Assessing the Presence, Extractability, and Function of Core Histones in the Perinuclear Theca of Murid Spermatozoa

Abstract

The perinuclear theca (PT) is a condensed cytosolic capsule that surrounds the eutherian sperm nucleus and contains various proteins involved in fertilization. Previously, proteomic analysis of the bovine PT revealed a novel population of four core histones residing in this distinctly non-nuclear location, despite the traditional role of histones in nuclear DNA stabilization. Utilizing immunocytochemistry, histones were localized to two sub-compartments of the murid PT, the post-acrosomal sheath and the murid-specific perforatorium. This finding created the framework to pursue our primary objective: to determine the role of PT core histones in fertilization and early embryogenesis. Specifically, given their inherent ability to bind DNA, we hypothesized that PT-associated histones stabilize sperm chromatin during male pronuclear formation in the developing zygote. To investigate this, we assessed the consequences of performing intracytoplasmic sperm injection (ICSI) with histone-depleted (HD) sperm. The mouse model is ideal for such functional assays because only the sperm nucleus and PT are required to achieve fertilization via ICSI. To create a valid depletion model, histones were selectively extracted from the mouse PT under high salt conditions paired with mild sonication. SDS-PAGE analysis of KCl-treated murid sperm extracts revealed four low molecular weight bands which, by immunoblotting with anti-core histone antibodies, corresponded to calf thymus core histones (H3, H2B, H2A, H4) run in adjacent lanes. Conversely, PT-bound activating factor, PAWP, remained largely intact following high salt extraction, indicating selectivity of the protocol. ICSI functional assays compared developmental patterns of mouse embryos fertilized with control or HD sperm. Using light microscopy, HD oocytes were found to initiate activation at the same rate as control groups but displayed significantly fewer 2-cell embryos and blastocysts at the appropriate developmental timepoints. This suggests that early embryonic development is delayed in the absence of PT-derived core histones, presumably as compensation for destabilization of the male chromatin following fertilization. While their direct influence on sperm chromatin was unclear by our methods, this thesis presents evidence for the influence of PT core histones on early embryogenesis, and proposes a model of PT histone depletion for future functional analyses.