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, 79 (6), 1038-45

A Phenotypic Spectrum of Sexual Development in Dax1 (Nr0b1)-deficient Mice: Consequence of the C57BL/6J Strain on Sex Determination

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A Phenotypic Spectrum of Sexual Development in Dax1 (Nr0b1)-deficient Mice: Consequence of the C57BL/6J Strain on Sex Determination

Susan Y Park et al. Biol Reprod.

Abstract

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1; hereafter referred to as Dax1) is an orphan nuclear receptor that regulates adrenal and gonadal development. Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (Dax1) mutations in the mouse are sensitive to genetic background. In this report, a spectrum of impaired gonadal differentiation was observed as a result of crossing the Dax1 knockout on the 129SvIm/J strain onto the C57BL/6J strain over two generations of breeding. Dax1-mutant XY mice of a mixed genetic background (129;B6Dax1(-/Y) [101 total]) developed gonads that were predominantly testislike (n = 61), ovarianlike (n = 27), or as intersex (n = 13). During embryonic development, Sox9 expression in the gonads of 129;B6Dax1(-/Y) mutants was distributed across a wide quantitative range, and a threshold level of Sox9 (>0.4-fold of wild-type) was associated with testis development. Germ cell fate also varied widely, with meiotic germ cells being more prevalent in the ovarianlike regions of embryonic gonads, but also observed within testicular tissue. Ptgds, a gene associated with Sox9 expression and Sertoli cell development, was markedly downregulated in Dax1(-/Y) mice. Stra8, a gene associated with germ cell meiosis, was upregulated in Dax1(-/Y) mice. In both cases, the changes in gene expression also occurred in pure 129 mice but were amplified in the B6 genetic background. Sertoli cell apoptosis was prevalent in 129;B6Dax1(-/Y) gonads. In summary, Dax1 deficiency on a partial B6 genetic background results in further modulation of gene expression changes that affect both Sertoli cell and germ cell fate, leading to a phenotypic spectrum of gonadal differentiation.

