Nucleolus disassembles in the pre-leptotene spermatocytes.

(A) RNA FISH detection of pre-rRNA in spermatogonia (c-Kit+) and pre-leptotene spermatocytes (SYCP3+). Representative images are shown at left. Spg, spermatogonia; Pre-L, pre-leptotene spermatocytes. Quantification of the pre-rRNA dispersion ratio is shown at right. (B) NPM1 staining in spermatogonia and pre-leptotene spermatocytes. Representative images are shown at left, with quantification of the NPM1 dispersion ratio at right. (C) Analysis of nascent pre-rRNA synthesis by 5-EU incorporation. Nascent RNA was detected by 5-EU labeling, and total pre-rRNA was visualized by RNA FISH. (D) Schematic representation of the nucleolar disassembly in the pre-leptotene spermatocytes. The nucleolus is integrity of FC, DFC, and GC in spermatogonia, whereas all three compartments exhibit widespread dispersion throughout the nucleoplasm in the pre-leptotene spermatocytes. Scale bars, 10 μm.

Nucleolar components dynamically fuse with the XY body.

(A) Distribution of pre-rRNA from leptotene to diplotene. Pre-rRNA was detected by RNA-FISH. SYCP3 staining was used to define meiotic stages. A schematic summary is shown at upper left, and representative images are shown below. The XY body is outlined by dashed circles. L, leptotene; Z, zygotene; e-P, early pachytene; m-P, middle pachytene; 1-P, late pachytene; D, diplotene. Quantification of pre-rRNA enrichment in the XY body is shown at right. (B) Distribution of NPM1 from leptotene to diplotene. Representative images of NPM1 staining are shown at left, with quantification of NPM1 enrichment in the XY body at right. (C) Pre-rRNA ChIRP-seq and NPM1 CUT&Tag-seq profiles in pachytene spermatocytes. Scale bars, 10 μm.

ATR-mediated DNA damage response signaling drives the sequestration of nucleolar components to the XY body.

(A) ATR inhibition and recovery assay in spermatocytes. The spermatocytes were treated with AZ20 (10 pM) for 24 h and collected at the indicated times after washout for immunofluorescence analysis. Representative γH2AX staining images are shown in the middle with a statistical analysis of γH2AX signal in the XY body at lower left. A schematic summary of γH2AX changes is provided at upper right. The XY body is outlined by dashed circles. (B) Analysis of pre-rRNA and NPM1 during ATR inhibition and recovery. Representative pre-rRNA and NPM1 staining images are shown at left with a statistical analysis of pre-rRNA and NPM1 signals in the XY body at lower left. A schematic summary of pre-rRNA and NPM1 changes is provided below. (C) Pre-rRNA ChIRP-seq and NPM1 CUT&Tag-seq profiles on sex chromosomes in WT and H2ax KO pachytene spermatocytes. Scale bars, 10 μm.

Pre-rRNA mediates MSCI.

(A) BMH-21 releases pre-rRNA from the XY body and promotes nascent RNA production in the XY body. Nascent RNA was measured by 5-EU incorporation. Representative images of pre-rRNA FISH and 5-EU staining are shown at left. The XY body is outlined by dashed circles. Quantification of relative 5-EU intensity in the XY body is shown at right. (B) RNA-seq analysis of pachytene spermatocytes treated with BMH-21 (2.5 μM, 12 h). Violin plots show transcriptional changes (BMH-21 versus mock) for autosomal and sex-linked genes. (C) Scatter plot of differentially expressed sex-chromosomal genes after BMH-21 treatment. Upregulated genes were defined by fold change ≥ 2 and adjusted P < 0.05 (DESeq2). (D) RT-qPCR validation of representative X- and Y-linked genes upregulated by BMH-21. Data are presented as mean ± s.e.m. (n=3 biological replicates).

Pre-rRNA excludes RNA Polymerase II from the XY body.

(A) BMH-21 releases pre-rRNA from the XY body and promotes relocalization of Pol II into this compartment. Pachytene spermatocytes were treated with BMH-21 (2.5 μM, 12 h). Pre-rRNA and Pol II were detected by RNA FISH and immunostaining, respectively. Representative images are shown at left, with quantification at right. The XYbody is outlined by dashed circles. Scale bar, 10 μm. (B) Genomic redistribution of pre-rRNA and Pol II after BMH-21 treatment. Pachytene spermatocytes treated with BMH-21 (2.5 μM, 12 h) were subjected to pre-rRNA ChIRP-seq and Pol II CUT&Tag-seq. Representative profiles across the X chromosome are shown at right. (C) Metaplot comparison of pre-rRNA ChIRP-seq and Pol II CUT&Tag-seq signals across the sex chromosome. (D) Representative pre-rRNA ChIRP-seq and RNA Pol II CUT&Tag-seq profiles at X-linked loci.

Pre-rRNA enforces a repressive chromatin state in the XY body.

