Targeted ablation of the WW domain-containing oxidoreductase tumor suppressor leads to impaired steroidogenesis

Abstract

The WW domain-containing oxidoreductase (WWOX) gene encodes a 46-kDa tumor suppressor. The Wwox protein contains two N-terminal WW domains that interact with several transcriptional activators containing proline-tyrosine motifs and a central short-chain dehydrogenase/reductase domain that has been suggested to play a role in steroid metabolism. Recently, we have shown that targeted deletion of the Wwox gene in mice leads to postnatal lethality and defects in bone growth. Here, we report that Wwox-deficient mice display impaired steroidogenesis. Mutant homozygous mice are born with gonadal abnormalities, including failure of Leydig cell development in testis and reduced theca cell proliferation in ovary. Furthermore, Wwox(-/-) mice displayed impaired gene expression of key steroidogenesis enzymes. Affymetrix microarray gene analysis revealed differentially expressed related genes in steroidogenesis in knockout mice testis and ovary as compared with control mice. These results demonstrate the essential requirement for the Wwox tumor suppressor in proper steroidogenesis.

Figure 1

Gonadal phenotype in Wwox KO male mice. A, Leydig cell defects. Histology section of testis showing normal Leydig cells in WT testis (a and c) stained with anti-WWOX antibody, whereas the KO testis (b and d) shows sparse interstitium and few Leydig cells and negative staining of Wwox. A positive marker for Leydig cells, anti-P450 scc (e–h), was used to confirm the marked reduction of Leydig cells in KO testis section. Arrows point at Leydig cells. B, Real-time PCR analysis of testis cDNA identifies impaired steroidogenesis in KO testis. Expression of key genes in the steroidogenesis pathway was analyzed. C, Semiquantitative RT-PCR analysis of testis cDNA identifies impaired steroidogenesis in KO testis. Expression of key genes in the steroidogenesis pathway was analyzed. The expression of cyclophilin A was used as a control.

Figure 2

Gonadal phenotype in Wwox KO female mice. A, Histology of the ovary shows multiple follicles at different stages in WT, whereas in KO mice follicles tended to be smaller in size. Staining with anti-WWOX antibody in a and b. c and d demonstrate reduced proliferation in theca cell in KO (d) compared with WT (c) after staining with anti-Ki67 antibody. B, Real-time PCR analysis of ovaries cDNA identifies impaired steroidogenesis in KO ovary. The expression of cyclophilin A was used as a control in B. C, Semiquantitative RT-PCR analysis of ovary cDNA identifies impaired steroidogenesis in KO testis. Expression of key genes in the steroidogenesis pathway was analyzed. The expression of cyclophilin A was used as a control.

Figure 3

Reduced expression of Fshb and Lhb in the pituitary gland of Wwox-null mice. A, Real-time PCR (TaqMan; Applied Biosystems, Foster City, CA) showing down-regulation of Fshb and Lhb in different Wwox KO mice compared with WT mice. B, Immunohistochemical staining of Fsh and Lh in the pituitary gland showing reduced expression in Wwox KO mice.

Figure 4

Wwox expression modulates steroidogenic gene expression in MLTC-1 Leydig cells in vitro. MLTC-1 mouse Leydig cells transiently expressing Wwox as indicated were harvested, and total RNA was extracted. Results show real-time PCR analysis of steroidogenic-related gene expression (as indicated) after the different transient expression relative to green fluorescent protein (GFP) expression. Results represent fold difference relative to expression of glyceraldehyde-3-phosphate dehydrogenase.