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Keywords:

  • backdoor pathway;
  • testosterone;
  • dihydrotestosterone;
  • disorders of sex development;
  • P450 oxidoreductase deficiency;
  • 21-OH deficiency

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

We review the current knowledge about the “backdoor” pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional “frontdoor” pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative “backdoor” pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 oxidoreductase deficiency and 21-hydroxylase deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17-hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions. Developmental Dynamics 242:320–329, 2013. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

Sex development is carried out by spatially, temporally, and hierarchically regulated genetic programs that define the sex phenotype of an individual, together with environmental factors that influence the sex phenotype (for detailed review, see Wilhelm and Koopman,2006; Achermann and Hughes,2011; Conte and Grumbach,2011) (Fig. 1). In the human, genetic sex is determined as 46,XY in males and 46,XX in females at the time of conception. Because SRY (sex determining region Y) on the Y chromosome has a dominant effect on the gonadal sex determination, the indifferent bipotential gonad develops as a fetal testis in the presence of SRY and as a fetal ovary in the absence of SRY. The fetal testis is associated with hormone producing somatic cells (Leydig cells and Sertoli cells) but is lacking meiotic cells, whereas the fetal ovary is associated with meiotic oocytes but is devoid of hormone producing somatic cells. Subsequent sex differentiation during the fetal life depends on the presence and the absence of fetal testis-derived hormones, i.e., testosterone, insulin-like factor 3 (INSL3), and anti-Müllerian hormone (AMH). Indeed, the testis descends into scrotum primarily because of the regression of the cranial suspensory ligament by testosterone and the development of the gubernaculum by INSL3, whereas the ovary stays within the abdomen due to the absence of such hormones. Of the sexual ducts that once appear in both males and females, the Wolffian duct develops into the epidydimis, the vas deferens, and the seminal vesicle in males due to testosterone effects and regresses in females lacking testosterone, whereas the Müllerian duct regresses in males because of AMH effects and develop into the uterus, the fallopian tube, and the upper third portion of the uterus in females lacking AMH. The external genitalia, and probably the brain as well, are masculinized when androgens such as testosterone and dihydrotestosterone (DHT) are present and are feminized when such androgens are absent. Sex differentiation further progresses with gametogenesis during puberty.

image

Figure 1. Schematic representation of sex development during the fetal life. SRY, sex determining region Y; AMH, anti-Müllerian hormone; T, testosterone; INSL3, insulin-like factor 3, AMHR, AMH receptor; INSL3R, INSL3 receptor; DHT, dihydrotestosterone; and AR, androgen receptor. Although T can also bind to AR, the affinity to AR is much higher for DHT than for T.

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Thus, androgens play an essential role in male sex development during the fetal life. In particular, because DHT is the most potent endogenous androgen, it is indispensable for masculinization of the external genitalia during fetal life as well as male sexual maturation during puberty (Matsumoto and Bremner,2011). Indeed, although both testosterone and DHT can bind to the androgen receptor, DHT has a much higher affinity to androgen receptor than testosterone (Matsumoto and Bremner,2011). In this regard, it is known that testosterone is produced in the fetal testis by the stimulation of placental chorionic gonadotropin around the critical period for sex development and by that of pituitary gonadotropins in the second to third trimesters as well as in the pubertal period (Grumbach and Gluckman,1994; Wilhelm and Koopman,2006; Dattani et al.,2011; Styne and Grumbach,2011). It is also known that DHT is converted from testosterone in the target tissue such as the external genital tissue (Wilhelm and Koopman,2006; Achermann and Hughes,2011), and this metabolic process from cholesterol to DHT by means of testosterone is called the conventional “frontdoor” pathway. Furthermore, recent studies have provided compelling evidence for the presence of an alternative “backdoor” pathway for the DHT biosynthesis that bypasses testosterone production. Here, we review the current knowledge about the “backdoor” pathway (for extensive review on steroidogenesis in general, see Miller and Auchus,2011). For convenience, terminology for steroid metabolites and enzymes described in this study is summarized in Table 1.

