Involvement of Classical Bipartite/Karyopherin Nuclear Import Pathway Components in Acrosomal Trafficking and Assembly During Bovine and Murid Spermiogenesis1

Abstract

This study arose from our finding that SubH2Bv, a histone H2B variant residing in the subacrosomal compartment of mammalian spermatozoa, contains a bipartite nuclear localization signal (bNLS) but in spite of this did not enter the spermatid nucleus. Instead, it associated with proacrosomic and acrosomic vesicles, which were targeted to the nuclear surface to form the acrosome. On this basis we proposed that SubH2Bv targets proacrosomic/acrosomic vesicles from the Golgi apparatus to the nuclear envelope by utilizing the classical bipartite/karyopherin alpha (KPNA) nuclear import pathway. To test the protein's nuclear targeting ability, SubH2Bv, with and without targeted mutations of the basic residues of bNLS, as well as bNLS alone, were transfected into mammalian cells as GFP-fusion proteins. Only the intact bNLS conferred nuclear entry. Subsequently, we showed that a KPNA, most likely KPNA6, occupies the same sperm head compartment and follows the same pattern of acrosomal association during spermiogenesis as SubH2Bv. Sperm head fractionation combined with Western blotting located this KPNA to the subacrosomal layer of the perinuclear theca, while immunocytochemistry of testicular sections showed that it associates with the surface of proacrosomic/acrosomic vesicles during acrosomal biogenesis. The identical sperm-localization and testicular-expression patterns between KPNA and SubH2Bv suggested a potential binding interaction between these proteins. This was supported by recombinant SubH2Bv affinity pull-down assays on germ cell extracts. The results of this study provide a compelling argument that these two nuclear homing proteins work in concert to direct the acrosomic vesicle to the nucleus. Their final residence in the subacrosomal layer of the perinuclear theca of spermatozoa indicates a role for SubH2Bv and KPNA in acrosomal-nuclear docking.

A unified phylogeny-based nomenclature for histone variants

“…This apparently rapidly evolving protein variant (Figure 2) is reportedly found in the subacrosome of primates, rodents, and marsupials as well as bovids [37,49]. The use of the prefix ‘sub’ for this family seems appropriate, since residence in the subacrosome rather than the nucleus is a distinctive property that appears to be distributed throughout mammals.…”

Nucleosome adaptability conferred by sequence and structural variations in histone H2A–H2B dimers

“…There are some other H2A and H2B variants, like mH2A variant, known to participate in X-chromosome inactivation, which tend to form heterotypic nucleosomes with canonical histones in vitro [39]. Furthermore, certain histone variants such as H2A.L and subH2B [51,53] were shown to be involved in the formation of protein assemblies in spermatids, which are distinct from nucleosomes, and whose exact structure is still unclear.…”

WBP2 shares a common location in mouse spermatozoa with WBP2NL/PAWP and like its descendent is a candidate mouse oocyte activating factor

“…The PT of mammalian spermatozoa is compositionally subdivided into two major regions, the subacrosomal layer (SAL-PT), including the outer periacrosomal layer lying over the equatorial segment, and the postacrosomal sheath (PAS-PT) [10][11][12][13]. It was initially observed that local PAS solubilization of the PT was sufficient to elicit full oocyte activation during IVF [14].…”