The masculinization programming window.

Concepts Hiort O, Ahmed SF (eds): Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel, Karger 2014, vol 27, pp 17–27 (DOI: 10.1159/000363609)

The Masculinization Programming Window Michelle Welsh a · Hiroko Suzuki b · Gen Yamada b

a

School of Life Sciences, West Medical Building, University of Glasgow, Glasgow, UK; b Department of Development of Genetics, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan

Abstract Sexual differentiation is a tightly regulated series of events which transform the indifferent gonads and genitalia into sex-specific structures. This is driven by hormones produced by the fetal testes, primarily testosterone (T). However, masculinization of each structure does not occur synchronously and, until recently, it was presumed that androgens also control this masculinization over a broad period of fetal life, coincident with the period of fetal T production. However, a common fetal masculinization programming window (MPW) has been identified in male and female rodent models in which androgens must act to masculinize all components of the reproductive tract and allow their later complete development. Impaired androgen action only within this MPW can induce cryptorchidism and hypospadias. This MPW is likely to occur between 8–14 weeks’ gestation in humans. Studies in transgenic mice have begun to investigate some of the underlying mechanisms involved. Anogenital distance is predictive of the incidence of disorders, such as azoospermia, hypospadias and cryptorchidism, and could provide a noninvasive, lifelong indicator of androgen action within this MPW, useful in clinical assessment of patients with disorders of sexual development. In addition, several diagnostic characteristics of the external genitalia are also useful in investigating this MPW. © 2014 S. Karger AG, Basel

Sexual differentiation is a genetically and hormonally regulated preprogrammed series of events which transform the indifferent gonads and genitalia into sex-specific structures. The mammalian reproductive system consists of the gonad, two genital duct systems, namely the Müllerian (MD) and Wolffian (WD) ducts, and a common opening for the genital and urinary tract to the outside through the genital folds. These structures are initially indistinguishable in both sexes but are predetermined to

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Reproductive Tract Development and Differentiation

T

T 5α-Reductase

DHT Fetal androgen (testosterone) Leydig cell

Testis

AR

Epididymis Testis Vas deferens Seminal vesicle Prostate

Penis Scrotum Male

develop along the female pathway; once termed the ‘default’ pathway. To become a male requires modification of this, initiated by Sry gene-driven testis formation [1]. Gonad development, however, does not itself cause a male phenotype; instead bodywide masculinization is driven by hormones produced by fetal testes, primarily androgens [2]. This relies on correct secretion of the hormones and expression of their receptors in target tissues, in the right place at the right time. The human fetal testes produce testosterone (T) from 8–37 weeks’ gestation [3] (embryonic day e15.5–21.5 in rats [4]), driving reproductive tract masculinization during this period. This T is locally converted into dihydrotestosterone (DHT) by 5α-reductase enzyme for the masculinization of the prostate and external genitalia (fig. 1), while T itself is believed to act on structures closer to the local source (namely the testis), such as the WD and its derivatives. However, these masculinization processes do not occur synchronously. Instead, the window of differentiation for each structure is distinct [summarized in fig. 7 of reference 5]. For example, the prostate forms at gestation weeks 10–13 [6] (rat, e18.5–19.5), the penis at gestation weeks 11– 13 [6] (rat, e17.5) and seminal vesicles at gestation weeks 14–16 [7] (rat, e19.5). Testis descent into the scrotum occurs much later (human, 27–35 weeks; rat, postnatal week 3) and brain masculinization even later. As a result of such observations, it had been presumed that masculinization occurs over a broad period of fetal life, coincident with the broad period of T production [3, 4].

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Welsh · Suzuki · Yamada Hiort O, Ahmed SF (eds): Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel, Karger 2014, vol 27, pp 17–27 (DOI: 10.1159/000363609)


Fig. 1. An illustration of how androgen signaling can be altered through modulation of: (1) androgen production by the fetal testis; (2) the conversion into DHT by 5α-reductase action, and/or (3) the AR.

