Wolffian duct differentiation by physiological concentrations of androgen delivered systemically.

In developing mammalian males, conversion of the Wolffian ducts into the epididymides and vasa deferentia depends on androgen secretion by the testes, whereas in females these ducts remain in a vestigial form or regress. However, there is continuing uncertainty whether the androgen needs to be delivered locally, either by diffusion from the adjacent testis or, by secretion into the lumen of the duct, or whether circulating androgens maintain and virilize the Wolffian ducts. To resolve this uncertainty, we transplanted either day 0-2 or day 8-9 post-partum testes beneath the flank skin of three groups of neonatal (days 0-1) female tammar wallabies, where they developed and secreted physiological levels of hormones. The Wolffian ducts of all these females were retained and had formed extensive epididymides when examined at days 25, 34 and 87 after birth. In the two older groups of females, sampled after the time of prostatic bud formation, the urogenital sinus was virilized and there was extensive prostatic development similar to that of normal males of the same age, showing that androgen secretion had occurred. Virilization of the Wolffian ducts occurred during an early but short-lived window of sensitivity. This study provides the first clear evidence that under physiological conditions virilization can be mediated by circulating androgen.

Formation of 5alpha-reduced androgens in the testes and urogenital tract of the grey short-tailed opossum, Monodelphis domestica.

Testicular 5alpha-reduced androgens, largely 5alpha-androstane-3alpha,17beta-diol (androstanediol), are responsible for virilisation of pouch young in one marsupial (the tammar wallaby), but are not formed until later in development in another marsupial (the brushtail possum) and in rodents. Because the mechanism of virilisation of the urogenital tract in the grey short-tailed opossum Monodelphis domestica has never been defined, androgen formation and metabolism were investigated in this species. Testis fragments from grey short-tailed opossums of a wide range of ages were incubated with [3H]-progesterone and the metabolites were separated by high-performance liquid chromatography (HPLC). The only 19-carbon metabolites identified in the youngest ages (5-26 days) and the major metabolites in adult testes were testosterone and androstenedione. At 30, 42 and 49 days of age, dihydrotestosterone and small amounts of androstanediol were present. Time-sequence studies indicated that dihydrotestosterone and androstanediol were formed from the 5alpha-reduction (and 3-keto reduction) of testosterone. In a second series of experiments, tissue fragments of a variety of urogenital tract tissues were incubated with [3H]-testosterone and the metabolites separated by HPLC. During the interval in which male urogenital tract differentiation takes place in this species (between Days 15 and 28), the major metabolite identified was dihydrotestosterone. We conclude that the timing of 5alpha-reductase expression in the testes of the grey short-tailed possum resembles that of rodents and the brushtail possum rather than that of the tammar wallaby and that dihydrotestosterone is probably the intracellular androgen responsible for virilisation of the urogenital tract in this species.

Report of fertility in a woman with a predominantly 46,XY karyotype in a family with multiple disorders of sexual development.

CONTEXT: We report herein a remarkable family in which the mother of a woman with 46,XY complete gonadal dysgenesis was found to have a 46,XY karyotype in peripheral lymphocytes, mosaicism in cultured skin fibroblasts (80% 46,XY and 20% 45,X) and a predominantly 46,XY karyotype in the ovary (93% 46,XY and 6% 45,X). PATIENTS: A 46,XY mother who developed as a normal woman underwent spontaneous puberty, reached menarche, menstruated regularly, experienced two unassisted pregnancies, and gave birth to a 46,XY daughter with complete gonadal dysgenesis. RESULTS: Evaluation of the Y chromosome in the daughter and both parents revealed that the daughter inherited her Y chromosome from her father. Molecular analysis of the genes SOX9, SF1, DMRT1, DMRT3, TSPYL, BPESC1, DHH, WNT4, SRY, and DAX1 revealed normal male coding sequences in both the mother and daughter. An extensive family pedigree across four generations revealed multiple other family members with ambiguous genitalia and infertility in both phenotypic males and females, and the mode of inheritance of the phenotype was strongly suggestive of X-linkage. CONCLUSIONS: The range of phenotypes observed in this unique family suggests that there may be transmission of a mutation in a novel sex-determining gene or in a gene that predisposes to chromosomal mosaicism.

