Abstract
Since the discovery in 1968 that dihydrotestosterone (DHT) is a major mediator of androgen action, a convincing body of evidence has accumulated to indicate that the major pathway of DHT formation is the 5α-reduction of circulating testosterone in androgen target tissues. However, we now know that DHT can also be formed in peripheral tissues by the oxidation of 5α-androstane-3α,17β-diol (adiol). This pathway is responsible for the formation of the male phenotype. We discuss the serendipitous discovery in the tammar wallaby of an alternate pathway by which adiol is formed in the testes, secreted into plasma and converted in peripheral tissues to DHT. This alternate pathway is responsible for virilisation of the urogenital system in this species and is present in the testes at the onset of male puberty of all mammals studied so far. This is the first clear-cut function for steroid 5α-reductase 1 in males. Unexpectedly, the discovery of this pathway in this Australian marsupial has had a major impact in understanding the pathophysiology of aberrant virilisation in female newborns. Overactivity of the alternate pathway appears to explain virilisation in congenital adrenal hyperplasia CAH, in X-linked 46,XY disorders of sex development. It also appears to be important in polycystic ovarian syndrome (PCOS) since PCOS ovaries have enhanced the expression of genes and proteins of the alternate pathway. It is now clear that normal male development in marsupials, rodents and humans requires the action of both the classic and the alternate (backdoor) pathways.
Introduction
The early seminal studies of Alfred Jost established the paradigm that genetic sex determines gonadal sex, which in turn determines phenotypic sex. In particular, testicular androgens were posited to be responsible for virilisation of the male reproductive tract (Jost 1953). A range of genetic, clinical and experimental studies confirmed the outline of this model in eutherian mammals (George & Wilson 1994). The classical pathway for virilisation involves the release of testosterone from the testis. This testosterone is carried around the body in the blood and at target organs it acts either directly via the androgen receptor, or it is converted by 5α-reductase to the more potent androgen dihydrotestosterone (DHT) which also acts through the same androgen receptor (Baulieu et al. 1968, Bruchovsky & Wilson 1968a,b, Wilson 1972, 2001). This model was abundantly established in the adult and was tacitly assumed to also apply during early virilisation. Direct testing of this assumption was challenging because the inaccessibility and small size of the eutherian fetus at the time of sexual differentiation made a direct assessment of circulating androgens a challenge.
Jean Wilson and his colleagues investigated the endocrinology of virilisation in an American marsupial, the North American opossum (Didelphisvirginiana) (George et al. 1985). They were able to confirm that the developing testis converts pregnenolone to testosterone at about the time when the scrotum first becomes grossly discernable, days or weeks before masculinisation of other parts of the urogenital tract. As in eutherian mammals, 5α reduction of testosterone to DHT is high in androgen target tissues – the urogenital tubercle, urogenital sinus and the developing Wolffian duct.
When Jean Wilson visited Australia shortly after the publication of this study, we invited him to collaborate with us to investigate early virilisation in an Australian marsupial. The real advantage of the marsupial is that gonadal differentiation and virilisation of the urogenital tract (which occur in utero in man and mouse) take place after birth (Fig. 1), so the young can be directly manipulated with hormones, growth factors or inhibitors without the complicating effects of the placenta or maternal physiology. Over the next many years, using our unique model marsupial, the tammar wallaby, our research led to the unexpected discovery of a new pathway of androgen biosynthesis that subsequently has been shown to also operate in humans, forming DHT via an alternate pathway in which the androstanediol (adiol), a pro-hormone with little or no intrinsic androgenic activity, is the key circulating steroid that is metabolised to the active androgen DHT within the target tissues.
Timing of sexual differentiation in the tammar. Tammars are altricial at birth (left panel), with indeterminate gonads. Testes become morphologically distinguishable by 2 days pp, and androgen production in the Leydig cells then continues until it falls between days 40 and 50. Androgen is responsible for virilisation of the Wolffian ducts to form the epididymis and vas deferens, the urogenital sinus to form the prostate and the genital tubercle to form the penis and penile urethra. These different androgen-dependent organs virilise at distinctly different times. However, even before the testis has differentiated, genetic male XY young can be distinguished by the presence of scrotal bulges, the precursor of the scrotum, structures that are never seen in XX female young. A full colour version of this figure is available at https://doi.org/10.1530/JOE-22-0296.
