CYP17A1 deficient XY mice display susceptibility to atherosclerosis, altered lipidomic profile and atypical sex development

Abstract

CYP17A1 is a cytochrome P450 enzyme with 17-alpha-hydroxylase and C17,20-lyase activities. CYP17A1 genetic variants are associated with coronary artery disease, myocardial infarction and visceral and subcutaneous fat distribution; however, the underlying pathological mechanisms remain unknown. We aimed to investigate the function of CYP17A1 and its impact on atherosclerosis in mice. At 4-6 months, CYP17A1-deficient mice were viable, with a KO:Het:WT ratio approximating the expected Mendelian ratio of 1:2:1. All Cyp17a1 knockout (KO) mice were phenotypically female; however, 58% were Y chromosome-positive, resembling the phenotype of human CYP17A1 deficiency, leading to 46,XY differences/disorders of sex development (DSD). Both male and female homozygous KO mice were infertile, due to abnormal genital organs. Plasma steroid analyses revealed a complete lack of testosterone in XY-KO mice and marked accumulation of progesterone in XX-KO mice. Elevated corticosterone levels were observed in both XY and XX KO mice. In addition, Cyp17a1 heterozygous mice were also backcrossed onto an Apoe KO atherogenic background and fed a western-type diet (WTD) to study the effects of CYP17A1 on atherosclerosis. Cyp17a1 x Apoe double KO XY mice developed more atherosclerotic lesions than Apoe KO male controls, regardless of diet (standard or WTD). Increased atherosclerosis in CYP17A1 XY KO mice lacking testosterone was associated with altered lipid profiles. In mice, CYP17A1 deficiency interferes with sex differentiation. Our data also demonstrate its key role in lipidomic profile, and as a risk factor in the pathogenesis of atherosclerosis.

Figure 1

Generation and characteristics of Cyp17a1 KO mice. (a) Targeting vector used to generate Cyp17a1 KO mice. (b) Genotyping of mice by PCR (1: XX, Cyp17a1(+/+)xApoe(d/d); 2: XX,Cyp17a1(d/d)xApoe(d/d); 3: XY,Cyp17a1(d/d)xApoe (d/d); 4: XY,Cyp17a1(+/+)xApoe(d/d); 5: XX, Cyp17a1(+/+)xApoe(+/+) control; 6: XX, Cyp17a1(d/d)xApoe(d/d) control; 7: H₂O; M: 100 bp marker). (c) The female appearance of XY KO mice. (d) Increased body weight of an XX KO mouse, compared with a control XX WT littermate. (e) Increased visceral fat in an XX KO mouse compared with a control littermate.

Figure 2

Body weight and visceral fat in Cyp17a1 KO and WT mice on an Apoe KO atherogenic background. Body weight and visceral fat were assessed in mice at 10 weeks old, before dietary differentiation, and after 8 weeks consuming a WTD or a standard chow diet. ns, not significant.

Figure 3

Atherosclerosis analyses in mice fed chow or WTD. (a) Quantification of plaque deposits in aortic roots and representative images after Oil Red O staining (red). (b) Oil Red O quantification and staining of atherosclerotic lesions in the whole aorta of WTD fed mice. ns, not significant.

Figure 4

Heatmap representing lipid species showing differences in levels between XY mice with a Cyp17a1 (d/d) x Apoe (d/d) (dbKO) and Cyp17a1 (+/+) x Apoe (d/d) (Apoe_KO) genotype. Lipids were extracted from plasma samples and evaluated by LC-MS. The levels of each lipid species were set from 0 to 1. The heatmap shows the difference in levels of lipid species between CYP17A1 deficient double KO mice (dbKO) and controls (Apoe_KO) mice. The asterisk (*) indicates lipid species with significantly different levels after correction for multiple testing (Bonferroni-corrected P-value threshold of 2.1 × 10⁻⁴ (0.05/234 all lipid species)).