Journal: bioRxiv

Article Title: Genome compartments guide protamine replacement and genome stability during spermiogenesis

doi: 10.64898/2026.03.08.710361

Figure Lengend Snippet: a , Fluorescence images of H3.3-mCherry and H4-Venus signals in seminiferous tubules from control ( Phf7 CA/+ ) and catalytic mutant ( Phf7 CA/CA ) testes. Spermiogenic stages are indicated in merged images. Asterisks indicate regions showing non-specific fluorescence signals. b, Immunofluorescence analysis of PRM1 and H3.3-mCherry signals in seminiferous tubules from Phf7 CA/+ and Phf7 CA/CA testes. Roman numbers indicated the spermatogenic stages of seminiferous tubules. Asterisks indicate regions showing non-specific fluorescence signals in the interstitial regions. Scale bar, 50 µm. c, Average ATAC-seq signal profiles across A/B compartment boundaries in Phf7 CA/+ and Phf7 CA/CA spermatids. Relative ATAC-seq signal is plotted as a function of genomic position within A and B compartments, illustrating impaired chromatin remodeling in the catalytic mutant.

Article Snippet: Approximately 2–4 pL of injection mixture containing 50 ng/μL Cas9 mRNA (TriLink Biotechnologies) and 0.4 pM each of guide RNA1 and guide RNA2 targeting Prm1 and Prm2 genome (Synthego) was injected into the cytoplasm of zygotes using a pneumatic microinjector (Narishige) equipped with a piezo micromanipulator (Prime Tech).

Techniques: Fluorescence, Control, Mutagenesis, Immunofluorescence

Journal: bioRxiv

Article Title: Genome compartments guide protamine replacement and genome stability during spermiogenesis

doi: 10.64898/2026.03.08.710361

Figure Lengend Snippet: a , Immunofluorescence analysis of PRM1 (magenta), and DNA (cyan) in representative spermatogenic cells. Scale bar, 10 μm. b , Integrated genome-wide view of three-dimensional genome organization, chromatin accessibility, and PRM1 incorporation during spermiogenesis. A Hi-C contact map of mouse chromosome 10 is shown together with the PC1 value of the Hi-C correlation matrix, indicating A/B compartment status. HOTs and MOTs and PRM1 preferentially associated partitions (PPAPs) are indicated. PRM1 CUT&Tag signal (normalized to IgG) and spike-in–normalized ATAC-seq signal tracks for steps 10–12, steps 13–14, and tSpm are shown. c , Overlap between PPAPs, HOTs, and MOTs defined in 50-kb genomic bins. PPAPs were defined as genomic bins exhibiting significant PRM1 CUT&Tag enrichment in steps 13–14 spermatids. Numbers indicate bin counts for each category and their intersections. d, Observed/expected (obs/exp) enrichment of PPAPs within HOTs, MOTs, and A/B compartment subclasses (strong A, weak A, strong B, weak B) in step 13–14 spermatids. e , PRM1 enrichment stratified by the magnitude of chromatin accessibility decrease. Genomic bins were ranked according to the change in spike-in–normalized ATAC-seq signal between steps 10–12 and steps 13–14 (deltaATAC = steps13–14 − steps10–12) and grouped into deciles. Violin plots show PRM1 CUT&Tag enrichment (log₂ PRM1/IgG) in steps 13–14 spermatids for each decile.

Article Snippet: Approximately 2–4 pL of injection mixture containing 50 ng/μL Cas9 mRNA (TriLink Biotechnologies) and 0.4 pM each of guide RNA1 and guide RNA2 targeting Prm1 and Prm2 genome (Synthego) was injected into the cytoplasm of zygotes using a pneumatic microinjector (Narishige) equipped with a piezo micromanipulator (Prime Tech).

Techniques: Immunofluorescence, Genome Wide, Hi-C

Journal: bioRxiv

Article Title: Genome compartments guide protamine replacement and genome stability during spermiogenesis

doi: 10.64898/2026.03.08.710361

Figure Lengend Snippet: a , Chromosome-wide occupancy of HOTs, MOTs, and PPAPs. Bars indicate the percentage of genomic bins assigned to each category across chromosomes 1–19. b , Observed/expected (obs/exp) enrichment of PRM1-high regions in tSpm across HOTs, MOTs, and A/B compartment subclasses (strong A, weak A, strong B, weak B). Values are shown as log₂ enrichment. c , PRM1 CUT&Tag enrichment (log₂ PRM1/IgG) in tSpm stratified by percentile groups (Q10 and Q100). P- value was calculated using a two-sided Welch’s t-test.