Figures

FIG. 1.
FIG. 1.
Phenotypic spectrum of sexual development in 129;B6Dax1−/Y animals. Adult male and female internal genitalia of wild-type mice are shown for comparison to mutants (AD). A) Wild-type males develop bilateral testis, epididymis (epi), and vas deferens (vas). C) Wild-type females have ovary (ov), oviduct (ovid), and uterus (uter). B, D) Cross-sectional histology of 12-wk-old testis (B) and ovary (D). In contrast, 129;B6Dax1−/Y animals (EN) displayed a spectrum of reproductive defects from male hypogonadism to complete sex reversal. E) Those that developed externally as males had testes that were reduced in size, but otherwise these animals resembled the overall male phenotype. F) Representative histology of a Dax1 mutant testis cross section. G) Complete sex reversal was evident in 129;B6Dax1−/Y animals that developed entirely with female reproductive tissue. H) In sex-reversed animals, oocyte-containing follicles were apparent by histology. IK) Gonadal dysgenesis was a feature of certain sex-reversed mutant animals (gonad, J). Defects in duct development were evident, shown by the presence of bilateral uteri (uter, K) with vestigial Wolffian structures (epi, J; vas, K). L) An example of a 129;B6Dax1−/Y intersex mouse in which evidence for both testis formation (M) and ovarian development (N) was apparent in the same animal. Bars = 0.5 cm. Original magnification ×200 (B); ×100 (D, F, H); ×25 (J, K); ×100 (M, N).
FIG. 2.
FIG. 2.
Sox9 expression in 129;B6Dax1−/Y gonads is modulated depending on genetic background. Sox9 expression levels of gonad pairs from 31 embryos of 129;B6Dax1−/Y were calculated with reference to wild-type (WT) male (y-axis) then plotted individually. Quantitative real-time RT-PCR was performed on transcripts of gonad-mesonephros complexes dissected at Embryonic Day 13.5 dpc. Gonad sex was determined at the time of dissection (male, n = 20, individual dashes; female, n = 11, triangles). Expression levels of male and female controls are indicated by separate horizontal lines.
FIG. 3.
FIG. 3.
Distribution pattern of ectopic meiotic germ cells in gonads susceptible to sex reversal. To investigate PGC fate in gonads of 129;B6Dax1/Y animals, germ cell sex was examined by immunodetection of gammaH2AX, a marker of phosphorylated histones that signifies meiotic entry. A) Female PGCs commence meiosis during embryonic development, and by 14.5 dpc meiotic germ cells are detected along the length of the gonad (arrow). BE) A representative spectrum of 129;B6Dax1/Y gonadal tissue demonstrates the variable manner in which meiotic germ cells appear during development. Micrographs B and C show two examples of Dax1 mutant gonad tissue resembling female development that contained gammaH2AX-positive germ cells dispersed in clusters along the gonad length (arrows), whereas D and E revealed meiotic germ cells (arrows) located in tissue with testislike morphology. In wild-type male gonads (F), meiotic germ cells were characteristically undetectable. Bar = 100 μm.
FIG. 4.
FIG. 4.
Embryonic gonadal expression of Ptgds is dependent on Dax1. In situ hybridization of Ptgds on whole-mount gonads at 13.5 dpc was performed on wild-type male gonads (A), in which expression is detected in testis cords. Ptgds expression was less distinct in 129Dax1−/Y gonads (B), and it was further abolished in 129;B6Dax1−/Y gonads (C) of mixed background. As a negative control (D), wild-type females had no detectable Ptgds expression. Quantitative expression levels of Ptgds transcripts were measured in embryonic gonads at 12.5 dpc by real-time RT-PCR (graph). In each case, relative expression (y-axis) was calculated with reference to wild-type (WT) male. There was a sexually dimorphic pattern of expression (compare WT male [black bar] vs. female [diagonally striped bar]). In 129Dax1−/Y gonads (gray bar), Ptgds transcript levels were markedly reduced, even though these mice mature as hypogonadal males. In 129;B6Dax1−/Y gonads (white bar) there was a further decrease in the expression levels of Ptgds. Bars represent mean ± SD. Statistical significance between genotype groups was evaluted by Student t-test; the asterisk symbol indicates a P value < 0.05.
FIG. 5.
FIG. 5.
Upregulation of Stra8 expression level in PGCs of Dax1 mutant gonads. Stra8 expression was measured to assess the commitment steps of the PGC lineage in Dax1-mutant gonads on both the pure 129 and the mixed 129;B6 backgrounds. Since Stra8 is a female-specific gene, the relative expression (y-axis) was measured with reference to wild-type female (white bars). Stra8 expression is sexually dimorphic during development; thus, males lack detectable levels of expression (diagonally striped bars). Quantitative real-time RT-PCR was performed at two timepoints: 13.0 and 14.5 dpc. In 129Dax1−/Y gonads, which develop as males, the level of Stra8 expression at 13.0 dpc (gray bars) was low, then increased significantly by 14.5 dpc, to reach a level nearly as high as the one in females. In 129;B6Dax1−/Y gonads (black bars), in which sex reversal was observed, Stra8 transcript abundance was markedly increased as early as 13.0 dpc, then exceeded female levels by 14.5 dpc. Bars represent mean ± SD. Statistical significance between genotype groups was evaluted by Student t-test; the asterisk symbol indicates a P value < 0.05.
FIG. 6.
FIG. 6.
Loss of Dax1 leads to Sertoli cell apoptosis. Apoptotic cells were identified in the developing gonads of 129;B6Dax1−/Y mice. Double staining of either AMH (red) and TUNEL labeling (green), or gammaH2AX (yellow) and TUNEL labeling revealed that apoptotic cells were of the Sertoli lineage. A) Cytoplasmic Sertoli cell staining of AMH in wild-type (WT) males demarcated testis cords, whereas TUNEL-positive cells were absent. B) A representative image of a 129;B6Dax1−/Y gonad section shows double staining of apoptotic cells with AMH-positive Sertoli cells (arrow). C) Wild-type female tissue lacked both AMH protein and apoptotic cells. DF) Double labeling of gammaH2AX, a germ cell marker, combined with TUNEL detection was used to distinguish apoptotic germ cells. D) Males lacked gammaH2AX positivity, as expected. E) In 129;B6Dax1−/Y gonads, apoptotic nuclei did not overlap with staining for gammaH2AX (arrow). F) Females had characteristically high levels of gammaH2AX-containing cells. Once again, both wild-type control tissues lacked apoptotic cells. The average number of apoptotic cells per gonad field is represented in the graph. Error bar represents mean ± SD. Bar = 50 μm.

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