Table 1. Terminology for steroid metabolites and enzymes
Steroid Metabolites
Chemical nameTrivial nameShort name
5-pregnen-3β-ol-20-onepregnenolone 
4-pregnen-3,20-dioneprogesterone 
5-pregnen-3β,17α-diol-20-one17-hydroxypregnenolone17-OH pregnenolone
4-pregnen-17α-ol-3,20-dione17-hydroxyprogesterone17-OH progesterone
5-androsten-3β-ol-17-onedehydroepiandrosteroneDHEA
4-androsten-3,17-dioneandrostenedione 
4-androsten-17β-ol-3-onetestosterone 
5α-androstan-17β-ol-3-onedihydrotestosteroneDHT
5α-pregnane-3,20-dionedihydroprogesteroneDHP
5α-pregnane-3α-ol-20-oneallopregnanolone 
5α-pregnane-17α-ol-3,20-dione17-hydroxydihydroprogesterone17-OH DHP (pdione)
5α-pregnane-3α,17α-diol-20-one17-hydroxyallopregnanolone17-OH allopregnanolone (pdiol)
3α-hydroxy-5α-androstan-17-oneandrosterone 
5α-pregnane-3,20-dione  
5α-pregnane-3α-ol-20-one  
5α-androstane-3,17-dioneandrostanedione 
5α-androstane-3α,17β-diolandrostanediol 
Enzymes
Protein/geneEnzymeShort name
AKR1C23α-hydroxysteroid dehydrogenase type 33α-HSD-3
AKR1C43α-hydroxysteroid dehydrogenase type 13α-HSD-1
AKR1C3/HSD17B53α-hydroxysteroid dehydrogenase type 2/17β-hydroxysteroid dehydrogenase type 53α-HSD-2/17β-HSD-5
RODH/HSD17B6Retinol dehydrogenase/17β-hydroxysteroid dehydrogenase type 6RODH/17β-HSD-6
HSD17B317β-hydroxysteroid dehydrogenase type 317β-HSD-3
SRD5A15α-reductase type 15α-reductase-1
SRD5A25α-reductase type 25α-reductase-2
CYP11A1cholesterol side chain cleavage 
CYP17A117α-hydroxylase, 17/20 lyase 
CYP19A1aromatase 
CYP21A221-hydroxylase21-OH
PORcytochrome P450 oxidoreductase 

FRONTDOOR PATHWAY TO DHT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

We first summarize the conventional frontdoor pathway from cholesterol to DHT (Fig. 2, upper part). This pathway is comprised of two major steps. The first step is the testosterone biosynthesis that is primarily performed in the testis, either through the Δ5 route via pregnenolone, 17-hydroxy (17-OH) pregnenolone, and dehydroepiandrosterone (DHEA), or through the Δ4 route via progesterone, 17-OH progesterone, and androstenedione (Flück et al.,2003). This process for testosterone production is mediated by sequential actions of several P450 and non-P450 enzymes, i.e., StAR, CYP11A1, CYP17A1, HSD3B2, and HSD17B3 (Flück et al,2003; Miller,2005; Matsumoto and Bremner,2011). Of these, the mitochondrial enzyme CYP11A1 is supported by ferredoxin (alias, adrenodoxin) and ferredoxin reductase (alias, adrenodoxin reductase), and the microsomal enzyme CYP17A1 by cytochrome P450 oxidoreductase (POR) (Miller,2005; Miller and Auchus,2011). The Δ4 route is primarily used in the mouse, whereas the Δ5 route is preferentially used in the human especially in the fetal life (Flück et al.,2003). Indeed, of 17α-hydroxylase and 17/20 lyase encoded by human CYP17A1, 17α-hydroxylase catalyzes both pregnenolone and progesterone with a similar efficiency, whereas 17/20 lyase has much lower activity for 17-OH progesterone (∼2%) than for 17-OH pregnenolone (Lee-Robichaud et al.,1995; Auchus et al.,1998; Miller and Auchus,2011). Furthermore, the 17/20 lyase activity for both 17-OH pregnenolone and 17-OH progesterone requires cytochrome b5 that mediates the electron transfer in concert with POR (Lee-Robichaud et al,1995; Auchus et al,1998, Miller and Auchus,2011). The second step is the conversion of testosterone into DHT. This step is primarily performed in androgen-target tissues such as the genital skin and the prostate by 5α-reductase-2 (SRD5A2) (Matsumoto and Bremner,2011), while a small amount of DHT is also synthesized in the testis (Hammond et al.,1977). The above metabolic process to DHT via testosterone is called the conventional “frontdoor” pathway.