Article Snippet: Approximately 2–4 pL of injection mixture containing 50 ng/μL Cas9 mRNA (TriLink Biotechnologies) and 0.4 pM each of guide RNA1 and guide RNA2 targeting Prm1 and Prm2 genome (Synthego) was injected into the cytoplasm of zygotes using a pneumatic microinjector (Narishige) equipped with a piezo micromanipulator (Prime Tech).

Techniques:

Journal: bioRxiv

Article Title: Genome compartments guide protamine replacement and genome stability during spermiogenesis

doi: 10.64898/2026.03.08.710361

Figure Lengend Snippet: a , Schematic illustration of the generation of Prm1/Prm2 double-knockout (dKO) mice. Guide RNAs targeting the Prm1 and Prm2 loci on chromosome 16 were used to generate a 6,976-bp deletion encompassing coding exons. Prm1/Prm2 dKO male mice were generated by elongated spermatid injection (ELSI) using Prm1/Prm2 double-heterozygous (dHET) oocytes and spermatids. b, Immunofluorescence analysis of PRM2 from WT and Prm1/Prm2 dKO testes. PRM2 signal was absent in step 15 dKO spermatids. Scale bar, 10 µm. c, Cell sorting plots and gate settings showing spermatid populations isolated from WT and Prm1/Prm2 dKO testes. Populations corresponding steps 8-9, 10-12, 13-14, 15-16, and tSpm populations are indicated. d, Quantification of tSpm frequency in WT and Prm1/Prm2 dKO testes. p -value was calculated using a two-sided statistical test. e, Genome browser snapshots showing Hi-C PC1 profiles, PRM1 CUT&Tag signal (normalized to IgG), and spike-in–normalized ATAC-seq signals in WT and Prm1/Prm2 dKO spermatids across late spermiogenic stages. ATAC-seq signals at A compartments remain elevated in Prm1/Prm2 dKO spermatids at steps 15–16, in contrast to WT spermatids. f , Stage-resolved dynamics of spike-in–normalized ATAC-seq signals within PPAPs, HOTs, and MOTs in dKO spermatids. g , Average ATAC-seq signal profiles across A/B compartment boundaries in WT and dKO spermatids.

Article Snippet: Approximately 2–4 pL of injection mixture containing 50 ng/μL Cas9 mRNA (TriLink Biotechnologies) and 0.4 pM each of guide RNA1 and guide RNA2 targeting Prm1 and Prm2 genome (Synthego) was injected into the cytoplasm of zygotes using a pneumatic microinjector (Narishige) equipped with a piezo micromanipulator (Prime Tech).

Techniques: Double Knockout, Generated, Injection, Immunofluorescence, FACS, Isolation, Hi-C

Journal: bioRxiv

Article Title: Genome compartments guide protamine replacement and genome stability during spermiogenesis

doi: 10.64898/2026.03.08.710361

Figure Lengend Snippet: a , Genotyping analysis of Prm1/Prm2 wild type (WT), dounle heterozygous (dHET), and double knockout (dKO mice). Representative electrophoresis images show diagnostic bands corresponding to WT (amplified with F1-R1 primer set in ) and KO (amplified with F1-R2 primer set in ) alleles, respectively. b, Immunofluorescence analysis of PRM1 (magenta) and DNA (cyan) in purified WT and dKO spermatids. Scale bar, 10 μm. c Immunofluorescence analysis of TNP1 in seminiferous tubules from WT and Prm1/Prm2 dKO testes. Representative images are shown for stage XII (step 12) and stages II–III (step 14). PNA and DNA counterstaining delineate acrosome morphology and nuclear structure. Scale bar, 10 µm. d, Flow cytometry–based gating strategy for quantification of tSpm populations.