Citation: Journal of Endocrinology 258, 3; 10.1530/JOE-22-0296
Sexual differentiation and direct genetic control of sexual dimorphisms
In humans, male and female external genitalia are indistinguishable until after week 8 of gestation (George et al. 1985, George & Wilson 1994). The initial sign of masculinisation is an increase in the distance between the anus and genital opening the anogenital distance (Baskin 2000). In both sexes, the genital tubercle elongates to form the phallus, genital swellings form around the base of the phallus, and a urethral groove develops as an invagination of the urethral plate epithelium along the ventral surface of the developing phallus. The urethral groove does not close in females, but in males, the phallus continues to grow and the urethral groove folds over and fuses to form the penile urethra (George & Wilson 1994, Baskin 2000, Yamada et al. 2003). Fusion of the urethral folds leaves a seam visible on the ventral surface of the scrotum and penis (the scrotal and penile raphe, respectively). This process is complete by week 16 in humans when the fetus has a crown-rump length of 115 mm (Altemus & Hutchins 1991, Butler et al. 1999, Baskin 2000, van der Werff et al. 2000).
Development of the penile urethra in the tammar (Butler et al. 1999, Leihy et al. 2011) conforms to the most widely accepted model of urethral formation in humans and other mammals (Penington & Hutson 2002, Yamada et al. 2003, Yiee & Baskin 2010). The urethral plate that is present in both sexes at birth forms the urethral groove by day 60 postpartum (pp) (Butler et al. 1999, Leihy et al. 2004). As in eutherians, in tammar males, the groove folds over and fuses in a proximal-to-distal direction in the midline leaving a visible seam on the ventral surface (the penile raphe). In females, it remains open. The urethra of the penile shaft appears to form by primary lumenisation as in eutherians. Like humans, but unlike mice and most mammals, there is no os penis in marsupials.
As noted above, marsupials complete their sexual differentiation after birth. Despite this, two key sexually dimorphic structures that are hormone-dependent in eutherian mammals, namely the scrotum and the mammary primordia, differentiate before the gonads in tammars (O et al. 1988) (Fig. 2). These hormone-independent structures (Shaw et al. 1988) are under the direct genetic control of a gene or genes on the X-chromosome (O et al. 1988, Renfree & Short 1988). This novel discovery of a direct genetic effect on sexual differentiation went against the well-established Jost paradigm that a testis was all that was needed for a functional male reproductive system. These observations opened up a new area of investigation in studies of sexual differentiation. We now know of numerous hormone-independent sexual dimorphisms in a range of mammals and other vertebrates (including mice and humans) that cannot be explained by gonadal hormones (Renfree et al. 2014). For many of these dimorphisms, the specific gene or genes involved have yet to be discovered. Nevertheless, our observation in marsupials led to the understanding that direct genetic (non-hormonal)-mediated sexual dimorphisms are widespread and diverse, including the growth rate of rodent blastocysts to sexual dimorphisms in the brains of finches (Renfree et al. 2014). The curious situation of the enlarged female phallus in the hyaena may also be due to direct genetic control or a non-androgenic hormonal pathway because the masculinised clitoris is formed before the ovary differentiates and is capable of synthesising androgens (Drea et al. 1998, Cunha et al. 2005, Glickman et al. 2005).
Early sexual development in tammars. The Y chromosome is testis determining. Thus, XY normal males and XXY females develop testes. The production of androgens virilises the Wolffian duct derivatives, whilst the anti-Mullerian hormone AMH causes regression of the Mullerian ducts. In the absence of a Y chromosome, an ovary develops, and in the absence of testicular hormones, the Wolffian duct regresses and the Mullerian duct persists to later form the vaginae and uteri. Gonadal hormones do not control formation of the scrotal bulges or mammary buds. If two X chromosomes are present (normal females and XXY), mammary buds form in the fetus and no scrotal bulges form. If only one X is present, (XY normal males and XO), then scrotal bulges develop and mammary buds do not form. If a Y chromosome is present, a penis develops, and in the case of XXY males, there is a penis and a pouch but no scrotum, while XO females have an empty scrotum and no pouch. A full colour version of this figure is available at https://doi.org/10.1530/JOE-22-0296.
