Sox3 is required for gonadal function, but not sex determination, in males and females

Abstract

Sox3 is expressed in developing gonads and in the brain. Evolutionary evidence suggests that the X-chromosomal Sox3 gene may be the ancestral precursor of Sry, a sex-determining gene, and Sox3 has been proposed to play a role in sex determination. However, patients with mutations in SOX3 exhibit normal gonadal determination but are mentally retarded and have short stature secondary to growth hormone (GH) deficiency. We used Cre-LoxP targeted mutagenesis to delete Sox3 from mice. Null mice of both sexes had no overt behavioral deficits and exhibited normal GH gene expression. Low body weight was observed for some mice; overgrowth and misalignment of the front teeth was observed consistently. Female Sox3 null mice (-/-) developed ovaries but had excess follicular atresia, ovulation of defective oocytes, and severely reduced fertility. Pituitary (luteinizing hormone and follicle-stimulating hormone) and uterine functions were normal in females. Hemizygous male null mice (-/Y) developed testes but were hypogonadal. Testis weight was reduced by 42%, and there was extensive Sertoli cell vacuolization, loss of germ cells, reduced sperm counts, and disruption of the seminiferous tubules. We conclude that Sox3 is not required for gonadal determination but is important for normal oocyte development and male testis differentiation and gametogenesis.

FIG. 1.

Expression of Sox3. Immunohistochemical staining was performed on a testis (A) (magnification, ×200; inset, ×400) and an ovary (D) (magnification, ×400). DAPI staining of testis (B) and ovary (E) was performed to identify cell nuclei, and merged images are presented for comparison (C and F). Arrows in panel A illustrate nuclear staining of Sertoli cells by Sox3. (G) Detection of Sox3 mRNA by RT-PCR, compared to the ribosomal gene RPL19. Controls are included in which RT was omitted to exclude amplification of contaminating genomic DNA.

FIG. 2.

Targeted mutagenesis of Sox3. (A) Sox3 is a single-exon gene located on the X chromosome. The upstream LoxP site (open triangle) was inserted into the 5′ UTR. The downstream LoxP site was inserted downstream of the poly(A) signal sequences adjacent to a Neo cassette. (B) To confirm correct 5′ recombination, genomic DNA from >350 ES cell clones was digested with EcoRV and BglII and subjected to Southern blotting with a probe (gray bar) immediately downstream of the Sox3 coding sequence. ES cells are male (XY) and Sox3 is X linked, so only a single band of either 5,040 bp (targeted) or 7,200 bp (WT) was observed for each clone. (C) Chimeric males were bred with WT females, and progeny that inherited the targeted allele were bred with CMV-Cre transgenic mice. WT and excised animal DNAs were detected by PCR as illustrated in the ethidium bromide-stained gel. The PCR products were eluted from the gel and sequenced to confirm the identities of the fragments and successful excision. Restriction enzymes are abbreviated as follows: RI, EcoRI; BII, BglII; RV, EcoRV; NI, NsiI.

FIG. 3.

Gross morphology of Sox3 KO mice. (A and B) Growth curves. Outliers are denoted by arrows. (C) Misaligned teeth in KO mice. (D) GH mRNA levels, presented as ratios to RPL19. n = 6 or 7.

FIG. 4.

Functional analysis of female Sox3 KO mice. (A) Fertility. WT and Sox3 KO females were paired and mated to WT males. (B) H&E-stained WT ovary. Magnification, ×50. Arrows, large antral follicles; CL, corpus luteum. (C) H&E-stained Sox3 KO ovary. Magnification, ×50. The small box surrounds atretic follicles that are enlarged to a higher magnification in the inset. (D) Hormone levels in sera (n = 5 to 8). (E) Ovulation induction. Six each of WT and KO females were subjected to superovulation, and ova for each genotype were harvested. Oocytes were categorized as dead-malformed or normal and then incubated with WT sperm to determine the rates of fertilization. Values within bars are percentages of the individual genotype pool (WT or KO). Within each category, data are presented as percentages of the WT level.

FIG. 5.

Characterization of male Sox3 KO mice. (A) Left testicle weight (n = 6 or 7); inset, left testicles from two WT and two KO males. (B) Cross section at midpoint of testis stained with H&E (magnification, ×50). (C to H, J, and K) Structural changes in seminiferous tubules of male Sox3 KO mice. Magnification, ×400, unless otherwise noted. Panels C to E illustrate degrees of damage observed. (C) Wild-type stage VII tubule. P, pachytene spermatocytes; S (box), round spermatids. (D) Mildly affected Sox3 KO tubule with detached cluster of germ cells. (E) Severely affected Sox3 KO tubule containing Sertoli cell bodies and projections only. Arrows, region in which the structural boundary of the tubule is missing. Note the presence of large Sertoli cell vacuoles. Panels F to H are serial sections of a Sox3 KO tubule stained with H&E (F), GATA-4 (G) (Sertoli cells), and espin (H) (Sertoli cell-germ cell junctions). Luminal elongated spermatids are joined by adherens junctions (H; inset magnification, ×1,000) in the absence of Sertoli cell bodies. (I) Hormone levels in sera (n = 5 to 8). Panels J and K show laminin staining of WT (J) and Sox3 KO (K) tubules, illustrating breakdown in the tubular boundary in KO mice.