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) Schematic depiction of murine PRM1 and PRM2 amino acid sequences (grey box = cP2). (B) Schematic depiction of CRISPR/Cas9-mediated gene editing of the Prm1-Prm2 locus. Black arrow heads indicate the target sites of CRISPR-Cas9 guide RNAs. (C) Schematic depiction of Prm1 and Prm2 gene-edited alleles and their respective amino acid predictions. (D) Genomic WT and dHET DNA amplified with PCRs targeting either Prm1 or Prm2 . L = ladder. (E) Average pregnancy frequency after mating WT and dHET males with WT C57BL/6J females. Dots represent different males (n = number of males). (F) Average litter size after mating WT and dHET males with WT females. Dots show each litter size (n = number of males).

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: CRISPR, Amplification

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) Separation of basic cauda epididymal sperm protein extractions on Coomassie-stained acid-urea (AU) and thiourea gels. PRMs are detected at the bottom of the gel. Precursor bands of PRM2 are prominent in dHET samples (dashed box). Thiourea gels were used for analyses shown in (B-I). (B) Relative level of protamines compared to total protein content extracted from WT and dHET sperm. (C) Relative level of total PRM2 compared to total protein content extracted from WT and dHET sperm. (D) Relative level of PRM1 compared to total protein content extracted from WT and dHET sperm. (E) Relative level of mP2 compared to total protein content extracted from WT and dHET sperm. (F) Relative level of PRM2 precursors compared to total protein content extracted from WT and dHET sperm. (G) Relative level of mP2 compared to total PRM content extracted from WT and dHET sperm. (H) Relative level of PRM2 precursors compared to total PRM content extracted from WT and dHET sperm. (I) Relative level of total PRM2 precursors compared to total PRM content extracted from WT and dHET sperm. (J) Relative amount of PRM2 precursors comared to total PRM2 extracted from WT and dHET sperm. (K) Separation of basic protein extractions from whole testis on thiourea gels stained with Coomassie. PRM2 precursors are marked with a dashed box. Thiourea gels were used for quantifications shown in (L-S). (L) Relative level of protamines compared to total protein content extracted from WT and dHET testis. (M) Relative level of total PRM2 compared to total protein content extracted from WT and dHET testis. (N) Relative level of PRM1 compared to total protein content extracted from WT and dHET testis. (O) Relative level of mP2 compared to total protein content extracted from WT and dHET testis. (P) Relative level of PRM2 precursors compared to total protein content extracted from WT and dHET testis. (Q) Relative level of mP2 compared to total PRM content extracted from WT and dHET testis. (R) Relative level of PRM2 precursors compared to total PRM content extracted from WT and dHET testis. (S) Relative level of total PRM2 precursors compared to total PRM content extracted from WT and dHET testis. (T) Relative amount of PRM2 precursors comared to total PRM2 extracted from WT and dHET testis.

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

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) Testis to body weight ratio of dHET and WT mice (n= number of males). (B) Representative stage VII-VIII seminiferous tubule section of dHET and WT stained with Hematoxylin and Eosin. (C) Percentages of viable and inviable mature sperm of WT, Prm1 +/- , Prm1 -/- , Prm2 +/- , Prm2 -/- and dHET mice. Part of the data has been published before , . Data are mean ± s.d. and was analyzed by ANOVA. (D) Percentages of motile and immotile sperm in WT and dHET mature swim-out samples (n= number of males). (E) Quantification of axonemal integrity. A minimum of 100 flagella cross sections were per animal were analyzed (n= number of males). Data are mean ± s.d. and were analyzed by two-tailed, unpaired Student’s t-test (***p<0.001). (F-N) Sperm stained with DAPI (blue), PNA-FITC (green) and MitoTracker (red). Turquoise arrows highlight excess residual cytoplasm. (C). Scale bars: 50 μm (B); 10 μm (F-N).

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

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) Representative images of DAPI-stained WT and dHET mature sperm. Sperm are visualized twice with different exposure times (8 ms and 33 ms), showing examples of DAPI-bright and DAPI-weak sperm in a dHET sample. Scale bars: 50 μm. (B) Sperm nuclei consensus shapes of WT and dHET epididymal sperm. Only DAPI-bright dHET cells were included (n = 3 males per genotype). (C-E) Violin plots depicting the area (C), minimum diameter (D) and length of hook (E) of WT and dHET epididymal sperm. (F) Agarose gel loaded with genomic DNA isolated from WT, Prm1 +/- (P1 +/- ), Prm1 -/- (P1 -/- ), Prm2 +/- (P2 +/- ), Prm2 -/- (P2 -/- ), and dHET cauda epididymal sperm. Part of the data has been published before . (G) IHC against 8-OHdG on epididymal (caput and cauda) tissue from WT and dHET males (Dapi counterstain in grey) Scale bars: 50 μm. (H) Percentage of 8-OHdG-positive sperm from WT and dHET sperm from caput and cauda tissue (n = 3 males per genotype). (I-K) Representative TEM images from (I) WT and (J-K) dHET mature sperm. Scale bars: 1 μm. (J) Ratio of slightly damaged (as shown in J) and heavily damaged (as shown in K) dHET sperm. Data are mean ± s.d. and were analyzed by two-tailed, unpaired Student’s t-test (***p<0.001). (M) Quantification of the difference in grey scale (grey scale range) measured for epididymal sperm nuclei from WT and dHET males (n = number of males).

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, Agarose Gel Electrophoresis, Isolation, Two Tailed Test

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