Distinct requirements for Sin3a in perinatal male gonocytes and differentiating spermatogonia

Abstract

Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through a variety of transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Mitotic germ cells expressing stimulated by retinoic acid gene 8 (Stra8) that lacked Sin3a exhibited increased DNA damage and apoptosis, yet collectively progressed through meiosis and spermiogenesis and generated epididymal sperm at approximately 50% of control levels, sufficient for normal fertility. In contrast, perinatal gonocytes lacking Sin3a underwent rapid depletion that coincided with cell cycle reentry, exhibiting 2.5-fold increased histone H3 phosphorylation upon cycling that suggested a prophase/metaphase block; germ cells were almost entirely absent two weeks after birth, resulting in sterility. Gene expression profiling of neonatal testes containing Sin3a-deleted gonocytes identified upregulated transcripts highly associated with developmental processes and pattern formation, and downregulated transcripts involved in nuclear receptor activity, including Nr4a1 (Nur77). Interestingly, Nr4a1 levels were elevated in testes containing Stra8-expressing, Sin3a-deleted spermatogonia. SIN3A directly binds to the Nr4a1 promoter, and Nr4a1 expression is diminished upon spermatogonial differentiation in vitro. We conclude that within the male germline, Sin3a is required for the mitotic reentry of gonocytes, but is dispensable for the maintenance of differentiating spermatogonia and subsequent spermatogenic processes.

Fig. 1

Testes of Stra8-cre; Sin3a^Δ/fl male mice have reduced weights but contain equivalent numbers of undifferentiated spermatogonia and cycling, premeiotic germ cells. A. Schematic illustration denoting the expression patterns of Stra8-cre and Vasa-cre transgenes in mouse spermatogonia: the former is initially expressed in a subset of Type A aligned (A_al) undifferentiated cells (dashed green line) and continues its expression through late Type B differentiating cells (solid green line), while the latter is expressed throughout all spermatogonial stages – undifferentiated A single (A_s), A paired (A_pr), A_al, and differentiating A₁–A₄, Intermediate (In), and B (solid red line). Matings of these cre driver mice with floxed Sin3a (Sin3a^fl/fl) mice generate SSKO (Stra8-cre; Sin3a^Δ/fl) and VSKO (Vasa-cre; Sin3a^Δ/fl) conditional gene-targeted mice, respectively. B. 6-wk-old SSKO male mice have testis weight/body weight ratios that are 46.5% reduced compared to littermate controls (N=7, ***p<0.001). C and D. Seminiferous tubule cross-sections of 6-wk-old testes stained with hematoxylin and eosin (H+E) reveal germ cells at all stages of development in SSKO and controls; arrowheads identify enlarged, multinucleated germ cells in SSKO tubules. E and F. Cross-sections of 6-wk-old seminiferous tubules immunostained for germ cell marker GCNA1. G and H. Cross-sections of 6-wk-old seminiferous tubules immunostained for germline stem cell marker PLZF (arrows). I and J. Seminiferous tubule cross-sections of 6-wk-old males injected with BrdU, examined 48 h later with anti-BrdU antibodies. Scale bars = 50μm.

Fig. 2

1-wk-old SSKO spermatogonia exhibit a modest increase in DNA double strand breaks and apoptosis. A and B. Seminiferous tubule cross-sections of 1-wk-old testes stained with H+E reveal equivalent numbers of cells in SSKO and controls. C and D. Cross-sections of 1-wk-old seminiferous tubules co-immunostained for GCNA1 (red) and SIN3A (green); arrowheads identify GCNA1⁺ cells containing SIN3A (C, D), while arrows denote GCNA1⁺ cells in SSKO sections lacking SIN3A (D). E and F. Cross-sections of 1-wk-old seminiferous tubules co-immunostained for GCNA1 (red) and γH2A.X (green); arrows identify GCNA1⁺ cells in SSKO sections that exhibit γH2A.X, signifying DNA double strand breaks. G and H. Cross-sections of 1-wk-old seminiferous tubules co-stained for DNA (DAPI, red) and apoptosis (TUNEL, green); arrows denote TUNEL⁺ cells in SSKO sections. Scale bars = 50μm.

Fig. 3

Testes of Vasa-cre; Sin3a^Δ/fl male mice are completely depleted of germ cells between 1 and 6 wks of age. A. VSKO testis weight/body weight ratios undergo a significant, progressive reduction from 27.7% to 84.9% between 1 and 6 wks of age, relative to littermate controls (N=3 for each time point; **p<0.01, ***p<0.001). B and C. Seminiferous tubule cross-sections of 1.5-wk-old testes stained with H+E; asterisk denotes meiotic spermatocytes in control section (B). Arrowhead identifies enlarged, abnormal germ cell and arrows denote cell remnants in VSKO section (C). D and E. Cross-sections of 6-wk-old seminiferous tubules stained with H+E; while control sections contain germ cells at all stages and in expected numbers (D), VSKO sections are completely devoid of germ cells, exhibiting a ‘Sertoli cell only’ phenotype (E). Scale bars = 50 μm.

