Ogata1* Abstract Mamld1 Knockdown Reduces Testosterone Production and Cyp17a1 Expression in Mouse Leydig Tumor Cells Michiko Nakamura1,2, Maki Fukami1, Fumihiro Sugawa 1 , Mami Miyado 1 , Katsuya Nonomura2, Tsutomu 1 Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan, 2 Department of Renal and Genitourinary Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan Background: MAMLD1 is known to be a causative gene for hypospadias. Although previous studies have indicated that MAMLD1 mutations result in hypospadias primarily because of compromised testosterone production around the critical period for fetal sex development, the underlying mechanism(s) remains to be clarified. Furthermore, although functional studies have indicated a transactivation function of MAMLD1 for the non-canonical Notch target Hes3, its relevance to testosterone production remains unknown. To examine these matters, we performed Mamld1 knockdown experiments. Methodology/Principal Findings: Mamld1 knockdown was performed with two siRNAs, using mouse Leydig tumor cells (MLTCs). Mamld1 knockdown did not influence the concentrations of pregnenolone and progesterone but significantly reduced those of 17-OH pregnenolone, 17-OH progesterone, dehydroepiandrosterone, androstenedione, and testosterone in the culture media. Furthermore, Mamld1 knockdown significantly decreased Cyp17a1 expression, but did not affect expressions of other genes involved in testosterone biosynthesis as well as in insulin-like 3 production. Hes3 expression was not significantly altered. In addition, while 47 genes were significantly up-regulated (fold change .2.06) and 38 genes were significantly down-regulated (fold change ,0.56), none of them was known to be involved in testosterone production. Cell proliferation analysis revealed no evidence for compromised proliferation of siRNA-transfected MLTCs. Conclusions/Significance: The results, in conjunction with the previous data, imply that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells and permit to produce a sufficient amount of testosterone for male sex development, independently of the Hes3-related non-canonical Notch signaling. Citation: Nakamura M, Fukami M, Sugawa F, Miyado M, Nonomura K, et al. (2011) Mamld1 Knockdown Reduces Testosterone Production and Cyp17a1 Expression in Mouse Leydig Tumor Cells. PLoS ONE 6(4): e19123. doi:10.1371/journal.pone.0019123 Editor: Michael Polymenis, Texas A&M University, United States of America Received January 31, 2011; Accepted March 17, 2011; Published April 29, 2011 Copyright: 2011 Nakamura et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Grant for Research on Intractable Diseases (H22-98) from the Ministry of Health, Labor and Welfare (http://www.mhlw.go.jp/english/), and Grants-in-Aid for Scientific Research (B) (20390265) and for Young Scientists (B-22791715) from the Japan Society for the Promotion of Science (JSPS) (http://www.jsps.go.jp/ english/), and by Grant-in-Aid for Scientific Research on Innovative Areas (22132004 A01) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (http://www.mext.go.jp/english/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tomogata@nch.go.jp Introduction MAMLD1 (mastermind-like domain containing 1, alias CXorf6) on human chromosome Xq28 is a causative gene for hypospadias, a mild form of 46,XY disorders of sex development (DSD) [1]. To date, multiple mutations have been identified in patients with various types of hypospadias [1–3]. In this regard, the mouse homologous gene Mamld1 is transiently expressed in fetal Sertoli and Leydig cells around the critical period for sex development [1], and transient Mamld1 knockdown using small interfering RNAs (siRNAs) reduces testosterone (T) production in cultured mouse Leydig tumor cells (MLTCs) [4]. Furthermore, the upstream region of MAMLD1/ Mamld1 harbors a putative binding site ‘‘CCAAGGTCA’’ for NR5A1 (alias, SF-1 and AD4BP) [4] that regulates the transcription of a vast array of genes involved in sex development [5], and NR5A1 protein has been shown to bind to the putative target site and exert a transactivation function for Mamld1 [4]. These findings imply that MAMLD1/Mamld1 is involved in fetal T production under the regulation of NR5A1, and that MAMLD1 mutations result in hypospadias primarily because of compromised T production around the critical period for sex development. However, the underlying mechanism(s) by which impaired MAMLD1/Mamld1 leads to compromised T production remains to be clarified, although there are several possibilities such as defective activities of enzyme(s) involved in T production and compromised proliferation of Leydig cells. Furthermore, although previous functional studies have indicated that MAMLD1 has a transactivation function for the non-canonical Notch target Hes3 [4], its relevance to biological function including T production remains unknown. To examine these matters, we performed detailed analyses in Mamld1 knockdown experiments using MLTCs. Methods Knockdown experiments MLTCs (ATCC, CRL-2065TM) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, and were PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e19123