J. Larry Jameson

Role of Ahch in gonadal development and gametogenesis

Ahch (also known as Dax1) encodes a transcription factor that has been implicated in sex determination and gonadal differentiation. Mutations in human AHC cause X-linked, adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Duplication of the Xp21 dosage-sensitive sex reversal (DSS) region, which contains the Ahch locus, and transgenic overexpression of Ahch (ref. 6) cause male-to-female sex reversal. Using Cre-mediated disruption of Ahch, we have generated a mouse model of AHC-HH 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, 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.

Precision Medicine — Personalized, Problematic, and Promising

This article considers the growing recognition of precision medicine by clinicians, patients, the pharmaceutical industry, and policymakers and summarizes how this rapidly accelerating field will leave a major imprint on the practice of medicine.

Estrogen Receptor Binding to DNA Is Not Required for Its Activity through the Nonclassical AP1 Pathway

In the classical signaling pathway, the estrogen receptor (ER) binds directly to estrogen response elements (EREs) to regulate gene transcription. To test the hypothesis that the nonclassical pathway involves ER interactions with other proteins rather than direct binding to DNA, mutations were introduced into the DNA binding domain (DBD) of the mouse ERα. The effects of these DBD mutations were examined in DNA binding assays using reporter constructs containing either EREs (classical) or AP1 (nonclassical) response elements. Using the AP1 reporter, there was a reversal of ER action relative to that seen with the ERE reporter. Estradiol induced suppression, and the antiestrogen ICI 182,780 stimulated transcription of the AP1 reporter. DBD mutations in the proximal (P-box) of the first zinc finger of the ER (E207A/G208A and E207G/G208S) eliminated ERE binding. These mutants were inactive using the ERE reporter but retained partial or full activity with the AP1 reporter. The DBD mutant ERs interacted with Jun when tested in mammalian cell two-hybrid assays. Two mutations (K366D and I362R) in the ER ligand binding domain known to alter coactivator interactions impaired transcriptional responses using either the ERE or AP1 reporters. We concluded that ER action through the AP1 response element involves interactions with other promoter-bound proteins instead of, or in addition to, direct binding to DNA. Interactions with coactivators were required for both pathways. These data supported a model in which ER-mediated transcriptional activation or repression is dependent on the ligand and the nature of the response element in the target gene.

A novel mutation in DAX1 causes delayed-onset adrenal insufficiency and incomplete hypogonadotropic hypogonadism

Mutations in the DAX1 gene cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). In affected boys, primary adrenal insufficiency occurs soon after birth or during early childhood; HHG is recognized at the expected time of puberty. In this report, we describe the novel phenotype of a man who presented with apparently isolated adrenal insufficiency at 28 years of age. Examination revealed partial pubertal development and undiagnosed incomplete HHG. Gonadotropin therapy did not improve his marked oligospermia, suggesting a concomitant primary testicular abnormality. Genomic analysis revealed a novel missense mutation, I439S, in DAX1. The mutant DAX-1 protein was studied for its ability to function as a transcriptional repressor of target genes. Consistent with the patients mild clinical phenotype, the I439S mutation conferred intermediate levels of repressor activity of DAX-1 when compared with mutations associated with classic AHC. This unique case extends the clinical spectrum of AHC to include delayed-onset primary adrenal insufficiency in adulthood and milder forms of HHG. Furthermore, in accordance with findings in Ahch (Dax1) knockout mice, the clinical features in this patient suggest that DAX-1 function is required for spermatogenesis in humans, independent of its known effects on gonadotropin production.