image

Figure 2. The frontdoor pathway (Δ5 and Δ4 routes via testosterone) and the backdoor pathway (5α and 5α,3α routes not via testosterone) in a physiological condition. In the frontdoor pathway, DHT is primarily converted in the target tissues from testosterone produced in the fetal testis, whereas in the backdoor pathway, DHT it is primarily converted in the target tissues from androstanediol produced in the fetal testis. The 17/20 lyase activity of CYP17A1 is low for 17-OH progesterone (shown with a dashed arrow) and high for 17-OH allopregnanolone (a double lined arrow). Several reactions are reversible, and the reactions opposite to the DHT productions are indicated with short arrows (the enzymes for the opposite reactions are not shown). DHEA, dehydroepiandrosterone; DHP, dihydroprogesterone; DHT, dihydrotestosterone; Fdx-FdR, ferredoxin and ferredoxin reductase; POR, cytochrome P450 oxidoreductase; and Cyt b5, cytochrome b5.

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BACKDOOR PATHWAY TO DHT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

The presence of the backdoor pathway was first demonstrated in the tammar wallaby pouch young testis (Shaw et al.,2000; Wilson et al.,2003). Tammar wallaby belonging to marsupials offers an advantage for studying male phenotypic development, because male sex development takes place over a relatively long period after birth when the young tammar wallaby is easily accessible in the pouch (Butler et al.,1999). For example, penile development commences around day 100. Masculinization process of the urogenital tract in the tammar wallaby is primarily dependent on the action of DHT that is converted in the target tissue from androstanediol rather than testosterone of testis origin (Shaw et al.,2000; Leihy et al.,2001; Wilson et al.,2003), although scrotal development is independent of the androgen effects (Renfree et al.,1995). Thus, while the circulating levels of testosterone (and DHT) were found to show no gross sex dimorphism at the time of prostatic and phallic development (Wilson et al.,1999), this is not inconsistent with androgen-dependent masculinization in the tammar wallaby. Furthermore, it was suggested that androstanediol as the major source for the DHT production in the target tissue could be formed via testosterone in the tammar wallaby pouch young testis (Wilson et al.,2003) and via 5α-reduced 17-OH progesterone in the immature rat testis (Echstein, 1985; Echstein et al.,1987). Thus, detailed steroidogenic studies have been performed for the tammar wallaby pouch young testis, confirming two different pathways for the production of DHT: the frontdoor pathway mediated by testosterone and the alternative pathway mediated by androstanediol produced from 17-OH progesterone, 17-OH dihydroprogesterone (17-OH DHP, or 5α-pregnane-17α-ol-3,20-dione), 17-OH allopregnanolone (5α-pregnane-3α,17α-diol-20-one, also frequently described as pdiol), and androsterone (Fig. 2, lower part) (Wilson et al.,2003). Furthermore, similar studies in the immature mouse testis have also indicated two different pathways to DHT: the frontdoor pathway mediated by testosterone and the alternative pathway mediated by androstanediol produced from progesterone via dihydroprogesterone (DHP, 5α-pregnane-3,20-dione), allopregnanolone (5α-pregnane-3α-ol-20-one), 17-OH allopregnanolone (= pdiol), and androsterone (Fig. 2, lower part) (Mahendroo et al.,2004). These findings have confirmed the presence of a metabolic route from cholesterol to DHT that does not involve testosterone, and this route has been named “backdoor pathway”.

ENZYMES INVOLVED IN THE BACKDOOR PATHWAY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

The backdoor pathway demands several enzymes (Fig. 2). First, the 5α-reductase activity is the core requirement for the operation of the backdoor pathway (reviewed in Auchus,2004; Ghayee and Auchus,2008; Miller and Auchus,2011). In this regard, both progesterone and 17-OH progesterone are excellent substrates for 5α-reductases, especially 5α-reductase-1 (Frederiksen and Wilson,1971). SRD5A1 for 5α-reductase-1 is well expressed in the human fetal testis and less clearly in the adult testis (Flück et al.,2011), and probably in the human fetal adrenal and the permanent adrenal at least in pathologic conditions (see below) (Fukami et al.,2009; Miller and Auchus,2011; Kamrath et al.,2012). Second, CYP17A1 involved in the frontdoor pathway is also required by the backdoor pathway. In this regard, it is known that DHP and allopregnanolone are excellent substrates for the 17α-hydroxylase activity of human CYP17A1, and that 17-OH allopregnanolone (= pdiol) is the most efficient substrate for the 17/20 lyase activity of CYP17A1 (Gupta et al.,2003; Miller and Auchus,2011). Furthermore, although the 17/20 lyase activity of CYP17A1 requires cytochrome b5 in the frontdoor pathway (Auchus et al.,1998), it is minimally dependent on cytochrome b5 in the backdoor pathway (Gupta et al.,2003; Miller and Auchus,2011). More importantly, CYP17A1 is expressed in the steroidogenic tissues that express SRD5A1 (Miller and Auchus,2011). Lastly, other enzymes such as AKR1C2/4, HSD17B3/AKR1C3, and AKR1C2/HSD17B6 are also necessary for the backdoor pathway. Reductive AKR1Cs are widely expressed including steroidogenic tissues (Penning et al.,2000), and HSD17B3 and HSD17B6 are expressed in testis (Flück et al.,2011). Taken together, the backdoor pathway is predicted to take place in steroidogenic tissues that express these enzymes in physiological and pathological conditions.

FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

Relative predominance between the frontdoor pathway and the backdoor pathway would be determined by several factors. The most important factor would be the relative function of CYP17A1 compared with SRD5A1, because this will determine whether 17-OH progesterone and progesterone are primarily used in the frontdoor pathway or in the backdoor pathway (Fig. 2) (Auchus,2004; Ghayee and Auchus,2008). The degree of the 17/20 lyase activity of CYP17A1 would also constitute an important factor. In this context, because the 17/20 lyase activity of CYP17A1 is relatively low in the tammar wallaby pouch young testis (Wilson et al.,2003; Auchus,2004), this would result in the accumulation of 17-OH progesterone in the frontdoor pathway and, consequently, activate the flux into the backdoor pathway. On the other hand, because the 17/20 lyase activity of CYP17A1 in the immature mouse testis is higher for 17-OH progesterone than for 17-OH allopregnanolone (= pdiol) (Auchus,2004), this would permit the flux of 17-OH progesterone into the frontdoor pathway. These characteristics of CYP17A1 in different species, together with the relative activity between SRD5A1 and CYP17A1, would explain why the backdoor pathway functions as a major route for DHT biosynthesis in the tammar wallaby pouch young testis and constitutes only a relatively minor source of DHT in the immature mouse testis (Auchus,2004). Indeed, because the 17α-hydroxylase activity of CYP17A1 remains low and 5α-reductase-1 is abundant in the immature mouse testis (Mahendroo et al.,2004), this may have driven the operation of the backdoor pathway beginning with progesterone (reviewed in Auchus,2004). Furthermore, gonadotropin secretary status may also constitute additional factor. Eckstein et al. (1987) have found that the minimal dose of human chorionic gonadotropin stimulates testosterone production via the frontdoor pathway and inhibits the biosynthesis of androstanediol via the backdoor pathway in the testicular microsomes of immature rats.

BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

The human backdoor pathway has recently been shown to be an indispensable source of androgens for male sex development. This is primarily based on the identification of mutations in genes involved in the backdoor pathway in two families with 46,XY disorders of sex development (DSD) accompanied by undermasculinized external genitalia due to compromised androgen biosynthesis (Flück et al.,2011). In family 1, DSD was inherited in a male-limited autosomal recessive manner, and linkage analysis indicated an association between disease phenotype and a genomic region harboring AKR1C1–4. Mutation analysis showed three missense mutations in AKR1C2 encoding 3α-HSD-3 (p.Asn300Thr, p.Ile79Val, and p.His90Gln), in combination with a splice mutation (r.85_252del) in AKR1C4 encoding 3α-HSD-1. In family 2, compound heterozygous mutations consisting of a missense mutation in AKR1C2 (p.His222Gln) and an AKR1C1-AKR1C2 fusion gene disrupting AKR1C1 and AKR1C2 were identified in a genetic male with DSD.

AKR1C2 functions as a ketosteroid reductase and a hydroxysteroid oxidase and mediates the critical steps in the backdoor pathway (Fig. 2) (Flück et al.,2011). Indeed, in vitro studies have indicated that AKR1C2 can efficiently convert androstanediol to DHT (Penning et al.,2000), and functional studies have demonstrated significantly impaired catalytic activity of the four AKR1C2 mutant proteins identified in the two families (p.Asn300Thr, p.Ile79Val, p.His90Gln, and p.His222Gln) (Flück et al.,2011). These data suggest that the enzymatic activity of AKR1C2 is critical for male sex development during the fetal period, and that hypomorphic mutations of AKR1C2 result in 46,XY DSD because of reduced androgen supply from the backdoor pathway. In addition, AKR1C4 and AKR1C3 would also play a certain role in male sex development, because AKR1C4 can also convert DHP to allopregnanolone and 17-OH DHP to 17-OH allopregnanolone (= pdiol), and AKR1C3 is involved in the conversion of androsterone to androstanediol (Fig. 2) (Penning et al.,2000; Penning,2010; Flück et al.,2011).

