Mouse Chd4-NURD is required for neonatal spermatogonia survival and normal gonad development

Abstract

Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of Chd4 and Chd3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of chd4 early in gametogenesis, but not chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that Chd4 controls expression of dmrt1 and its downstream target plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of Chd4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

Keywords: Chromatin remodeling; Mouse gametogenesis; NURD; Spermatogonia.

Fig. 1

Chd4 expression and formation of different NURD complexes during gametogenesis. A Expression of chd4 monitored by immunofluorescence in testis sections of 14 dpp and 2 months (adult) old mouse using two distinct antibodies against Chd4, a rabbit polyclonal anti-Chd4 and anti-Plzf (top panel) and mouse monoclonal anti-Chd4 and anti-Sox9 (bottom panel). Arrows indicate examples of Chd4 positive spermatogonia (Plzf) or Sertoli (Sox9) cells. Cells within the punctuated line are pachytene spermatocytes. Similar results were observed independently using three different mice. B Western blot analysis of different NURD components in samples of cells enriched in undifferentiated spermatogonia (Thy1.2+) (Plzf positive), differentiating spermatogonia (c-Kit+) (Stra8 positive) and the flow-through (FT), the latter from the spermatogonia enrichment procedure, which contains mostly somatic cells, including a large amount of Sertoli cells (Sox9 positive). Lamin B (Lam B) and α-tubulin (Tub.) were used as loading standards. The scheme represents the composition of a canonical NURD complex. C Chd4-participating NURD complexes in enriched fractions of spermatogonia or cells in the flow-through assessed by co-immunoprecipitation with anti-Chd4 as a bait. Non-specific IgG was used as a bait control. Experiments were repeated twice, and the star marks an unspecific reactive band

Fig. 2

Chd4 and chd3 gene targeting design and testis developmental defects in chd4 mutant mice. A, B Testis specific Cre knockout strategy for deletion of chd4 and chd3. See description in “Materials and methods”. C H&E-stained paraffin testis sections of wild type, ddx4-chd4^−/−, and ddx4-chd3^−/− mice. D Quantification of testis weight for wild type and homozygous knockout mice is shown. Images and testis weigh measurements were obtained independently from three mice. E Chd3 transcription levels were measured in ddx4-chd3^−/− total testis and compared to wild-type littermates (2 months old mice, n = 3). Chd4 transcription level in enriched fractions of spermatogonia obtained from 7 dpp ddx4-chd4^−/− and control wild-type litter mate mice (mice, biological replicate n = 3). *** represents P < 0.0001 (two-tailed Student t test)

Fig. 3

Ddx4-chd4^−/− mice show profound defects in gametogenesis. A H&E-stained histological sections of wild type and ddx4-chd4^−/− testis. Stars mark seminiferous tubules with absent germ cells. Note unchanged number and morphology of Sertoli cells (indicated by green arrows). B Histological sections of wild type and ddx4-chd4^−/− testis showing seminiferous tubules immunolabeled with Sox9 (a marker of Sertoli cells) and Tra98 (to mark germ cells). P pachytene cells, Se sertoli cells, Rs rounded spermatids. C H&E-stained histological sections of wild type and ddx4-chd4^−/− ovaries. F follicles, CL corpora lutea

