The external genital anlagen is in a bipotent stage around the seventh week of gestation. If no sex steroids are acting on the tissue, female external structures will arise with a clitoris, labia minora and majora. Under the influence of androgens, namely dihydrotestosterone con verted peripherally from testosterone through 5alpha- reductase type 2, the phallic structure arising from the genital tubercle will form into a penis with elongation of the urethra to the tip of the phallus. Likewise, the urogenital folds will fuse and form the scrotal folds, while the labia minora are equivalent to parts of the shaft of the phallus in the male. This differentiation will be finished by the end of the twelfth week of gestation and thereafter, high amounts of dihydrotestosterone are needed for the growth of the phallus and of the prostate. Interestingly, these differentiation processes can only take place during very defined and strict time intervals, which represent the previously described ‘male programming window’. Elaborate animal model experiments demonstrate that the lack of androgens will lead to a female phenotype regardless of karyotype while androgenization leads to a male phenotype regardless of karyotype.
Thus, human sex development depends to a large extent on the systematic and local synthesis and action of specific hormones, which act in a very distinct spatial and time- dependent manner. The precursor of sex steroids is cholesterol, which is metabolized through a variety of enzymes, which may in part be dependent on adequate co- enzymes and co- factors. The majority of steroidogenic enzymes are P450 (CYP) enzymes, which catalyse redox reactions relying on electron supply via specific electron transfer. Typically, these enzymes are localized in the mitochondria. This applies for instance to the first critical enzyme P450 side- chain cleavage (CYP11A1), 11beta- hydroxylase (CYP11B1), and aldosterone synthase (CYP11A1); in contrast 17alpha- hydroxylase (CPY17A1), 21- hydroxylase (CYP21A2), and aromatase (CYP19A1) are localized to the endoplasmic reticulum. The latter enzymes depend on P450 oxidoreductase (POR) for electron transfer.
The initial step of cholesterol conversion to pregnenolone is crucial and of special interest, because it not only relies on the P450 side- chain cleavage enzyme (P450scc), but in selected organs such as adrenal and testis it needs a specific transporter, the Steroidogenic Acute Regulatory (StAR) protein; StAR pro motes a rapid trans- membranous crossing of cholesterol into the mitochondrium. Two very rare conditions have been described due to mutations of either P450scc or StAR. The description of deleterious mutations in P450scc came as a surprise, because it had been thought that this would also hamper severely the placental steroid production and thus not be compatible with life. However, recently mutations have been described which only partially affect P450scc function and lead to minor phenotypes in the DSD spectrum. StAR defects have initially been described in infants with grossly enlarged adrenals, ‘congenital lipoid adrenal hyperplasia’, due to accumulation of cholesterol within the cytoplasm, but these findings have been challenged in part by other case descriptions.
CYP17A1 is an interesting protein with actually two enzymatic capacities, namely the 17alpha- hydroxylase activity and an additional 17,20 lyase activity. The first converts pregnenolone or progesterone to their 17alpha- hydroxylated products, the latter metabolizes these compounds into dehydroepiandrosterone (DHEA) or androstenedione, respectively. The differentiation of the two enzymatic capacities of CYP17A1 is important in the diagnosis of DSD, because of the presence or absence of alterations of the mineralo- and glucocorticoid pathways. Isolated 17,20 lyase deficiency is mostly due to defects in the cytochrome b5, which is an allosteric factor promoting the interaction with POR and CYP17A1 and thus enhancing the 17,20 lyase activity without influencing 17alpha- hydrolyse activity.
The 3beta- hydroxysteroid dehydrogenase type 2 (HSD3B2) enzyme is critical, because of its ability to convert pregnenolone and its 17alpha- hydroxylated form into progesterone and 17- OH progesterone, therefore playing an important role in the adrenal for mineralo- and glucocorticoid synthesis. In addition, HSD3B2 con verts DHEA directly into androstenedione and thus controls the complete Δ4 steroid synthesis pathway. Mutations affecting HSD3B2 are associated with rare forms of congenital adrenal hyperplasia, but due to its capacity to convert DHEA to androstenedione it should be considered in the differential diagnosis of DSD.
Mainly in 46,XY DSD, defects of the down- stream cascade of testosterone synthesis play an important role. The enzymes are crucial and necessary for male phenotypic development. 17beta- hydroxysteroid dehydrogenase (HSD) type 3 (HSD17B3) synthesizes testosterone from androstenedione. This enzyme is mostly expressed in the testes and primarily acts during fetal development.
