have completed histone-to-protamine exchange. Finally, we present a model for centromere positioning in mature sperm nuclei and propose that centralised clustering of centromeres may serve a protective function during histone-to-protamine exchange.