Chd4 expression during mouse germ cell development
To investigate a potential role for CHD4 in germ cells, we assessed CHD4 expression in newborn and adult mouse testes by immunofluorescence. CHD4 was highly expressed in spermatogonia cells (marked by PLZF, aka ZBTB16) and in Sertoli cells (marked by SOX9) (Fig. 1A and Fig. S1) but not in the negative control (PLZF-positive spermatogonia from Ddx4-Chd4−/− testes, Fig. S2A). PLZF is expressed in undifferentiated spermatogonia [16, 17] (Fig. 1A). In agreement with a previous report , immunosignal of CHD4 was detected in late-pachytene stages (Fig. 1A, selected area on adult mouse top panel). In sum, CHD4 is detected in spermatogonia, Sertoli cells, and primary spermatocytes.
We confirmed that CHD4 is expressed at pre-meiotic stages of male gamete development by analyzing CHD4 protein levels by western blot in enriched fractions (see methods for details) of undifferentiated and differentiating spermatogonia (obtained from wild type 7dpp mice and using THY1.2+ and c-KIT+ affinity columns, respectively) (Fig. 1B). CHD4 was also detected in the affinity column flowthrough (which was enriched mostly in Sertoli cells, here demonstrated by immunoblotting of Sox9). The level of cell population enrichment was assessed by western blot and markers specific for undifferentiated spermatogonia (PLZF) and differentiating spermatogonia (STRA8) (Fig. 1B). Additionally, we analyzed cell fractions enrichment by immunofluorescence (Fig. S3).
Composition of CHD4-NURD complexes during spermatogenesis
NURD function is influenced by its subunit composition [18, 19]. To determine whether the expression of NURD composition might change during spermatogenesis, we analyzed the levels of representative NURD subunits in enriched fractions of undifferentiated and differentiating spermatogonia, as well as in the flow-through (enriched in Sertoli cells) after spermatogonia enrichment. NURD subunits HDAC2A, MTA1, RBBP4, RBBP7 and MBD2 were present in enriched fractions of undifferentiated (THY1.2+) and differentiating (c-KIT+) spermatogonia (Fig. 1B), suggesting a particular composition and perhaps a different function for CHD4-NURD in this cell type.
To determine the composition of CHD4-NURD complexes, we used co-immunoprecipitation analysis to uncover NURD subunits that interact with CHD4 in enriched fractions of THY1.2 and c-KIT spermatogonia cells together and the flow-through. The NURD subunits HDAC2A, MTA1, and RBBP4/RBBP7, but not MBD2, coimmunoprecipitated with CHD4 from all fractions (Fig. 1C). HDAC2A coimmunoprecipitated with CHD4 from wild type and Chd3−/− spermatogonia cells (Fig. S2B). These data suggest that: i) CHD4 forms a NURD complex independently of CHD3, ii) that loss of CHD3 does not perturb CHD4-NURD complex formation in spermatogonia cells.
Deletion of Chd4 but not Chd3 results in testis developmental defects
To examine the potential functions of Chd4 and Chd3 during spermatogenesis, we generated a series of Chd4 and Chd3 germline conditional knockout mice (Fig. 2). To delete the floxed allele in gonocytes (embryonic day 15.5, Fig. 2C) , male Ddx4-Cre; Chd4WT/Δ were crossed with Chd4fl/fl females to generate Ddx4-Cre; Chd4fl/Δ conditional knockout mice (here called Ddx4-Chd4−/−) (Fig. 2A). A similar strategy was used to generate Ddx4-Chd3−/− mice (Fig. 2B).
Ddx4-Chd4−/− adult mice (2 months old) appeared normal in all aspects except in the reproductive tissues. Testes were significantly smaller in Ddx4-Chd4−/− males (mean: 0.017g ± SD: 0.005, number of quantified mice n = 4 (8 testes), P≤0.0001, t test) compared to wild type (0.104 g ± 0.015, n = 6 mice (12 testes)) littermates (Fig. 2C and D), indicating severe developmental defects in the testis. We confirmed deletion of Chd3 and Chd4 by RT-qPCR (Fig. 2E). Immunofluorescence analysis of neonatal Ddx4-Chd4-/- testis CHD4 was absent at 1ddp (Fig. S2A).
We found that adult Ddx4-Chd4−/− males develop testicular hypoplasia with hyperplasia of interstitial cells and lack spermatozoa (Fig. 3A). The number of seminiferous tubules is similar between wild type and mutant animals, but the diameter is reduced (wild type, mean ± SD, 287 ± 34, n=400 seminiferous tubules cross sections (3 different mice, 2-month-old) versus Chd4−/− 148 ± 21.3, n=210, P<0.0001 t test).
Analysis of Ddx4-Chd4−/− testes revealed a total loss of germ cells (marked by TRA98) in seminiferous tubules (Fig. 3B). No developing gametes were observed, including cell types at early stages (e.g., spermatogonia) (Fig. 3A and B). Sertoli cells develop normally in Ddx4-Chd4−/− mice, consistent with the specific loss of Chd4 in germ cells at early stages of development. We did not observe differences in germ cell development between wild type and Ddx4-Chd3−/− mice (Fig. S3), consistent with their similar testis sizes (Fig. 2C and D).
We also analyzed H&E-stained histological sections of ovaries from 45-day-old wild type and Ddx4-Chd4−/− female mice. We noted a significant reduction in ovary size, an increase in stromal cells, a reduced number of follicles (wild type, 10 ± 3, n=6 mice versus Ddx4-Chd4−/− 0.6 ± 0.9, n=5, P<0.0001 t test) and absent corpora lutea (wild type, 9 ± 1, n=6 mice versus Ddx4-Chd4−/− 0 ± 0, n=5, P<0.0001 t test) in the Ddx4-Chd4−/− mice compared to wild type (Fig. 3C).
