Chd4-floxed mice (chd4fl/fl) have been described [30]. chd3-floxed mice were generated by Cyagen Biosciences using homologous recombination of a targeting vector in C57Bl/6 embryonic stem cells. The targeting vector incorporated a 5′ LoxP site inserted between exons 12 and 13 and a 3′ LoxP site inserted between exons 20 and 21 of the wild-type chd3 allele. Transgenic Cre recombinase mice ddx4-CreFVB−Tg(ddx4−cre)1Dcas/J was purchased from The Jackson Laboratory (Bar Harbor, ME). chd3 or chd4 gonad-specific knockouts and wild-type heterozygotes littermates were obtained from crosses between female homozygous flox/flox mice with male heterozygous Cre/+; chd3 wt/flox and/or chd4 wt/flox mice.

All experiments conformed to relevant regulatory standards guidelines and were approved by the Oklahoma Medical Research Foundation-IACUC (Institutional Animal Care and Use Committee).

Mice genotyping

Characterization of wild type and floxed alleles was carried out by PCR using the following oligonucletides: chd3 forward 5′-GGGTGGAGGTGGAAAGTGTA, chd3 reverse 5′-AGAGGACAGGTCACAGGACAA, chd4 forward 5′-TCCAGAAGAAGACGGCAGAT and chd4 reverse 5′-CTGGTCATAGGGCAGGTCTC. The presence of cre recombinase allele were determine by PCR using the following primers: ddx4-Cre forward 5′-CACGTGCAGCCGTTTAAGCCGCGT, ddx4-Cre reverse 5′-TTCCCATTCTAAACAACACCCTGAA.

Real-time PCR

Total RNA was isolated from adult testis or from enriched fractions of spermatogonia with the Direct-zol RNA MiniPrep Plus kit (Zymo Research). RNA (2.0 μg) was oligo-dT primed and reverse-transcribed with the high-capacity RNA-to-cDNA kit (Applied Biosystems). Exon boundaries of chd4 and chd3 were amplified using TaqMan Assays (Applied Biosystems) as directed by the manufacturer using Beta-2 macroglobulin as standard. TaqMan Mm01190896_m1 (chd4), Mm01332658_m1 (chd3), Mm00437762_m1 (Beta-2 microglobulin), and Mm00443809_m1 (dmrt1). Gene expression was normalized with respect to wild type with wild-type expression levels considered to be 1.

Western blot cell lysates

Total testis or enriched cells fractions were lysed in ice-cold protein extraction buffer containing 0.1% Nonidet P-40, 50 mM Tris–HCl, pH 7.9, 150 mM NaCl, 3 mM MgCl2, 3 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF and protease inhibitors (ThermoFisher Scientific, A32965) followed by sonication (3 pulses of 10 s) using micro ultrasonic cell disrupter (Kontes). The relative amount of protein was determined measuring absorbance at 260 nm using NanoDrop 2000c spectrophotometer (ThermoFisher Scientific). Proteins were solubilized with 2× sample buffer (4% SDS, 160 mM Tris–HCl, pH 6.8, 20% glycerol, 4% mM β-mercaptoethanol, and 0.005% bromophenol blue) and 30 µg/lane of sample were separated by 4–15% gradient Tris–acetate SDS–PAGE and electro transferred to PVDF membrane (Santa Cruz Biotechnology, sc-3723). The blots were probed with individual primary antibodies, and then incubated with HRP-conjugated goat anti-mouse or rabbit antibody as required. In all blots, proteins were visualized by enhanced chemiluminescence, and images acquired using Western Blot Imaging System c600 (Azure Biosystems). ImageJ software were used for quantification of non-saturated bands and α-tubulin were used for normalization. Antibodies used are detailed in Additional file 11: Table S4.

Histology and immunostaining

Testes and ovaries were dissected, fixed in 10% neutral-buffered formalin (Sigma) and processed for paraffin embedding. After sectioning (5–8 µm), tissues were positioned on microscope slides and analyzed using hematoxylin and eosin using standard protocols. For immunostaining analysis, tissue sections were deparaffinized, rehydrated and antigen was recovered in sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) by heat/pressure-induced epitope retrieval. Incubations with primary antibodies were carried out for 12 h at 4 °C in PBS/BSA 3%. Primary antibodies used in this study are detailed in Additional file 11: Table S4, following three washes in 1× PBS, slides were incubated for 1 h at room temperature with secondary antibodies. A combination of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Jackson laboratories) with Rhodamine-conjugated goat anti-mouse IgG and Cy5-conjugated goat anti-human IgG each diluted 1:450 were used for simultaneous triple immunolabeling. Slides were subsequently counterstained for 3 min with 2 µg/ml DAPI containing Vectashield mounting solution (Vector Laboratories) and sealed with nail varnish. We use Zen Blue (Carl Zeiss, inc.) for imaging acquisition and processing.