Fig. 4

Spermatogonia cell survival requires Chd4. A Histological sections of wild type and ddx4-chd4^−/− testis cord from 9 days old mice stained with H&E (a, b) and Hematoxylin and immunostained with Stra8 antibodies (marking differentiating spermatogonia) (c, d). Arrows mark Stra8 positive cells present in wild type but reduced in number or absent in the mutant. Experiments were done in at least three different mice showing similar results. B Immunostaining of sections of developing testis reveals severe loss of germ cells (Tra98) in chd4^−/− testis cords. C Quantitation of number of cell positive for Tra98, Plzf, and Sox9. 4 dpp testis (Plzf, wild type, 9.9 ± 4.1, n = 47 seminiferous tubules; chd4^−/− 2.2 ± 1.4, n = 56, P < 0.0001, t test. Tra98, wild type 12.6 ± 5.3, n = 35; chd4^−/− 3.0 ± 1.9, n = 37, P < 0.0001, t test). 7 dpp testes (Plzf, wild type, 14.6 ± 5.0, n = 28; chd4^−/− 3.4 ± 2.4, n = 45, P < 0.0001, t test. Tra98, wild type 19.6 ± 5.9, n = 48; chd4^−/− 3.0 ± 2.1, n = 24, P < 0.0001, t test). Experiments were done in three different mice showing similar results combined in our quantification. D Higher cell death in chd4^−/− knockout testes than in wild-type controls at 4 days post-partum. Apoptotic cells were visualized using TUNEL staining in three mice of each genotype. Series of images (tiles) were used to construct the lower magnification (top panel) and the magnification showed a single tile in the bottom panel. The percentage of apoptotic cells was counted only in the tubule cross sections that contained apoptotic cells and was normalized to total number of germ cells. 19.05 ± 11.17, n = 468 (mean ± SD, n = number of seminiferous tubules counted) wild type and 38.25 ± 13.72 chd4^−/−, n = 344; P < 0.0001 (two-tailed Student t test). The arrows indicate apoptotic cells and arrowhead non-apoptotic germ cells

Fig. 5

Chd4 genome-wide chromatin binding profile and analysis. A Heatmaps showing Chd4 occupancy centered in transcription start site (TSS) for all genes. The left and right panel correspond to heatmaps obtained from enriched spermatogonia fractions and whole 7 dpp testis, respectively. B Examples of Chd4 occupancy at or near dmrt1 promoter assessed by ChIP-seq. The top and bottom panels correspond to profiles obtained from enriched spermatogonia fractions and whole 7 dpp testis, respectively. C Pie chart depicting genomic features associated with Chd4 peaks. D Gene ontology analysis of genes associated to Chd4 ChIP-seq signals. E RT-PCR analysis for dmrt1 expression at exon 4–5 boundary in wild type and chd4 knockout testis

Fig. 6

Effect of chd4 deletion in Dmrt1 and Plzf expression. A Histological sections of wild type and ddx4-chd4^−/− testis from 1, 4, and 7 dpp mice stained with Chd4 and Dmrt1 antibodies. Arrows indicate examples of Chd4 positive spermatogonia (Wt) or Chd4 negative staining in ddx4-chd4^−/− spermatogonia and the correspondent cell stained for Dmrt1. B Quantitation of fluorescence intensity of Dmrt1 expression on spermatogonia is shown for wild type (Wt) and ddx4-chd4^−/− knockout at ages of 1 dpp (Wt = 82.17 ± 22.02, n = 272; and KO = 37.20 ± 10.01, n = 287, P < 0.0001, Student t test) and 4 dpp (Wt = 71.09 ± 20.93, n = 242; and KO = 43.47 ± 14.88, n = 198, P < 0.0001, Student t test). Values represent the median fluorescence intensity ± standard deviation, n = total number of cells analyzed from 3 biological replicates using 3 different mice. C Histological sections of wild type and ddx4-chd4^−/− testis from 1, 4, and 7 dpp mice stained with antibodies against Plzf. D Quantitation of fluorescence intensity of Plzf expression on spermatogonia is shown for wild type and ddx4-chd4^−/− knockout at ages of 1 dpp (wt = 17.94 ± 4.10, n = 300; and ddx4-chd4^−/−  = 10.89 ± 4.07, n = 339, P < 0.0001, Student t test) and 4 dpp (Wt = 17.78 ± 4.70, n = 282; and ddx4-chd4^−/− = 9.11 ± 3.75, n = 230, P < 0.0001, Student t test). Values represent the median fluorescence intensity ± standard deviation, n = total number of cells analyzed from 3 biological replicates using 3 different mice. E Diagram representing Chd4, Dmrt1, and Plzf relationship in spermatogonia stem cell maintenance. Note that our studies do not address the possibility that Plzf may be a direct target of CHD4