Long Non-Coding RNA Expression Profiling of Mouse Testis during Postnatal Development

Jin Sun,Ji Wu,Yi Lin,Wei Yan

doi:10.1371/journal.pone.0075750

Abstract

Mammalian testis development and spermatogenesis play critical roles in male fertility and continuation of a species. Previous research into the molecular mechanisms of testis development and spermatogenesis has largely focused on the role of protein-coding genes and small non-coding RNAs, such as microRNAs and piRNAs. Recently, it has become apparent that large numbers of long (>200 nt) non-coding RNAs (lncRNAs) are transcribed from mammalian genomes and that lncRNAs perform important regulatory functions in various developmental processes. However, the expression of lncRNAs and their biological functions in post-natal testis development remain unknown. In this study, we employed microarray technology to examine lncRNA expression profiles of neonatal (6-day-old) and adult (8-week-old) mouse testes. We found that 8,265 lncRNAs were expressed above background levels during post-natal testis development, of which 3,025 were differentially expressed. Candidate lncRNAs were identified for further characterization by an integrated examination of genomic context, gene ontology (GO) enrichment of their associated protein-coding genes, promoter analysis for epigenetic modification, and evolutionary conservation of elements. Many lncRNAs overlapped or were adjacent to key transcription factors and other genes involved in spermatogenesis, such as Ovol1, Ovol2, Lhx1, Sox3, Sox9, Plzf, c-Kit, Wt1, Sycp2, Prm1 and Prm2. Most differentially expressed lncRNAs exhibited epigenetic modification marks similar to protein-coding genes and tend to be expressed in a tissue-specific manner. In addition, the majority of differentially expressed lncRNAs harbored evolutionary conserved elements. Taken together, our findings represent the first systematic investigation of lncRNA expression in the mammalian testis and provide a solid foundation for further research into the molecular mechanisms of lncRNAs function in mammalian testis development and spermatogenesis.

Highlights

The mammalian testis is the site of spermatogenesis and testosterone production, so it plays a central role in the male reproductive system
Overview of long (.200 nt) non-coding RNAs (lncRNAs) and mRNA profiles in neonatal and adult mouse testes To examine the lncRNA expression profiles of the mouse testis during post-natal development, we interrogated a commercial mouse lncRNA microarray with RNA isolated from neonatal (6-day-old, N) and adult (8-week-old, A) mouse testes
We found that 56% of lncRNAs on the microarray (8,265 out of 14,724) exhibited expression above background (Table S1), and that 37% of these (3,025 out of 8,265) were significantly differentially expressed between neonatal and adult mouse testes (Table S2)

Summary

Introduction

The mammalian testis is the site of spermatogenesis and testosterone production, so it plays a central role in the male reproductive system. Spermatogenesis is the primary biological process in the testis and produces mature haploid spermatozoa from diploid spermatogonia This developmental process is complicated, and involves a series of cellular differentiation and cell biological events, including spermatogonial proliferation, meiosis of spermatocytes and morphological changes of round spermatids [1,2]. Elucidation of the molecular mechanisms underlying spermatogenesis is important for our understanding of the genetic regulation of normal male germ cell development. Investigations into the molecular mechanisms of testis development and spermatogenesis are prominent in the field of reproductive biology. To date, these investigations have largely focused on the role of protein-coding genes and small non-coding RNAs, including microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs). Gene expression profiling, proteome profiling, miRNA profiling, piRNA profiling during testis development or spermatogenesis in mouse have been investigated [3,4,5,6,7]

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