Targeted disruption of the Ahch (Dax-1) gene: knockout of old concepts.

Mutations in the human transcription factor AHC cause the X-linked recessive form of adrenal hypoplasia congenita (1–3). Boys with adrenal hypoplasia congenita present with cortisol and mineralocorticoid deficiency in early childhood, and they fail to undergo puberty due to hypogonadotropic hypogonadism (HHG) (3, 4). In addition, a subset of these patients has a contiguous gene deletion syndrome involving several X-chromosomal genes (glycerol kinase deficiency, Duchenne muscular dystrophy, ornithine transcarbamoyl transferase deficiency, mental retardation). Duplication of the AHC locus on chromosome Xp21 also has dramatic consequences and results in a female phenotype in 46XY males, a mechanism referred to as ‘dosage sensitive sex reversal’. The AHC gene, initially called DAX-1 (dosage-sensitive sex reversal – adrenal hypoplasia congenita on the X chromosome gene 1), consists of two exons and encodes an orphan nuclear receptor. The aminoterminus is formed of three and a half copies of a repeated motif, but lacks a typical zinc-finger DNA binding domain, while the carboxyterminal domain displays high homology to the ligand domain of nuclear receptors. AHC is expressed in the three layers of the adrenal cortex, ovarian granulosa and theca cells, testicular Sertoli and Leydig cells, pituitary gonadotropes, and in the hypothalamus (5, 6). Remarkably, there is developmental and spatial coexpression with another orphan nuclear receptor, the steroidogenic factor-1 (Sf-1), a key regulator in the development and function of the reproductive axis (7). Functionally, AHC was found to be an inhibitor of Sf-1-mediated transcription in transfection assays (8), and it can also directly repress certain target genes (9). Consistent with the human phenotype, transgenic overexpression of the mouse homologue Ahch resulted in sex reversal by antagonizing the Y-chromosomal Sry gene (10). In the December 1998 issue of Nature Genetics, Richard Yu and colleagues present the targeted disruption of Ahch in transgenic mice. Several of their observations are surprising and challenge our concepts on sexual development (11). Using a conditional knockout with a Cre-loxP strategy, the carboxyterminal domain of Ahch was deleted. Exon 2 of Ahch was first flanked by loxP sites and a neomycin cassette was inserted downstream of it. This transgene was introduced in embryonic stem cells for homologous recombination. Mice expressing this transgene were then mated with transgenic mice overexpressing the recombinase Cre, an enzyme that recognizes the loxP sites. By this strategy, exon 2 could be excised in preimplantation embryos. In males, this results in a deletion of the X-chromosomal Ahch gene (AhchD2/Y), while females are hemizygous for the mutation (AhchD2/Ahch). At birth, the transgenic AhchD2/Y males did not differ from their wild-type littermates. However, at the onset of sexual maturation, the fetal adrenal cortex failed to regress. The zona glomerulosa and fasciculata were found to be normal, and corticosterone levels were within the normal range. This contrasts, in part, with the findings in humans with adrenal hypoplasia congenita. In these patients, the adrenal cortex also contains fetal cells, but the permanent zone of the adrenal cortex does not develop and it is replaced by large vacuolated cells (12). Expression of the P450 side-chain cleavage enzyme was present in the adrenal cortex of both the wild-type and the mutant animals, but reduced in the zona fasciculata. These results suggest that Ahch is required for the involution of the fetal adrenal cortex, but is not necessary for the development of the mature cortex and steroidogenesis in mice. The testicles of DAhch/Y mice were reduced by about 50% in volume, but there were no other malformations of the reproductive organs. Histologically, the germinal epithelium was not stratified normally, and older mice showed epithelial dysgenesis and degeneration. After 14 weeks, there was a complete loss in germ cells. This suggests that Ahch is not involved in the initiation of spermatogenesis, but essential for the maintenance of the germinal epithelium as well as Sertoli cell function. Moreover, Leydig cell hyperplasia and hypertrophy were observed adjacent to degenerated seminiferous tubules. Pituitary glands from wild-type and mutant animals were similar in size and there were no differences in serum gonadotropin levels, cell numbers, or immunopositivity for pituitary hormones. Serum levels for luteinizing hormone, follicle-stimulating hormone and testosterone were not different in transgenic mice in comparison to their wild-type littermates. These findings are distinct from the human phenotype with combined hypothalamic-pituitary HHG (4). They suggest that the basis for the infertility in the transgenic DAhch/Y mice is the result of a primary testicular defect affecting the germinal epithelium rather than a hypothalamic or pituitary defect. European Journal of Endocrinology (1999) 140 291–292 ISSN 0804-4643 H IG H L IG H T