Enrichment of spermatogonia populations

Our procedure of cell enrichment followed [31, 32]. Briefly, testis from 7 dpp mice (or any other indicated age) were removed from mice and placed in a Petri dish containing Dulbecco’s Modified Eagle Medium (DMEM without phenol red). After detachment of the tunica albuginea, the seminiferous tubules were loosen using forceps and incubated in a 15 mL tube containing DMEM containing 1 mg/mL of collagenase, 300 U/mL of hyaluronidase and 5 mg/mL DNAse I (StemCell Technologies) under gentile agitation for 10 min. The seminiferous tubule clumps were pelleted by gravity and the cell suspension containing interstitial cells was discarded. The tubules were then incubated of with 0.05% Trypsin–EDTA solution (Mediatech Inc) for 5 min and the reaction was stopped by adding 10% volume of 10% BSA in PBS. Single cell suspension was obtained by mechanical resuspension followed by filtration through a 40-µm-pore-size cell strainer and dead cells were removed using Dead Cell Removal Kit (Miltenyi Biotec 130-090-101). Differentiating c-KIT+ neonate spermatogonia cells were magnetically labeled with CD117 (c-KIT+) MicroBeads (Miltenyi Biotec 130-091-224) and isolated using MS columns (Miltenyi Biotec 130-042-201) according to manufacturer’s instructions. After the depletion of the c-KIT+ cells, the population of undifferentiated neonate spermatogonia cells were separated using CD90.2 (THY1.2+) MicroBeads (Miltenyi Biotec 130-121-278). Relative enrichment of cell populations was evaluated by STRA8 (c-Kit fractions) or PLZF (THY1.2 fractions) western blots (Fig. 1B). After c-kit and THY1.2 separation, the flow-through mostly contained Sertoli cells (SOX9 positive, Additional file 3: Fig. S3). The number of cells obtained from a pool of 4 mice testis at 7 dpp was approximately 3.43 × 105 in THY1.2 fractions and 5.71 × 105 in c-Kit fractions.

Primary spermatocyte enrichment

Synchronized pachytene spermatocytes from the first spermatogenic wave were purified as described in [33]. Briefly, 2 dpp mice were injected for 7 consecutive days with WIN 18,446 to arrest germ cells as spermatogonia. The next day (9 dpp), mice were injected with retinoic acid (RA) to induce their coordinated maturation. 2 Mice were killed at 13 days after RA injection (22 dpp). Testes were disaggregated using proteases and meiocytes were then purified by fluorescence activated cell sorting (FACS). Unlike Romer et al., after testes disaggregation and prior to FACS, cells were not washed (by centrifugation and resuspension) to avoid breakage of fragile pachytene cells. Instead, cells were sorted from the protease-containing buffer. Purity and stage of cells was assessed by immunofluorescence of chromosome spreads using anti SYCP1 and anti SYCP3 antibodies. More than 80% of cells were at pachytene stage.


Co-immunoprecipitation experiments were performed using testis of wild type or ddx4chd3−/− mouse (adult—2 months). After detunication, seminiferous tubules were loosen using forceps, washed twice with cold 1× PBS and lysed using ice-cold RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.5% Deoxycholate) containing protease inhibitors (ThermoFisher Scientific, A32965), sheared using 23 G needle, incubated on ice for 15 min and centrifugated at 1000×g for 10 min at 4 °C. Supernatant were collected in a separate tube, the pellet was resuspended in RIPA buffer, disrupted by sonication (3 pulses of 10 s) and centrifuged 12,000×g. This second supernatant was combined with the previous one and protein concentration was determined. We used 1 mg of protein for each immunoprecipitation. Lysates were pre-cleared with protein G magnetic beads (BioRad, 161-4023) for 1 h at room temperature and incubated with rabbit anti-Chd3 (5 μg, Bethyl A301-220A), rabbit anti-Chd4 (2 μg, Abcam ab72418), or rabbit IgG (5 μg Jackson ImmunoResearch, 011-000-003). Lysates were rotated overnight at 4 °C and immune complexes were collected with protein G magnetic beads (2 h at 4 °C). Beads were washed 4 times with washing buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% TX100, 5% glycerol) and two times with PBS. Proteins were eluted by boiling the beads with 2× sample buffer and analyzed by SDS–PAGE as described above.

EdU-based proliferation assay

Mice at indicated age received subcutaneous injection of EdU (50 mg/kg) (Invitrogen, A10044) 3 h prior euthanasia. After that, testes were removed and processed for whole-mount immunohistochemistry. EdU was detected by incubation of testis samples with reaction mix (2 mM CuSO4, 50 mM ascorbic acid and 2 mM Alexa Azide conjugates (488 or 647) in PBS) for 3 h at room temperature.

