Joshua J. Meeks

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.

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.

Risk Factors and Management of Urine Leaks After Partial Nephrectomy

PURPOSE As nephron sparing surgery is used more frequently for select renal tumors, the incidence of urine leaks will likely increase. To our knowledge the risk factors of and management strategies for urine leaks have not been studied. We report our experience with the risk factors of and management for urine leaks after open and laparoscopic partial nephrectomy. MATERIALS AND METHODS We retrospectively reviewed the records of 127 consecutive patients who underwent partial nephrectomy between 2001 and 2007, including 70 with open and 57 with laparoscopic partial nephrectomy, as performed by 3 surgeons. Urine leak was defined as drain output consistent with urine greater than 48 hours after laparoscopic partial nephrectomy. RESULTS Of the patients 21 experienced a urine leak after partial nephrectomy, including 13.3% overall, and 10.5% after laparoscopic and 18.5% after open partial nephrectomy. Patients with a urine leak had significantly greater tumor size (3.2 vs 2.4 cm, p <0.044), endophytic locations (57% vs 19%, p <0.00027) and repair of collecting system defects during partial nephrectomy (95% vs 56%, p <0.00072). There was no association with the number of tumors removed, estimated blood loss, ischemia time, body mass index, age or other surgical complications. The median duration of urine leak was 20 days. While most urine leaks resolved with prolonged drainage, 38% of cases required further intervention. Patient age at surgery was the only factor that correlated with prolonged (greater than 30 days) urine leak. CONCLUSIONS Urine leak is a complication unique to partial nephrectomy that is more commonly noted when a larger endophytic mass involves the renal collecting system. Most leaks resolve with prolonged drainage or replacement of a ureteral stent.

Nuclear receptors Sf1 and Dax1 function cooperatively to mediate somatic cell differentiation during testis development

Mutations of orphan nuclear receptors SF1 and DAX1 each cause adrenal insufficiency and gonadal dysgenesis in humans, although the pathological features are distinct. Because Dax1 antagonizes Sf1-mediated transcription in vitro, we hypothesized that Dax1 deficiency would compensate for allelic loss of Sf1. In studies of the developing testis, expression of the fetal Leydig cell markers Cyp17 and Cyp11a1 was reduced in heterozygous Sf1-deficient mice at E13.5, consistent with dose-dependent effects of Sf1. In Sf1/Dax1 (Sf1 heterozygous and Dax1-deleted) double mutant gonads, the expression of these genes was unexpectedly reduced further, indicating that loss of Dax1 did not compensate for reduced Sf1 activity. The Sertoli cell product Dhh was reduced in Sf1 heterozygotes at E11.5, and it was undetectable in Sf1/Dax1 double mutants, indicating that Sf1 and Dax1 function cooperatively to induce Dhh expression. Similarly, Amh expression was reduced in both Sf1 and Dax1 single mutants at E11.5, and it was not rescued by the Sf1/Dax1 double mutant. By contrast, Sox9 was expressed in single and in double mutants, suggesting that various Sertoli cell genes are differentially sensitive to Sf1 and Dax1 function. Reduced expression of Dhh and Amh was transient, and was largely restored by E12.5. Similarly, there was recovery of fetal Leydig cell markers by E14.5, indicating that loss of Sf1/Dax1 delays but does not preclude fetal Leydig cell development. Thus, although Sf1 and Dax1 function as transcriptional antagonists for many target genes in vitro, they act independently or cooperatively in vivo during male gonadal development.

