Otoacoustic emissions,auditory evoked potentials and self-reported gender in people affected by disorders of sex development (DSD)

Both otoacoustic emissions (OAEs) and auditory evoked potentials (AEPs) are sexually dimorphic, and both are believed to be influenced by prenatal androgen exposure. OAEs and AEPs were collected from people affected by 1 of 3 categories of disorders of sex development (DSD) — (1) women with complete androgen insensitivity syndrome (CAIS); (2) women with congenital adrenal hyperplasia (CAH); and (3) individuals with 46,XY DSD including prenatal androgen exposure who developed a male gender despite initial rearing as females (men with DSD). Gender identity (GI) and role (GR) were measured both retrospectively and at the time of study participation, using standardized questionnaires. The main objective of this study was to determine if patterns of OAEs and AEPs correlate with gender in people affected by DSD and in controls. A second objective was to assess if OAE and AEP patterns differed according to degrees of prenatal androgen exposure across groups. Control males, men with DSD, and women with CAH produced fewer spontaneous OAEs (SOAEs) – the male-typical pattern – than control females and women with CAIS. Additionally, the number of SOAEs produced correlated with gender development across all groups tested. Although some sex differences in AEPs were observed between control males and females, AEP measures did not correlate with gender development, nor did they vary according to degrees of prenatal androgen exposure, among people with DSD. Thus, OAEs, but not AEPs, may prove useful as bioassays for assessing early brain exposure to androgens and predicting gender development in people with DSD.     

Novel and recurrent mutations in patients with androgen insensitivity syndromes

BACKGROUND/AIMS: Androgen insensitivity syndrome (AIS) caused by mutations within the androgen receptor gene represents a variety of phenotypes from females with 46,XY karyotype over individuals with ambiguous genitalia to infertile males. METHODS: We studied 24 patients with AIS by sequencing androgen receptor gene. 19 of the investigated patients were affected by complete androgen insensitivity syndrome (CAIS) and 5 suffered from partial androgen insensitivity syndrome (PAIS). RESULTS: So far we have detected 12 unreported mutations as well as 9 recurrent mutations (3 recurrent mutations were detected twice) in exons 2-8 of the androgen receptor gene. Three of the novel mutations cause a frameshift with subsequent premature termination and were found in patients with CAIS. These frameshifts were induced by single nucleotide deletion or insertion, or in one case by a 13-bp deletion, respectively. Another premature stop codon found in a CAIS patient results from an already reported nucleotide substitution in exon 5. Furthermore, in a CAIS patient we found a novel duplication of codon 788. All other mutations caused single base substitutions spread through exons 2-8 and were associated with CAIS or PAIS. CONCLUSIONS: We report a broad spectrum of different mutations within the AR gene leading to various manifestations of AIS. Apart from truncating mutations, a reliable genotype/phenotype correlation cannot be established. Therefore, modifying factors must be effective.     

Androgen insensitivity in forty-nine patients: classification based on clinical and androgen receptor phenotypes

Androgen receptor binding was studied in genital skin fibroblasts from 49 patients with androgen insensitivity syndrome (AIS) classified as complete (CAIS) or partial (PAIS) based on the clinical phenotype. The majority (64%) of CAIS and a minority (7%) of PAIS patients were receptor negative. Only 3 receptor-positive AIS cell strains of 30 studied failed to show an increase in specific receptor binding after prolonged androgen exposure in vitro. The gene coding for the androgen receptor in such patients appears intact.     

L712V mutation in the androgen receptor gene causes complete androgen insensitivity syndrome due to severe loss of androgen function

Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C > G mutation (CTT–GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein.     

Point mutation in the steroid-binding domain of the androgen receptor gene in a family with complete androgen insensitivity syndrome (CAIS)

An exonic single nucleotide substitution in the human androgen receptor gene (hAR) could be detected in an Italian family with two children affected by complete androgen insensitivity syndrome (CAIS), also called testicular feminization. This mutation leads to a guanine to adenine transition in exon 5, changing the sense of the codon from methionine (ATG) to valine (GTG). As this mutation abolishes a NcoI restriction site, a rapid test for the mutation can be performed by digestion of the polymerase chain reaction products with this enzyme. Previous results of indirect gene diagnosis in this family could be confirmed by this method.     