The backdoor pathway appears to be operating primarily in the fetal testis in a physiological condition. As motioned above, the human fetal testis contains sufficient SRD5A1 and variable degrees of the remaining enzymes required for the backdoor pathway such as AKR1Cs, CYP17A1, HSD17B3, and HSD17B6 (Flück et al.,2011), although the conversion of androstanediol to DHT is primarily carried out in peripheral tissues (Shaw et al.,2000). Thus, it is likely that fetal testis produces DHT precursors from both the frontdoor and the backdoor pathways, and that reduced androgen production in genetic males can be caused by (1) impaired activity of enzymes common to both pathways such as StAR, CYP11A1, HSD3B, CYP17A1, and HSD17B3, (2) defective activity of enzymes specific to the frontdoor pathway such as SRD5A2 (if 5α-reduction of 17-OH progesterone is sufficiently performed by SRD5A1); and (3) compromised activity of enzymes specific to the backdoor pathway such as AKR1C2 (Fig. 2). Furthermore, it is notable that impaired activity of the backdoor pathway does not affect estradiol production in affected females, whereas that of the frontdoor pathway compromises estradiol production in affected females because of reduced testosterone production. This explains why the mutation positive females in family 1 had normal phenotype with fertility.

BACKDOOR PATHWAY IN POR DEFICIENCY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

The operation of the backdoor pathway has been indicated by urine steroid profile analysis in POR deficiency (PORD). PORD is a rare autosomal recessive disorder caused by mutations in the POR gene encoding an electron donor to all microsomal enzymes including CYP17A1, CYP21A2, and CYP19A1 (reviewed in Miller,2004; Scott and Miller,2008). PORD is characterized by various clinical features, including skeletal malformations and adrenal steroidogenic dysfunction with increased 17-OH progesterone in both 46,XX and 46,XY patients, undermasculinization during fetal and pubertal periods in 46,XY patients, and virilization during fetal life and poor pubertal development without worsening of virilization in 46,XX patients (Fig. 3), together with maternal virilization during pregnancy (Miller,2004).

image

Figure 3. Steroidogenic pathways in the adrenals and in the placenta. Several reactions are reversible, and the reactions opposite to the DHT productions are indicated with short arrows (the enzymes for the opposite reactions are not shown). In a pathologic condition accompanied by an accumulation of 17-OH progesterone, the backdoor pathway is driven and produces DHT presumably because of the interaction between the fetal and the permanent adrenals. In contrast to the metabolic pathway in the fetal testis (Fig. 2), the conversions mediated by HSD17B3, and probably by HSD17B6 as well, are unlikely to occur in the permanent adrenals, because of lack of such enzymes. However, because AKR1C3 (HSD17B5) is present in the fetal adrenal, this would permit a certain because of conversion of androstenedione to testosterone primarily during the fetal life because of the interaction between the fetal and the permanent adrenals. The backdoor pathway beginning with progesterone is unlikely to be operating in such a pathologic condition with an accumulation of 17-OH progesterone rather than progesterone. While 5α-reduction appears to be primarily carried out by SRD5A1, the relevance of SRD5A2 remains tenable in the adrenal. For the placental steroidogenesis, the metabolic pathway from DHEA to estradiol or estrone mediated by POR is shown. DHEA is converted into DHEA-sulfate (DHEAS) by sulfotransferase in the fetus (and the mother), and transferred into the placenta where it is catalyzed into DHEA by sulfatase. Metabolic pathway from 16α-OH DHEAS of fetal origin to estriol is not shown. DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; POR, cytochrome P450 oxidoreductase; PORD, POR deficiency.