Age-related synaptic changes in the central nucleus of the inferior colliculus of Fischer-344 rats

The central nucleus of the inferior colliculus (ICc) is a major processing center for the ascending auditory pathways. Gamma‐aminobutyric acid (GABA) and excitant amino acids (EAAs) are essential for coding many auditory tasks in the IC. Recently, a number of neurochemical and immunocytochemical studies have suggested an age‐related decline in GABAergic inhibition in the ICc, and possibly excitant‐amino‐acid‐mediated excitation as well. The objective of this study was to compare quantitatively changes in the synaptic organization of the ICc among three age groups (3, 19, and 28 months) of Fischer‐344 rats. Immunogold electron microscopic methods were used to determine if there were age‐related changes in the density, distribution, or morphology of GABA‐immunoreactive (+) and GABA‐immunonegative (–) synapses in the ICc. The data suggest similar losses of excitatory and inhibitory synapses in the ICc. There were significant reductions in the densities of GABA+ and GABA– synaptic terminals (∼30% and ∼24%, respectively) and synapses (∼33% and ∼26%, respectively) in the ICc of 28‐month‐old rats relative to 3‐month‐olds. The numeric values, which were adjusted to consider changes in volume of the IC with age, depict similar effects, although the effect magnitude for the adjusted values was reduced by approximately 9%. For both types of synapses, the decreases did not differ significantly from each other. The reductions in synaptic numbers appeared to be related to a similar numeric decline in dendrites, in particular those with calibers of between 0.5 and 1.5 μm. The number and distribution of synaptic terminals on the remaining dendrites of GABA– neurons appeared not to undergo major age‐related changes. GABA+ neurons, on the other hand, may have evolved patterns of synaptic and dendritic change during aging in which the distribution of synaptic terminals shifts to dendrites of larger caliber. In the 19‐month group, the synaptic areas were elevated in terminals apposed to dendrites with calibers of 1.5 μm or less. However, this increase in synaptic size did not persist in the aged animals. No neuronal losses were detectable among the three age groups. Thus, the decrease in GABA and EAAs identified in the IC by previous studies may be attributable to synaptic and dendritic declines, rather than cell loss. J. Comp. Neurol. 406:285–298, 1999.

SF1 in the Development of the Adrenal Gland and Gonads

SF1 (steroidogenic factor-1; NR5A1) is an orphan nuclear receptor that is expressed in the adrenal gland, gonads, spleen, ventromedial hypothalamus and pituitary gonadotroph cells. Combined approaches of targeted mutagenesis in mice and examination of the effects of naturally occurring mutations in humans have clarified the role of SF1 in steroidogenesis and development. Targeted disruption of Sf1 (Ftzf1) in mice prevents gonadal and adrenal development and causes male-to-female sex reversal. A heterozygous loss-of-function human SF1 mutation (G35E) was described in a patient with adrenal failure and complete 46,XY sex reversal, indicating that haploinsufficiency of this transcription factor is sufficient to cause a severe clinical phenotype. In an infant with a similar clinical phenotype, a homozygous SF1 mutation (R92Q) was identified. In functional assays, this mutant SF1 protein exhibited partial loss of DNA binding and transcriptional activity when compared with the more severe G35E P-box mutant. These patients reveal the exquisite sensitivity of SF1-dependent developmental pathways to gene dosage and function in humans.

Tumor Characteristics of Carriers and Noncarriers of the deCODE 8q24 Prostate Cancer Susceptibility Alleles

PURPOSE In collaboration with deCODE Genetics Inc. we previously reported on the association between genetic variants on chromosome 8q24 and prostate cancer susceptibility. Several prior studies have examined the relationship between these 8q24 alleles and clinical tumor features. In this study we examine the differences in clinical and pathological tumor features between carriers and noncarriers of these 8q24 alleles in patients with prostate cancer. MATERIALS AND METHODS We genotyped 551 white men who underwent radical prostatectomy or radiation therapy for clinically localized prostate cancer at single institution between 2002 and 2005. Of these men 177 (32.1%) were carriers of the -8 allele of the microsatellite marker DG8S737, the A allele of the single nucleotide polymorphism rs1447295 and/or the A allele of the rs16901979 (a surrogate single nucleotide polymorphism of HapC) 8q24 alleles. We used statistical analyses to compare the distribution of clinical characteristics and pathological outcomes between carriers and noncarriers. RESULTS The -8, A and HapC surrogate single nucleotide polymorphism alleles were present in 77 (14%), 128 (23%) and 61 (14%) patients with prostate cancer, respectively. Carriers of the -8 or multiple 8q24 alleles were significantly more likely to have a Gleason score of 7 or greater and lymph node metastases. Among men with a family history of prostate cancer, carriers of the -8 allele had a significantly greater risk of high grade disease (64% vs 39%, p = 0.04). CONCLUSIONS In our predominantly surgically treated population there was a significant association between 8q24 prostate cancer susceptibility alleles, particularly the -8 allele, and high grade disease. In men with a family history of prostate cancer the presence of 1 or more of these alleles also conferred a greater risk of some adverse pathological features.

