Figure 3. Cell proliferation assay. The yellow, the green, and the blue line graphs and bars indicate the data obtained from MLTCs transfected with non-targeting RNA, siRNA1, and siRNA2, respectively. *: P,0.05; and {: P,0.001. A. Endogenous Mamld1 expression levels. B. Absorbance values. doi:10.1371/journal.pone.0019123.g003 However, a straightforward explanation appears to be difficult between impaired 17a-hydroxylase activity and reduced Cyp17a1 expression. Indeed, 17/20 lyase activity was well preserved in siRNA-transfected MLTCs, although the same Cyp17a1 enzyme is utilized for both17a-hydroxylase and 17/20 lyase reactions [14]. In addition, defective 17a-hydroxylase activity occurred in the presence of ,70% of Cyp17a1 expression, despite 17a-hydroxylase deficiency being an autosomal recessive disease in which 50% of enzyme reduction has no major effect on the steroid metabolism [14]. In this context, it is notable that MLTCs originally have a markedly low 17a-hydroxylase activity and a well preserved 17/20 lyase activity for both D4- and D5-pathways (Figure 1) [6]. Such a unique property of MLTCs may be relevant to the preferential impairment of 17a-hydroxylase activity in siRNA-transfected MLTCs. Mamld1 knockdown had no discernible effect on the Hes3 expression. In addition, while a Notch-related gene Hey1 [15–17] References Mamld1 Knockdown in Mouse Leydig Tumor Cells was up-regulated in siRNA-transfected MLTCs, there are no data suggesting a possible interaction between Hes3 and Hey1 in the T production process. Furthermore, while Hes3 is weakly expressed in the MLTCs [4], Hes3 expression is apparently absent from mouse fetal gonads around the critical period for sex development [18]. Thus, it is unlikely that Hes3-related non-canonical Notch signaling underlies a link between Mamld1 and Cyp17a1. In addition, while microarray analysis revealed multiple up-regulated and down-regulated genes in siRNA-transfected MLTCs, none of them is known to be involved in the T production at present. It remains to be clarified, therefore, how Mamld1 enhances Cyp17a1 expression and T production. The cell proliferation analysis revealed no clear evidence for the reduced number of viable MLTCs transfected with siRNAs. This implies that the reduced T and several other steroid metabolite concentrations observed at 48 hours after the transfection (Figure 1) are inexplicable by impaired proliferation of MLTCs. However, since the cell doubling time of MLTCs is 35–40 hours [8], a slight difference in cell proliferation would not be detected by the present analysis. Thus, it might remain tenable at this time that impaired cell proliferation becomes discernible after multiple cell divisions, and that such a possibly reduced cell proliferation underlies the development of hypospadias phenotype in patients with MAMLD1 mutations, in addition to compromised T production in Leydig cells. In summary, the present study implies that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells and permit to produce a sufficient amount of T for male sex development, independently of the Hes3-related non-canonical Notch signaling. Although the data were obtained from in vitro studies using MLTCs, they provides a useful clue to clarify the underlying factors for the development of hypospadias and other forms of 46,XY DSD. Supporting Information Figure S1 Cell proliferation assay by the colorimetric method, using non-transfected MLTCs. The absorbance value is well correlated with cell number until the absorbance value of ,2.0, but does not reflect the cell number after the absorbance value of ,2.0. (TIF) Table S1 List of up-regulated genes in MLTCs trasnfected with siRNAs for Mamld1. (DOC) Table S2 List of down-regulated genes in MLTCs trasnfected with siRNAs for Mamld1. (DOC) Author Contributions Conceived and designed the experiments: MF KN TO. Performed the experiments: MN MF FS MM. Analyzed the data: MN MF. Contributed reagents/materials/analysis tools: MF TO. Wrote the paper: TO. 1. Fukami M, Wada Y, Miyabayashi K, Nishino I, Hasegawa T, et al. (2006) CXorf6 is a causative gene for hypospadias. Nat Genet 38: 1369–1371. 2. Kalfa N, Liu B, Klein O, Audran F, Wang MH, et al. (2008) Mutations of CXorf6 are associated with a range of severities of hypospadias. Eur J Endocrinol 159: 453–458. 3. Chen Y, Thai HT, Lundin J, Lagerstedt-Robinson K, Zhao S, et al. (2010) Mutational study of the MAMLD1-gene in hypospadias. Eur J Med Genet 53: 122–126. 4. Fukami M, Wada Y, Okada M, Kato F, Katsumata N, et al. (2008) Mastermind- like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence. J Biol Chem 283: 5525–5532. 5. Lin L, Achermann JC (2008) Steroidogenic factor-1 (SF-1, Ad4BP, NR5A1) and disorders of testis development. Sex Dev 2: 200–209. 6. Panesar NS, Chan KW, Ho CS (2003) Mouse Leydig tumor cells produce C-19 steroids, including testosterone. Steroids 68: 245–251. PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e19123