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Of these clinical findings, virilization of female patients was initially regarded as the consequence of defective placental CYP19A1 (aromatase) activity leading to the accumulation of testosterone and androstenedione that would be transferred into the fetal circulation as well as into maternal circulation (Fig. 3) (Arlt et al.,2004; Flück et al.,2004). However, it has become obvious that 46,XX DSD in PORD is inexplicable by compromised placental CYP19A1 activity alone. First, although only genetic female infants with nearly complete loss of an enzyme activity exhibit virilization in congenital CYP19A1 deficiency (Grumbach and Auchus,1999), genetic female patients with residual POR activities manifest virilization in PORD. Indeed, genotype-phenotype analyses in 35 Japanese patients have revealed that severe virilization is almost invariably present in female patients with two hypomorphic Japanese founder mutations (p.R457H) as well as in those with compound heterozygous mutations with p.R457H and an apparently null mutation (Fukami et al.,2009), although p.R457H yields ∼1% of supporting activities for CYP19A1 (Pandey et al.,2007) (Fig. 4). Furthermore, although the p.A287P mutation, which is frequent in Caucasian patients with PORD (Miller,2004; Scott and Miller,2008), provides grossly normal supporting activities for CYP19A1 (Pandey et al.,2007), virilization has been reported in some females with this mutation (Huang et al.,2005). Second, the supply of DHEA, the precursor of androstenedione and testosterone, from the fetus to the placenta would be reduced in PORD, because of compromised CYP17A1 activity (Fig. 3). Third, urine 16α-OH androsterone characteristic of CYP19A1 deficiency is not elevated in the mothers bearing PORD fetuses (Shackleton et al.,2004). These data suggest the presence of another mechanism for virilization in female patients with PORD. Because worsening of virilization has not been observed in genetic female patients with PORD after birth (Fukami et al.,2005), such a mechanism appears to take place primarily during the fetal life.

image

Figure 4. External genitalia in patients with PORD caused by homozygosity for the p.R457H mutation. Shown are severely virilized external genitalia in a 46,XX individual and mildly undermasculinized external genitalia with micropenis in a 46,XY individual. PORD, cytochrome P450 oxidoreductase deficiency.

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Thus, the relevance of the backdoor pathway has been postulated in PORD, because the backdoor pathway is driven by the accumulation of 17-OH progesterone. In this regard, urine steroid profile analysis in PORD has revealed characteristic findings shared by both genetic male and female patients (Fig. 5) (Fukami et al., 2006; Homma et al.,2006). They include persistently decreased DHEA metabolites consistent with compromised CYP17A1 activity and persistently increased 17-OH progesterone metabolites (pregnanetriolone and 17-OH allopregnanolone) compatible with defective CYP17A1 and CYP21A2 activities and resultant elevation of serum 17-OH progesterone. More importantly, while urine etiocholanolone derived from androstenedione in the frontdoor pathway remains grossly normal from infancy to adulthood probably because of co-existence of low DHEA and high 17-OH progesterone values, urine androsterone derived from both androstenedione in the frontdoor pathway and 17-OH allopregnanolone (= pdiol) in the backdoor pathway is increased during early infancy and remains grossly normal thereafter (Fig. 6). These findings suggest a transient supply of androsterone from the backdoor pathway in the early infancy. Furthermore, because 17-OH allopregnanolone (= pdiol) has a much higher affinity for human CYP17A1 than 17-OH progesterone (Gupta et al.,2003), it is likely that the backdoor pathway functions better than the frontdoor pathway in PORD with elevated 17-OH progesterone. Consistent with this, Shackleton et al. (2004) have performed sequential urinary steroid profile analyses in a pregnant woman with a PORD fetus, and found disproportionate elevations of 17-OH allopregnanolone (= pdiol) and androsterone. Thus, it is likely that DHT derived from the backdoor pathway would have contributed to virilization in genetic female patients with PORD, although the increased level of DHT itself has not been confirmed in such patients.

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Figure 5. Simplified schematic representation indicating urine steroid metabolites analyzed by gas chromatography/mass spectrometry (indicated with double line squares). The cross (×) symbols represent metabolic processes compromised in PORD. Although the activities of gonadal and placental POR-dependent enzymes are also compromised in PORD, urine steroid metabolite data primarily reflect the steroid metabolites of adrenal origin. POR, cytochrome P450 oxidoreductase; PORD, POR deficiency.

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Figure 6. Representative urine steroid profile data in PORD patients with an accumulation of 17-OH progesterone (see Fig. 5). The light blue circles and the pink circles show the data obtained from control subjects. The triangles indicate the data obtained from patients with homozygosity for the p.R457H mutation, and the circles represent those obtained from patients with compound heterozygosity for the p.R457H mutation and one apparently null mutation. Note that all the data are expressed using a logarithm scale. PORD, cytochrome P450 oxidoreductase deficiency.