Internal Reproductive Organ Differentiation

Reproductive tract differentiation begins with the internal genitalia developing from the MD and WD, which are initially present in male and female embryos. MD and WD differentiation depends on T and anti-Müllerian hormone (AMH) secretion from the fetal testis [2]. In males, AMH signals through the AMH receptor in the MD mesenchyme and drives MD regression craniocaudally [8]. T initially stabilizes the male WD then drives subsequent differentiation; namely, elongation and convolution of the cranial WD to form the epididymis, elongation of the middle portion to form the vas deferens and distension and folding of the caudal WD to form the seminal vesicles [8]. The prostate develops as branching and budding of the urogenital sinus (UGS), which arises at around 7 weeks gestation in humans from the cloaca and remains sexually indistinguishable at 10–12 weeks. Androgens drive UGS epithelium to bud and grow out into the UGS mesenchyme in a specific spatial pattern, establishing the prostate lobar (rodents) or zonal (humans) subdivisions, and canalize and differentiate to form the prostate [9]. Unlike males, the female does not depend on the ovary or hormones to mediate differentiation. In the absence of AMH, the female MD is programmed to differentiate into the oviducts, uterus, cervix and upper vagina, while the lack of androgens causes the female WD to regress craniocaudally, until it has completely regressed by e19 in rats [10].

Unlike the internal genitalia, which develop from different ducts in males and females, the external genitalia develop from common primordial structures, namely the genital tubercle (GT), genital swellings and urethral folds [11]. Phallus masculinization requires hormonal stimulus unlike the female external genitalia, which develop without androgens [12]. The GT elongates to form the penis shaft, the urethral folds elongate and fuse to form the penile urethra while the urogenital swellings fuse to form the scrotum, which will ultimately house the testes outside the body cavity [13]. Hypospadias results when the urethra is not completely enclosed by the urethral folds with the site of fusion failure dictating the position of the abnormal opening of the urethra [14]. In rodents, the male fetal external genitalia do not grow significantly so, at birth, the phallus size does not differ greatly between males and females [11]. Conversely, human sex can easily be determined externally at birth. However, in both rodents and humans, the distance between the anus and the base of the GT (anogenital distance; AGD) is approximately twice as long in males as females, due to fetal androgen action [15]. Female external genitalia in humans undergo considerable differentiation by 26 weeks gestation, with the GT differentiating into the clitoris while the genital swellings and folds give rise to the labia majora and minora, respectively [11].

The MPW Hiort O, Ahmed SF (eds): Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel, Karger 2014, vol 27, pp 17–27 (DOI: 10.1159/000363609)

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Differentiation of the External Genitalia

Reduced fetal androgen action impairs masculinization, and can result in disorders of sex differentiation or more rarely phenotypic sex reversal. Disorders of sex differentiation, such as hypospadias, are amongst the most common human congenital disorders, and are related to and risk factors for low sperm counts and testicular cancer. These disorders comprise a ‘testicular dysgenesis syndrome’ with a proposed common origin in fetal life, linked to deficient androgen action/production [16]. This has been demonstrated using both transgenic and chemical models of impaired fetal androgen action. For example, rat exposure to the antiandrogen flutamide between e15.5–21.5, the time window of reproductive development, impairs masculinization of the reproductive system, resulting in no prostate, reduced AGD and impaired WD development [17–19]. These abnormalities persist into adulthood, confirming that the patterning and establishment of the reproductive tract is set up fetally, and interfering with androgens during this period permanently alters reproductive tissues. It was once believed that androgens induce and complete reproductive masculinization during the time when morphological differentiation is observed in these tissues, i.e. late in fetal life for the rat prostate and penis. However, studies exposing animals to antiandrogens during specific narrow time windows have questioned this, highlighting that androgens ‘preprogram’ masculinization before morphological changes are observed. For example, exposing pregnant mice to hydroxyflutamide on e11–15 resulted in smaller epididymides and infertility, whereas exposure only on e19–20, when the future epididymis is coiling, had no obvious reproductive effect [20]. Similarly, rat exposure to flutamide on e16 or 17 resulted in missing epididymides in adults, whereas later exposure (on e18 or 19) only resulted in smaller epididymides, and the peak incidence of abnormal prostate development was following flutamide on e17 or 18 [21]. Furthermore, hypospadias was maximally induced by exposure of rats to finasteride, a 5α-reductase inhibitor, before e18 [22]. These studies were substantiated by Welsh et al. [23], demonstrating that flutamide exposure between e19.5 and e21.5, the period when the WD is differentiating, did not obviously alter WD development, whereas exposure between e15.5 and 17.5, the period of WD stabilization, inhibited WD differentiation to the same extent as exposure from e15.5 to 21.5, and resulted in a similar high incidence of epididymal loss in adulthood. This demonstrated that the pattern of WD differentiation/coiling and its subsequent ability to develop postnatally is established early in fetal reproductive development (e15.5– 17.5), before any sign of this morphological differentiation, and that high levels of androgen action later in fetal development are not essential for maintaining this male phenotype. Similar findings were seen in other reproductive organs with early flutamide exposure (e15.5–19.5; termed ‘masculinization programming window’; MPW) resulting in a similar phenotype as exposure to flutamide during the entire window of male fetal reproductive development (e15.5–21.5); namely, males presented with a vaginal pouch instead of a prostate, impaired WD and phallus development, and re-