Whole-mount seminiferous tubules

Immunohistochemistry of whole-mount seminiferous tubules was performed as described [34]. Briefly, after detachment of the tunica albuginea, the seminiferous tubules were loosen using forceps and incubated in a 15 mL tube containing DMEM containing 1 mg/mL of collagenase, 300 U/mL of hyaluronidase and 5 mg/mL DNAse I (StemCell Technologies) under gentile agitation for 10 min. The seminiferous tubules clumps were pelleted by gravity and the cell suspension containing interstitial cells was discarded. Seminiferous tubules were fixed for 4 h in 4% PFA (pH7.2 in PBS) at 4 °C. After extensively wash in PBS, the tubules were permeabilized with series of MeOH/PBS (25, 50, 75, 95%, and twice in 100% MeOH) for 15 min at room temperature, treated with MeOH: DMSO: H2O2 (4:1:1), and rehydrated with MeOH/PBS (50, 25% and twice in PBS). Samples were incubated in ice-cold blocking solution PBSMT (PBS with 2% non-fat dry milk and 0.5% triton X-100) for 3 h and then over-night at 4 °C with indicated primary antibodies under gentle rotation. Seminiferous tubules were washed in PBSMT (5 × 1 h) and incubated with dye conjugated (Alexa488 or TRITC) goat anti-mouse or rabbit antibody as required. The tubules were mounted in raised coverslips glass slides.


We used whole 7 dpp testes or undifferentiated and differentiating spermatogonia cell fractions obtained by affinity purification (C-kit and Thy1, respectively) of total wild-type testes. ChIP was performed as previously described with modifications [35]. Cells were fixed for 5 min 1% methanol-free formaldehyde, quenched for 5 min with 125 mM glycine, washed twice with PBS 1× and suspended in 5 mL of PBS 1× containing 1 mM of PMSF. Cells were dounced 20 strokes with a ‘B’ pestle, and then, pelleted at 2000 rpm for 5 min at 4 °C. The cells were suspended in in 5 mL of Lysis buffer 1 containing 1 mM PMSF (10 mM Tris–HCl pH8, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and centrifuged at 2000 rpm for 5 min at 4 °C. The same procedure was repeated with 5 mL of Lysis buffer 2 containing 1 mM PMSF (10 mM Tris–HCl pH8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). The cells were finally resuspended in 1 mL of ChIP buffer containing 1X protease inhibitor cocktail, Roche (50 mM Tris–HCl pH8, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS) and sonicated for 15 or 24 min for spermatogonia or whole testes, respectively, using the Covaris E220 evolution (peak power 140, duty factor 5, 200 cycles per burst). The sample was cleared at 12,000×g for 10 min at 4 °C, and the chromatin was incubated with anti-Chd4 antibody (ab 70469, Abcam) overnight at 4 °C. Antibody/chromatin complexes were captured with ChIP-grade protein A/G magnetic beads (ThermoFisher) for 2 h at 4 °C and washed 2 times with increasing salt concentration (20 mM Tris–HCl pH8, 2 mM EDTA, 150–500 mM NaCl, 1% Triton X-100, 0.1% Na deoxycholate) and once with a lithium buffer (10 mM Tris–HCl pH8, 1 mM EDTA, 250 mM LiCl, 1% Igepal, 0.7% Na deoxycholate). The beads were washed twice with TE buffer pH7.4 (10 mM Tris–HCl, 1 mM EDTA) and DNA was eluted at 65 °C with agitation for 30 min using 150 ~ µL 1% SDS + 100 mM NaHCO3 made freshly (twice). Cross-links were reversed overnight by adding 5 µL of 5 M NaCl and incubating overnight at 65 °C. DNA was treated with 3 µL RNaseA (Qiagen cat # 19,101) for 30 min at 37 °C and then with 5 µL of proteinase K (approximately 3 U, Qiagen, cat. # 19131) for 1 h at 56 °C. The DNA was purified using the miniElute PCR purification kit using 7 volumes of PB buffer, washing with ethanol twice and eluting twice in 12 µL (Qiagen, cat. # 28004) and quantified using Qubit (Life Technologies) before library.

Library preparation and sequencing

Spermatogonia-purified ChIP and its input libraries were prepared using an in-house method (see below), and sequenced single read 85 bp on an Illumina NextSeq 500 instrument. Whole testes ChIP and its input libraries were prepared using ACCEL-NGS® 1S PLUS DNA LIBRARY KIT (cat. # 10024) in conjunction with Swift unique dual indexing kit (cat. # X9096), following manufacturer’s instructions, and sequenced as paired end 150 bp on an Illumina Novaseq 6000 instrument. Both sequencings were done at OMRF Clinical Genomics Core.

