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

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.

Differential Expression of Kisspeptin System and Kisspeptin Receptor Trafficking during Spermatozoa Transit in the Epididymis

“…In this respect, the intracellular trafficking of Kiss1R during sperm maturation recapitulates the trafficking occurring during sperm elongation. In mature spermatozoa, the post acrosomal sheet and perforatorium of the perinuclear theca represent a storage site for critical factors in early fertilization and zygotic events [ 48 , 49 ]. Consistently, the functional inhibition of Kiss1R by kisspeptin antagonist Kp-234 decreased the success of in vitro fertilization in mice [ 17 ].…”

High Resolution Proteomic Analysis of Subcellular Fractionated Boar Spermatozoa Provides Comprehensive Insights Into Perinuclear Theca-Residing Proteins

“…Within this protocol, the final alkaline extraction step is thought to extract all the covalently bound PT proteins. Similarly, some proteins that interact with polar bonds to the PT, such as histones, are detached during the saline extraction step (Tovich and Oko, 2003;Hamilton et al, 2021). Somatic histones have been identified as major constituents of the PT of bovine (Tovich and Oko, 2003) and murid spermatozoa (Hamilton et al, 2021), and those somatic histones are extractable using 1 M KCl.…”

“…Similarly, some proteins that interact with polar bonds to the PT, such as histones, are detached during the saline extraction step (Tovich and Oko, 2003;Hamilton et al, 2021). Somatic histones have been identified as major constituents of the PT of bovine (Tovich and Oko, 2003) and murid spermatozoa (Hamilton et al, 2021), and those somatic histones are extractable using 1 M KCl. However, the extractability of the histones differs in our study whereby somatic histones were still abundantly present in the final alkaline step after high salt treatments.…”

“…This is likely due to previous limitations in isolating PT proteins and the relatively slow uptake of proteomic technologies to investigate reproductive cells. However, even prior to the widespread use of mass spectrometry to characterize sperm proteins, pioneering work by Oko and Sutovsky and their respective colleagues revealed that the PT houses an unexpected array of proteins including somatic (core) histones (Tovich and Oko, 2003) that appear to be de novo-synthesized during the round spermatid stage of development and are then assembled during spermatid elongation (Hamilton et al, 2021). Remarkably, these histones then appear to play an important role postfertilization as intra-cytoplasmic sperm injection (ICSI) of sperm cells depleted of PT-histones resulted in a delay in embryonic development (Hamilton et al, 2021).…”

“…However, even prior to the widespread use of mass spectrometry to characterize sperm proteins, pioneering work by Oko and Sutovsky and their respective colleagues revealed that the PT houses an unexpected array of proteins including somatic (core) histones (Tovich and Oko, 2003) that appear to be de novo-synthesized during the round spermatid stage of development and are then assembled during spermatid elongation (Hamilton et al, 2021). Remarkably, these histones then appear to play an important role postfertilization as intra-cytoplasmic sperm injection (ICSI) of sperm cells depleted of PT-histones resulted in a delay in embryonic development (Hamilton et al, 2021). Similarly, the PT-residing protein glutathione S-transferase omega 2 (GSTO2) has recently been shown to accelerate nuclear decondensation of sperm cells during fertilization (Hamilton et al, 2019), implying that multiple PTenriched proteins may contribute to functions in the zygote.…”