Somatic Histones Are Components of the Perinuclear Theca in Bovine Spermatozoa

Abstract

The perinuclear theca is a non-ionic detergent-resistant, electron-dense layer surrounding the condensed nucleus of mammalian sperm. The known proteins originating from the perinuclear theca have implicated the structure in a variety of important cellular processes during spermiogenesis and fertilization. Nonetheless, the composition of the perinuclear theca remains largely unexplored. We have isolated a group of low molecular mass (14 -19 kDa) perinuclear theca-derived proteins from acrosome-depleted bovine sperm heads by salt (1 M KCl) extraction and have identified them as core somatic histones. N-terminal sequencing and immunoblotting with anti-histone antibodies confirmed the presence of both intact and proteolytically cleaved somatic histones H3, H2B, H2A, and H4. Identical proteins were isolated using 2% SDS or 1 N HCl extractions. Subsequent acid and SDS extractions of intact bovine sperm revealed the presence of all four intact histone subtypes, with minimal proteolysis. Two-dimensional acid/urea/Triton-SDS-PAGE, coupled with immunoblotting analysis, confirmed the somatic nature of these perinuclear theca-derived histones. Estimates of the abundance of perinuclear theca-derived histones showed that up to 0.2 pg per sperm of each histone subtype was present. Immunogold labeling at the ultrastructural level localized all four core somatic histones to the postacrosomal sheath region of bovine epididymal sperm, when probed with affinity-purified anti-histone antibodies. Little immunoreactivity was detected in residual perinuclear theca structures following the extractions. Taken together, these findings indicate the unprecedented and stable localization of non-nuclear somatic histones in bovine sperm perinuclear theca.The perinuclear theca (PT) 1 is a specialized cytoskeletal component residing under the acrosome and almost completely surrounding the nucleus of mammalian sperm heads. Based on morphological and protein composition assessments, the PT is divided into two structurally continuous regions, termed the subacrosomal layer, and the post-acrosomal sheath (reviewed in Refs. 1 and 2). Although numerous proteins have been characterized from these PT regions (reviewed in Ref. 3), few of the known PT proteins to date resemble traditional cytoskeletal proteins. Rather, the PT contains a range of proteins of varying function, including Calicin, a basic structural protein that binds actin (4, 5); Stat4, a transcriptional activator hypothesized to contribute to zygotic gene activation (6); SubH2Bv (PT15), a histone H2B-like variant proposed to contribute to acrosome-nuclear docking (3); and PERF 15, a fatty acid-binding protein predominating in the perforatorium of murid sperm (7-9). The PT structure has been implicated in a number of essential cellular processes, such as nuclear shaping during spermiogenesis (2, 10), and egg activation and pronuclear formation during fertilization (11-15), although the PT proteins involved in these processes remain elusive. Their identification and characterization is theref...

Molecular morphology and function of bull spermatozoa linked to histones and associated with fertility

“…4); this pattern was also similar to that reported in previous experiments in swines (Flores et al 2011) and humans (Zhang et al 2006). Additionally, one of the few immunocytochemical studies conducted on bull sperm chromatin has demonstrated that histones are also located in the perinuclear theca (Tovich & Oko 2003). This result is consistent with our findings, where we observed that the histone signals were mainly present in the pre-equatorial region of the sperm head.…”

Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa

“…The 2D basic protein system used in this study did not resolve and identify modified protamines because they are extremely basic and rich in disulphide bridges; however, histones were present on the gels. It is tempting to speculate that these may be residual nuclear histones, although somatic histones are known also to be present in the subacrosomal sheath of the perinuclear theca (PT) (Tovich and Oko, 2003). The PT, a specialized cytoskeletal structure under the acrosome and surrounding sperm nuclei, has been implicated in acrosome-nuclear docking and nuclear shaping during spermiogenesis (Aul and Oko, 2002;Oko, 1995), as well as in oocyte activation and pronuclear formation (Sutovsky et al, 1997;Kimura et al, 1998;Manandhar and Toshimori, 2003;Sutovsky et al, 2003); the potential exists for PT-derived histones to contribute to male pronuclear development.…”

PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization

“…sonication, Triton X-100, and alkaline carbonate, pH 11.5) were all able to trigger Ca 2ϩ oscillations comparable with those observed at natural fertilization. These investigators emphasized that only in the case of high pH treatment was the Ca 2ϩ releasing activity fully released indicating that the factor was most likely a PT component according to our definition (4,20). Perry et al (21) proposed a so-called trans-complementation scheme of sperm-borne oocyte activating factors, detailing that full oocyte activation and embryo development can be achieved only with the combination of a DTT-soluble fraction obtained from Triton-demembranated mouse sperm head and the heat-stable component of the head after DTT extraction.…”

“…For example, alkaline extractable SubH2Bv, exclusive to the subacrosomal layer, is implicated in acrosome-nuclear docking during spermiogenesis (1). Salt-extractable non-nuclear somatic core histones, residing in the PAS, may be involved in stabilizing the chromatin of the decondensing sperm nucleus soon after oocyte entry (4,5). The DTT salt or alkaline extractable calicin and cylicin II share a basic pI with the histones and bind to actin in vitro (6 -8).…”