Staged Reconstruction of Long Segment Urethral Strictures in Men With Previous Pediatric Hypospadias Repair

PURPOSE Reconstruction of long segment urethral stricture disease in adults with a history of pediatric hypospadias repair remains complex secondary to poor urethral blood supply, extensive penile scarring and the need for significant amounts of graft or flap tissue. We describe our experience with staged urethroplasty in this cohort of men. MATERIALS AND METHODS A total of 15 males underwent staged urethroplasty for urethral stricture disease following previous hypospadias repair in childhood. All men underwent 2-stage repair with a minimum of 6 months separating each operation. Bulbar urethral stricture disease due to previous dilation was seen in 2 of the 15 men (14%). Graft sources included buccal mucosa in 12 cases, full-thickness hairless abdominal wall skin in 2, penile skin in 1 and posterior auricular tissue in 1. RESULTS Median patient followup was 22 months (range 5 to 62) after stage 2 closure. Median patient age at presentation was 31 years (range 19 to 57). Mean stricture length was 8 cm (range 5 to 14) and mean graft area was 22 cm(2) (range 10 to 38). There was no clinical evidence of stricture recurrence, fistula or wound dehiscence at last followup in 13 of the 15 men (86%). Two men with a congenital hypoplastic glans were left with distal hypospadias. Minor voiding symptoms were described in 3 of the 15 men (21%) with resolution in all by 6 months postoperatively. CONCLUSIONS We describe outcomes after staged reconstruction for extensive urethral stricture disease in men after previous pediatric hypospadias repair in childhood. At intermediate followup staged urethroplasty provided acceptable outcomes.

Leydig Cell-Specific Expression of DAX1 Improves Fertility of the Dax1-Deficient Mouse

Abstract Dax1 is an orphan nuclear receptor expressed in both Leydig and Sertoli cells of the testis. Mutation of DAX1 in humans causes adrenal failure and hypogonadotropic hypogonadism. Targeted mutagenesis of Dax1 in mice reveals a primary gonadal defect characterized by overexpression of aromatase and cellular obstruction of the seminiferous tubules and efferent ductules, leading to germ cell death and infertility. Transgenic expression of DAX1 under the control of the müllerian-inhibiting substance promoter, which is selectively expressed in Sertoli cells, improves fertility but does not fully correct the histological abnormalities in the testes of Dax1 knockout (Dax1KO) mice. We therefore hypothesized that Dax1 may also play a crucial role in other somatic cells of the testis, namely the Leydig cells. A 2.1-kilobase fragment of the murine LH receptor 5′-promoter (LHR-DAX1) was used to generate transgenic mice that selectively express DAX1 in Leydig cells. Expression of the LHR-DAX1 transgene caused no observable phenotype in wild-type mice but improved fertility when expressed in Dax1KO males (rescue [RS]). Although testicular size was not increased in LHR-DAX1 RS animals, aromatase expression was restored to normal levels, and sperm production was increased. Testicular pathology was only slightly improved in RS mice compared to Dax1KO animals. Taken together with the result of previous studies of DAX1 expression in Sertoli cells, we conclude that the testis phenotype of Dax1KO mice reflects the combined effects of Dax1 deficiency in both Sertoli and Leydig cells.