During foetal development, androgens play a central role in the development of male external genitalia. This second phase of male sexual development is called sex differentiation and is mechanistically distinct from the initial phase of sexual determination, which is mainly regulated by transcription factors and signalling molecules other than steroids.
Genetically male (46,XY) and female (46,XX) foetuses do not differ morphologically until about 6 weeks post conceptionem (wpc), when sexual dimorphism occurs due to the differentiation of the bipotential gonadal anlage into
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either testes or ovaries. In 1990, a gene on the Y-chromosome was identified as being responsible for the initiation of male sex determination, and called ‘sex- determining region of the Y-chromosome’ (SRY) (Berta et al., 1990; Sinclair et al., 1990). Since the discovery of SRY, numerous rodent and human studies have unravelled complex developmental pathways that mediate the determination of the gonadal phenotype in mammals, with the majority of these genes being discovered because of genetic abnormalities found in patients with disordered sexual development (DSD); see (Eggers and Sinclair, 2012) for an extensive review. In brief, the presence of SRY, which is expressed in the bipotential gonad, initiates the expression of the transcription factor SOX9, which stimulates in turn FGF9; both SOX9 and FGF9 suppress WNT4 and establish a testis-specific pathway. In ovarian development, WNT4 initiates canonical Wnt-signalling in the gonadal anlage, crucial for the establishment of the ovarian-specific pathway.
Both mammalian steroidogenic tissues, the gonads and adrenals, are derived from a common embryological origin, the adreno-gonadal primordium (AGP). This has been suggested by histological studies as well as the detection of distinct steroidogenic enzymes (CYP11A1, HSD3B2, CYP19A1, CYP17A1) in both the gonadal and adrenal anlage during early mammalian development (Val et al., 2006; Val and Swain, 2010). Studies in mice implicate certain genes in the development of the AGP, and knock-out mice lacking these factors do not develop either gonads or adrenals. Steroidogenic factor-1 (SF1), a nuclear receptor from the subfamily 5 (NR5A1), acts as a key regulator in steroidogenic cell-differentiation and function, with important implications for male sex differentiation (El-Khairi and Achermann, 2012). SF1 knock-out mice are not able to maintain adrenal primordia and show a male-to-female sex reversal (Luo et al., 1994). Upstream of SF1, the Wilms’ tumour
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gene 1 (WT1) has been implicated in murine SF1 transcription, modulating adrenal cortex differentiation (Val et al., 2007).
In the course of male sex determination, the bipotential gonad develops into a testis consisting of functional Sertoli and Leydig cells. The Sertoli cells secrete the Muellerian inhibitory substance (MIS) at 8 wpc, inhibiting the development of the para-mesonephric ducts (‘Muellerian’ ducts) into uterus and vagina. At 9 wpc, testicular Leydig cells start to secrete testosterone (Siiteri and Wilson, 1974). Androgen actions are mediated by the androgen receptor (AR), which is expressed in the urogenital tract from 8 wpc. There are no differences in AR expression between male and female foetuses at this stage (Sajjad et al., 2004b; 2004a). AR activation by androgen action results in growth and differentiation of the male external genitalia and the prostate gland, respectively. From a stage of phenotypic ambiguity, androgens support the fusion of the two bilateral genital swellings, which forms the scrotum; in addition, the genital tubercle enlarges and forms the penis shaft (Werner et al., 2010). The critical developmental time window for the external male genitalia to masculinise opens between 8 and 10 wpc only (Goto et al., 2006) (see Figure 11).
Figure 11: External male genitalia at 8 wpc, showing an indifferent/ambiguous stage (panel A), which
masculinizes under the influence of testicular androgens at 10 wpc (panel B). gs: genital swelling; uf: urethral fold; gt: genital tubercle. Asterisks indicate patency of the scrotal fusion. Bar: 500 µm. With kind permission from Goto et al., 2006.
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1.2.3. Adrenarche
Adrenarche refers to the developmental maturation of the adrenal gland, observed only in the human, chimpanzee, and gorilla (Dhom, 1973; Cutler et al., 1978; Conley et al., 2012; Arlt et al., 2002). At adrenarche, the innermost layer of the human adrenal cortex, the ZR, starts to produce increasing amounts of DHEA and DHEAS (Rege and Rainey, 2012; Auchus and Rainey, 2004; de Peretti and Forest, 1978; 1976). The term “adrenarche” was coined by Fuller Albright and Nathan Talbot in the 1940s when they linked the developmental rise in adrenal androgens to the appearance of pubertal and axillary hair, which they called “sexual hair” (Marshall and Tanner, 1969; Albright, 1947; Marshall and Tanner, 1970; Talbot et al., 1943).
