Deletion of the mouse P450c17 gene causes early embryonic lethality.

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-hydroxylase and 17,20lyase 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 C17,20-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. The synthesis of steroid hormones in the adrenals, gonads, placenta, and brain requires the expression of several steroidogenic enzymes. In all tissues, steroidogenesis is initiated by conversion of cholesterol to pregnenolone by the mitochondrial cholesterol side chain cleavage enzyme, P450scc. Thereafter, the specific steroid that is synthesized by a particular tissue depends upon the differential expression of additional steroidogenic enzymes. The conversion of pregnenolone and progesterone to their 17-hydroxylated products and then to either dehydroepiandrosterone (DHEA) or androstenedione, respectively, is mediated by a single microsomal enzyme, P450c17 (33, 34, 48), encoded by a single gene (22, 39). The pattern of P450c17 expression in steroidogenic tissues is species specific: it is expressed in the human adrenal and gonad but not placenta (9, 11, 15, 44), and it is expressed in the rodent gonad and placenta but not adrenal (19, 23, 25). P450c17 is also expressed in the fetal mouse brain beginning at embryonic day 9.5 (E9.5) (12). At this time, P450c17 is found in cells migrating from the neural crest, and subsequently, P450c17 is found in many cells derived from the neural crest. P450c17 is also expressed in the neocortical subplate, a region that receives thalamic projections, produces signals for cortical projections, and may produce signals for efferent thalamic projections from the cortex (32, 41). We hypothesized that DHEA, a steroid product of P450c17, may be an endogenous signal in the subplate to target axons coming from this region to specific sites in the developing cortex and showed that DHEA increased axonal outgrowth while DHEA-sulfate (DHEAS) increased dendritic growth (13). DHEA, but not DHEAS, induced other morphological indices of synaptic contacts, increased mRNAs for Tau-1 and type 1 and 2 dopamine receptors (14), mediated increases in intracellular calcium via N-methyl-D-aspartic acid receptors (13), protected hippocampal neurons from glutamate toxicity (27), and stimulated neurogenesis in the hippocampus (26). Thus, DHEA may serve as an endogenous neurotrophic agent. However, all these studies have been performed with cultured neuronal cells derived from specific regions of the nervous system at particular times in development. Thus, we sought to determine the function of DHEA and DHEAS in vivo during the development of the nervous system by deleting the gene encoding P450c17 in genetargeted mice.