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Background  Tight regulation of testicular gene expression is a prerequisite for male reproductive success, while differentiation of gene activity in spermatogenesis is important during speciation. Thus, comparison of testicular transcriptomes between closely related species can reveal unique regulatory patterns and shed light on evolutionary constraints separating the species.    Methodology/Principal Findings  Here, we compared testicular transcriptomes of two closely related mouse species,  Mus musculus  and  Mus spretus , which diverged more than one million years ago. We analyzed testicular expression using tiling arrays overlapping Chromosomes 2, X, Y and mitochondrial genome. An excess of differentially regulated non-coding RNAs was found on Chromosome 2 including the intronic antisense RNAs, intergenic RNAs and premature forms of Piwi-interacting RNAs (piRNAs). Moreover, striking difference was found in the expression of X-linked  G6pdx  gene, the parental gene of the autosomal retrogene  G6pd2 .    Conclusions/Significance  The prevalence of non-coding RNAs among differentially expressed transcripts indicates their role in species-specific regulation of spermatogenesis. The postmeiotic expression of  G6pdx  in  Mus spretus  points towards the continuous evolution of X-chromosome silencing and provides an example of expression change accompanying the out-of-the X-chromosomal retroposition.

Differential Expression of Non-Coding RNAs and Continuous Evolution of the X Chromosome in Testicular Transcriptome of Two Mouse Species