Transgenic expression of androgen receptors improves spatial memory retention in both sham-irradiated and 137Cs gamma-irradiated female mice

Using a water maze, it has been shown that both wild-type and apoE4-expressing female mice are at greater risk of developing age-related hippocampal-dependent impairments in spatial learning and memory than age-matched male mice of the same genotype. In addition, apoE4-expressing female mice were more sensitive to 137Cs gamma-radiation-induced impairment in spatial learning and memory than age-matched male mice of the same genotype. These findings imply that androgen receptors (ARs) contribute to spatial learning and memory, posing the question as to whether transgenic expression of AR in female mice might modulate hippocampal-dependent learning and memory under baseline conditions and after local brain irradiation. Hippocampal-dependent novel location recognition was comparable in wild-type and AR-Tg female mice. This function was impaired after irradiation in AR-Tg but not wild-type mice. In contrast, sham-irradiated wild-type and AR-Tg female mice showed hippocampal-independent novel location recognition, and this was not affected by radiation. After the second day of hidden platform training, in a water maze probe trial, sham-irradiated and irradiated AR-Tg female mice showed spatial memory retention but irradiated wild-type mice did not. After the third day of hidden platform training, only irradiated wild-type female mice did not show spatial memory retention in the water maze probe trial. Both sham-irradiated and irradiated wild-type and AR-Tg female mice showed passive avoidance learning and memory. These data support an important role for AR in spatial memory retention in water maze probe trials in female mice under baseline conditions and after cranial irradiation.     

Photoaffinity labelling with [3H]R1881 of androgen receptors from human cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities

Androgen receptors were partially purified by affinity chromatography of cytosols prepared from cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities. Partially purified receptors were covalently labelled with [3H]R1881 (triated methyltrienolone) by ultraviolet photoactivation. Gel electrophoresis of cytosols from normal individuals showed a single radioactive peak, Mr approximately 97 K. Cytosols from patients with decreased nuclear transfer showed a similar peak, Mr approximately 97 K; cytosols from patients with partial androgen insensitivity (PAIS) or receptor positive complete androgen insensitivity (CAIS, R+) showed a peak Mr approximately 30 K-43 K.     

Puberty in subjects with complete androgen insensitivity syndrome

BACKGROUND: Androgen receptor defects affect the regulation of the gonadotropic axis. However, little is known about the timing of pubertal maturation in complete androgen insensitivity syndrome (CAIS). AIMS: To evaluate growth, skeletal maturation and gonadotropin and sex steroid secretion in patients with CAIS and intact gonads at puberty. METHODS: Clinical, auxological and hormonal evaluation of 9 patients with CAIS from birth up to 17 years of age, prior to gonadectomy, in a single institution, retrospective study. RESULTS: Breast development occurred at a median age of 11.1 years, thumb sesamoid appeared at 11.5 years, and peak height velocity at 12.3 years, all consistent with average female values. However, median adult male height (+1.2 SDS) was closer to the patients' male target height (-0.3 SDS). Plasma testosterone levels rose early compared to normal boys. LH (basal and GnRH-stimulated) increased rapidly, above normal male values, in early puberty. CONCLUSIONS: This retrospective evaluation of a limited number of cases with a heterogeneous pattern of follow-up suggests that patients with CAIS may enter puberty at an age closer to female standards. These results imply a major role of direct androgen action, in utero or in early life, in determining the pattern of pubertal gonadotropin maturation.     