Citation: Journal of Endocrinology 258, 3; 10.1530/JOE-22-0296
Discovery of the alternate pathway
Another curiosity in tammars, in contrast to humans and other mammals, is that different aspects of virilisation start at very different times (Fig. 1). The testis is recognisable with differentiation of Leydig cells and synthesis of androgens by 2 days after birth. Differentiation of the Wolffian duct to form the epididymis and vas deferens starts soon after, by day 7 pp. However, the start of formation of prostate tissue in the urogenital sinus only commences about day 25 pp, and sexual dimorphism of the urogenital tubercle is not visible until about day 50 pp. This difference in timing is intriguing given they are all androgen mediated.
Attempting to clarify these questions, we measured testosterone in developing testes and ovaries. Since the testis begins its differentiation 2 days after birth, and the ovary is recognisable by day 8 after birth, we assumed that there would be differences in circulating testosterone levels between males and females. Surprisingly, this was not the case (Renfree et al. 1992). Gonadal testosterone levels were low or unmeasurable in ovaries at all stages but rose in testes by day 5, soon after the formation of seminiferous cords, remaining high until after day 40, but had fallen to low levels by day 50 pp (Renfree et al. 1992). Thus, through the time when the phallus became sexually dimorphic, testicular testosterone levels remained low. Further complicating the picture, when we measured androgens in the plasma of developing male and female young through the period of sexual differentiation, we found no differences between male and female young in the levels of testosterone or DHT, which remained low throughout (Wilson et al. 1999). These surprising results seemed at odds with the paradigm that androgens from the testis are responsible for virilisation of the prostate and phallus. How was virilisation controlled during this time frame?
Jean had previously reported in a series of studies that adiol had potent androgenic effects, stimulating prostate development in dogs and it was involved in prostate formation in eight species of mammals including humans (Jacobi et al. 1978, Jacobi & Wilson 1979) so he suggested that this could be the circulating androgenic steroid. To further clarify, we investigated androgen formation in tammar gonads. In a series of incubations of tiny gonadal samples with radioactive precursors, Jean tested this hypothesis. He recruited his colleagues Cedric Shackelton and Esther Roitman from UT Dallas to conduct the sensitive HPLC assays. These studies demonstrated that the testes made adiol via an alternate pathway of testosterone metabolism (Wilson et al. 2003) (Figs 3 and 4). In this alternate, or backdoor, pathway (Auchus 2004), the testes make a 3α-reduced derivative of DHT 5α-androstane-3α,17β-diol (adiol) (Shaw et al. 2000, Wilson et al. 2003). Adiol itself is a very weak androgen – binding to the androgen receptor is 5 orders of magnitude less than binding with DHT, so of itself, it is a very weak androgen (Penning 1997). However, we showed that the tissues of the urogenital sinus and genital tubercle are able to convert adiol to DHT (Fig. 4). We then treated developing young from day 20 to day 45 and examined the urogenital sinus for prostatic buds. Adiol treatment (but not control) induced the formation of prostatic tissue in females and increased prostatic buds in males (Shaw et al. 2000). Thus, adiol is acting as a circulating prohormone that is converted in the target tissues to the active androgen DHT.
Simplified outline of the classical and alternate pathway of androgen metabolism via adiol in the tammar. Enzyme names in italics. For more complete pathways, see Wilson et al. 2003, Auchus 2004, Mahendroo et al. 2004, Auchus & Miller 2012, Saito et al. 2016, Sharpe 2020. A full colour version of this figure is available at https://doi.org/10.1530/JOE-22-0296.
Citation: Journal of Endocrinology 258, 3; 10.1530/JOE-22-0296
The testis produces testosterone (T) via the conventional pathway. This drains into the Wolffian duct where it is taken up and binds to the androgen receptor (AR). The activated androgen receptor (T-AR) binds to the DNA regulating the genes directing differentiation of the epididymis, vas deferens and seminal vesicles. The testis also releases androstanediol (adiol) made via the alternate pathway. Adiol is carried in the blood to target cells in the urogenital sinus and urogenital tubercle. Adiol is converted to the potent androgen 5α-dihydrotestosterone (DHT), which binds and activates the androgen receptor (DHT-AR) to control the genes that virilise these structures. A full colour version of this figure is available at https://doi.org/10.1530/JOE-22-0296.
