Richard N. Yu, Masafumi Ito, Thomas L. Saunders, Sally A. Camper& J. Larry Jameson Nature Genetics volume 20 december 1998
Ahch (also known as Dax1) encodes a transcription factor that has been implicated in sex determination and gonadal differentiation (13) . Mutations in human AHC cause X-linked, adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (4,5) (HH). Duplication of the Xp21 dosage-sensitive sex reversal (DSS) region, which contains the Ahch locus 1 , and transgenic overexpression of Ahch (ref. 6) cause male-to-female sex reversal. Using Cremediated disruption of Ahch, a mouse model of AHC-HH has generated that allows the function of Ahch to be examined in both males and females. Although Ahch has been postulated to function as an ovarian determination gene (2,6) , the loss of Ahch function in females does not affect ovarian development or fertility. Ahch is instead essential for the maintenance of spermatogenesis. Lack of Ahch causes progressive degeneration of the testicular germinal epithelium independent of abnormalities in gonadotropin and testosterone production and results in male sterility. Ahch is thus not an ovarian determining gene, but rather has a critical role in spermatogenesis.
Ahch encodes Dax-1, a member of the nuclear hormone receptor superfamily that lacks a typical zinc-finger DNA-binding domain (4 ). It is expressed in the developing urogenital ridge, ovary, testis, adrenal cortex, hypothalamus and anterior pituitary gland (2) , and it colocalizes with another orphan nuclear receptor, steroidogenic factor 1 (Sf1; ref. 7). Sf-1 activates Ahch, and its gene product, Dax-1, inhibits the transcriptional activity of Sf-1 through protein-protein interactions (811) , suggesting that they act in a common genetic pathway. Disruption of mouse Ftzf1, which encodes Sf-1, leads to complete adrenal and gonadal agenesis, persistence of Müllerian structures in male mice and hypothalamic and pituitary abnormalities (1214). AHC mutations in humans cause an X-linked syndrome with AHC and HH. This disorder is characterized by adrenal insufficiency, which usually presents in early infancy, and reflects the abnormal development of the adult zone of the adrenal cortex (15 ). Later in life, affected males fail to undergo puberty. They have low serum gonadotropin levels and there is evidence for hormonal defects at both the hypothalamic and pituitary levels (15,16) . Females who are heterozygous carriers of AHC mutations are normal and there are no descriptions of female homozygotes with AHC mutations, presumably because males who would transmit the disease are infertile. Although the salient clinical and hormonal manifestations ofAHC have been well characterized in humans, it has not been possible to study the effects of AHC mutations during organ development. Several lines of evidence indicate that AHC may have a role in ovarian development.
1- A duplication of the DSS locus at Xp21, which encompasses AHC, causes phenotypic male-to-female sex reversal in XY genetic males (1) .
2-it was also shown that transgenic overexpression of Ahch antagonizes the function of Sry, preventing normal testes development and converting the bipotential gonad towards the ovarian lineage (6)
CreloxP recombination 17 allows targeting of the Ahch locus in ES cells (linea di cellule indifferenziate) and to permit transmission of a disrupted X-linked Ahch to both male and female offspring. Recombination sites were introduced on either side of exon 2 of the mouse Ahch locus . Deletion or point mutations of this Ahch exon are known to cause AHC and HH (ref. 15). Cremediated excision of the loxP-targeted ('floxed') gene in transfected ES cells generated multiple cell lines exhibiting deletion. None of the isolates, however, contained a deleted Ahch second exon, consistent with the idea that Ahch function is necessary to sustain growth of the undifferentiated ES cells.
Four targeted ES cell lines were used to generate chimaeric, floxed Ahch male mice (Ahch flox2/Y ). Transmission of the floxed Ahch second exon was confirmed in the offspring of chimaeric animals by PCR and Southern-blot analyses. No phenotypic abnormalities in mice heterozygous or homozygous for the loxP-modified Ahch locus were seen. Female offspring carry both normal and Ahch-deleted alleles (Ahch Ahch/del2 ). Male offspring have only a single Ahch-deleted allele (Ahch del2/Y ). There is no detectable expression of full-length Ahch transcript in tissues in which it is normally produced, and the deleted gene is transmitted through the germ line.
Ahch del2/Y mice are externally indistinguishable from their wild-type litter mates and are similar in size. Development of the fetal and adult cortical zones in Ahch del2/Y adrenal glands are similar to wild-type mice until sexual maturation). The outer adult cortical zone of Ahch del2/Y mice have normal zonae glomerulosa and fasciculata, but the fetal X-zone fails to regress as normally occurs after puberty 18 . The retention of the fetal zone in Ahch del2/Y males resembles the adrenal defect in humans with ACH, in that the adrenal cortex contains fetal-type cells, but it differs in that the adult zone is absent in humans with ACH (ref. 4). Immunohistochemical staining of P450 side-chain cleavage enzyme is present in both wild-type and mutant adrenal glands, but the reaction is weaker in the less well-developed zona fasciculata of mutant mice. Consistent with the relatively normal structure of the adrenal gland in mutant mice, the serum corticosterone levels are similar to those of wild-type animals. These results suggest that Ahch function is required for the initiation of fetal adrenal degeneration, but it is not necessary for the formation of the definitive cortex or steroidogenesis in mice.