Cognitive performance in rhesus monkeys varies by sex and prenatal androgen exposure

Men and women differ on performance and strategy on several spatial tasks. Rodents display similar sex differences, and manipulations of early hormone exposure alter the direction of these differences. However, most cognitive testing of nonhuman primates has utilized sample sizes too small to investigate sexually differentiated behaviors. This study presents an investigation of sex differences and the effects of prenatal androgen on spatial memory and strategy use in rhesus monkeys. Monkeys prenatally exposed to vehicle, testosterone, or the androgen receptor blocker flutamide performed a search task in which 5 of 12 goal boxes contained food rewards. Spatial consistency and the presence of local landmarks were varied. Performance when both spatial and marker cues were available did not differ by sex or prenatal treatment. Contrary to predictions, females easily solved the task when local markers were removed, and their performance outscored males. Although eliminating spatial consistency and requiring subjects to use local markers impaired performance by all monkeys, females continued to locate correct goal boxes at higher than chance levels and scored better than males. Blocking prenatal androgen exposure in males improved use of local markers. These findings suggest that the tendency to attend to landmarks and to use them in solving spatial problems is typical of females across many species, including rodents, humans, and rhesus monkeys. In rhesus monkeys and rodents, developmental androgen eliminates this specialization. However, these results are the only known example of better performance of females than males when salient markers are removed.     

A large deletion/insertion-induced frameshift mutation of the androgen receptor gene in a family with a familial complete androgen insensitivity syndrome

Androgen insensitivity syndrome (AIS) is an X-linked recessive genetic disorder with a normal 46, XY karyotype caused by abnormality of the androgen receptor (AR) gene. One Chinese family consisting of the proband and 5 other members with complete androgen insensitivity syndrome (CAIS) was investigated. Mutation analysis by DNA sequencing on all 8 exons and flanking intron regions of the AR gene revealed a unique large deletion/insertion mutation in the family. A 287 bp deletion and 77 bp insertion (c.933_1219delins77) mutation at codon 312 resulted in a frameshift which caused a premature stop (p.Phe312Aspfs*7) of polypeptide formation. The proband's mother and grandmother were heterozygous for the mutant allele. The proband's father, uncle and grandfather have the normal allele. From the pedigree constructed from mutational analysis of the family, it is revealed that the probably pathogenic mutation comes from the maternal side.     

The effect of rapid and depot testosterone and estradiol on spatial performance in water maze

Men and women differ in some cognitive functions including spatial abilities. These differences seem to be affected by sex steroids, but the results are controversial. The aim of this work is to describe the effects of rapid or depot testosterone and estradiol on spatial memory in rats. Thirty-two adult male Wistar rats were divided into 6 groups. Five groups were gonadectomized, and one group was left as control. Castrated groups received sterile oil, testosterone isobutyras, testosterone propionate, estradiol dipropionate or estradiol benzoate. We evaluated spatial performance (escape latency, overall improvement, and time in the quadrant after platform removal) of the rats in a spatial water maze. Animals receiving exogenous sex steroids showed higher plasma concentrations of the particular hormones. Experimental groups improved during the acquisition spatial trials in the water maze. No significant differences between the groups during probe trial were found. In overall improvement, the testosterone depot and estradiol depot groups showed less improvement in comparison to the control groups (P<0.05). No differences in respect to administered hormones were found in corresponding receptor gene expression in hippocampus. In conclusion, exogenous testosterone affects spatial memory of adult castrated males.     

Transactivation properties of wild-type and mutant androgen receptors in transiently transfected primary human fibroblasts

BACKGROUND: Stromal cells play key roles during androgen-mediated male sexual differentiation. Our objective was to establish a transient transfection method for primary human fibroblasts enabling functional characterization of wild-type (wt) and mutant androgen receptor (AR) plasmid constructs, corresponding to partial and complete androgen insensitivity syndrome (PAIS/CAIS). METHODS: An AR-negative fibroblast strain (ARD842) was established from the gonads of a CAIS patient. Wt-AR or either mutants L712F (PAIS), R774C or V866M (CAIS) were transfected using a polyamine-based procedure. Alternatively, two AR-positive male foreskin fibroblast strains were investigated. Androgen-induced activation of two co-transfected reporter plasmids ((ARE)(2)TATA-, MMTV-luciferase) was measured. RESULTS: All three fibroblast strains showed a ligand-dependent rise of luciferase activity after transfection of wt-AR. Mutant plasmids were assessed in AR-negative ARD842 cells. While L712F showed high partial activity, R774C and V866M were nearly inactive. The intrinsic AR of normal foreskin fibroblasts revealed no measurable ligand-inducible reporter gene activity. CONCLUSIONS: Polyamine-based transfection of AR plasmids into cultured fibroblasts provides a promising tool for analysis of AR transactivation, thereby considering a stromal cellular background. This is supported by the mutant ARs which showed the expected levels of impaired transactivation with respect to the corresponding AIS phenotypes. The role of the intrinsic AR in normal male human foreskin fibroblasts needs further exploration.     