Blasts from the past.

With this issue of the JCI, we celebrate the 80th anniversary of the Journal. While 80 years is not a century, we still feel it is important to honor what the JCI has meant to the biomedical research community for 8 decades. To illustrate why the JCI is the leading general-interest translational research journal edited by and for biomedical researchers, we have asked former JCI editors-in-chief to reflect on some of the major scientific advances reported in the pages of the Journal during their tenures.

Identification of the Underlying Androgen Receptor Defect in the Dallas Reifenstein Family.

CONTEXT: The Dallas Reifenstein family - first described in 1965 - includes 14 known members with partial androgen insensitivity syndrome (PAIS). However, the underlying molecular defect was never identified. OBJECTIVE: To identify the underlying genetic defect for PAIS in the Dallas Reifenstein family. DESIGN: DNA was purified from scrotal skin fibroblasts, and whole exome sequencing was then performed in four affected men in the family. Additional family members - both affected and unaffected - were subsequently recruited to confirm segregation of the candidate mutations with the PAIS phenotype. PATIENTS: The affected men have PAIS with infertility associated with azoospermia, hypospadias, and gynecomastia. RESULTS: All four men harbored an intronic variant NC_000023.10:g.66788676A>C between exon 1 and exon 2 of the androgen receptor (AR) canonical transcript NM_000044 (complementary DNA position NM_000044: c.1616+22072A>C) predicted to cause an alternatively spliced AR transcript. Reverse transcription (RT) polymerase chain (PCR) experiments detected the predicted PCR product of the alternatively spliced AR transcript, and the mutation segregated with the PAIS phenotype in this family. The transcript includes the insertion of 185 nucleotides with a premature stop codon at chrX:66863131-66863133, likely resulting in a reduction in AR protein expression due to nonsense-mediated decay. CONCLUSIONS: An intronic AR mutation was identified in the Dallas Reifenstein family. The findings suggest that in cases of PAIS without identifiable AR mutations in coding regions, intronic AR mutations should be considered.

Reprint of "Steroid 5α-reductase 2 deficiency".

Dihydrotestosterone is a potent androgen metabolite formed from testosterone by action of 5α-reductase isoenzymes. Mutations in the type 2 isoenzyme cause a disorder of 46,XY sex development, termed 5α-reductase type 2 deficiency and that was described forty years ago. Many mutations in the encoding gene have been reported in different ethnic groups. In affected 46,XY individuals, female external genitalia are common, but Mullerian ducts regress, and the internal urogenital tract is male. Most affected males are raised as females, but virilization occurs at puberty, and male social sex develops thereafter with high frequency. Fertility can be achieved in some affected males with assisted reproduction techniques, and adults with male social sex report a more satisfactory sex life and quality of life as compared to affected individuals with female social sex.

Role of the alternate pathway of dihydrotestosterone formation in virilization of the Wolffian ducts of the tammar wallaby, Macropus eugenii.

Dihydrotestosterone in androgen target tissues is formed under most circumstances by the 5alpha-reduction of testosterone, but an alternate pathway involves the oxidation of androstanediol to dihydrotestosterone. To investigate the mechanism by which androgens virilize the Wolffian ducts in the tammar wallaby, [(3)H]progesterone was incubated with testes from d 10 and 19 pouch young, and radioactivity was recovered in testosterone and androstanediol at both ages. Analysis of the intermediates indicates that androstanediol was formed both from testosterone via 5alpha-reduction and 3alpha-keto reduction and directly from 5alpha-reduced progestogens. 5alpha-Reductase activity was high in minces of mesonephros/epididymis from d 6-21 pouch young. When minces of urogenital tract tissues from d 19 pouch young were incubated with [(3)H]testosterone, [(3)H]dihydrotestosterone, and [(3)H]androstanediol, dihydrotestosterone was the principal androgen formed in the mesonephros/epididymis, urogenital sinus, and urogenital tubercle, whereas androstanediol was the principal androgen formed by the testis. In intact pouch young studied between d 10 and 34, administration of the 5alpha-reductase inhibitor, 17beta-(N,N-diethyl)carbamoyl-4-methyl-4-aza-5alpha-androstan-3-one, blocked virilization of the Wolffian ducts in males, and administration of androstanediol caused virilization of the Wolffian ducts in females. We conclude that dihydrotestosterone, largely formed in the tissue by the oxidation of androstanediol derived from the testes and also the 5alpha-reduction of testosterone, is responsible for Wolffian duct virilization in this species.

