Core Histones Are Constituents of the Perinuclear Theca of Murid Spermatozoa: An Assessment of Their Synthesis and Assembly during Spermiogenesis and Function after Gametic Fusion.

Lauren E Hamilton,Peter Sutovsky,Mark Baker,Luis Aguila,Richard Oko,Nicole Protopapas,João Suzuki,Genevieve Acteau,Wei Xu,Morgan Lion

doi:10.3390/ijms22158119

Abstract

The perinuclear theca (PT) of the eutherian sperm head is a cytoskeletal-like structure that houses proteins involved in important cellular processes during spermiogenesis and fertilization. Building upon our novel discovery of non-nuclear histones in the bovine PT, we sought to investigate whether this PT localization was a conserved feature of eutherian sperm. Employing cell fractionation, immunodetection, mass spectrometry, qPCR, and intracytoplasmic sperm injections (ICSI), we examined the localization, developmental origin, and functional potential of histones from the murid PT. Immunodetection localized histones to the post-acrosomal sheath (PAS) and the perforatorium (PERF) of the PT but showed an absence in the sperm nucleus. MS/MS analysis of selectively extracted PT histones indicated that predominately core histones (i.e., H3, H3.3, H2B, H2A, H2AX, and H4) populate the murid PT. These core histones appear to be de novo-synthesized in round spermatids and assembled via the manchette during spermatid elongation. Mouse ICSI results suggest that early embryonic development is delayed in the absence of PT-derived core histones. Here, we provide evidence that core histones are de novo-synthesized prior to PT assembly and deposited in PT sub-compartments for subsequent involvement in chromatin remodeling of the male pronucleus post-fertilization.

Highlights

Four bands in the range of 14–20 kDa were identified and determined to be resistant to extraction with non-ionic detergent (TX-100) but fully extractable with ionic detergent (SDS), suggesting that they were ionically bound. Verification of their identity as histones was accomplished by probing with affinity-purified antibodies specific for each of the core histones on Western blots containing purified calf thymus core histones
As was found in bull spermatozoa, these non-nuclear histones assemble as part of the postacrosomal sheath (PAS)
The perforatorium is an apical extension of the perinuclear theca (PT), found in falciform spermatozoa of murids, which shares developmental and compositional similarities with the post-acrosomal sheath (PAS) [15]

Summary

Introduction

There are three main compartments within the sperm head: the nucleus, the acrosome, and the perinuclear theca (PT) (Figure 1). The acrosome is a secretory vesicle that encapsulates the anterior region of the head and contains proteins required for oocyte penetration. Underneath the acrosome and surrounding the nucleus is the PT, a condensed cytosolicprotein-rich layer [1]. Based on compositional and functional considerations, the PT can be divided into two compartments: the subacrosomal layer (SAL), which houses proteins involved in the assembly and attachment of the acrosome to the spermatid nucleus during 4.0/).

Methods

Results

Conclusion