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Androgen insensitivity syndrome (AIS) is an X-linked disorder in which defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. This survey reports the analysis of 11 AIS subjects. The androgen receptor gene of these subjects was analyzed using polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis and sequencing or sequencing of PCR-amplified androgen receptor gene fragments alone. In total, 10 single base changes and one partial gene deletion were detected. Seven single base changes resulted in an amino acid change, one resulted in the introduction of a premature stop codon, one event represented a single base insertion resulting in a frame-shift, and one single base change affected a donor splice site. The androgen receptor protein in genital skin fibroblasts from several patients was studied with respect to molecular mass after immunoprecipitation and SDS-PAGE. Two patients expressed a truncated receptor protein in agreement with the established genomic mutation. Pedigree analysis was performed to identify possible carriers for the syndrome in families of AIS patients using single-strand conformation polymorphism and restriction site analysis of PCR products. In one case, the polymorphic (CAG)n(CAA) repeat in exon 1 encoding a polyglutamine stretch was used to identify the mutant allele in a family with X-linked partial androgen insensitivity before the identification of the actual genomic mutation. PCR-single-strand conformation polymorphism analysis proved to be a fast and reliable technique to screen for androgen receptor gene mutations and to study the androgen receptor gene of family members of AlS-affected individuals.

Observations on hair follicles presented in this paper show that boundaries to junctional communication are formed between groups of cells following different pathways of differentiation. The patterns of junctional communication in the bulbs of rat vibrissa follicles and human hair follicles were studied by microinjection of the fluorescent tracer dye Lucifer Yellow CH. Dye spread was extensive between undifferentiated cells of the hair bulb matrix but communication boundaries were found between groups of morphologically distinct cells. For example, boundaries to dye spread were observed between undifferentiated matrix cells and cells in the early stage of differentiation into the inner root sheath, between Huxley's and Henle's layers in the early inner root sheath and between cells of the cuticle and cortex of the hair. Dye did not spread between epithelial cells of the hair bulb and mesenchymal cells of the connective tissue sheath or dermal papilla. The patterns of dye spread became more complex (increased boundary formation and subcompartmentation) as differentiation progressed in higher regions of the hair bulb. The observed communication can be related to previous ultrastructural studies by others on the distribution of gap junctions in the wool follicle. These results show that junctional communication, with its consequent intercellular spread of small ions and molecules, is associated with uniformity of expression and behaviour within cell populations and that interruption of communication through the formation of boundaries and communication compartments is temporally and spatially related to the production of subpopulations of cells committed to the expression of different phenotypes.

Fresh scalp, genital, chest and axillary skin from human foetuses of 12–41 weeks' maturity was incubated in Krebs' improved Ringer I medium with [7α-3H]dehydroepiandrosterone, [7α-3H]testosterone and [7α-3H]androstenedione. The metabolites identified were androstenedione, 5α-androstane-3,17-dione, androsterone, 3-epiandrosterone, 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5-androstene-3β,17β-diol and testosterone. The results provide evidence for the presence of 3β-hydroxysteroid dehydrogenase, Δ4–5isomerase, 17β-hydroxysteroid dehydrogenase, Δ4-3-oxosteroid-5α-reductase and 3α-hydroxysteroid dehydrogenase in human foetal skin. There were quantitative differences in the various enzyme activities between different body sites and skin specimens of different gestational age. 5α-Reductase activity was particularly high in genital skin. 3β-Hydroxysteroid dehydrogenase Δ4–5isomerase activity was low in skin from a 12-week foetus, but high in skin specimens from 28-, 38- and 41-week foetuses. 17β-Hydroxysteroid dehydrogenase activity was already high in the skin of the 12-week foetus and remained so in the older foetuses. These results were correlated with the development of the foetal sebaceous glands, and were in general agreement with a parallel enzyme histochemical study. The role of androgen metabolism in human foetal skin is discussed.

Human skin from forehead, cheek and axilla was incubated in vitrowith [7α-3H]dehydroepiandrosterone (DHA), [7α-3H]DHA sulphate, [7α-3H]-androstenedione and [7α-3H]testosterone. The following enzyme activities were detected: 3β-hydroxysteroid dehydrogenase Δ4-5isomerase, 17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, 5α-reductase, 5β-reductase, sulphotransferase, sulphatase, steroid hydroxylase. 5α-Reduced steroids were the major metabolites. All four substrates were converted to 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol. In axillary skin, conversion of 17-oxosteroids to 17β-hydroxysteroids was favoured, 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol being major metabolites. In facial skin, formation of 17-oxosteroids predominated with little accumulation of 5α-dihydrotestosterone or 5α-androstane-3α,17β-diol. 5α-Androstane-3β,17β-diol was a metabolite of DHA, androstenedione and testosterone but was found in lower amounts than 5α-androstane-3α,17β-diol. Similarly conversions to epiandrosterone were much lower than to androsterone in all the skin specimens.It was concluded that the differences in accumulation of 5α-dihydrotestosterone were determined by the differences in 17β-oxidoreduction rather than differences in 5α-reductase, the activity of which was high in all skin specimens.

