The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells

Abstract

The switch from mitosis to meiosis is a unique feature of germ cell development. In mammals, meiotic initiation requires retinoic acid (RA), which activates meiotic inducers, including Stra8, but how the switch to meiosis is controlled in male germ cells (spermatogonia) remains poorly understood. Here we examine the role of the Doublesex-related transcription factor DMRT1 in adult spermatogenesis using conditional gene targeting in the mouse. Loss of Dmrt1 causes spermatogonia to precociously exit the spermatogonial program and enter meiosis. Therefore, DMRT1 determines whether male germ cells undergo mitosis and spermatogonial differentiation or meiosis. Loss of Dmrt1 in spermatogonia also disrupts cyclical gene expression in Sertoli cells. DMRT1 acts in spermatogonia to restrict RA responsiveness, directly repress Stra8 transcription, and activate transcription of the spermatogonial differentiation factor Sohlh1, thereby preventing meiosis and promoting spermatogonial development. By coordinating spermatogonial development and mitotic amplification with meiosis, DMRT1 allows abundant, continuous production of sperm.

Figure 1. DMRT1 is expressed in spermatogonia and Sertoli cells, but not in meiotic and postmeiotic germ cells

Immunofluorescence (IF) of testes from 28 day control testes. (A,B) Section IF showing DMRT1 in GFRA1-positive undifferentiated spermatogonia (filled arrowhead; primarily A_s and A_pr, and a small proportion of A_al), and Sertoli cells (open arrowheads). (C,D) Section IF showing DMRT1 in PLZF-positive undifferentiated spermatogonia (filled arrowheads) but not postmeiotic cells (spermatids; “PM”). Arrow: differentiating spermatogonium. (E,F) Whole-mount IF of seminiferous tubules showing DMRT1 in c-KIT-positive differentiating spermatogonia. SC: Sertoli cells. (G,H) Section IF of DMRT1 at different tubule stages. DMRT1-positive germ cells in stage VI tubules (G) are mainly type B spermatogonia. In stage VII tubules (H) these have become preleptotene spermatocytes and no longer express DMRT1 (DMRT1-positive cells are Sertoli cells and A spermatogonia). (I) Section IF showing higher DMRT1 expression in type A spermatogonia relative to type B spermatogonia and Sertoli cells. Scale bars: 20 micron. (J) Schematic diagram showing progression of spermatogonial development and expression of markers used in panels A-F.

Figure 2. Loss of DMRT1 in early spermatogonia disrupts spermatogenesis

(A-D) Whole-mount analysis of seminiferous tubules. ECAD IF alone (A,B) and merged with DMRT1 IF (C,D). DMRT1-positive Sertoli cells (with dark spots), are not affected by Ngn3-cre conditional mutation. Scale bars: 20 microns. (E-H) Section IF staining for pan-germ cell marker TRA98 and DAPI at low magnification (E,F; scale bars: 50 microns) and higher magnification (G,H; scale bars: 20 microns). (I,J) Hematoxylin and eosin staining. Scale bars: 100 microns. (K,L) TUNEL labeling. Arrows: apoptotic cells. Scale bars: 20 microns. (M,N) Periodic acid/Schiff (PAS-H) staining of adult (eight-week-old) testes. Insets: darkly staining elongating spermatids. Scale bars: 20 microns.

Figure 3. Precocious and uncontrolled meiotic initiation in Dmrt1 mutant germ cells

(A-D) Whole-mount IF staining of seminiferous tubules for ECAD and STRA8. Arrows indicate examples of single, paired, and aligned double-positive germ cells found only in mutant. Scale bars: 20 microns. (E,F) Section IF for STRA8 and TRA98. Control tubules at stage VII are indicated (“VII”). White dots: mutant tubules with germ cells. Scale bars: 50 microns. (G-N) Section IF two hours after BrdU labeling. Arrow: STRA8- and BrdU-positive cell with spermatogonial DAPI morphology. Scale bars: 20 microns. (O-T) Section IF 24 hr after BrdU labeling. Insets: BrdU-labeled SYCP3-positive leptotene spermatocytes in control and similar cells in mutant. Scale bars: 20 microns. (U-Z) Section IF eight days after BrdU labeling. Insets: BrdU-labeled pachytene spermatocytes with SYCP3 localized to synaptonemal complexes. Scale bars: 20 microns. (AA, AB) Whole mount IF of seminiferous tubules for c-KIT. Bars indicate diameters of seminiferous tubules. Scale bars: 50 microns.