Steroid 5α-reductase 2 deficiency.

Dihydrotestosterone is a potent androgen metabolite formed from testosterone by action of 5α-reductase isoenzymes. Mutations in the type 2 isoenzyme cause a disorder of 46,XY sex development, termed 5α-reductase type 2 deficiency and that was described forty years ago. Many mutations in the encoding gene have been reported in different ethnic groups. In affected 46,XY individuals, female external genitalia are common, but Mullerian ducts regress, and the internal urogenital tract is male. Most affected males are raised as females, but virilization occurs at puberty, and male social sex develops thereafter with high frequency. Fertility can be achieved in some affected males with assisted reproduction techniques, and adults with male social sex report a more satisfactory sex life and quality of life as compared to affected individuals with female social sex.

The marsupial model for male phenotypic development.

In all mammals, androgen formed in the developing testes is responsible for the aspects of male development in which the Wolffian ducts, urogenital sinus and urogenital tubercle are transformed into the epididymis/vas deferens, prostate and penis. That these events take place after birth in the marsupial makes it possible to examine male phenotypic development during pouch life. In the tammar wallaby, Macropus eugenii, the testicular androgen 5 alpha-androstane-3 alpha,17 beta-diol (5 alpha-adiol) is formed in the developing testis, is secreted into plasma and has the capacity to virilize female young pouch when administered exogenously. 5 alpha-Adiol is formed by immature testes in many species and appears to act in target tissues once it has been converted to dihydrotestosterone.

Ontogeny and pathway of formation of 5alpha-androstane-3alpha,17beta-diol in the testes of the immature brushtail possum Trichosurus vulpecula.

The testicular androgen 5alpha;-androstane-3alpha,17beta-diol (androstanediol) mediates virilisation in pouch young of a marsupial, the tammar wallaby, and is the principal androgen formed in immature rodent testes. To chart the pattern of androstanediol formation in another marsupial species, the testes or fragments of testes from brushtail possums (Trichosurus vulpecula) that spanned the age range from early pouch young to mature adults were incubated with (3)H-progesterone and the products were identified by high-performance liquid chromatography. The only 19-carbon steroids identified in pouch young and adult testes were the Delta(4)-3-keto-steroids testosterone and androstenedione. However, androstanediol and another 5alpha-reduced androgen (androsterone) were synthesised by testes from Day 87-200 males and these appeared to be formed from the 5alpha-reduction and 3-keto reduction of testosterone and androstenedione. In the prostate and glans penis of the immature male, (3)H-androstanediol was converted to dihydrotestosterone. We conclude that the timing of androstanediol formation in the possum testis resembles the process in rodents rather than in the tammar wallaby and that any androstanediol in the circulation probably acts in target tissues via conversion to dihydrotestosterone.

Penile development is initiated in the tammar wallaby pouch young during the period when 5alpha-androstane-3alpha,17beta-diol is secreted by the testes.