Fig. 4

VSKO germ cells begin to exhibit DNA double strand breaks upon cell cycle reentry. A and B. Seminiferous tubule cross-sections of postnatal day (P)0 testes stained with H+E; arrows identify quiescent gonocytes in the lumen of control (A) and VSKO (B) tubules. C and D. Cross-sections of P0 seminiferous tubules co-immunostained for GCNA1 (red) and Ki67 (green); note the absence of co-localization. E and F. Cross-sections of P0 seminiferous tubules co-immunostained for GCNA1 (red) and γH2A.X (green); no GCNA1⁺ cells exhibit γH2A.X in either control (E) or VSKO (F) sections. G and H. Cross-sections of P2 seminiferous tubules co-immunostained for GCNA1 (red) and SIN3A (green); control sections exhibit GCNA1⁺ cells containing SIN3A (G), while GCNA1⁺ cells in VSKO sections lack SIN3A (H). Inset features cells within the dashed lines in separate channels for SIN3A and GCNA1. I and J. Cross-sections of P2 seminiferous tubules co-immunostained for GCNA1 (red) and Ki67 (green); arrows identify GCNA1⁺ cells exhibiting Ki67, signifying reentry into the cell cycle. K and L. Cross-sections of P2 seminiferous tubules co-immunostained for GCNA1 (red) and γH2A.X (green); arrows identify GCNA1⁺ cells exhibiting γH2A.X in VSKO (L), but not in control (K) sections. The outlines of distinct seminiferous tubules in C-F and I-L are demarcated with dashed lines, representing basement membranes. Scale bars = 50μm.

Fig. 5

P3 VSKO germ cells exhibit a G2/M phase block and increased apoptosis that contribute to rapid cell depletion. A and B. Seminiferous tubule cross-sections of P3 testes stained with H+E; arrowheads identify germ cells that have migrated to the basement membranes in control (A) but not in VSKO (B) tubules. Asterisks denote VSKO cross-sections already devoid of germ cells (B). C and D. Cross-sections of P3 seminiferous tubules co-immunostained for GCNA1 (red) and Ki67 (green); almost one-third of GCNA1⁺ cells exhibit Ki67 in both control (C) and VSKO (D) sections. E and F. Cross-sections of P3 seminiferous tubules co-immunostained for GCNA1 (red) and phosphorylated histone H3 (pH3, green); arrows identify GCNA1⁺ cells containing pH3. G and H. Cross-sections of P3 seminiferous tubules co-immunostained for GCNA1 (red) and activated caspase 3 (Casp3; green); arrows identify GCNA1⁺ cells that exhibit Casp3 in VSKO (H), but not in control (G) sections. I. top, GCNA1⁺ cells per complete testis section in P3 VSKO males was significantly diminished when compared to controls (N=27 sections representing 3 animals for control, VSKO; ***p<0.001). I. bottom, Percentages of GCNA1⁺ cells that exhibited Ki67 (% Ki67⁺; GCNA1⁺, black), pH3 (% pH3⁺; GCNA1⁺, red), and Casp3 (% Casp3⁺; GCNA1⁺; blue) in P3 control (solid bars) and VSKO (hashed bars) testis sections (N=9 complete sections representing 3 animals for control, VSKO; *p<0.05, ***p<0.001). The outlines of distinct seminiferous tubules in E-H are demarcated with dashed lines, representing basement membranes. Scale bars = 50μm.

Fig. 6

VSKO testicular transcriptome at P0 exhibits an upregulation of genes important for development and patterning and a downregulation of Nr4a1, a gene conversely upregulated in P9 SSKO testes. A. Gene ontology (GO) categories and p-values for the top up- and downregulated genes expressed in P0 VSKO testes relative to wild type (Sin3a^+/+) testes. B. Heat map of an individual cluster (hierarchically generated from 2 VSKO and 2 wild type testicular transcriptomes) of downregulated genes of interest. This specific cluster contains Cxcr4, Pla2g3, and Nr4a1. C and D. Quantitative (q)RT-PCR validation of microarray data, showing a 6.82-fold downregulation of Nr4a1 for P0 VSKO (C) and a 1.95-fold upregulation of Nr4a1 for P9 SSKO (D), relative to control samples (N=3, *p<0.05, **p<0.01). E. qRT-PCR revealing a 7.44-fold upregulation of Nr4a1 in THY1⁺/GFRA1⁺ cells (enriched from P7 wild type testes by magnetic-activated cell sorting, representing undifferentiated spermatogonia) relative to KIT⁺ cells (representing differentiating spermatogonia) (N=4, *p<0.05). F. qRT-PCR revealing a 3.03-fold downregulation of Nr4a1 in cultured wild type undifferentiated spermatogonia treated with 1 μM retinoic acid (RA) for 24 h (N=4, ***p<0.001). G. ChIP for SIN3A and IgG control on THY1⁺/GFRA1⁺ cells, showing SIN3A enrichment at the Nr4a1 transcription start site (TSS), but not at the Actb TSS or at a region −1.6kb upstream of Nr4a1 (N=3, ***p<0.001). 18s rRNA is the internal control transcript for C–F.