Figure 4. Kinetics of the switch from mitosis to meiosis in mutant spermatogonia

Conditional deletion of DMRT1 in adult testes using the tamoxifen inducible cre transgene Ubc-cre/ERT2. (A,B) Section IF for DMRT1two days after tamoxifen injection showing DMRT1 in spermatogonia and Sertoli cells (SC) in control but only in Sertoli cells in mutant. (C-F) Section IF six days after tamoxifen injection. BC7-positive cells at stage VI are primary spermatocytes (late zygotene to mid-pachytene) with underlying layer of DMRT1-positive B spermatogonia (SC: Sertoli cell). Inset shows missing layer of spermatogonial cells in mutant; DMRT1-positive cells in mutant are Sertoli cells. (G-L) Section IF eight days after tamoxifen injection. In stage VI control tubule the germ cells underlying the layer of BC7- and SYCP3-positive primary spermatocytes are negative for SYCP3. In mutant tubules with abundant BC7- and SYCP3-positive primary spermatocytes, underlying cells have SYCP3 distribution typical of leptotene spermatocytes (arrowheads), not normally found together with BC7-positive spermatocytes. Scale bars: 20 microns.

Figure 5. Meiosis of mutant spermatogonia requires retinoic acid

(A,B) Section IF for STRA8 in adult vitamin A sufficient (VAS) testes. (C-H) Section IF from vitamin A deficient (VAD) testes at fifteen weeks. Arrested undifferentiated A spermatogonia in VAD mice have low expression of SYCP3 (C,E,G), whereas mutant VAD tubules contain germ cells with intense foci of SYCP3 similar to those of preleptotene spermatocytes. SYCP3 channel in panels E and G is intentionally overexposed to show faint puncta in A spermatogonia of control. (I-N) Section IF six days after vitamin A replenishment. Differentiating spermatogonia of control have weak expression of γH2AX and SYCP3. Leptotene spermatocytes in mutant have intense and distributed SYCP3 localization and punctate accumulation of γH2AX at presumptive double-strand DNA breaks (for comparison, see control leptotene spermatocytes in Figure 3Q). Scale bars: 20 microns.

Figure 6. Regulation of Stra8 transcription and RA activity by DMRT1

(A,B) Section IF for DMRT1 and STRA8. At stage VII DMRT1 is expressed in Sertoli cells but not preleptotene spermatocytes (A) and STRA8 is robustly expressed in preleptotene spermatocytes (B). Scale bars: 20 microns. (C) ChIP-Chip of DMRT1 in P9 testis (Murphy et al., 2010) detects binding of DMRT1 to the Stra8 promoter. Red dots: positions of RAREs. Black dot: DMRT1 consensus DNA binding site, sequence of which is at top of panel, aligned to in vivo-derived DNA binding consensus. More distal peak of DMRT1 binding lacks a clear consensus element and is presumably bound either indirectly or via a non-canonical binding site. (D) qChIP of DMRT1 at P28 detects DMRT1 bound to DNA near Stra8 transcriptional start site (0) but not 3 kb upstream or downstream. B2m is negative control promoter. (E) qChIP of DMRT1 on Stra8 promoter in testes with germ cell specific deletion of Dmrt1 (GCKO) or Sertoli cell-specific deletion of Dmrt1 (SCKO). (F-I) Expression of RA-dependent RAREhsplacZ reporter transgene in control and mutant testes at P28. (F,G) Beta-galactosidase (BGAL) IF. Expression in control is higher in meiotic germ cells relative to surrounding mitotic spermatogonia (arrowheads). Reporter expression in mutant is higher in the spermatogonia that ring the seminiferous tubule (arrowheads). (H,I) DMRT1 IF showing that control spermatogonia with high DMRT1 expression in spermatogonia have low reporter expression and germ cells with elevated reporter expression in mutant testes lack DMRT1. Scale bars: 20 microns. (J,K) Expression of mRNAs involved in RA-dependent transcription. qRT-PCR reveals decreased Cyp26a1 and Tbx1 transcript levels at P28 and increased Crabp2 levels in mutant testes (J). Similar expression changes are detected by qRT-PCR in c-KIT sorted mutant germ cells (K). Error bars: SD.

Figure 7. Regulation of spermatogonial development and meiosis by DMRT1

(A-D) IF of DMRT1 and SOHLH1 in adult testis sections. Control spermatogonia are double-positive (A,C), and mutant spermatogonia are double-negative (B,D). Cells positive for DMRT1 alone are Sertoli cells. Scale bars: 20 microns. (E,F) Binding of DMRT1 to Sohlh1 promoter in vivo. (E) ChIP-chip at P9 (Murphy et al., 2010) detects DMRT1 bound at the Sohlh1 promoter. Black dot: DMRT1 consensus DNA binding site, sequence shown at top of panel aligned to in vivo-derived DNA binding consensus. (F) DMRT1 binds Sohlh1 in both germ cells and Sertoli cells. GCKO: Deletion of Dmrt1 in spermatogonia. SCKO: deletion of Dmrt1 in Sertoli cells. B2m is negative control promoter. Error bars: SD. (G) Model for control by DMRT1 of spermatogonial development and meiosis. Solid lines indicate direct transcriptional control and dashed lines indicate undefined regulatory interactions.