Sox3 Is Required for Gonadal Function, but Not Sex Determination, in Males and Females

ABSTRACT Sox3 is expressed in developing gonads and in the brain. Evolutionary evidence suggests that the X-chromosomal Sox3 gene may be the ancestral precursor of Sry, a sex-determining gene, and Sox3 has been proposed to play a role in sex determination. However, patients with mutations in SOX3 exhibit normal gonadal determination but are mentally retarded and have short stature secondary to growth hormone (GH) deficiency. We used Cre-LoxP targeted mutagenesis to delete Sox3 from mice. Null mice of both sexes had no overt behavioral deficits and exhibited normal GH gene expression. Low body weight was observed for some mice; overgrowth and misalignment of the front teeth was observed consistently. Female Sox3 null mice (−/−) developed ovaries but had excess follicular atresia, ovulation of defective oocytes, and severely reduced fertility. Pituitary (luteinizing hormone and follicle-stimulating hormone) and uterine functions were normal in females. Hemizygous male null mice (−/Y) developed testes but were hypogonadal. Testis weight was reduced by 42%, and there was extensive Sertoli cell vacuolization, loss of germ cells, reduced sperm counts, and disruption of the seminiferous tubules. We conclude that Sox3 is not required for gonadal determination but is important for normal oocyte development and male testis differentiation and gametogenesis.

Regulation of Kiss1 and Dynorphin Gene Expression in the Murine Brain by Classical and Nonclassical Estrogen Receptor Pathways

Kisspeptin is a product of the Kiss1 gene and is expressed in the forebrain. Neurons that express Kiss1 play a crucial role in the regulation of pituitary luteinizing hormone secretion and reproduction. These neurons are the direct targets for the action of estradiol-17β (E2), which acts via the estrogen receptor α isoform (ERα) to regulate Kiss1 expression. In the arcuate nucleus (Arc), where the dynorphin gene (Dyn) is expressed in Kiss1 neurons, E2 inhibits the expression of Kiss1 mRNA. However, E2 induces the expression of Kiss1 in the anteroventral periventricular nucleus (AVPV). The mechanism for differential regulation of Kiss1 in the Arc and AVPV by E2 is unknown. ERα signals through multiple pathways, which can be categorized as either classical, involving the estrogen response element (ERE), or nonclassical, involving ERE-independent mechanisms. To elucidate the molecular basis for the action of E2 on Kiss1 and Dyn expression, we studied the effects of E2 on Kiss1 and Dyn mRNAs in the brains of mice bearing targeted alterations in the ERα signaling pathways. We found that stimulation of Kiss1 expression by E2 in the AVPV and inhibition of Dyn in the Arc required an ERE-dependent pathway, whereas the inhibition of Kiss1 expression by E2 in the Arc involved ERE-independent mechanisms. Thus, distinct ERα signaling pathways can differentially regulate the expression of identical genes across different brain regions, and E2 can act within the same neuron through divergent ERα signaling pathways to regulate different neurotransmitter genes.

Phenotypic spectrum of mutations in DAX-1 and SF-1.

SF-1 (steroidogenic factor-1) (NR5A1) and DAX-1 (dosage-sensitive sex-reversal, adrenal hypoplasia congenital, X chromosome) (NR0B1) are orphan nuclear receptors that are expressed in the adrenal gland, gonads, ventromedial hypothalamus (VMH), and pituitary gonadotrope cells. The function of these genes has been clarified by examining the consequences of naturally occurring mutations in humans, as well as targeted disruption of the genes in mice. Mutations in DAX1 cause adrenal hypoplasia congenita (AHC), an X-linked disorder characterized by adrenal insufficiency and failure to undergo puberty because of hypogonadotropic hypogonadism. Most DAX1 mutations introduce frameshifts and/or cause premature termination of the protein. Relatively few missense mutations have been described and all are located within the carboxy-terminal half of the protein. Transfection assays demonstrate that AHC-associated DAX1 mutations abrogate its ability to act as a transcriptional repressor of SF-1. Most boys affected with AHC present with adrenal insufficiency in early infancy, although a significant fraction present in later childhood or even as young adults. The degree of gonadotropin deficiency is also variable. With the exception of one mild missense DAX1 mutation, genotype-phenotype correlations have been elusive, suggesting an important role for modifier genes. Targeted mutagenesis of Dax1 (Ahch) in mice reveals an additional role in testis development and spermatogenesis. Similar abnormalities appear to be present in humans. Targeted mutagenesis of Sf1 (FtzF1) prevents gonadal and adrenal development, and causes male-to-female sex-reversal. A human XY individual with a heterozygous SF1 mutation presented with adrenal insufficiency and complete sex-reversal; this DNA-binding domain mutation prevents SF-1 stimulation of its target genes. In addition to their clinical relevance, studies of SF1 and DAX1 are proving useful for unraveling the genetic pathways that govern adrenal and gonadal development.