Adrenarche is a physiological mystery because it is not well understood how the development of the ZR is initiated or controlled [see (Arlt et al., 1999; Auchus and Rainey, 2004) for review] nor why adrenal androgens are significant for human pre- pubertal development. Adrenal androgens contribute to changes in body composition and transient growth acceleration but without having a major impact on final height or subsequent developmental milestones like puberty. From the evolutionary perspective it has been suggested that adrenarche is a key component of ‘juvenility’, a period that emerges during evolution in the late Hominides and prolongs the transition from childhood to adolescence and adult life; juvenility may serve the adaptation of body composition and metabolic status to environmental conditions (Binder et al., 2009; Hochberg, 2010). Another interesting hypothesis refers to the neuro-modulatory effects of DHEAS that may help to protect more metabolically active regions of the cerebral cortex to support brain maturation in the developing pre-pubertal child (Remer et al., 2004; Campbell, 2011; Voutilainen et al., 1983; Sopher et al., 2011).
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The postnatal reappearance and increase in circulating DHEAS has been traditionally perceived as a relatively sudden surge, physiologically occurring between six to eight years of age (Counts et al., 1987; de Peretti and Forest, 1976; Sklar et al., 1980; de Peretti and Forest, 1978). However, previous studies employing immunoassays for determination of serum DHEAS only identified increasing levels once they went above the lower limit of detection. In a recent study that applied highly sensitive steroid mass-spectrometry indicates that “biochemical” adrenarche starts with a detectable increase in DHEA and related androgenic steroids at about three years of age (Martin, 2004; Remer et al., 2005). In addition, histological studies in the 1970s from human adrenal samples taken throughout childhood, revealed that focal ZR cell islets are present from the age of three years onwards (Arlt, 2006; Dhom, 1973) (Figure 12).
Figure 12: Schematic representing the morphological changes of the adrenal cortex with the rise of
adrenal plasma androgens. DHEA and DHEAS slowly increase during mid-childhood, which correlates with the development of adrenal ZR cells present as focal islets in mid-childhood and then forming a continuous layer over a time span of several years until adolescence. Adapted from (de Peretti and Forest, 1976; 1978; Rege and Rainey, 2012).
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These islets progressively form a continuous layer of ZR cells until early adolescence. These findings suggests that adrenarche, both as a biochemical and morphological phenomenon, is a long lasting developmental process rather than a sudden event.
There are no sex-differences in androgen excretion in pre- and early-pubertal age groups (Remer et al., 2005) and consequently, these results may challenge the well-accepted cut-off for the definition of premature adrenarche including sex differences (age 8 in girls and 9 in boys), derived from Marshall and Tanner’s clinical observations that the appearance of pubic hair occurs about one year earlier in girls than in boys (Marshall and Tanner, 1969; 1970) (see section 1.3.2). The reason for earlier pubarche in girls might be that girls are more susceptible to peripheral androgen action and develop clinical signs earlier than boys.
In normal development, the first appearance of pubic hair, i.e. pubarche, from the age of 8 years onwards is the direct result of the physiological rise in adrenal androgen production during adrenarche (Figure 12). DHEA is converted to active androgens in the gonads and peripheral target tissues of androgen action, including the skin, resulting in the development of pubic and also axillary hair. Women with acquired adrenal insufficiency without physiologic DHEA production suffer from a lack of axillary and pubic hair, which reappears after initiation of DHEA replacement therapy (Arlt et al., 1999). Physiologically, increasing androgen production during adrenarche manifests with distinct changes in body odour, oily skin and hair, followed by the first appearance of pubic and axillary hair. In addition, the rise in adrenal androgens can result in transient growth acceleration and contributes to bone maturation (Voutilainen et al., 1983; Remer et al., 2004).
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Generally, adrenarche appears to represent a developmental process independent of the maturation of the gonads (Sklar et al., 1980; Counts et al., 1987) Gonadarche, i.e. the onset of sex steroid production by the gonads, manifests with testicular enlargement and penile growth in boys and breast development and menarche in girls. Children with precocious puberty have no corresponding advance in the timing of adrenarche; their basal and ACTH-stimulated adrenal androgens are lower than in children matched for pubertal stage and only slightly higher than in age- matched children (Wierman et al., 1986). Conversely, children with isolated hypogonadotropic hypogonadism and subsequent lack of spontaneous puberty were found to have no corresponding delay in adrenarche (Counts et al., 1987; Sklar et al., 1980).
1.3. A
NDROGENS AND HUMAN DISEASE1.3.1. Monogenic causes of impaired androgen synthesis and metabolism