FGF are essential signaling molecules for embryogenesis [10-14]. FGF8 and FGF10 are closely linked to embryonic development, especially to formation of limbs and of the genital tubercle at an early, androgen independent stage in embryonic development via the regulation of SHH [14]. FGF signaling has been shown to work directionally and reciprocally across epithelial-mesenchymal borders, prompting the induction of overlying epithelial differentiation by mesenchymal FGF expression [15]. Although FGF10, via its receptors FGFR1/2b, is linked to a lot of signaling pathways, many of its known implications in human pathology are connected to differences in expression of the molecule partly regulated by circular RNAs [16].
In a recent mouse study, expression of FGF8 was seen outside the urogenital sinus epithelium in early embryogenesis, and expression gradually came to be confined to the distal urethral epithelium [6,7]. FGF8 expression gradually weakens and begins to be replaced by FGF10 as the reproductive nodule mesenchyme differentiates. Initial extension of the genital tubercle progresses by a series of processes. It is thought that prepuce formation will also progress as the external genitalia extend. Therefore, suppression of FGF8 or FGF10 in the distal urethral epithelium, which is important for the initial elongation of the genitalia, could conceivably cause hypospadias, with severity being correlated with the degree of suppression. This is exactly what we identified in this study; i.e., decreased expression of FGF10 with increasing severity of hypospadias. Of note is the seemingly stable expression of FGF8 identified in all groups. This hints that FGF8, which is expressed in early embryogenesis, may not be a causal factor for hypospadias per se; rather, it is a precursor for FGF10 and it is FGF10 that may cause hypospadias by affecting migration of the urethral meatus or elongation of the external genitalia or both.
Studies using various animal models of hypospadias have also shown the importance of malformations of the genital tubercle. Unlike FGF8, which plays an early role in genital tubercle formation, FGF10 and its receptor (FGFR2 IIIb) appear to be involved in a hormone dependent phase [9]. In FGF10 deficient mutant male mice, ventral fusion of the urethral plate fails, resulting in a deformity consistent with hypospadias [8]. However, FGF10 deficiency appears to be incompatible with life, because FGF10 deficient mutant male mice die immediately after birth. On autopsy there is no evidence of lung, salivary gland, or kidney development and absence of definitive limbs. Studies of the mouse homolog suggest that FGF10 is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation and that FGF10 signaling is required for epithelial branching. Therefore, all development reliant on morphogen induced branching such as occurs in the lungs, skin, ears, salivary glands, and urethra require constant expression of FGF10. Furthermore, there is a strain of mice with loss of FGF10 expression in the urethra and phenotypic hypospadias [9], suggesting that FGF10 may also be sensitive to androgen stimulation.
Separate to the activity of FGF10 is the process of urethral closure, already well known to be exquisitely responsive to the hormonal environment. It is well known that estrogenic and anti-androgenic compounds can induce hypospadias in humans and mice. In fact, the increasing incidence of hypospadias, particularly in developed countries, has led to the hypothesis that elevated exposure to estrogenic and anti-androgenic environmental factors may be etiologic [17]. This may include exposure to molecules that interfere with the synthesis, transport, and metabolism of endogenous hormones, such as xenoestrogens or endocrine disrupting compounds.
To the best of our knowledge, this is the first study to investigate the expression of FGF8 and FGF10 in human prepuce. From animal studies, suppression of FGF8 or FGF10, or both, or disruption of the transition from FGF8 to FGF10 have attracted attention as possible etiologic processes for the onset and severity of hypospadias. Development of the external genitalia is very complicated and while FGF10 is involved in fusion of the urethral folds and foreskin formation by controlling mesenchymal differentiation of the genital tubercle resulting in various phenotypes, the expression of FGF10 would appear to be important in humans although its role may not necessarily be similar to that in mice.
Cohn’s extensive report [18] on the development of the external genitalia mentions the role of sex steroids in maturing the FGF10 mediated differentiation of the genital tubercle and that FGF8 is not involved in the development of the external genitalia. Our results confirm their findings based on animal experiments by identifying stable similar FGF8 expression in phimosis and hypospadias prepuce tissue and suppressed FGF10 expression correlated with severity in human hypospadias. A similar study to our current report in females would be valuable to establish FGF10 as the definitive causal factor of external genitalia malformation.
In conclusion, the significant down-regulation in FGF10 observed in human prepuce specimens from hypospadias patients in this study provides strong evidence that altered expression of FGF10 during embryogenesis prevents normal urethral development that in males manifests as hypospadias and persists as a structural fault in hypospadias prepuce. Our study provides a valuable link between animal models of hypospadias and human hypospadias, enhancing the understanding of urethral formation and may have some possible application for developing new methods to detect or prevent hypospadias.