Dax1 regulates testis cord organization during gonadal differentiation.

Mutations of the DAX1 nuclear receptor gene cause adrenal hypoplasia congenita, an X-linked disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism. Targeted deletion of Dax1 in mice also reveals primary testicular dysgenesis, which is manifest by obstruction of the rete testis by Sertoli cells and hyperplastic Leydig cells, leading to seminiferous tubule dilation and degeneration of germ cells. Because Dax1 is expressed early in gonadal development, and because Sertoli and Leydig cells are located ectopically in the adult, we hypothesized that these testis abnormalities are the result of an early defect in testis development. In Dax1-/Y males, the gonad develops normally until 12.5 dpc. However, by 13.5 dpc, the testis cords are disorganized and incompletely formed in Dax1-deficient mice. The number of germ and Sertoli cells is unchanged, and the expression of Sertoli-specific markers appears to be normal. However, the number of peritubular myoid cells, which normally surround the testis cords, is reduced. BrdU labeling of peritubular myoid cells is low, consistent with decreased proliferation. The basal lamina produced by peritubular myoid and Sertoli cells is disrupted, leading to open and incompletely formed testis cords. Leydig cells, which normally reside in the peritubular space and extend from the coelomic surface to the dorsal surface of the gonad, are restricted to the coelomic surface of Dax1-deficient testis. We conclude that Dax1 plays a crucial role in testis differentiation by regulating the development of peritubular myoid cells and the formation of intact testis cords. The developmental abnormalities in the Dax1-deficient testis lay the foundation for gonadal dysgenesis and infertility in adult mice and, potentially in humans with DAX1 mutations.

Estradiol Increases Proliferation and Down-Regulates the Sodium/Iodide Symporter Gene in FRTL-5 Cells

Goiter (increased thyroid gland size) is more prevalent in women than men, even in areas where iodine levels in the diet are sufficient. We investigated a possible role of estrogen on thyroid follicular cell growth using rat FRTL-5 thyroid follicular cells as a model. Estrogen receptor-α (ERα) messenger RNA was present in FRTL-5 cells using a RT-PCR assay and was confirmed by Western blot analysis. An estrogen-responsive reporter gene was transfected into FRTL-5 cells to test the functionality of the endogenous ERs. Estradiol increased the activity of the reporter gene, and the antagonist, ICI182780, inhibited ER-dependent transcription. To extend this analysis, we examined the effect of estradiol on FRTL-5 cell growth. Estradiol increased FRTL-5 cell growth in a time- and concentration-dependent manner in either the absence or presence of TSH. Because iodine is known to inhibit thyroid cell growth, the effect of estradiol on the expression of the sodium/iodide symporter (NIS) was assessed as a potential ...

Genetic rescue of nonclassical ERα signaling normalizes energy balance in obese Erα-null mutant mice

In addition to its role in reproduction, estradiol-17β is critical to the regulation of energy balance and body weight. Estrogen receptor α-null (Erα-/-) mutant mice develop an obese state characterized by decreased energy expenditure, decreased locomotion, increased adiposity, altered glucose homeostasis, and hyperleptinemia. Such features are reminiscent of the propensity of postmenopausal women to develop obesity and type 2 diabetes. The mechanisms by which ERα signaling maintains normal energy balance, however, have remained unclear. Here we used knockin mice that express mutant ERα that can only signal through the noncanonical pathway to assess the role of nonclassical ERα signaling in energy homeostasis. In these mice, we found that nonclassical ERα signaling restored metabolic parameters dysregulated in Erα-/- mutant mice to normal or near-normal values. The rescue of body weight and metabolic function by nonclassical ERα signaling was mediated by normalization of energy expenditure, including voluntary locomotor activity. These findings indicate that nonclassical ERα signaling mediates major effects of estradiol-17β on energy balance, raising the possibility that selective ERα agonists may be developed to reduce the risks of obesity and metabolic disturbances in postmenopausal women.