(A) BMH-21 increases chromatin accessibility on the sex chromosomes in pachytene spermatocytes. Chromatin accessibility was assessed by ATAC-seq after BMH-21 treatment (2.5 μM, 12 h). Violin plots show ATAC-seq changes (BMH-21 versus mock) across autosomes and sex chromosomes. (B) Metaplot comparison of ATAC-seq signals across the sex chromosomes. (C) Heat map of ATAC-seq signals across the sex chromosomes. (D) Scatter plot of sex-chromosomal genes with significantly altered chromatin accessibility after BMH-21 treatment. (E) Overlap between sex-linked genes with increased chromatin accessibility by ATAC-seq and reduced pre-rRNA occupancy by ChIRP-seq after BMH-21 treatment. (F) Working model of pre-rRNA-mediated MCSI. In the XY body, pre-rRNA restricts chromatin accessibility and excludes RNA polymerase II, thereby maintaining transcription repression in the sex chromosomes.

Nucleolus disassembly in the pre-leptotene spermatocytes.

Examination of the nucleolar proteins POLR1E (A) and FBL (B) in spermatogonia (c-Kit+) and pre-leptotene spermatocytes (SYCP3+). Representative images are shown at left. Nuclei were counterstained with DAPI (blue). Spg, spermatogonia; Pre-L, pre-leptotene spermatocytes. Quantification of the dispersion ratio of POLR1E or FBL is shown at right. Scale bars, 10 μm.

Nucleolar components dynamically fuse with the XY body during meiotic prophase I.

Distribution of the nucleolar proteins POLR1E (A) and FBL (B) from leptotene to diplotene. Representative images of POLR1E and FBL staining are shown. L, leptotene; Z, zygotene; e-P, early pachytene; m-P, middle pachytene; l-P, late pachytene; D, diplotene. The XY body is indicated with dashed circles. Scale bars, 10 μm.

The relocation of nucleolar components from spermatogonia to diplotene spermatocytes.

Upper, statistical analysis of the dispersion ratio of pre-rRNA and NPM1 from spermatogonia to diplotene spermatocytes. Spg, spermatogonia; Pre-L, pre-leptotene spermatocytes; L, leptotene; Z, zygotene; e-P, early pachytene; m-P, middle pachytene; l-P, late pachytene; D, diplotene. Lower, schematic summary of nucleolar component dynamics. Following nucleolar disassembly, the nucleolar components are reaggregated and fused into the XY body during meiotic prophase 1.

Detection of the nuclear envelope and rDNA loci in pachytene spermatocytes.

(A) Detection of the nuclear envelope by LaminB1 immunostaining. (B) Detection of rDNA loci by DNA FISH. White arrows indicate rDNA signals. The XY body is indicated with dashed circles. Scale bars, 10 μm.

Persistent RAD51 and BRCA1 signals on the sex chromosomes in ATR-inhibited or H2ax KO pachytene spermatocytes.

(A, B) RAD51 and BRCA1 are still localized on the elongated X chromosome after ATR inhibition in pachytene spermatocytes. Pachytene spermatocytes were treated with the ATR inhibitor AZ20 (10 μM, 24 h). The distribution of RAD51 (A) and BRCA1 (B) was detected by immunostaining. The XY body is indicated with dashed circles. Sex chromosomes are indicated by arrows. (C, D) RAD51 and BRCA1 are still localized on the elongated X chromosome in H2ax KO pachytene spermatocytes. The distribution of RAD51 (C) and BRCA1 (D) in WT and H2ax KO pachytene spermatocytes was detected by immunostaining. Sex chromosomes are indicated by arrows. Scale bars, 10 μm.

BMH-21 releases pre-rRNA from the XY body.

(A) Schematic workflow for the ex vivo drug treatment assay. Mouse pachytene spermatocytes were isolated by FACS and were then cultured with the indicated concentration of BMH-21 (12 h) for further analysis. (B) Analysis of cell viability after BMH-21 treatment. (C) Distribution of pre-rRNA after BMH-21 treatment. Representative images are shown at left. The XY body is outlined by dashed circles. Quantification of pre-rRNA localization in the XY body is shown at right. Scale bars, 10 μm.

XY body architecture and NPM1 retention are maintained after BMH-21 treatment.

Analysis of γH2AX (A) and NPM1 (B) in pachytene spermatocytes with or without BMH-21 treatment (2.5 μM, 12 h). Representative images are shown at left. The XY body is outlined by dashed circles. Quantification of relative γH2AX and NPM1 signal intensity in the XY body is shown at right. Scale bars, 10 μm.

Pre-rRNA is required for spermatogenesis.

BMH-21 (10 μL, 50 uM) was delivered to spermatocytes by rete testis microinjection in 8-week-old male mice. Testes were collected 1 week later for analysis. (A) Examination of pre-rRNA in the XY body. (B) Representative pictures of the testes. (C) H&E staining of the testes. Round spermatids are indicated by arrows. (D) Immunostaining of testis sections. Pachytene spermatocytes were identified based on SYCP3 and γH2AX staining and are indicated by arrows. PNA-positive cells denote haploid spermatids. Scale bar, 50 μm.