Ahch del2/Y males are hypogonadal, and paired testicular weights are reduced by approximately one-half in comparison with wild-type males. Cryptorchidism does not occur and reproductive organs other than the testes are normal. Testosterone production during embryonic and early postnatal development is sufficient for the formation of male internal and external genitalia, for testicular descent and for the normal development of the testosterone-sen-sitive seminal vesicles.
Testes from immature Ahch del2/Y animals demonstrate a lack of stratification of the germinal epithelium. Early pachytene stage spermatocytes fill the tubules, obscuring the central lumen. At 10 weeks, seminiferous tubules exhibit a spectrum of epithelial dysgenesis and degeneration. Some tubules show thin, irregular epithelia with sloughing of germ cells into the lumen, and a small number of type A spermatogonia and Sertoli cells are retained. Occasional seminiferous tubules exhibit active spermatogenesis with stratified germinal layers and a relatively normal appearance. Complete loss of germ cells is evident after 14 weeks. Ahch function is therefore not required for the initiation of spermatogenesis, but it is essential for the maintenance of germinal epithelial integrity and gametogenesis in the adult.
The degeneration of large segments of the seminiferous tubules may reflect a primary Sertoli cell defect, consistent with the proposed role of Dax-1 function in the regulation of spermatogenesis (19 ). Leydig cell hyperplasia and hypertrophy are also observed adjacent to severely degenerated tubules, suggesting either a primary defect due to loss of Dax-1 function in Leydig cells or a secondary Leydig cell response to the inactivation of Ahch in Sertoli cells.
The spermatogenic defect could also arise from altered production of hypothalamic gonadotropin-releasing hormone (GnRH), or from deficiencies of the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (16) (FSH). Pituitary glands from wild-type and mutant animals are identical in size and there are no differences in the number of cells or intensity of immunohistochemical staining for LHb, FSHb, glycoprotein hormone a-subunit, or ACTH . In contrast to human males with AHC mutations (15) , serum hormone measurements of LH and FSH from Ahch del2/Y males are indistinguishable from those of wild-type mice. Hypogonadism is therefore unlikely to reflect deficiencies of LH or FSH, but rather stems from primary testicular failure.
Homozygous mutant females (Ahch del2/del2 ) were generated by mat-ing females heterozygous for the Ahch deletion and with males carrying the loxP-modified Ahch locus. Disruption of Ahch function in females does not affect sexual maturation, ovulation or fertility. The macroscopic appearance of the female internal reproductive organs, including the ovaries, is normal. Histological analyses of ovaries from mature, Ahch del2/del2 females show a normal complement of follicles at different stages of maturation, as well as the presence of corpora lutea. Ahch del2/del2 females mated with wild-type males produce normal litter sizes with equal transmission of the mutation to male and female offspring.
Despite apparently normal fertility in females, a subset of follicles exhibit an abnormality characterized by the presence of multiple oocytes.. These results implicate Ahch in follicular recruitment, granulosa cell proliferation, or in the formation of structures that normally segregate different follicles. It is possible that the abnormal granulosa cell organization surrounding the oocyte is functionally related to a defect in Sertoli cell sup-port of germ cells in the male. Ahch function is not required for ovarian formation and it is not an ovarian determining factor (3) . As Ahch mutations are transmitted in vivo, it is an unexpected result that targeted disruption of the Ahch locus impairs the survival of undifferentiated ES cells. RT-PCR analysis of RNA isolated from wild-type ES cells reveals abundant Ahch mRNA, suggesting that Dax-1 may function in the survival of ES cells.
The design of the conditional mutation of the second exon ofAhch was based on reports indicating that frameshift or nonsensemutations of the C-terminal region of Dax-1 (which is encoded by the second exon) are sufficient to cause fully penetrant AHC-HHG (refs 9,15,20). Cre-mediated excision of the floxed second exon also ablates the intronic splice acceptor site and the down-stream polyadenylation signal, resulting in low levels of an abnormal, unspliced transcript. As some residual Ahch transcript remains, the possibility that a truncated Dax-1 is produced that might exhibit partial or altered function cannot entirely rule out. Such a protein product might account for the incomplete adrenal insufficiency and retained gonadotropin and ovarian function in mice. There is no evidence to date, however, for a correlation of AHC phenotype with the locations of AHC mutations (15) , suggesting that N-terminal Dax-1 protein products are unlikely to be functional.
The data indicate that Ahch is essential for the integrity of the testicular germinal epithelium, which ultimately affects gameto-genesis and fertility. Spermatogenesis is not severely impaired until later in adulthood, raising the possibility that alterations in Ahch function might represent one of several factors that cause defects in spermatogenesis and male infertility. Dax-1, acting in a dose-dependent manner, may function to restrict the inductive activity of testis-promoting factors such as Sry (ref. 6) and Sf-1 (ref. 9), thereby modulating their effects during testicular development . In addition, Ahch is not required for ovarian development, in combination with the reported sex-reversal phenotype that results from Ahch overexpression, supports a role for Ahch as an 'anti-testis' factor 6 as opposed to an ovarian determination gene.