Journal: bioRxiv
Article Title: H2B.W2, a Spermatocytes-specific Histone Variant, disrupts nucleosome stability and reduces chromatin compaction
doi: 10.1101/2024.12.12.626759
Figure Lengend Snippet: ( A ) Sections of human testis were immunostained with an (a) anti-H4, (b) anti-H2B.C1, (c) anti-H2B.W1, or (d) anti-H2B.W2 antibody. The region bounded by the black rectangle in (d) is shown at higher magnification in (d’), where H2B.W2-positive spermatocytes are indicated by black arrows. Scale bars are 50 µm (a-d) and 10 µm (d’). ( B ) (upper panel) Representative immunofluorescence staining examples of human testis sections with anti-H2B.W2 (red) and anti-phospho-histone H2A.X (γ-H2A.X; green) antibodies. The DNA was counter-stained with Hoechst 33342 (blue). Scale bars = 50 μm. (lower panel) Enlarged images of representative cells at different stages of spermatogenesis, which were extracted from the merged image shown in the upper row panel or merged images of samples treated with the same immunofluorescence staining. These images show: an early spermatogonia located at the outermost part of seminiferous tubule without any γ-H2A.X signal in its oval-shaped nucleus (i); a mid-late spermatogonia with very weak γ-H2A.X signal and weak H2B.W2 signal that appear as numerous small foci (ii); an early spermatocyte with strong and globally distributed γ-H2A.X signal and strong H2B.W2 signal (iii); a pachytene spermatocyte with strong γ-H2A.X-staining in the sex bodies and weaker H2B.W2 signal (iv); a round spermatid without γ-H2A.X staining and weak H2B.W2 signal (v); an elongating spermatid with condensed nuclei and almost disappeared H2B.W2 signal (vi); an elongated spermatid with conically-shaped condensed nuclei (vii). H2B.W2 (red), γ-H2A.X (green), and DNA (blue). Scale bars = 2 μm. ( C ) UMAP (Uniform Manifold Approximation and Projection) plots of 2,854 human testicular cells (left panel). This plot is the same as the figure from (Ding et al., 2024) . The expression heat map of stage marker genes and histone variants according to different stages (right panel). The single cell RNA sequencing data of human testicular cells were rebuilt from a data set previously published by Wang M. et al., (). ( D ) Schematic view of H2B.W2 expression stages in spermatogenesis.
Article Snippet: A monoclonal mouse anti-phospho-histone H2A.X (Ser139) antibody, clone JBW301 (Sigma-Aldrich, 05-636, at 1:50 dilution) was added to the anti-H2B.W1 antibody solution.
Techniques: Immunofluorescence, Staining, Expressing, Marker, RNA Sequencing Assay