The metabolism of [7α-3H]dehydroepiandrosterone (DHA), [7α-3H]-androstenedione and [7α-3H]testosterone was studied in the ventral sebaceous gland patch of the Mongolian gerbil in vitro. The main enzyme activities found were 17β-hydroxysteroid dehydrogenase, 5α-reductase, 17α-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, sulphotransferase and hydroxylase. The active androgen 5α-dihydrotestosterone was formed in appreciable amounts from both testosterone and androstenedione. The 17β-hydroxysteroid dehydrogenase actively formed both 17-oxo and 17β-hydroxysteroids in this tissue. The conversion of DHA to C4–5unsaturated steroids and 5α-steroids was not observed presumably due to a lack of 3β-hydroxysteroid dehydrogenase Δ4–5isomerase. The metabolism was compared with that in human and rat skin and its significance discussed.

Binding of [3H]testosterone and 5α-dihydro[3H]testosterone ([3H]DHT) to specific androgen-receptor sites of 5α-reductase-deficient human genital skin fibroblasts (five cell-lines) was studied in the intact cultured cells at 37 °C. Under the conditions of the experiments, conversion of [3H]testosterone into [3H]DHT was negligible. Both steroids bound to the same set of high-affinity saturable sites in cytoplasmic and nuclear fractions of the cells. Unlabelled testosterone, DHT and methyltrienolone competed effectively with the labelled steroids. Progesterone and oestradiol were weaker competitors; cortisol did not compete. The dissociation constant (Kd) for high-affinity complexes with [3H]testosterone (0·44 ± 0·035 nmol/l) was higher than that for [3H]DHT complexes (0·20 ± 0·090 nmol/l). Unlabelled DHT was more effective than unlabelled testosterone in competing with either radioactive steroid. Complexes of [3H]DHT and receptor dissociated more slowly than [3H]testosterone-receptor complexes and [3H]DHT bound more extensively to low-affinity non-saturable sites in fibroblasts. As judged by competition with the radioactive androgens, progesterone bound to the androgen receptor with a Kdof about 7 nmol/l. 5α-Pregnane-3,20-dione had an approximately fivefold lower affinity than progesterone for androgen receptors; 3α/β- or 20α-reduction lowered its affinity further. It is suggested that in 5α-reductase deficiency in man, progesterone in amniotic fluid and blood could effectively inhibit testosterone binding to androgen receptors in the male embryonic external genitalia. One function of the high levels of 5α-reductase activity normally found in embryonic external genitalia and urogenital sinus may be to protect these tissues from the potentially antiandrogenic action of progesterone.

The distribution of androgen metabolism in human skin was studied using tissues isolated either by direct dissection of axillary skin or by dissection of collagenase-digested forehead and axillary skin. All tissues (epidermis, sweat glands, sebaceous glands, hair follicles and dermis) were found to contain 17β-, 3β- and 3α-hydroxysteroid dehydrogenase (HSD) activities, 3β-hydroxysteroid dehydrogenase-Δ4–5isomerase (Δ5-3β-HSD) activity and 5α-reductase activity. All tissues converted testosterone into 5α-dihydrotestosterone. In confirmation of previous histochemical studies, over 90% of the Δ5-3β-HSD of forehead skin was found in the sebaceous glands. In forehead skin, 40–66% of the 5α-reductase activity was in the sebaceous glands, while in axillary skin 50–70% was in the sweat glands, especially the apocrine glands. There was a more even distribution of 17β-HSD activity in skin tissues than histochemical studies have indicated previously. Knowledge of the distribution of these enzymes has helped in the understanding of the function of androgen metabolism in skin.

Human forehead skin incubated in vitrois known to metabolize testosterone to 17-oxosteroids faster than the reverse reaction, while axillary skin rapidly metabolizes androstenedione to 17β-hydroxysteroids, such as testosterone and 5α-dihydrotestosterone, While this has been confirmed using a larger number of patients, some indication has been found that 17β-hydroxysteroid oxidoreductase activity declines with age in the axilla. The relative rates of 17β-oxidation and reduction (direction of operation of skin 17β-hydroxysteroid oxidoreductase activity) were not altered by a variety of incubation conditions. Large amounts of a membrane-bound 17β-hydroxysteroid oxidoreductase, showing preference for NAD as coenzyme and testosterone (rather than androstenedione) as steroid substrate, were found in forehead skin from one patient. On the other hand, the main axillary skin enzyme in skin from another patient was soluble and showed preference for NADP and androstenedione. It is postulated that 17β-oxidation and reduction in skin is controlled by the relative amount, the coenzyme preferences and the kinetic properties of these two enzymes.

Testosterone metabolism was studied in tissues associated with a keratin-filled cutaneous cyst. Instead of the 5α-reduced metabolites usually associated with skin steroid metabolism, considerable amounts of 5β-reduced steroids were found. These included 5β-androstane-3,17-dione, 17β-hydroxy-5β-androstan-3-one, and 5β-androstane-3β,17β-diol. This change in metabolic pattern is discussed.