Citation: Journal of Endocrinology 258, 3; 10.1530/JOE-22-0296
The male programming window of sensitivity
Whilst we had established a pathway for virilisation occurring in the absence of sexually dimorphic levels of testosterone and DHT in the circulation, we still had the open question of the control of penis development. This occurs much later than prostate formation at a time when circulating T, DHT and adiol were all low. We, therefore, began a series of studies of phallus development in male and female young with Jean and PhD student Michael Leihy, These studies clarified the timing. Male phalluses become larger than female phalluses only by day 48 and the major growth occurs after day 60. Closure of the urethral groove to form the penile urethra does not start until about day 60 and was not complete until day 150 (Shaw et al. 2000, Leihy et al. 2004). Thus, the majority of differentiation of the penis and in particular formation and closure of the urethral groove to form the penile urethra occurs when there is no measurable sex difference in concentrations of testosterone, DHT or adiol in the gonads or in the circulation. We investigated this with a series of experiments using castration and exogenous androgen treatment of developing male and female young (Leihy et al. 2004, 2011).
Castration of male young at day 25 prevents closure of the urethral groove, whereas partial closure of the urethral groove still occurs in males castrated at day 40 or later (Leihy et al. 2004). Transplantation of testes into neonatal female pouch young results in Wolffian duct differentiation and the development of a prostate and a penis with complete urethral closure (Tyndale-Biscoe & Hinds 1989, Renfree 2019). Partial fusion of the urethral groove occurs by day 150 in females treated with adiol from day 20 day 40 but not in females treated from day 40 to day 80 or from day 80 to day 120 (Leihy et al. 2004). In brief, transient androgen treatment of females causes hypospadias, and castration of males after day 40 does not prevent closure. Our experiments showed there was a critical window of time between day 20 and day 40 pp where androgen action is critical for normal masculinisation and, in particular, later differentiation of the phallus and development of the penile urethra (Shaw et al. 2000, Leihy et al. 2002, Leihy et al. 2004, 2011, Renfree 2019).
This was the first demonstration of androgen imprinting in which exposure to the hormone during a short window of sensitivity caused later sexual differentiation events (Fig. 5). We followed up by investigating the expression of the genes responsible for phallus differentiation, notably SHH (Chew et al. 2014). We found that SHH, along with Gli2 and AR were markedly upregulated in the urethral epithelium at day 50 pp, a time when testicular androgens are falling. These genes were downregulated in female pouch young treated with adiol from day 24 to day 50 but not when treatments were begun at day 29, suggesting an early window of androgen sensitivity. SHH, GLI2 and AR expression in the phallus of males castrated at day 23 did not differ from controls, but there was an increase in SHH and GLI2 and a decrease in FGF8 and BMP4 expression when the animals were castrated on day 29. These results suggest that an androgen-dependent window of sensitivity between about days 24 and 50 regulates later SHH expression in the developing male phallus (Chew et al. 2014).
Comparison of timing of some key events in male urogenital development in tammar, rat and human. Androgen levels (blue-shaded area) rise soon after testis differentiation. In human and rat, differentiation of prostate and phallus both start soon after. In tammars onset of prostate development occurs well after onset of androgen production, and phallus differentiation starts even later, after androgen levels have fallen. In all three species, there is a ‘masculinisation programming window’ (MPW) where androgen action is essential for later virilisation of the urogenital system especially prostate and penis. A full colour version of this figure is available at https://doi.org/10.1530/JOE-22-0296.
Citation: Journal of Endocrinology 258, 3; 10.1530/JOE-22-0296
Failure to recognise the asynchrony between the androgen signal and the downstream morphological changes undoubtedly slowed progress in this area. This finding of an early programming window in the tammar (Leihy et al. 2002, 2004) was described a few years later in 2008 in rats. This male programming window (MPW) (Welsh et al. 2007, 2008, 2009, 2010, Macleod et al. 2010) in rats and a similar study in mice (Zheng et al. 2015) show the same necessity for androgen exposure for later masculinisation to occur as in the tammar (Fig. 5). Rats treated with androgen or with flutamide, an androgen receptor antagonist, during both an early (E15.5 days to E17.5) and middle (E17.5–19.5) programming window failed to virilise (Welsh et al. 2008). The common early programming window in rats was defined as E15.5 to E18.5, and reducing androgen during this window causes hypospadias and cryptorchidism (Macleod et al. 2010, van den Driesche et al. 2011). There are similar effects in mice in which interfering with androgen receptor action at different stages of genital development induces penile abnormalities (Zheng et al. 2015). A number of authors have now demonstrated similar masculinisation programming windows for the male reproductive tract and sexually dimorphic brain development in a range of species including rats (Welsh et al. 2008, 2010, Dean et al. 2012) and mice (Ghahramani et al. 2014) and is likely to occur between week 8 and 14 of gestation in humans (Welsh et al. 2008, Fisher et al. 2016) (Fig. 5). The androgens essential for masculinisation are primarily produced by the alternate pathway (O'Shaughnessy et al. 2019, Sharpe 2020).