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Masculinization Programming Window in Males

duced AGD [5]. This phenotype was not observed in male fetuses exposed to flutamide later in development (e19.5–21.5). Similar results were seen following exposure to di(n-butyl) phthalate, which reduces fetal testicular T production, linking di(n-butyl) phthalate exposure in the MPW and testicular dysgenesis syndrome [24]. Together, these studies demonstrate that androgen-driven reproductive tract masculinization is mediated during a common early MPW (before e19.5 in rats), as opposed to during the later ‘differentiation window’ (after e19.5 in rats) when morphological differentiation of tissues is observed. This agrees with previous studies identifying a similar window in individual tissues [21, 25].

Interestingly, cryptorchidism was also induced by interfering with early (∼e15.5– 19.5), but not late fetal androgen action [5, 26]. Trans-inguinal testis descent, which occurs in humans around gestation weeks 27–35 [27] and rats around postnatal week 3 [28], is androgen dependent and is the stage most commonly affected in cryptorchid boys [28]. If these rat studies apply to humans, incomplete or delayed transinguinal testis descent likely reflects impaired androgen action much earlier in pregnancy than was previously suspected. Penis formation and subsequent growth is also mediated by androgens. It is thought that androgen-dependent penile growth occurs at three time points in humans: in late gestation, the first 4 years after birth, and at puberty [29]. Penile disorders, such as micropenis, a normally formed but abnormally small penis, or hypospadias, an altered penile formation, are linked to deficient androgen action, possibly during the MPW, and doubts exist over their optimal treatment [29, 30]. Rat studies have demonstrated that penis formation is critically dependent on androgens during the early MPW, and also that late fetal and/or postnatal androgen action is required for phallus growth [31, 32]. This is consistent with current understanding in humans and nonhuman primates [29]. Rat studies have also demonstrated that fetal deficiencies in androgen-dependent penile length cannot be rescued by postnatal T treatment [32, 33]. This might explain the poor response to androgen treatment in some boys with micropenis, and why most boys with micropenis ultimately have a smaller than average penis in adulthood [29, 34]. Interestingly, exposure of normal male rats to postnatal T treatment will advance, but not ultimately enhance, penile size [33]. The human penis is responsive to growth stimulation by exogenous androgens at comparable stages of development to rats, with boys with isolated micropenis experiencing a bigger increase in penile length in response to T treatment than do boys with micropenis and hypospadias [35]. It could be suggested from these rat studies that androgen action in the MPW is essential for formation of a penis with some unknown factors predetermining the ultimate length the penis can reach. However, androgen action later in fetal life and postnatally is essential to achieve this potential. It should be not-


21


Clinical Implications of the MPW in Males

ed that these studies examined penile size, urethral closure and penile bone ossification, but in future studies other histological parameters of GT masculinization, such as the formation of the urethral plate, canalization of the urethra and bilateral mesenchymal development, should also be evaluated.

