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Mamld1 knockdown reduces testosterone production and Cyp17a1 expression in mouse Leydig tumor cells.

ABSTRACT: 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.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 >2.0×) and 38 genes were significantly down-regulated (fold change <0.5×), none of them was known to be involved in testosterone production. Cell proliferation analysis revealed no evidence for compromised proliferation of siRNA-transfected MLTCs.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.

SUBMITTER: Nakamura M

PROVIDER: S-EPMC3084764 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Mamld1 knockdown reduces testosterone production and Cyp17a1 expression in mouse Leydig tumor cells.

Nakamura Michiko M Fukami Maki M Sugawa Fumihiro F Miyado Mami M Nonomura Katsuya K Ogata Tsutomu T

PloS one 20110429 4

<h4>Background</h4>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 r ...[more]

PMID: 21559465

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Project description: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. To date, multiple mutations have been identified in patients with various types of hypospadias. In this regard, the mouse homologous gene Mamld1 is transiently expressed in fetal Sertoli and Leydig cells around the critical period for sex development, and transient Mamld1 knockdown using small interfering RNAs (siRNAs) reduces testosterone production in cultured mouse Leydig tumor cells (MLTCs). We performed detailed analyses in Mamld1 knockdown experiments using MLTCs. We used microarrays to display the global gene expression profile change, after knockdown of Mamld1 expression in MLTCs, to find the gene set regulated by Mamld1. To compare changes in gene expression profile in MLTCs, MLTCs were transfected with control siRNA or Mamld1 siRNA, 48h later, total RNA was subjected to Q-PCR to confirm a significantly decreased Mamld1 mRNA, and then these total RNA samples were for microarray analysis. The microarray data was analyzed to explore gene sets/pathways, which were significantly impacted by knockdown of Mamld1. Real-time RT-PCR and microarray analyses showed significantly decreased Cyp17a1 expression (~70%) in both siRNA1- and siRNA2-transfected MLTCs. The siRNAs knockdown did not affect the expressions of Nr5a1 (Sf1), Star, Por, and Insl3. 47 genes including a Notch-related gene Hey1 were significantly up-regulated (fold change >2.0) and 38 genes were significantly down-regulated (fold change <0.5) in both siRNA1- and siRNA2-transfected MLTCs (Table S1 and Table S2). However, Mamld1 knockdown had no discernible effect on the Hes3 expression level (siRNA1: fold change 0.92, P=0.80; siRNA2: fold change 1.43, P=0.35). MLTCs were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, and were transiently transfected with two siRNAs against Mamld1 or with non-targeting control RNA. To compare changes in gene expression profile in MLTCs, 48h later, total RNA was subjected to Q-PCR to confirm a significantly decreased Mamld1 mRNA, and then these total RNA samples were for microarray analysis. Supplementary file: t-test unpaired [i1] Vs [control], [i2] Vs [control], P <= 0.05 FC >=2.0

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Mamld1 knockdown reduces testosterone production and Cyp17a1 expression in mouse Leydig tumor cells.

Publications

Mamld1 knockdown reduces testosterone production and Cyp17a1 expression in mouse Leydig tumor cells.

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