Disorders of sexual differentiation and the alternate pathway
Elucidating the alternate pathway in the Australian tammar wallaby opened up new fields of investigation and this pathway has now been demonstrated to be physiologically important in several other mammals including humans (Mahendroo et al. 2004, Wilson et al. 2005). The alternate/backdoor pathway is crucial to understanding some disorders of sexual differentiation and has been incorporated into an Endocrine Society (USA) clinical guideline for congenital adrenal hyperplasia (Speiser et al. 2010). Androgens produced via the alternate pathway appear to control the virilisation seen in female infants with P450 oxidoreductase deficiency that occurs in >90% of cases of congenital adrenal hypoplasia (Arlt et al. 2004, Homma et al. 2006). We now know that both the classic and the alternate pathways are also essential for normal human male sexual differentiation (Fluck et al. 2011, Kamrath et al. 2012, Biason-Lauber et al. 2013, Lee & Kim 2022, Naamneh Elzenaty et al. 2022). Genes encoding the 3α-reductases, AKRIC2 and AKRIC3, produce alloprenanolone only by the alternate pathway, and mutations in AKRIC2 and aberrant splicing of AKRIC3 explain some DSDs in 46,XY individuals. The essential role of these genes confirmed that both the classic and alternate pathways of testicular androgen synthesis are necessary for normal human male sexual development, and since it occurs in widely divergent mammals, namely marsupial, rodent and human fetal testis, it suggests this is common to all mammals (Fluck et al. 2011, Lee & Kim 2022). Recently, the conclusion that virilisation in the human male fetus is mediated by both the fetal testis and non-gonadal tissues has been reinforced. Measuring plasma and tissue levels of steroids implicated in both pathways shows that placental progesterone and several other tissues including the liver and adrenal act as a substrate to produce alternate androgens of which aldosterone is the key precursor to adiol and primary androgen in the human alternate pathway and then to DHT synthesis in the genital skin (O'Shaughnessy et al. 2019). It also appears to be important in polycystic ovarian syndrome (PCOS) since PCOS ovaries have enhanced expression of genes and proteins of the alternate pathway (Marti et al. 2017). The aldo-keto reductases (AKR1C1/AKR1C4), 5α-reductases (SRD5A1/2) and retinol dehydrogenase (RoDH) are all expressed in the human ovary, and their proteins are all specifically localised in ovarian theca cells and overexpressed in PCOS ovaries (Marti et al. 2017). However, there was no expression of AKRIC4 in the ovary. Women with PCOS also have a higher level of 17-OH-progesterone which promotes androgen production through the alternate pathway (Homma et al. 2006).
Conclusions
Studies of the endocrinology of marsupial mammals were of key importance in both the US and Australia in the early twentieth century because of their accessibility to investigate sexual differentiation since it occurred after the birth of the altricial young, rather than in utero as in other mammals. We revived this interest in recent decades. Using the Australian tammar wallaby as a model, we showed that some aspects of virilisation are controlled directly by genes rather than gonadal hormones; we identified an alternate pathway of androgen metabolism where androstanediol is the main circulating virilising steroid; and we identified a window of androgen sensitivity early in development where androgen action programmed later virilisation at a time when testicular androgen production is low. These three aspects of sexual differentiation have now been demonstrated in a range of eutherian mammals including humans, opening up a new understanding of normal and disordered sexual development and avenues for clinical investigation and treatments. Collectively these data highlight how our results with the tammar wallaby in Australia continue to contribute to a new understanding of the control of virilisation.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Acknowledgements
The authors dedicate this review to the memory of our late colleague Prof Jean D Wilson, a clinician and researcher who recognised the power of alternative animal models. His enthusiasm, insight and expertise made possible many of the studies reported here.
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