Alterations of sex determination pathways in the genital ridges of males with limited Y chromosome genes†

Abstract

We previously demonstrated that in the mouse only two Y chromosome genes are required for a male to produce an offspring with the help of assisted reproduction technologies (ART): testis determinant Sry and spermatogonial proliferation factor Eif2s3y. Subsequently, we have shown that the function of these genes can be replaced by transgenic overexpression of their homologs, autosomally encoded Sox9 and X-chromosome encoded Eif2s3x. Males with Y chromosome contribution limited to two (XEif2s3yOSry), one (XEif2s3yOSox9 and XOSry,Eif2s3x), and no genes (XOSox9,Eif2s3x) produced haploid germ cells and sired offspring after ART. However, despite successful assisted reproductive outcome, they had smaller testes and displayed abnormal development of the seminiferous epithelium and testicular interstitium. Here we explored whether these testicular defects originated from altered pro-testis and pro-ovary factor signaling in genital ridges at the time of sex determination. Timed pregnancies were generated to obtain transgenic XEif2s3yOSry, XEif2s3yOSox9, XOSry,Eif2s3x, XOSox9,Eif2s3x, and wild-type XX and XY fetuses at 12.5 days post coitum. Dissected genital ridges were assessed for their morphology and anatomy, and expression of pro-testis and pro-ovary transcripts. All transgenic males displayed incomplete masculinization of gonadal shape, impaired development of testicular cords and gonadal vasculature, and decreased expression of factors promoting male pathway. Fetal gonad masculinization was more effective when sex determination was driven by the Sry transgene, in the presence of Y chromosome genes, and to a lesser extent a double dosage of X genes. The study adds to the understanding of the role of Y chromosome genes and their homologs during sex determination.

Keywords: X chromosome; Y chromosome; sex determination; sex differentiation; testis.

Figure 1.

Breeding schemes. The breeding schemes that were used to produce male progeny with limited Y chromosome genes. The primary genotypes of interest are shown in yellow boxes: X^EOSry (A), X^EOSox9 (B), XOSry,Eif2s3x (C), XOSox9,Eif2s3x (D). Female genotypes and male genotypes are shown in pink and blue font, respectively. X and Y represent wild-type sex chromosomes. X^E is an X chromosome carrying the Eif2s3y transgene. Y^Tdym1 is a Y chromosome carrying a deletion of endogenous Sry. Eif2s3x, Sry, and Sox9 are autosomally encoded transgenes. Embryos lacking an X chromosome do not survive (crossed). Progeny genotype distribution is shown as a number and an average percentage (mean ± SEM; with n = number of litters: 18, 8, 15, and 14 for A, B, C, and D, respectively) of pups of each genotype. One-way ANOVA analysis revealed significant differences in progeny genotype distribution: P = 0.003 (A); P = 0.0052 (B); P = 0.0032 (C); and P < 0.0001 (D). For other analyses, see Supplementary Figures S2–S4.

Figure 2.

Morphometric evaluation of gonadal shape in the context of XO. Four types of males with limited Y chromosome gene complement (tested: X^EOSry, X^EOSox9, XOSry,Eif2s3x, and XOSox9,Eif2s3x) were compared to wild-type XY and XX controls. (A) Diagrammatic presentation of measures for morphometric analysis; (B) gonadal width; (C) gonadal length: width ratio. (D) Exemplary images of genital ridges. Graphs are mean ± SDev (n ≥ 4, except X^EOSox9 n = 2). Statistical significance (t-test, P < 0.05): graph bars marked with different letters are significantly different from each other while those marked with the same letters do not differ. tg = transgene. Bar = 100 μm.

Figure 3.