Virilization of the urogenital tract is under the control of testicular androgens in all mammals. In tammar young, prostate differentiation begins between d 20 and d 40 under the control of the testicular androgen 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol), but uncertainties exist about the control of penile development. We performed longitudinal studies up to d 150 of pouch life to define normal penile development and the effects of androgen administration and castration. In control animals the male phallus was longer than the female phallus by d 48. Closure of the urethra in males begins around d 60 and continues to at least d 150. Administration of supraphysiological doses of testosterone to females caused penile development equivalent to that of the male and also induced partial closure of the urethral groove by d 150. Castration of male pouch young at d 25 prevented penile development, whereas the penis in males castrated at d 40, 80, or 120 had partial closure of the urethral groove. Administration of 5alpha-adiol to females from d 20-40 also caused partial closure of the urethral groove and some growth of the phallus at d 150, whereas 5alpha-adiol treatment from d 40-80 or 80-120 caused some penile growth but had little effect on urethral development. These findings, together with the fact that we found no sex differences in plasma levels of testosterone, dihydrotestosterone, 5alpha-adiol, dehydroepiandrosterone, or androstenedione from d 51-227, clearly indicate that the action of 5alpha-adiol between d 20 and 40 imprints later differentiation of the male penis.

Administration of 5alpha-androstane-3alpha,17beta-diol to female tammar wallaby pouch young causes development of a mature prostate and male urethra.

Secretion of 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) by the testes of the tammar wallaby is responsible for initiation of prostatic development after d 20 in male pouch young. To ascertain the role of this hormone in the subsequent growth and differentiation of the prostate and in the development of the male phallus, 5alpha-adiol was administered to tammar female pouch young in two regimens. Administration of the hormone by mouth (8 microg/g body weight.wk) between d 70 and 150 of pouch life caused prostate development equivalent to that in d 150 males and promoted growth and differentiation of the penis, but not masculinization of the urethra. Treatment with a small dose of 5alpha-adiol enanthate (1 microg/g body weight.wk) from d 20-150 produced similar results. However, administration of larger doses of 5alpha-adiol enanthate (10 or 100 microg/g body weight.wk) from d 20-150 caused supraphysiological growth of the prostate, development of a male-type urethra, and penile growth. These results indicate that prostatic development and penile growth can be initiated over a wide time period, but that formation of a male urethra requires androgen action before d 70, when male penile differentiation begins. This further strengthens the hypothesis that 5alpha-adiol is the circulating androgen responsible in this species for virilization during development.

5alpha-androstane-3alpha,17beta-diol is formed in tammar wallaby pouch young testes by a pathway involving 5alpha-pregnane-3alpha,17alpha-diol-20-one as a key intermediate.

The synthetic pathway by which 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20-40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [(3)H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5alpha-adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5alpha-pregnane-3alpha,17alpha-diol-20-one (5alpha-pdiol) and androsterone as intermediates. Formation of 5alpha-adiol from both [(3)H]testosterone and [(3)H]progesterone was blocked by the 5alpha-reductase inhibitor 4MA. The addition of nonradioactive 5alpha-pdiol blocked the conversion of [(3)H]progesterone to 5alpha-adiol, and [(3)H]5alpha-pdiol was efficiently converted to androsterone and 5alpha-adiol. We conclude that expression of steroid 5alpha-reductase in the developing wallaby testes allows formation of 5alpha-reduced androgens by a pathway that does not involve testosterone as an intermediate.

Androgen physiology: unsolved problems at the millennium.

Androgen physiology differs from that of other steroid hormones in two major regards. First, testosterone, the predominant circulating testicular androgen, is both an active hormone and a prohormone for the formation of a more active androgen, the 5alpha-reduced steroid dihydrotestosterone. Genetic evidence indicates that testosterone and dihydrotestosterone work via a common intracellular receptor, and studies involving in vitro reporter gene assays and intact mice in which both steroid 5alpha-reductase isoenzymes have been disrupted by homologous recombination indicate that dihydrotestosterone acts during embryonic life to amplify hormonal signals that can be mediated by testosterone at higher concentrations. However, in post-embryonic life dihydrotestosterone plays unique roles that have not been elucidated. Studies of other 5alpha-reduced steroids, including the plant hormone brassinolide, the hog pheromones androstanol and androstenol, and 5alpha-dihydroprogesterone (in horses and elephants) indicate that this reaction serves different functions in different systems. Second, during embryonic life androgen causes the formation of the male urogenital tract and hence is responsible for development of the tissues that serve as the major sites of androgen action in postnatal life. It has been generally assumed that androgens virilize the male fetus by the same mechanisms as in the adult, namely by the conversion of circulating testosterone to dihydrotestosterone in target tissues. However, in marsupial mammals there is no sexual dimorphism in the levels of testosterone or dihydrotestosterone at the time the male phenotype forms, and in the pouch young of one marsupial, the tammar wallaby, the testes secrete another 5alpha-reduced steroid, 5alpha-androstane-3alpha, 17beta-diol (5alpha-adiol), into plasma. The administration of 5alpha-adiol to female pouch young causes profound virilization of the urogenital sinus and external genitalia, but within target tissues 5alpha-adiol appears to work after oxidation to dihydrotestosterone. Thus, two separate mechanisms evolved for the formation of dihydrotestosterone in target tissues. 5alpha-adiol is the predominant androgen in neonatal testes in several placental mammals, but it is unclear whether it plays a similar role in other mammalian species.