It is well established that inappropriately high androgen exposure in both animal models and humans can masculinize the female fetus. For example, exposure to 0.5– 10 mg of T propionate/pregnant rat permanently increased female AGD, masculinized external genitalia, and induced prostate and seminal vesicle development, but was unable to rescue the WD in these female offspring [36]. Interestingly, rescue and differentiation of the WD in females requires higher levels of T exposure than does masculinization of the UGS or external genitalia [36]. Further evidence for extraneous T exposure masculinizing female fetuses comes from studies in Wnt-4 knockout mice [37]. Their ovaries synthesize T, which in turn rescues and differentiates the WD but not the external genitalia [37]. This difference is likely to be due to the source of T, i.e. local T from the gonads more easily masculinizes WD structures, while circulating androgens are converted to DHT in the more distal structures, and so masculinize the prostate and part of the external genitalia. Interestingly, in females, as in males, androgens are considered to act within the MPW to masculinize the reproductive tract. Females exposed to T during the MPW developed prostates and seminal vesicles, had an increased AGD and increased phallus length, whereas later exposure (i.e. e19.5–21.5) did not masculinize the female reproductive tract, although it did increase the size of the phallus [5]. Importantly, exposure to exogenous T any time between e15.5 and 21.5 did not enhance development of reproductive structures in the male; for example, penis size was no larger than in control males [5]. This demonstrates that reproductive tract masculinization in normal males is not limited by T availability. Fetal androgen exposure in females was unable to induce a male-sized phallus when measured at postnatal day 17; however, this is due to a lack of androgen action postnatally [5]. The female phallus (clitoral) weight and length both remained sensitive to exogenous androgen action after the MPW, but for a more restricted period than in males [33]. This is consistent with monkey and human data showing that cliteromegaly can be induced by androgens in late fetal life [38, 39], and suggests that there could be basic differences in the responsiveness of the undifferentiated male and female phallus. This could have important clinical implications for XX individuals with altered phallus development. It is not only the reproductive tract which is sensitive to androgen action in fetal life – brain development can also be affected. T exposure can also affect neonatal growth and induce intrauterine growth retardation [36]. For example, polycystic

22



Masculinization Programming Window in Females

ovarian syndrome patients have an increased prevalence of babies born small for gestational age, but it is unclear if this is due to the T directly or because it is converted into estradiol [40].

Anogenital Distance

Many previous animal studies have used AGD as an index of altered fetal androgen action [18, 21, 23]. For example, human AGD at birth correlates with gestational exposure to phthalates which, in rat studies, reduce fetal T production [41, 42]. However, rat AGD has recently been demonstrated to be altered by manipulation of androgen action only during the MPW [5]. Furthermore, animal and human studies have demonstrated that subnormal AGD correlates with hypospadias and its severity, cryptorchidism, reduced phallus length, reduced seminal vesicle weight, and reduced adult testis size, T production and fertility [5, 32, 33, 43, 44]. Other human studies found no such correlation with hypospadias [45], although this study did not correct AGD for bodyweight, an important confounder in babies. Fetally induced changes in AGD are permanent [18, 46, 47]; therefore, AGD measured at any time in humans or animal models could provide insight into androgen action during the critical MPW, and so act as a noninvasive measure of androgen action specifically within the MPW. AGD can therefore be used to provide insight into androgen action during the MPW, but may not be a reliable guide in decision making about ‘sex of rearing’ in cases of ambiguous genitalia at birth, as androgen-induced brain masculinization is a late fetal event in humans and monkeys, and is likely to be independent of the MPW.

Much of the research discussed above relies upon animal models in which steroid hormone action was pharmacologically manipulated. However, conditional genetic strategies should be used to complement these and fully elucidate the masculinization processes. Recent progress in conditional mutant mouse technology has allowed new opportunities to address the tissue and temporal specific role for genes postulated to regulate masculinization of the reproductive tract. This technology now enables mutation of the ligand and/or receptor using Cre recombinase to target genes in specific cells/tissues at an appropriate embryonic timing. Such time-dependent gene modification is possible using tamoxifen or tetracycline-inducible gene modulation, and will allow for further analysis of the MPW. Mutations in genes involved in production of steroid hormone ligands, such as the steroidogenic enzymes, can result in impaired masculinization. For example, mutations in steroidogenic acute regulatory protein induces several abnormalities, including reduced AGD [48]. However, to fully understand the factors controlling the MPW,


