Differential gene expression in Tmem203 null testes
Expression profiling studies were carried out to identify potential molecular consequences of loss of Tmem203. We compared the expression profiles as described in the methods for testes and skeletal muscle, the highest and lowest sites of Tmem203 expression levels as seen in the tested tissues (S6 Fig). The expression profile comparisons between Tmem203 wild type and null mice showed significant changes in testes (Fig 6A) and little or no significant alterations in skeletal muscle (data not shown). 163 and 129 known genes were found to be increased or decreased by > 1.5 fold (S2 and S3 Tables). Differentially expressed genes were analyzed for pathway enrichment using Ingenuity Pathway Analysis (IPA) (Fig 6B). The most significantly up-regulated pathways were those involved in fibrosis and inflammation. It is unclear if these changes were due directly to Tmem203 deletion or secondary responses due to altered development of the testes. Interestingly, the most significantly down-regulated pathway was that composed of proteins annotated to regulate intracellular calcium levels. This set of genes included increased expression of inositol 1, 4, 5-triphosphate receptor 1 (Ip3r1) and ATPase, Ca++ transporting, plasma membrane 1 (Atp2b1 / Pmca1) RNAs and reduction ofTrpv6, Trpm5 and Trpm8 calcium channel RNAs in Tmem203 deficient testes. The differential expression of these genes was further validated by real-time quantitative RT PCR (Fig 6C). The differential expression of channels and transporters suggest that Tmem203 deficient spermatogenic cells would have altered calcium handling capacity. It should also be noted that the IPA also suggested a significant up-regulation in Germ Cell-Sertoli Cell Junction signaling genes {Ras Homolog Family Member Q (Rhoq), P21 Protein Activated Kinase 6 & 3 (Pak6, Pak3), Rho family GTPase 3 (Rnd3), integrin, alpha 6 (Itga6), catenin (cadherin-associated protein), alpha 1 (Ctnna1), junction plakoglobin (Jup)} The up-regulation of this gene-set was not pursued further as there was no obvious relevant morphological defects seen in ultra-structural studies of testes from Tmem203 null mice.
10.1371/journal.pone.0127480.g006 Fig 6  Gene expression profiling in Tmem203 null mouse testes indicates aberrant expression of key calcium channels and pumps.
(A) A spotfire based visualization of differential gene expression displaying fold changes in gene expression versus FDR corrected P value obtained from a microarray based RNA expression analysis from a set of five Tmem203 null and wild type mice. (B) Ingenuity based pathways enrichment of differentially expressed genes in Tmem203 null mouse testes. For the analysis the pathways showing a significant change with a p value of >2 were considered. (C) Quantitative real time PCR analysis of RNA obtained from WT and Tmem203 null mice testes for genes differentially expressed in the calcium signaling pathway selected from (B). Expression level of mentioned genes in Tmem203 null mice testes relative to WT testes expression level after normalization to Gapdh. Data represents 4 replicates (+/- Std Dev) and validated in 2 or more RNA preparations from Tmem203 null and WT testes. *—Transcript not detected in Tmem203 null mice testes.