Molecular analysis of the androgen receptor gene in 52 patients with complete or partial androgen insensitivity syndrome: a collaborative study.

In patients with androgen insensitivity syndrome (AIS), RFLP study of the androgen receptor gene made it possible to analyze whether deletions or mutations could be responsible for abnormalities in androgen responsiveness. We studied RFLPs of DNA from 25 46,XY patients with partial AIS (PAIS), defined as a concentration of androgen receptor in genital-skin fibroblasts less than 340 fmol/mg DNA, and DNA from 27 46,XY patients with complete AIS (CAIS) with no detectable androgen receptor site. DNA samples were digested with BamHI, EcoRI, HindIII and TaqI restriction enzymes and hybridized with three cDNA probes covering the three domains of the androgen receptor. When we had the maternal and an unaffected brother's DNA, we analyzed the two androgen receptor gene polymorphisms described, the HindIII and the exon 1 CAG repeat polymorphisms, in order to distinguish the two maternal X chromosomes, and to detect carriers of AIS. We did not find any large deletion among the 52 patients. We observed a heterozygous mother in 3 of 14 families studied with the HindIII polymorphism, and in 12 of 25 families using the exon 1 CAG repeat polymorphism. This study suggests that in AIS, abnormalities in androgen receptor response could be related to point mutations or microdeletions rather than to gross structural alterations of the androgen receptor gene. Furthermore, unless the point mutation has been described, exon 1 and HindIII polymorphism studies would enable the identification of carriers in 50% of families, and the prenatal diagnosis of AIS.     

Cognitive outcome in adult women affected by congenital adrenal hyperplasia due to 21-hydroxylase deficiency

BACKGROUND: Some research suggests that girls with congenital adrenal hyperplasia (CAH), who are exposed to higher than normal levels of prenatal androgens, perform better on spatial tasks, worse on verbal tasks and have a greater incidence of left-handedness than unaffected controls, all of which suggests the development of a more male-typical cognitive pattern. However, research in all three areas has produced inconsistent findings. OBJECTIVES: To determine if prenatal androgen exposure has an organizing effect on female cognitive development and to what extent. METHODS: 24 women, 21-71 years, with either the salt-losing (SL) or simple virilizing (SV) forms of CAH due to 21-hydroxylase deficiency, and 18 controls, 21-73 years, who were unaffected female relatives or women with polycystic ovary syndrome, were assessed with IQ, handedness, executive function, verbal learning and memory, non-verbal learning and memory, perceptual speed, visuospatial processing and visuomotor ability measures. The battery included tests known to elicit sex differences and control measures. RESULTS: The findings did not support the hypothesis that women with CAH develop a more male-typical cognitive pattern. CONCLUSION: This study differs from others in the older age of its participants, grouping by SL/SV diagnosis and assessment of medical treatment and compliance as determined through hormone assays. Our findings provide additional support for the conclusion that, in adult women with CAH, previous prenatal androgen exposure does not enhance spatial abilities, impair verbal abilities nor alter hand preferences in a long-lasting way.     