However, currently, a number of isoenzymes are known, for instance 17beta HSD type 5 (AKR1C3, aldo- keto reductase family 1 member C3) which may be involved in substantial testosterone syn thesis in the testes postnatally, especially at the time of puberty. This may support virilization in patients with 17beta HSD type 3 deficiency during pubertal development despite underlying deleterious mutations. Conversion of testosterone to the more po tent dihydrotestosterone (DHT) is mainly not localized in the testes, but directly in the androgen target tissues. Here 5- alpha reductase type 2 (SRD5A2) is the main enzyme, in particular during prenatal development. Lack of DHT due to mutation of SRD5A2 will lead to variable phenotypes of under- androgenization from almost completely female external genitalia to only slightly diminished penile size. These patients have a high virilization potential at the time of puberty, mainly due to the effects of testosterone; however, some of DHT may also be synthesized via the 5- alpha reductase type 1 (SRD5A1) pathway in these patients. Genetic alterations of HSD17B3 and SRD5A2 play a role only in 46,XY male development; in females with homozygous or compound heterozygous mutations, no abnormality in sex development is seen and these women are presumably fertile.
It has recently been demonstrated that 5- alpha reduction of testosterone is not the only pathway of DHT synthesis. Via the so- called backdoor pathway DHT may be produced from androstanediol, a compound that is converted through several steps directly from 17- alpha- hydroxy progesterone and requires CYP17A1 and 17- beta HSD type 3 and type 5, with the latter also known as 3alpha hydroxysteroid dehydrogenase type 2. This alternative mode for DHT synthesis could explain some of the virilization in patients with congenital adrenal hyperplasia due to 21 hydroxylase deficiency, because of the very high substrate levels of 17- OH progesterone (Figure 1).
Fig1. The ‘backdoor’ pathway of dihydrotestosterone synthesis. Reproduced with permission from Steroid Biochemisty by Kamrath, Wudy, Krone, in Hiort, Ahmed (eds.): Understanding Differences and Disorders of Sex Development (DSD), Karger (Series: Endocrine Development) ISSN 1421- 7082; vol. 27. Copyright © 2014 S. Karger AG, Basel.
In humans and other mammals, androgens act via a single androgen receptor (AR) in a very specific manner. Both sexes express the AR, therefore the appropriate levels of androgens promote male sex development of an individual. The AR is a single- copy gene localized on the X- chromosome at Xq11– 12. Due to the hemizygous state of the gene in 46,XY individuals, mutations in the AR gene directly affect male sex development. The AR acts as a typical nuclear receptor via transcriptional regulation of defined target genes. The genetic and functional structure of the AR is of interest as numerous studies have been performed to explain a genotype- phenotype correlation in androgen insensitivity syndrome (OMIM #300068), but also in conditions relating to other functions of the AR, such as spinobulbar muscular atrophy (SBMA, OMIM #313200) or the role of the AR in prostate cancer. The gene is com posed of 8 exons, where the first large exon encodes for the variable N- terminal domain, exons 2 and 3 for the DNA- binding domain, and exons 5– 8 for the ligand- binding domain. The N- terminal do main contains two variable repeat regions, a CAG and a GGN re peat, which have been associated with aspects of androgen action. Elongation of the CAG- repeat has been associated with sub- or in fertility, and very expanded CAG repeats may lead to spinal and bulbar muscular atrophy.
The AR initially resides in the cytoplasm and is bound to a complex of heat- shock proteins, chaperones, and co- chaperones. Ligand- binding induces a conformational switch, involving N- and C- terminal interaction, which eventually unmasks a nuclear localization signal. The ligand- bound AR translocates rapidly into the nucleus, which again may involve a number of heat- shock proteins and immunophilins. Within the nucleus, the AR forms a homodimer and binds to androgen- response elements within the target DNA. Some of these elements may also be recognized by other steroid receptors, but the AR can bind to selective androgen- response elements, also involving specific co- regulator proteins, which enhance or supress AR- dependent transcription of target genes. Many co factors possess enzymatic activities that lead to post- translational modifications including phosphorylation, acetylation, methylation, ubiquitination, sumoylation, and ADP- ribosylation. More than 300 different AR- interacting proteins have been described, but their role in DSD conditions remains unclear. Analysis of AR- dependent transcription of target genes demonstrates that cell- specific classes of genes may be up- or downregulated.
Interestingly, different androgens can elicit different effects via the single AR, as has been shown in elegant cellular model studies. This is also seen in normal sex development with the different actions of testosterone and DHT or even androstenedione and is exemplified by DSD conditions due to mutations in the aforementioned enzymatic pathways.
This may be due to the different binding capacities of the various androgens, but also due to cellular metabolism within the target cells, as demonstrated by DHT synthesis in cells harbouring the 5- alpha reductase enzyme. Furthermore, AR action is, in common with other nuclear receptors, dependent on cell- or tissue- specific coregulators, which may also be expressed in a time- dependent manner [91]. Hence, androgen- related genital and sex development will arise from very specific gene expression patterns regulated through the AR [92, 93]. This effect is irreversible, and genetic signa tures will be present throughout life representing the sexual phenotype of an individual. Each individual may have a specific ‘androgen response index’, which may reflect the variability in all humans, but may be specifically altered in people with DSD conditions.