Before sequencing, samples were quantified by qPCR using Kapa library quantification kit (cat # KK4854), and size and quality of DNA were assessed using Agilent Tape station.

In-house library preparation method

20 µL of the eluted DNA was incubated with 30 µL of end-repair mix (0.66 mM dNTP mix (NEB, cat. # N0447S), 100 U/mL T4 DNA polymerase (NEB, cat. # M0203L), 33 U/mL Klenow fragment (NEB, cat. # M0210S), 333 U/mL T4 PNK (cat. # M0201L), and 1.67× T4 PNK buffer) at 20 °C for 30 min. DNA was purified using Qiagen Minelute kit using 7 volumes of PB buffer and eluted in 12 µL of EB buffer. 10 µL of eluted DNA were mixed with 40 µL of A-tailing mix (0.25 mM dATP, 125 U/mL Klenow fragment (cat. # NEB M02105S), and Klenow buffer 1.25×) and incubated at 37 °C for 30 min. DNA was purified again using Qiagen Minelute kit using 7 volumes of PB buffer and eluted in 12 µL of EB buffer. Truseq single index adaptors (Illumina, cat. # 20015960 or 20015961) were diluted according to the DNA concentration (insert:adaptor molar ratio of 1:2, with maximal dilution of 1:50) and 1 µL of a specific diluted adaptor was added to each sample. 20 µL of ligation mix (30 U/µL T4 DNA ligase (cat. # NEB M0202L), and 1.58× ligase buffer) was added to 10 µL of the insert:adaptor mix and incubated for 30 min at 20 °C. DNA was purified using Qiagen Minelute kit using 7 volumes of PB buffer and eluted in 12 µL of EB buffer. Finally, DNA was amplified using the Kapa HiFi Hot Start library amplification kit (cat. # KK2621) according to manufacturer’s instructions.

ChIP-seq data processing and analysis

Alignment and quality filtering

For samples prepared using adaptase technology (whole testes and its input), reads 1 and 2 were trimmed 10 bases at the beginning using fastp [36] (version 0.23.2), as recommended by the library preparation kit manufacturer. Further processing was done in parallel for all samples. Reads were adapter-trimmed and quality-pruned using fastp with default settings. Then, reads were aligned to mm10 genome using BWA–MEM [37] (version 0.7.15) with default settings except for option ‘-M’ for Picard compatibility. Picard (version 2.21.2, http://broadinstitute.github.io/picard/) and SAMtools (version 1.11) [38] were used to obtain mapping quality metrics, remove duplicates and filter reads. Only primary alignment reads that were not duplicated, properly paired, with a MAPQ > 30, and not placed in mitochondrial or unplaced-chromosomes were kept.

Greylist regions

Greylist regions were prepared for each input using the GreyListChIP R-package (R package version 1.24.0, https://bioconductor.org/packages/release/bioc/html/GreyListChIP.html) together with the Bsgenome.Mmusculus.UCSC.mm10 package (https://bioconductor.org/packages/release/data/annotation/html/BSgenome.Mmusculus.UCSC.mm10.html) using the following parameters: reps = 10, sampleSize = 10,000, p = 0.9999. These lists were merged and to mouse blacklist to create a single black-greylist, which was later used to filter peaks.

Peak calling

Peaks were called using MACS2 [39] (version using broad mode and default values for the rest of the parameters.

Peak annotation and gene ontology analysis

Common peaks to both ChIPs were filtered by black-greylist and then annotated using HOMER’s annotatePeaks.pl script (22, version 4.10). To assign a score to the common peaks, we averaged the −log(q value) for both peaks. In cases in which a peak from one experiment was intersected by more than one peak from the other experiment, the −log(q values) from the same experiment were averaged first. Annotated peaks can be found at Additional file 8: Table S1. Functional annotation analysis was done using DAVID with default parameters and the list of annotated genes in which the promoter was closer than 5000 bp. Further processing and plotting were done using R (https://www.r-project.org/, version 4.1.1) and packages within tidyverse (https://joss.theoj.org/papers/10.21105/joss.01686). Functional analysis results can be found at Additional file 12: Table S5.

Heatmaps and IGV snapshots

Bigwig coverage tracks were generated using bamCoverage tool from deepTools (version 3.4.3, [40]) with a bin size of 10 bp, 40 bp smoothing and RPKM normalization. Average aggregate profiles and heatmaps were plot using deepTools as well (computeMatrix and plotHeatmap). Mapping statistics can be found in Additional file 12: Table S5.

Statistical analyses

Results are presented as mean ± standard deviation (SD). Statistical analysis was performed using Prism Graph statistical software. Two-tailed unpaired Student’s t test was used for comparisons between 2 groups. P < 0.05 was considered statistically significant.

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