Deletion of the mouse P450c17 gene causes early embryonic lethality

Abstract

Dehydroepiandrosterone (DHEA), a 19-carbon precursor of sex steroids, is abundantly produced in the human but not the mouse adrenal. However, mice produce DHEA and DHEA-sulfate (DHEAS) in the fetal brain. DHEA stimulates axonal growth from specific populations of mouse neocortical neurons in vitro, while DHEAS stimulates dendritic growth from those cells. The synthesis of DHEA and sex steroids, but not mouse glucocorticoids and mineralocorticoids, requires P450c17, which catalyzes both 17 alpha-hydroxylase and 17,20-lyase activities. We hypothesized that P450c17-knockout mice would have disordered sex steroid synthesis and disordered brain DHEA production and thus provide phenotypic clues about the functions of DHEA in mouse brain development. We deleted the mouse P450c17 gene in 127/SvJ mice and obtained several lines of mice from two lines of targeted embryonic stem cells. Heterozygotes were phenotypically and reproductively normal, but in all mouse lines, P450c17(-/-) zygotes died by embryonic day 7, prior to gastrulation. The cause of this early lethality is unknown, as there is no known function of fetal steroids at embryonic day 7. Immunocytochemistry identified P450c17 in embryonic endoderm in E7 wild-type and heterozygous embryos, but its function in these cells is unknown. Enzyme assays of wild-type embryos showed a rapid rise in 17-hydroxylase activity between E6 and E7 and the presence of C(17,20)-lyase activity at E7. Treatment of pregnant females with subcutaneous pellets releasing DHEA or 17-OH pregnenolone at a constant rate failed to rescue P450c17(-/-) fetuses. Treatment of normal pregnant females with pellets releasing pregnenolone or progesterone did not cause fetal demise. These data suggest that steroid products of P450c17 have heretofore-unknown essential functions in early embryonic mouse development.

FIG. 1.

Targeted disruption of the P450c17 gene. (A) Partial restriction endonuclease map of the murine P450c17 genomic locus showing the locations of the probe used in cloning the genomic fragment and the approximate positions of primers 1 and 2. Restriction endonuclease sites: E, EcoRI; B, BamHI; A, AvaI; S, SacI. (B) P450c17 targeting vector containing the neomycin resistance (NEO) gene under the control of the PGK promoter. (C) Structure of the recombinant P450c17 mutant allele, showing the location of primer 3. (D) Southern blot of DNA prepared from the offspring of a P450c17^+/− × P450c17^+/− mating. DNA was digested with EcoRI and hybridized to the radiolabeled P450c17 genomic probe shown in panel A. The positions of the wild-type (WT; 10-kb) and targeted (knockout [KO]; 6.5-kb) alleles are indicated with arrows.

FIG. 2.

Expression of the P450c17 gene in the postimplantation mouse embryo. Histological and immunocytochemical analyses were performed on E7.0 mouse embryos. (A and C) Hematoxylin- and eosin-stained sections through two E7 embryos. (B and D) Immunocytochemical analysis of the sections with an antibody to human P450c17. RBC, red blood cells.

FIG. 3.

P450c17 enzyme activities in mouse embryos. (A) 17α-Hydroxylase activity. E7 embryos from timed pregnancies were removed from the deciduae, or entire E5-E6 decidual tissues containing embryos were incubated with [¹⁴C]pregnenolone, NADPH, and cyanoketone for 1, 2, and 4 h, and the resulting steroids were analyzed by thin-layer chromatography and phosphorimaging in comparison to the migration of known standards. DHP, dihydroprogesterone. (B) C_17,20-lyase activity. E7 embryos (n = 10) from timed pregnancies were removed from the decidua and incubated with 17-OH-[³H]progesterone, NADPH, and finasteride for 1 and 3 h. Rat ovarian microsomal protein (Ov; 25 μg) and E7 decidual homogenate (Decid; 25 μg of protein) were used as controls. The resulting steroids were analyzed by thin-layer chromatography and phosphorimaging in comparison to the migration of known standards.

FIG. 4.

Laser catapult microdissection of mouse embryo sections. Embryos from timed pregnancies were fixed, sectioned, and analyzed for P450c17 expression by immunocytochemistry (left panels). Embryos that were not immunostained (indicated by white arrowheads), as well as control embryos that were positively immunostained (indicated by white arrowheads), were counterstained with hematoxylin and eosin and collected by laser catapult microdissection (middle panels). Black arrowheads indicate the laser cut outline that was made to ensure that no maternal tissue was collected. DNA was prepared from the tissue and analyzed by PCR amplification (right panels). The size of the wild-type (WT) allele DNA fragment is 500 bp, and the size of the knockout (KO) allele DNA fragment is 270 bp. M, markers.