Steroid 5alpha-reductase 1 promotes 5alpha-androstane-3alpha,17beta-diol synthesis in immature mouse testes by two pathways.

5alpha-Androstane-3alpha,17beta-diol (androstanediol) is the predominant androgen in immature mouse testes, and studies were designed to investigate its pathway of synthesis, the steroid 5alpha-reductase isoenzyme involved in its formation, and whether testicular androstanediol is formed in embryonic mouse testes at the time of male phenotypic development. In 24-26-day-old immature testes, androstanediol is formed by two pathways; the predominant one involves testosterone --> dihydrotestosterone --> androstanediol, and a second utilizes the pathway progesterone --> 5alpha-dihydroprogesterone --> 5alpha-pregnane-3alpha-ol-20-one --> 5alpha-pregnane-3alpha,17alpha-diol-20-one --> androsterone --> androstanediol. Formation of androstanediol was normal in testes from mice deficient in steroid 5alpha-reductase 2 but absent in testes from mice deficient in steroid 5alpha-reductase 1, indicating that isoenzyme 2 is not expressed in day 24-26 testes. The fact that androstenedione and testosterone were the only androgens identified after incubation of day 16 and 17 embryonic testes with [3H]progesterone implies that androstanediol formation in the testis plays no role in male phenotypic differentiation in the mouse.

Unsolved problems in male physiology: studies in a marsupial.

Testicular androgens induce formation of the male urogenital tract in all mammals. In marsupials male development occurs after birth and over a prolonged period. For example, in the tammar wallaby virilization of the Wolffian ducts begins by day 20, prostate formation begins about day 25, and phallic development starts after day 80 of pouch life. Between days 20 and 40 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in tammar testes and secreted into plasma. Administration of 5alpha-adiol to pouch young females induces urogenital sinus virilization by day 40 and formation of a mature male prostate and phallus by day 150. 5alpha-Adiol is synthesized in pouch young testes by two pathways, one involving testosterone and dihydrotestosterone and the other 5alpha-pregnane-3alpha,17alpha-diol-20-one and androsterone as intermediates, both utilizing steroid 5alpha-reductase. In target tissues 5alpha-adiol acts via the androgen receptor after conversion to dihydrotestosterone but may have other actions as well. Whether 5alpha-adiol plays a role in male development in placental mammals is uncertain.

The hormonal control of sexual development.

The formation of the testis or ovary is a critical step in development. The pioneering studies of Professor Alfred Jost showed that the hormones produced by the embryonic rabbit testis are essential for development of the male phenotype. Sexually dimorphic hormones play a key role in the transition from an undifferentiated gonad into the mature testis and ovary. Marsupials, with their altricial young, provide an accessible model for the study of sexual differentiation because most of these events occur postnatally, while the young are attached to teats within their mothers' pouches. The relatively long time-course for the marsupial sexual differentiation has provided an excellent opportunity to correlate morphological changes with the genes and hormones that control them. Using this model species we have demonstrated that not all sexual dimorphisms are controlled by hormones. Virilization of the prostate and phallus is androgen dependent but appears to rely on circulating 5alpha-androstane-3alpha, 17beta-diol which is converted to dihydrotestosterone in these target tissues. Collectively these studies have led to the development of new paradigms to explain the hormonal mechanisms mediating sexual differentiation.