Testis cord and gonadal vasculature development evaluation in the context of XO. Four types of males with limited Y chromosome gene complement (tested: X^EOSry, X^EOSox9, XOSry,Eif2s3x, and XOSox9,Eif2s3x) were compared to wild-type XY controls in terms of testis cord and gonadal vasculature development. (A) Exemplary images of XY (top) and XOSox9,Eif2s3x (bottom) genital ridges displaying parameters used in morphometric analyses of testis cord and gonad vasculature development. Black dashed lines are outlines of the testis cords; blue arrows show testis cord width; red arrowheads point to coelomic artery; red arrows show interstitial arterial branches; bar = 100 μm; (B) testis cord number; (C) testis cord width; (D) coelomic artery development expressed as percentage of genital ridge coelomic surface coverage; (E) overall vascular score expressed using 6 point system, with maximum two points derived from each category: coelomic artery visibility, coelomic artery surface coverage, and number of vascular clusters in the interstitial domain (see Methods section for more details). Graphs are mean ± SDev (B, D, and E, n > 3, except X^EOSox9 n = 2) and mean ± SEM (C, n > 4). Statistical significance (t-test, P < 0.05): graph bars marked with different letters are significantly different from each other while those marked with the same letters do not differ. tg = transgene.

Figure 4.

Transcript expression in the genital ridges of XO males. Four types of males with limited Y chromosome gene complement (tested: X^EOSry, X^EOSox9, XOSry,Eif2s3x, and XOSox9,Eif2s3x) were compared to wild-type XY and XX controls. Transcript expression was defined for a master regulator Sox9 (A), male-specific targets of SOX9: Amh, Fgf9, Cyp26b1 (B), and ovarian markers known to be downregulated by SOX9 signaling: FoxL2, Rspo1, Wnt4 (C). The loading controls were with three ubiquitously expressed genes (Sdha, Rps29, and Tbp), and normalization was achieved by geometric averaging of these genes. The reference controls were XY (A and B) and XX (C). Values are mean ± SDev with n > 3 (except X^EOSox9, n = 2). Statistical significance (t-test, P > 0.05): graph bars marked with different letters are significantly different from each other while those marked with the same letters do not differ.

Figure 5.

The effects of X chromosome number on male gonad development. Four sets of male genotypes carrying an analogous transgene complement in the context of either XO or XX were compared (tested pairs: X^EOSry vs X^EXSry, X^EOSox9 vs X^EXSox9, XOSry,Eif2s3x vs XXSry,Eif2s3x, and XOSox9,Eif2s3x vs XXSox9,Eif2s3x). (A) Exemplary images of genital ridges. Black arrows point to representative testis cords, which are poorly resolved and lacking in width in males carrying the Sox9 transgene when compared to the Sry transgenics. (B) Gonadal width; (C) gonadal length/width ratio; (D) testis cord number; (E) testis cord width; (F) coelomic artery development expressed as percentage of genital ridge coelomic surface coverage; (G) overall vascular score expressed using 6 point system, with maximum two points derived from each category: coelomic artery visibility, coelomic artery surface coverage, and number of vascular clusters in the interstitial domain (see Methods section for more details). Graphs are mean ± SDev (A–C and E–F, n > 3, except X^EOSox9 n = 2) and mean ± SEM (D, n > 3). Statistical significance (t-test, P < 0.05): ^a different from XX; ^b different from XY; the differences between XX vs XO are marked by the horizontal lanes with * P < 0.05, ** P < 0.01, *** P < 0.001.Bar = 100 μm.

Figure 6.

The effects of X chromosome number on transcript expression in the genital ridges. Four sets of male genotypes carrying an analogous transgene complement in the context of either XO or XX were compared (tested pairs: X^EOSry vs X^EXSry, X^EOSox9 vs X^EXSox9, XOSry,Eif2s3x vs XXSry,Eif2s3x, and XOSox9,Eif2s3x vs XXSox9,Eif2s3x). Transcript expression was defined for a master regulator Sox9 (A), male-specific targets of SOX9: Amh, Fgf9, Cyp26b1 (B), and ovarian markers known to be downregulated by SOX9 signaling: FoxL2, Rspo1, Wnt4 (C). The loading controls were with three ubiquitously expressed genes (Sdha, Rps29, and Tbp), and normalization was achieved by geometric averaging of these genes. The reference controls were XY (A and B) and XX (C). Values are mean ± SDev with n > 3 (except X^EOSox9, n = 2). Statistical significance (t-test, P < 0.05): ^a different from XX; ^b different from XY; the differences between XX vs XO are marked by the horizontal lanes with * P < 0.05, ** P < 0.01, *** P < 0.001.