DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary

Abstract

Dmrt1 belongs to the DM domain gene family of conserved sexual regulators. In the mouse Dmrt1 is expressed in the genital ridge (the gonadal primordium) in both sexes and then becomes testis-specific shortly after sex determination. The essential role of DMRT1 in testicular differentiation is well established, and includes transcriptional repression of the meiotic inducer Stra8. However Dmrt1 mutant females are fertile and the role of Dmrt1 in the ovary has not been studied. Here we show in the mouse that most Dmrt1 mutant germ cells in the fetal ovary have greatly reduced expression of STRA8, and fail to properly localize SYCP3 and γH2AX during meiotic prophase. Lack of DMRT1 in the fetal ovary results in the formation of many fewer primordial follicles in the juvenile ovary, although these are sufficient for fertility. Genome-wide chromatin immunoprecipitiation (ChIP-chip) and quantitative ChIP (qChIP) combined with mRNA expression profiling suggests that transcriptional activation of Stra8 in fetal germ cells is the main function of DMRT1 in females, and that this regulation likely is direct. Thus DMRT1 controls Stra8 sex-specifically, activating it in the fetal ovary and repressing it in the adult testis.

Figure 1. DMRT1 is required to activate STRA8 in fetal ovarian germ cells

(A–F) Immunofluorescence (IF) staining for DMRT1 and DNA staining with DAPI. (AC) DMRT1 is expressed in the nucleus of germ cells at E13.5. (D–F) DMRT1 is expressed in germ cell cytoplasm at E14.5. (G–R) Double staining for germ cell marker TRA98 and meiotic inducer STRA8 in (G–L) wild type and (M–R) Dmrt1^−/− ovary sections at E13.5 (G–H, M–N), E14.5 (I–J, O–P) and E15.5 (K–L, Q–R). Scale bars: 50 microns. (S) Percentage of STRA8-positive germ cells in wild type versus Dmrt1^-/− ovaries at E13.5–E15.5 (n≥3 animals, **P<0.005, ***P<0.0005).

Figure 2. Abnormal prophase I in Dmrt1 mutant ovaries

(A–F) Double staining for SYCP3 and TRA98 by IF in wild type (A–C) and Dmrt1^−/− (D–F) ovaries. (A–F) SYCP3 is expressed in the nucleus of wild type germ cells at E13.5 and in (D–F) the cytoplasm of Dmrt1^−/− germ cells. (G–L) SYCP3 is properly loaded onto chromosomes in wild type (G–I), but not in Dmrt1^−/− (J–L) germ cells at E15.5. DNA is marked with DAPI. (M–R) Double staining for the germ cell marker MVH and γH2AX in wild type (M–O) and Dmrt1^−/− (P–R) ovaries at E15.5. Most germ cells in the wild type ovary (M–R) are γH2AX positive, whereas Dmrt1^−/− germ cells (P–R) are only rarely γH2AX positive. (S–X) Double staining for MVH and the diplotene marker MSY2 in wild type (S–U) and Dmrt1^−/− (V–X) ovaries at P4. Scale bars: 25 micron.

Figure 3. Identification of DMRT1 direct targets in the E13.5 ovary

DMRT1 binding to the proximal promoter region of (A) Dpf3 and (B) Zfp384 detected by ChIP-chip in three independent experiments. Gold dot indicates position of in vitro defined DMRT1 consensus binding motif (Murphy et al., 2007) and brown bar indicates position of polyC tract. (C) Validation of promoter binding by qChIP for 12 genes detected by ChIP-chip, using an independent chromatin sample. Eleven of twelve promoter regions show greater than 2.5 fold enrichment. B2m and Pgk1 are negative controls. Error bars: SD from technical replicates.

Figure 4. In vivo binding of DMRT1 to the proximal promoter region of Stra8

(A) ChIP-chip showing enrichment in Stra8 proximal promoter with DMRT1 in vitro defined motif (Murphy et al., 2007) in fetal ovaries in three separate experiments. (B). qChIP validating enrichment for Stra8 promoter in fetal ovaries (pink), but not fetal testes (blue). Error bars: SD from technical replicates. Gold dot indicates position of in vitro defined DMRT1 consensus binding motif (Murphy et al., 2007) and peak from P9 testis.

Figure 5. mRNA expression profiling in Dmrt1 mutant ovaries

(A) Heat map showing seven mRNAs elevated greater than two-fold and two mRNAs reduced greater than two-fold in Dmrt1^−/− ovaries at E13.5 (P<0.05). (B) qRT-PCR of Stra8 at E13.5 normalized to HPRT. Error bars: SD from 3 animals of each genotype. (*P<0.05).

Figure 6. Reduction in number of follicles at P8

(A,B) IF staining for germ cell marker MVH in wild type (A) and Dmrt1^−/− (B) ovaries at P8. DNA is stained with DAPI. Scale bars: 50 microns. (C) Number of primordial and (D) primary and secondary follicles per ovary at P8. (E) Percentage of oocytes found in nests at P8. Error bars: SD from 3 animals of each genotype (*P<0.05, **P<0.005).