DMRT1 Is Required for Mouse Spermatogonial Stem Cell Maintenance and Replenishment

Abstract

Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion.

Fig 1. Oct4-CreER is active in SSCs and efficiently deletes Dmrt1.

(A-C) Immunofluorescence (IF) of wholemount seminiferous tubules. Oct4-CreER; Rosa26-tdTomato; Id4-Gfp testes eight hours (hr) post-tamoxifen (PT) treatment. (A) GFP (green). (B) GFRα1 (gray). (C) Merge with RFP (red). Anti-RFP labels all tdTomato-positive cells (labeled “tdT” here and in subsequent figures). A_s spermatogonia are indicated by white arrowheads, A_pr are indicated by magenta arrowheads and A_al are indicated by yellow arrowheads. (D-I) IF of wholemount seminiferous tubules of Dmrt1^flox/+;Oct4-cre/+; tdTomato; Id4-Gfp (hereafter, “control”) mice to the same strain homozygous for Dmrt1^flox (hereafter, “mutant”) stained for DMRT1 (green) and tdTomato (red) four days post-tamoxifen (DPT). In the control (D-F) DMRT1 protein expressed in tdTomato-positive A_s, A_pr (inset) and A_al cells. In mutant (G-I) DMRT1 is depleted from tdTomato-positive A_s, A_pr (inset) and A_al cells. Scale bars: 20 μm.

Fig 2. Dmrt1 mutant undifferentiated spermatogonia can differentiate.

IF of sectioned control (A-C, G-I, M-O) and conditional mutant (D-F, J-L, P-R) seminiferous tubules 10 and 15 days post-tamoxifen (DPT) detecting tdTomato (red) and three stage-specific germ cell markers (green). DAPI (blue) stains DNA. Anti-PLZF (A-F) was used to detect undifferentiated spermatogonia, anti-SOHLH1 (G-L) for differentiating spermatogonia and anti-SYCP3 (M-R) for meiotic cells. Double-positive cells are labeled by white arrowheads. Scale bars: 20μm.

Fig 3. Dmrt1 is required in undifferentiated spermatogonia to maintain spermatogenesis.

(A-H) IF of sectioned testes showing tdTomato (red) at 10, 15, 21 and 40 DPT. DAPI DNA stain (blue). In controls (A-D) tdTomato-positive cells are maintained for a full round of spermatogenesis (40d). In mutant testes (E-H) tdTomato-positive cells were progressively lost, with more than 60% of tubules (N = 101/167) having no tdTomato-positive cells by 40 DPT (indicated by asterisks). Scale bars: 20 μm.

Fig 4. Dmrt1 mutant SSCs are not maintained.

(A-F) IF of sectioned 15 DPT testes showing tdTomato (red) and GFP (green). DAPI DNA stain (blue). All tdTomato/GFP double-positive cells are labeled with white arrowheads. In control testes (A-C) many tdTomato/GFP double-positive cells were present, while in mutant testes (D-F) double-positive cells were rare. Higher magnifications are shown in insets. GFP-positive meiotic cells represent previously described ectopic expression of the Id4-Gfp transgene (11). (G, H) IF of wholemount seminiferous tubules 15 DPT showing tdTomato (red) and GFP (green). In control (G) all tdTomato-positive cells were GFP-positive (white arrowhead; inset), while in mutant (H), GFP-positive cells almost all lacked tdTomato (yellow arrowhead; inset) indicating that they were wild-type. Scale bars: 20μm. (I) Quantification of Id4-GFP and tdTomato double-positive cells in control and mutant testes 10 (yellow) and 15 (blue) DPT. Values are average from >300 tubules. Error bars indicate standard deviation. * indicates P < 0.05 (Student’s T test). ** indicates P<0.005.

Fig 5. Mutant SSCs are lost due to defective maintenance.

(A-C) Controls treated with BrdU for 4 hours. IF of sectioned testes showing BrdU (red) and spermatogonial markers: Id4-GFP, PLZF or c-KIT (green). DAPI DNA stain (blue). Double-positive cells are indicated by the white arrowhead. Higher magnifications are shown in insets. (D) Quantification of BrdU and GFP⁺/PLZF⁺; GFP^-/PLZF⁺ and c-KIT⁺ positive cells. Values are average from >300 tubule sections for GFP staining, and >100 for PLZF and c-KIT staining. Error bars indicate standard deviation. ** indicates P < 0.005 (Student’s T test). (E-G) Diagram of the BrdU chase experiment in the control (E) and two possible outcomes in the mutant: failure to maintain SSCs (F) and SSC death (G). (H, I) IF of sectioned testes showing tdTomato (red) and BrdU (green) in control (H) and mutant (I) 18 DPT with BrdU chase for 12 days. DAPI DNA stain (blue). Double-positive cells are indicated by the white arrowheads and tdTomato single-positive cells are indicated by yellow arrowheads. Scale bars: 20 μm.

Fig 6. PLZF expression requires DMRT1.

(A-F) IF of sectioned testes showing PLZF (green), SALL4 (red), and DMRT1 (gray) staining in controls (A-C) and mutant (D-F) at 15 DPT. DAPI DNA stain (blue). DMRT1-positive germ cells are indicated by blue arrowheads, and DMRT1-negative germ cells by yellow arrowheads. DMRT1-negative and SALL4-positive germ cells consistently have reduced PLZF relative to DMRT1-positive cells. Scale bars: 20μm. (G) ChIP-seq showing that DMRT1 binds near Plzf. Wild-type adult (blue track) and adult testes treated with 30 mg/kg busulfan for 4-weeks (green track) to deplete germ cells were analyzed. The data range shown for each binding profile is 0 to 2 read counts/million. DNA sequence centered under the binding site is shown, with a DMRT1 consensus half-site underlined.

Fig 7. Dmrt1 is required for the recovery of spermatogenesis post-busulfan treatment.

IF of sectioned testes 60 days after busulfan treatment double stained with tdTomato (red) and TRA98, PLZF, SOHLH1 or SYCP3 (green). DAPI DNA stain (blue). In the control (A-D) all stages of germ cells that are tdTomato-positive were detected (white arrowhead). In the mutant (E-H), no germ cells were observed. Scale bars: 20 μm.

Fig 8. Dmrt1 is required for SSC regeneration.

IF of sectioned adult testes showing tdTomato (red) and GFP (green) 60 days after busulfan treatment (20mg/kg). DAPI DNA stain (blue). In Dmrt1^flox/+; Ngn3-cre/+; tdTomato; Id4-Gfp control testes (A) tdTomato and GFP double-positive cells were observed after 60 days (inset) but in Dmrt1^flox/flox; Ngn3-cre/+; tdTomato; Id4-Gfp mutant (B) no double-positive cells were observed. Scale bars: 20μm. (C) Quantification of Id4-GFP and Id4-GFP/tdTomato double-positive cells in control and mutant testes. Double-positive cells are shown in yellow, GFP-positive cells are shown in green. Values are average from >200 tubule sections. Error bars indicate standard deviation. *** indicates P < 0.0005 (Student’s T test). (D) Model of DMRT1 during SSC homeostasis. SSCs are indicated in green, NGN3-positive undifferentiated spermatogonia are indicated in red.