A comparative study of the performance of individuals with fragile X syndrome and Fmr1 knockout mice on Hebb-Williams mazes

Fragile X syndrome (FXS) is the most prevalent form of heritable mental retardation. It arises from a mutation in the FMR1 gene on the X chromosome that interferes with expression of fragile X mental retardation protein (FMRP) and leads to a wide range of behavioural and cognitive deficits. Previous studies have shown a deficit in basic visual perceptual processing as well as spatial abilities in FXS. How such a deficit may impact spatial navigation remains unknown. The current study extended previous research by evaluating spatial learning and memory using both virtual and physical versions of Hebb-Williams mazes, which allows for testing of humans and animals under comparable conditions. We compared the performance of individuals affected by FXS to typically developing individuals of equivalent mental age as well as the performance of Fmr1 knockout mice to wild-type control mice on the same maze problems. In human participants, performance of the comparison group improved across trials, showing expected significant decreases in both errors and latency. In contrast, the performance of the fragile X group remained at similar levels across trials. Although wild-type control mice made significantly fewer errors than the Fmr1 knockout mice, latencies were not statistically different between the groups. These findings suggest that affected humans and mice show similar spatial learning deficits attributable to the lack of FMRP. The implications of these data are discussed including the notion that Hebb-Williams mazes may represent a useful tool to examine the impact of pharmacological interventions on mitigating or reversing the symptoms associated with FXS.     

The cholinesterase inhibitor, phenserine, improves Morris water maze performance of scopolamine-treated rats

Male Fischer-344 rats (n = 38) at 5 months old were tested in a Morris water maze to determine if treatment with the cholinesterase inhibitor, phenserine (PHEN), would overcome a learning impairment induced by scopolamine (SCOP), a muscarinic cholinergic receptor antagonist. Each rat was randomly assigned to one of five groups to receive two intraperitoneal injections 60 and 30 min, prior to testing, respectively, as follows: (1) saline-saline (SAL); (2) saline-1.0 mg/kg (SCOP); (3) 2 mg/kg PHEN- SCOP (PHEN2); (4) 4 mg/kg PHEN-SCOP (PHEN4); and (5) 1 mg/kg PHEN-SAL (PHEN1). Maze testing occurred across 5 days with 4 days of acquisition trials (4 trials per day) and a fifth day consisting of a single 120 sec probe trial. PHEN1 and SAL were combined into one control (CON) group for purposes of statistical analysis for both acquisition and probe trials as comparison of the two groups revealed that they did not significantly differ on any measure. SCOP-treated rats were significantly impaired compared to CON in learning the location of the submerged platform as measured by latency to locate the platform and the distance traversed to find the platform across days of testing. The PHEN4 group had significantly lower latencies and traveled a shorter distance to reach the submerged platform when compared to SCOP on the fourth day of trials while the PHEN2 group traveled more directly to the submerged platform but did not have shorter latencies than the SCOP group. For probe trials, CON rats swam closer to the target area (a measure of proximity to the removed platform) than did all other groups, and the PHEN4 group swam in an area more proximate to the target area than did the SCOP-treated group. These findings demonstrate the ability of this drug to improve learning when cholinergic function has been impaired in a spatial memory task.     

Testosterone modulates performance on a spatial working memory task in male rats

Gonadal hormones have been shown to modulate memory retention in female rats. The current experiments examine the role of testicular hormones in modulating the performance of male rats on two spatial water maze tasks. In the first study, castrated and intact rats were trained on the visible platform and hidden platform versions of the Morris water maze task. Castration did not affect performance on either version of this reference memory task with castrated and intact rats demonstrating similar performance both during acquisition and on post-training probe trials. In the second experiment, castrated and intact rats were tested on a delayed-matching-to-place version of the water maze. Rats received a series of trial pairs in the maze with a hidden platform located in the same pool location on the exposure and retention trials of each pair; between pairs of trials, however, the platform was repositioned to a novel pool location. The interval between trials was either 10- or 60-min and memory retention, taken as the difference between the pathlengths on the exposure and retention trials, declined as the interval increased. Relative to intact males, castrated males demonstrated impaired working memory retention at 60-min but not at 10-min retention intervals. This interval-dependent impairment in working memory retention was reversed by physiologic levels of testosterone replacement. These findings indicate that castration does not significantly affect acquisition or probe trial performance on a classic reference memory task but does impair spatial working memory retention, an effect that is reversed by exogenous testosterone.