23


Modulation of Androgen Signal through Conditional Mutant Mice

24



fetal T production must be understood. A recent study has shown that, unlike adult Leydig cells, the fetal Leydig cells can only synthesize androstenedione and that the fetal Sertoli cells convert this androstenedione into T [49]. Future studies should therefore investigate the possible role for both the fetal Leydig and Sertoli cells in androgen production and hence the MPW. The MPW can also be analyzed through modulating the expression of the androgen receptor (AR), and therefore altering AR signaling. AR conventional null knockout mice (or testicular feminization; termed tfm) fail to masculinize, as seen in humans with complete androgen insensitivity syndrome due to AR mutations. When investigating androgen signaling, the androgen-estrogen balance should also be considered, for example through AR and estrogen receptor double and triple knockout models [50–52]. Since the generation of the AR gene Flox allele, it is now possible to perform conditional ablation of AR in specific cells or tissues [53]. For example, cellspecific ablation of AR in the WD epithelium revealed that epithelial AR is essential for epithelial cell differentiation in the future epididymis but not for initial WD stabilization, which is presumably controlled by mesenchymal AR around e16.5 in mice [54]. Furthermore, mesenchymal AR has been shown to be critical in the development of the GT in mice at e14.5 [55, 56]. AR is broadly expressed in the entire GT mesenchyme, albeit its expression is stronger in the bilateral/lower mesenchyme close to the urethra [55, 56]. This bilateral mesenchyme is considered essential to mediate urethral formation, including midline fusion to form the penile raphe. The bilateral mesenchyme can be labeled on the basis of Hedgehog signal responsiveness (Gli1-positive mesenchyme). Less is understood of the genes regulating the lateral and upper GT development. Recent analysis suggests that upper GT and the ventral body wall development are linked, as mutations in aristaless-like 4 cause both ventral wall and GT abnormalities [57]. However, how the upper and lateral GT mesenchyme contributes to the adult penis and the regulatory genes involved requires further investigation to enable accurate characterization of GT masculinization and the MPW. It has been suggested that locally produced growth factor signals could interact with androgen signals to mediate masculinization [55, 56], but this remains to be elucidated. There have been some reports on the isolation of putative mesenchymal masculinization effector genes in the GTs, some of which are strongly expressed in this bilateral mesenchyme [58]. Such effector genes include growth factor signals like canonical Wnt/β-catenin and Hedgehog signals [55, 56]. Some effector genes have been suggested as locating ‘downstream’ of androgen signaling. The bilateral mesenchyme of GT is also expected to be influenced by Shh, which is expressed in the urethral plate epithelia [59]. Genetic studies, including gain of function mutants, for those ‘downstream’ effector genes, such as Wnt/β-catenin, showed ‘masculinization’ phenotypes; for example, enhanced Wnt/β-catenin signaling results in the development of a more masculine GT in females with prepuce hyperplasia [56]. Wnt/β-catenin is known to play central roles in organogenesis and cell growth control, differentiation and tumor formation [60]. Canonical-Wnt signals have been identified as augmented in the male

bilateral mesenchyme of GT around e15 before the morphological dimorphisms [56]. Many Wnt ligands have been identified for the canonical signals, some of which are expressed in the urethral plate epithelia, the GT ectoderm and in the mesenchyme [56]. It is possible that such epithelia-derived ligands signal to the nearby mesenchyme to mediate their effects. Regulators such as these may mediate masculinization at various times during embryonic development; thus, further investigation of effector functions is required to identify their roles.

Conclusion

In conclusion, a common early MPW has been identified in male and female rodent models in which androgen action must occur to masculinize all components of the reproductive tract and enable their correct later development. Impaired androgen action only within this MPW can induce cryptorchidism and hypospadias. Based on parallels with such models, this programming window is likely to be between 8 and 14 weeks’ gestation in humans. This discovery of a common MPW raises new questions, such as what regulates this programming and can it be affected by other risk factors, such as intrauterine growth restriction. Studies in transgenic mice suggest a possible role for signaling pathways, such as the canonical Wnt signal in mediating the effects of androgens during the MPW, but further investigation is still required to elucidate the factors controlling the MPW and mediating the downstream effects. Measuring AGD could provide a noninvasive, lifelong ‘read-out’ of androgen action within this MPW and predicts the incidence of disorders, such as low sperm count, hypospadias and cryptorchidism. AGD could be clinically useful in assessing androgen exposure early in gestation and predicting adult-onset reproductive disorders.

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Michelle Welsh, BSc hons, PhD School of Life Sciences, West Medical Building University of Glasgow Glasgow G12 8QQ (UK) E-Mail [email protected]


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Pericardial window.

Computer programming.

Sexually dimorphic expression of Mafb regulates masculinization of the embryonic urethral formation.

Masculinization and defeminization of female rats by males located caudally in the uterus.

Radiological evaluation of the aortic-pulmonic window.

Developmental programming: the role of growth hormone.

Python scripting: the return to programming.

Programming: Pick up Python.

Pupillometry: A Window to the Preconscious?

Process versus outcome: the sugar window.