We conclude that deletion of Chd3 has no apparent effect on gamete development. However, germ cell specific deletion of Chd4 results in severe male and female germ cell developmental defects, possibly originated at premeiotic stages of development.
CHD4 is required for neonate spermatogonia survival
The severe phenotypes observed in Ddx4-Chd4−/− mice (Fig. 2 and 3) prompted us to investigate spermatogonial differentiation during testis development in newborns. Testis sections from 9 dpp Ddx4-Chd4−/− mice stained with H&E showed a markedly reduced number of germ cells (Fig. 4A) as well as differences in cell composition, compared to those from age-matched wild type mice. To analyze this in detail, we examined the presence of cells expressing STRA8 (Fig. 4A), which marks differentiating spermatogonia, SYCP3 and yH2AX which are markers of primary spermatocytes and TRA98, a marker for germ cells (Fig. S5). Whereas tubules from 9 dpp wild type mice contained cells expressing TRA98 (45 ± 10, n=66 seminiferous tubules counted obtained from 3 mice) and STRA8 (18.8 ± 8.4, n=36 obtained from 3 mice), tubules from Chd4−/− mice showed a near absence of cells expressing these markers (TRA98 4.6 ± 3, n=60 obtained from 3 mice, P<0.0001, t test; STRA8 2.5 ± 3.5, n=42 obtained from 3 mice, P<0.0001, Student t test) (Fig. 4A). Testes sections from 9 dpp Chd4−/− mice also showed a reduction in primary spermatocytes expressing the meiotic prophase I markers SYCP3 and γH2AX compared to those from 9 dpp wild type mice (Fig. S5A and B). Together, the results further suggest that testis defects in Chd4−/− mice begin early, during pre-meiotic stages of postnatal development, leading to an absence of germ cells in adults.
To pinpoint when the testes defects originate in Ddx4-Chd4−/− mice, we stained testes sections from 1-21 dpp mice for the expression of TRA98 (all germ cells). We observe that as general trend, in all analyzed stages, the number of TRA98 positive cells was reduced in Chd4−/− mice compared to wild type, progressing to total absence of germ cells (Fig. S6). Chd4 mutant sections displayed a small reduction in the number of cells expressing TRA98 at both 1dpp and 3dpp (Fig. 4B and 4C and Fig. S6). We observed equal numbers of SOX9-positive Sertoli cells in testes from wild type and Chd4−/− mice at 3, 4, and 7 dpp (Fig. 4C), as expected for the specific loss of Chd4 in spermatogonia cells. Both PLZF-positive and TRA98-positive cells were substantially reduced in Chd4−/− testis compared to wild type testis at 4 dpp and 7dpp (Fig. 4C).
Given that Chd4 may act as a regulator of cell-cycle progression, we then examined whether the rapid loss of PLZF-positive neonate spermatogonia in Chd4−/− testes was due to altered proliferative activity. We conducted EdU incorporation study to test this possibility. 4 dpp mice were injected with EdU and analyzed 3 h later, after which we assayed its incorporation in PLZF-positive spermatogonia in whole mounts of seminiferous tubules (Fig. S7A). We found that spermatogonia cell proliferation (PLZF/EdU+ cells) in wild type and Ddx4-Chd4−/− is proportionally the same (Fig. S7C). In addition, reduced amount of total PLZF-positive cells was found in Ddx4-CHD4−/− compared to wild type in the whole-mounting experiment (Fig. S7B).
To determine whether cell death contributed to the loss of Ddx4-Chd4−/− neonate spermatogonia (Fig. 4C), we performed TUNEL assay in staining paraffin embedded testis sections of wild type and Chd4−/− four days old testis. At this age the testis is mostly constituted by spermatogonia and Sertoli cells, which can be easily distinguished by DAPI nuclear staining patterns. We found a significant increase in the percentage of apoptotic cells in Chd4−/− testis compared to wild type mice (Fig. 4D).
We conclude that the possible cause of spermatogonia failure in Chd4−/− mice is in the survival/maintenance of neonate undifferentiated spermatogonia.
CHD4, DMRT1, and PLZF work together in a regulatory axis involved in spermatogonia cell survival
Our results show that CHD4 is required for spermatogonia maintenance/survival. We then reason that CHD4 may interact with genes that have been described to work in spermatogonia maintenance. Indeed, DMRT1 has been show to function in spermatogonia stem cells maintenance, and this function seems to be mediated by direct regulation of Plzf gene expression, another transcription factor required for spermatogonia maintenance . To test our hypothesis, we immunostained paraffined testes sections from 1, 4, and 7 dpp mice for the presence of DMRT1 (Fig. 5A and B). Compared to wild type, Chd4 mutant showed a clear reduction in DMRT1 immunosignal.
Recent work described that Dmrt1 controls Plzf expression, which is a transcription factor required for spermatogonia maintenance . We then tested the effect of CHD4 depletion in Plzf expression. PLZF immunostaining of testes sections from 1, 4, and 7 dpp mice revealed a significantly reduction of this protein in Chd4 knockout cells respect to wild type (Fig. 5C and D). This is consistent with a model by which CHD4 control of Dmrt1 and downstream targets influences cell survival/maintenance (Fig. 5E).
We concluded that Chd4 participates in the maintenance/survival of neonate spermatogonia stem cell possibly through transcriptional regulation of genes participating in these critical processes. We note, however, that the dramatic phenotype observed in CHD4−/− spermatogonia likely reflect CHD4 targeting a wide spectrum of genes participating in different pathways.