ABSTRACT: microRNAs (miRNAs) are important post-transcriptional regulators of gene expression. However, comprehensive expression profiles of miRNAs during spermatogenesis in mammals is lacking. In the present study, we sequenced small RNAs in highly purified spermatogonial stem cells (SSCs), spermatocytes in the pre-leptotene, leptotene, zygotene and pachytene stages, haploid round spermatids and spermatozoa. We found that a family of X-linked miRNAs, named spermiRs, is predominantly expressed in spermatogonia and early meiotic phases. Profiling of small RNAs in different mammalian tissues revealed a conserved high expression pattern of spermiRs in testes. spermiR appears to originate from THER1 in marsupial, a retrotransposon that is extinct in current mammals. It has expanded rapidly and has diversified into two clusters, spermiR-L and spermiR-R, by tandem duplication, which was partially mediated by flanking retrotransposable elements. Notably, despite the highly frequent lineage-specific duplication events, all spermiR family members are strictly located between two conserved protein coding genes, Slitrk2 and Fmr1. Intriguingly, spermiR-Ls and spermiR-Rs have evolved different expression preferences during spermatogenesis in different mammals. Moreover, the sequences of spermiRs and their precursors, including the seed regions, are highly divergent within and among mammals. However, their target genes are largely overlapping in male reproduction-related pathways. When miR-741, the most highly expressed spermiRs during mouse spermatogenesis, was knocked out in SSCs, another spermiR, miR-465, was dramatically up-regulated and became the most abundant one, indicating functional compensation between spermiR family members. All these observations indicate a strong co-evolution between spermiRs and their targets.