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A major problem is the organ(s)/tissue(s) for the backdoor pathway in PORD. Although the organ(s)/tissue(s) remains to be clarified, one possible candidate would be the fetal and/or the permanent adrenal (Fig. 3). Indeed, adrenals appear to contain 5α-reductases (Miller and Auchus,2011), and the 17-OH progesterone is excellent substrates for 5α-reductase but not for 17/20 lyase (Auchus et al.,1998). Thus, the accumulated 17-OH progesterone would preferentially converted into 17-OH allopregnanolone (= pdiol) that is the most efficient substrate for 17/20 lyase (Miller and Auchus,2011). Furthermore, because the fetal adrenal is common to both males and females and disappears shortly after birth (Grumbach and Gluckman,1994; Dattani et al.,2011), this is consistent with the transient activation of the backdoor pathway in PORD patients of both sexes. One may argue that the fetal adrenal alone is incapable of producing a sufficient amount of 17-OH progesterone to drive the backdoor pathway, because HSD3B2 activity is low in the fetal adrenal (Dattani et al.,2011). Furthermore, Flück et al. (2011) have shown that AKR1C2 expression remains low in the fetal adrenal. However, the fetal adrenal has abundant CYP17A1 (especially 17/20 lyase activity) and AKR1C4, and the fetal adrenal and/or the permanent adrenal possesses enzymes involved in the backdoor pathway including AKR1Cs (Fig. 3) (Voutilainen and Miller,1986; Flück et al.,2011). Taken together, the interaction between the fetal and the permanent adrenals may play a primary role in the operation of the backdoor pathway in PORD. In addition, other tissue(s)/organ(s) such as the liver may also be relevant to the backdoor pathway, because SRD5A1 and AKR1Cs are expressed in the liver (Penning et al.,2000; Penning,2010).

In contrast to severe DSD phenotype in 46,XX patients with PORD, the DSD phenotype remains relatively mild in 46,XY patients with PORD (Fig. 4) (Fukami et al.,2009). This would be explained by three factors. First, because of the compromised CYP17A1 activity, both testosterone production from the frontdoor pathway and androstanediol production from the backdoor pathway would be compromised in the fetal testis (Fig. 2). This would result in reduced DHT production in the target tissue. Second, as in the 46,XX female patients with PORD, DHT would be produced from the backdoor pathway during fetal to early infancy because of the interaction between the fetal and the permanent adrenals (Fig. 3). Third, defective CYP19A1 activity would more or less accumulate testosterone and androstenedione in the placenta (Grumbach and Auchus,1999) that would be transferred into the fetal circulation as well as into the maternal circulation. Thus, the effects of compromised androgen production in the fetal testis would be considerably compensated for by those of the extra androgen production in the fetal and permanent adrenals and in the placenta.

BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

The backdoor pathway has also been implicated in virilization of genetic female patients with 21-OHD associated with high 17-OH progesterone that would drive the backdoor pathway in the fetal and the permanent adrenals (Fig. 3). This condition is an autosomal recessive disorder caused by loss-of-function mutations in CYP21A2, and represents the most common form of congenital adrenal hyperplasia (Conte and Grumbach,2011; Stewart and Krone,2011).

Kamrath et al. (2012) have examined urine steroid metabolites in untreated patients with 21-OHD, and found significantly increased 17-OH allopregnanolone (= pdiol) levels and the androsterone to etiocholanolone ratios. Notably, while the elevation of 17-OH allopregnanolone (= pdiol) persisted after 1 year of age, the androsterone to etiocholanolone ratio was highest in the neonatal period and became less remarkable after 1 year of age, as observed in PORD. These results are consistent with the backdoor pathway being activated in patients with 21-OHD, primarily during the infancy. Thus, the interaction between the fetal and the permanent adrenals may play a primary role in the operation of the backdoor pathway in 21-OHD as well as in PORD. However, as in PORD, it remains to be determined whether DHT is actually produced from the backdoor pathway in 21-OHD.

