Mamld1 Knockdown in Mouse Leydig Tumor Cells Figure 1. Steroid metabolite concentrations. A. Steroid metabolic pathway from cholesterol to testosterone and enzymes involved in each conversion. Pregnenolone, 17-OH pregnenolone, and DHEA (dehydroepiandrosterone) are D4-steroid metabolites (D4-pathway), and progesterone, 17-OH progesterone, and androstenedione are D5-steroid metabolites (D5-pathway). Hsd3b1 also functions as D5 4 isomerase. B. Steroid metabolite concentrations in culture media and endogenous Mamld1 expression levels in MLTCs. The yellow, the green, and the blue bars indicate the data obtained from MLTCs transfected with non-targeting RNA, siRNA1, and siRNA2, respectively. {: P,0.01; and {: P,0.001. The conversion factor to the SI unit: cholesterol 0.026 (mmol/L), pregnenolone 3.16 (nmol/L), progesterone 3.18 (nmol/L), 17-OH pregnenolone 3.00 (pmol/L), 17-OH progesterone 3.03 (nmol/L), DHEA 3.46 (pmol/L), androstenedione 3.49 (nmol/L), and testosterone 3.46 (nmol/L). doi:10.1371/journal.pone.0019123.g001 this was not reproduced in siRNA1-transfected MLTCs. After 72 hours of incubation, the MLTCs became confluent, and the absorbance values became a plateau phase around ,2.0. In this Figure 2. Gene expression analysis. The yellow, the green, and the blue bars indicate the data obtained from MLTCs transfected with non- targeting RNA, siRNA1, and siRNA2, respectively. *: P,0.05; {: P,0.01; and {: P,0.001. A. Real-time RT-PCR analysis. B. Microarray analysis. doi:10.1371/journal.pone.0019123.g002 plateau phase, although the absorbance values at 96 hours after the transfection were significantly low in both siRNA1- and siRNA2-treated MLTCs, this was not reproduced at 120 hours after the transfection. Discussion Mamld1 knockdown with two siRNAs resulted in compromised T production, together with reduced 17a-hydroxylase activity and Cyp17a1 expression in MLTCs. This provides further support for a positive role of Mamld1 in T production [4], and implies for the first time a possible interaction between Mamld1 and Cyp17a1, at least in MLTCs. In this regard, it is noteworthy that Mamld1 is clearly expressed in fetal Leydig and Sertoli cells and is barely expressed in adrenal cells [1,13], and that Cyp17a1 expression is indispensable for T production in Leydig cells [14]. Thus, it appears likely that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells, permitting the production of a sufficient amount of T for male sex development. In addition, since the expressions of other genes involved in T production and insulin-like 3 biosynthesis were not clearly affected in siRNA-transfected MLTCs, this would argue against the possibility that Mamld1 knockdown causes a global dysfunction of MLTCs, resulting in T hyposecretion. PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e19123