Article Snippet: Approximately 2–4 pL of injection mixture containing 50 ng/μL Cas9 mRNA (TriLink Biotechnologies) and 0.4 pM each of guide RNA1 and guide RNA2 targeting Prm1 and Prm2 genome (Synthego) was injected into the cytoplasm of zygotes using a pneumatic microinjector (Narishige) equipped with a piezo micromanipulator (Prime Tech).

Techniques: Double Knockout, Electrophoresis, Diagnostic Assay, Amplification, Immunofluorescence, Purification, Flow Cytometry

Journal: bioRxiv

Article Title: Genome compartments guide protamine replacement and genome stability during spermiogenesis

doi: 10.64898/2026.03.08.710361

Figure Lengend Snippet: a , Schematic illustration of the experimental workflow for detecting genome-wide DNA damage in epididymal sperm. Spermatozoa were collected from the caput, corpus, and cauda regions of the epididymis from Prm1/Prm2 dHET males, followed by isolation of genomic DNA, recovery of low-molecular-weight DNA fragments, and next-generation sequencing. b, Cell sorting plots showing sperm populations collected from the caput, corpus, and cauda epididymis of WT and Prm1/Prm2 dHET males using H4-Venus and H3.3-mCherry reporters. c, Flow cytometry histograms showing fluorescence intensity distributions of H4-Venus and H3.3-mCherry signals in epididymal sperm populations. d, Size distributions of total genomic DNA isolated from caput, corpus, and cauda sperm from WT and Prm1/Prm2 dHET males. e, Size distributions of purified low-molecular-weight DNA fragments recovered from caput and corpus sperm. f, Enrichment of PPAPs among genomic bins ranked by the DNA fragment sequencing signal intensity in the caput sperm. Genomic bins were ordered from highest to lowest sequencing signal, and the fraction overlapping PPAPs was calculated for each rank fraction. g, Schematic model illustrating the relationship between PRM1 incorporation, chromatin accessibility, and DNA damage in epididymal sperm. In dHET sperm, DNA damage in cauda sperm is broadly distributed across the genome, whereas caput sperm exhibits preferential DNA damage accumulation within A compartments.

Article Snippet: Approximately 2–4 pL of injection mixture containing 50 ng/μL Cas9 mRNA (TriLink Biotechnologies) and 0.4 pM each of guide RNA1 and guide RNA2 targeting Prm1 and Prm2 genome (Synthego) was injected into the cytoplasm of zygotes using a pneumatic microinjector (Narishige) equipped with a piezo micromanipulator (Prime Tech).

Techniques: Genome Wide, Isolation, Molecular Weight, Next-Generation Sequencing, FACS, Flow Cytometry, Fluorescence, Purification, Sequencing

Journal: bioRxiv

Article Title: Genome compartments guide protamine replacement and genome stability during spermiogenesis

doi: 10.64898/2026.03.08.710361

Figure Lengend Snippet: Schematic model summarizing step-resolved chromatin remodeling during spermiogenesis. Early spermatids (steps 1–8) retain histone-rich chromatin with localized accessibility at transcription start sites. During mid-spermiogenesis (steps 10–12), chromatin undergoes a transient genome-wide hyper-relaxed state characterized by extensive histone eviction and the emergence of HOTs, predominantly within A compartments. These hyper-accessible regions constitute PPAPs and serve as major sites of PRM1 incorporation during late spermiogenesis (steps 13–14). Protamine deposition is coupled to rapid chromatin closing, resulting in progressive compartment-biased condensation. In the absence of protamines, hyper-accessible A-compartment regions fail to undergo proper closure, leading to persistent accessibility and preferential DNA fragmentation. This non-monotonic remodeling trajectory establishes a spatially organized transition from histone-based to protamine-based chromatin architecture during sperm maturation.

Article Snippet: Approximately 2–4 pL of injection mixture containing 50 ng/μL Cas9 mRNA (TriLink Biotechnologies) and 0.4 pM each of guide RNA1 and guide RNA2 targeting Prm1 and Prm2 genome (Synthego) was injected into the cytoplasm of zygotes using a pneumatic microinjector (Narishige) equipped with a piezo micromanipulator (Prime Tech).

Techniques: Genome Wide