Journal: bioRxiv

Article Title: Male mice heterozygous for Protamine-1 and Protamine-2 are infertile displaying sperm damage and retention of Protamine-2 precursors, transition proteins and histones

doi: 10.64898/2026.03.15.711850

Figure Lengend Snippet: (A) CMA3 staining of mature WT and dHET sperm counterstained by DAPI. Scale bars: 30 μm. (B) Quantification of CMA3-stained nuclei in WT and dHET samples. Data are mean ± s.d. and were analyzed by two-tailed, unpaired Student’s t-test (***p<0.001). (C) Representative image of a Coomassie stained AU-PAGE of basic nuclear-enriched proteins isolated from WT and dHET epididymal sperm. Bands corresponding to ODF2 and protamines are marked. Bands containing predominantly PRM1 and mature PRM2 (mP2) are labelled and areas of bands containing Prm2 precursors are marked (red dashed box). Non-protamine, nuclear-enriched proteins are found to be differentially abundant in dHET compared to WT sperm (red box). (D) Upper part of image shown in (C) compared to merged Western Blots against ODF2, histone H3 and histone H4 (pan-H3 and pan-H4 antibodies). (E) Western blots against histone H3 and H4 basic protein extractions of WT testis lysate and sperm samples form WT, dHET, Prm1 +/- (P1 +/- ), Prm1 -/- (P1 -/- ), Prm2 +/- (P2 +/- ), Prm2 -/- (P2 -/- ). ODF2 was used as loading control, as described before . (F) IHC against pre-PRM2 (antibody epitope in cP2) on WT and dHET epididymal caput tissue sections. (G) IHC against transition proteins TNP1 and TNP2 on WT and dHET epididymal caput tissue sections. Scales (F-G): 50 µm

Article Snippet: Primary antibodies diluted in blocking solution were added to the membrane and incubated overnight at 4 °C [PRM1 (Briar Patch Biosciences; Mab-Hup1N-150; 1:1000), PRM2 (Briar Patch Biosciences; Mab-Hup2B-150; 1:1000), ODF2 (proteintech; 12058-1-AP; 1:500-1:1000), histone H3 (abcam; ab1791; 1:1000), histone H4 (abcam; ab177840 ; 1:500)].

Techniques: Staining, Two Tailed Test, Isolation, Western Blot, Control