In 21-OHD, androstenedione derived from the frontdoor pathway is also markedly increased (Lin-Su et al.,2008). In this context, because AKR1C3 (HSD17B5) is present in the fetal adrenal (Goto et al.,2006), this may permit a certain degree of conversion of androstenedione to testosterone primarily during the fetal life because of the interaction between the permanent adrenal (the conversion of DHEA to androstenedione) and the fetal adrenal (the conversion of androstenedione to testosterone). Furthermore, a novel biosynthetic pathway from androstenedione to DHT has been postulated in the prostate of gonadectomized males; in such males, DHT levels in the prostate are reduced only by ∼50% whereas blood testosterone levels are decreased by 90–95% (reviewed in Luu-The et al.,2008). Thus, it has been proposed that DHT can be produced from androstenedione either through 5α-androstanedione (i.e., androstenedione [RIGHTWARDS ARROW] 5α-androstanedione [RIGHTWARDS ARROW] DHT) or through 5α-androstanedione, androsterone, and androstanediol (i.e., androstenedione [RIGHTWARDS ARROW] 5α-androstanedione [RIGHTWARDS ARROW] androsterone [RIGHTWARDS ARROW] androstanediol [RIGHTWARDS ARROW] DHT) in the human prostate (Luu-The et al.,2008). Indeed, SRD5A1/2 and AKR1C2 presumably involved in this pathway are clearly expressed in the human prostate (Luu-The et al.,2008). By analogy, DHT may also be produced in 21-OHD from androstenedione through such an aberrant pathway. In addition, while HSD17B3 essential for the conversion of androstenedione to testosterone in the frontdoor pathway is almost exclusively expressed in the testis (Geissler et al.,1994), variably elevated blood testosterone values are usually observed in patients with 21-OHD (Stewart and Krone,2011). Thus, a certain degree of testosterone may be produced via a hitherto unknown route in 21-OHD.

46,XX DSD IN PORD AND 21-OHD

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

Both PORD and 21-OHD are associated with variable but definitive virilization in genetic female patients. In this regard, blood 17-OH progesterone levels are usually higher in 21-OHD than in PORD (Fukami et al., 2006,2009; Conte and Grumbach,2011; Stewart and Krone,2011), and the 17/20 lyase activity of CYP17A1, though it is physiologically low for 17-OH progesterone, is normally preserved in 21-OHD and compromised in PORD (Miller,2004; Stewart and Krone,2011). Thus, both the frontdoor and the backdoor pathways would be more activated in 21-OHD than in PORD. By contrast, PORD, but not 21-OHD, is accompanied by testosterone overproduction in the placenta (and possibly in other tissues such as the liver) (Scott and Miller, 2008). Because the placental testosterone transferred to maternal circulation often causes maternal virilization during pregnancy (Achermann and Hughes,2011), such testosterone transferred to fetal circulation would also contribute to virilization of female fetuses. These findings would explain why both PORD and 21-OHD are associated with obvious virilization.

PERSPECTIVES AND CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES

Recent studies indicate that the backdoor pathway to DHT that bypasses testosterone production is operating in the human as well as in other mammals. The backdoor pathway appears to be involved in normal masculinization in a physiological condition and in abnormal virilization in pathological conditions.

However, several matters remain to be clarified in the human backdoor pathway. First, although the data currently available argue for the backdoor pathway starting with 5α-reduction of 17-OH progesterone, it is unknown whether the backdoor pathway beginning with 5α-reduction of progesterone is present (Fig. 2). Second, although a small amount of DHT would be produced from the backdoor pathway in normal female subjects because of an interaction between the fetal and the permanent adrenals, the physiological significance of DHT derived from the extra-gonadal backdoor pathway remains to be elucidated. Third, although the backdoor pathway may also be involved in the virilization of other pathologic conditions such as polycystic ovary syndrome, no studies have been performed in such conditions. Further studies will permit a better clarification of the biological role of the human backdoor pathway.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FRONTDOOR PATHWAY TO DHT
  5. BACKDOOR PATHWAY TO DHT
  6. ENZYMES INVOLVED IN THE BACKDOOR PATHWAY
  7. FRONTDOOR PATHWAY VERSUS BACKDOOR PATHWAY
  8. BACKDOOR PATHWAY IN HUMAN MALE SEX DEVELOPMENT
  9. BACKDOOR PATHWAY IN POR DEFICIENCY
  10. BACKDOOR PATHWAY IN 21-HYDROXYLASE DEFICIENCY (21-OHD)
  11. 46,XX DSD IN PORD AND 21-OHD
  12. PERSPECTIVES AND CONCLUSIONS
  13. REFERENCES
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