STUDY QUESTION - Is the noncoding transcriptional landscape during spermatogenesis conserved between human and rodents?SUMMARY ANSWERWe identified a core group of 113 long noncoding RNAs (lncRNAs) and 20 novel genes dynamically and syntenically transcribed during spermatogenesis.WHAT IS KNOWN ALREADY - Spermatogenesis is a complex differentiation process driven by a tightly regulated and highly specific gene expression program. Recently, several studies in various species have established that a large proportion of known lncRNAs are preferentially expressed during meiosis and spermiogenesis in a testis-specific manner. STUDY DESIGN, SIZE, DURATION - To further investigate lncRNA expression in human spermatogenesis, we carried out a cross-species RNA profiling study using isolated testicular cells.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman testes were obtained from post-mortem donors (N = 8, 51 years old on average) or from prostate cancer patients with no hormonal treatment (N = 9, 80 years old on average) and only patients with full spermatogenesis were used to prepare enriched populations of spermatocytes, spermatids, Leydig cells, peritubular cells and Sertoli cells. To minimize potential biases linked to inter-patient variations, RNAs from two or three donors were pooled prior to RNA-sequencing (paired-end, strand-specific). Resulting reads were mapped to the human genome, allowing for assembly and quantification of corresponding transcripts.MAIN RESULTS AND THE ROLE OF CHANCEOur RNA-sequencing analysis of pools of isolated human testicular cells enabled us to reconstruct over 25 000 transcripts. Among them we identified thousands of lncRNAs, as well as many previously unidentified genes (novel unannotated transcripts) that share many properties of lncRNAs. Of note is that although noncoding genes showed much lower synteny than protein-coding ones, a significant fraction of syntenic lncRNAs displayed conserved expression during spermatogenesis.LARGE SCALE DATARaw data files (fastq) and a searchable table (.xlss) containing information on genomic features and expression data for all refined transcripts have been submitted to the NCBI Gene Expression Omnibus under accession number GSE74896.LIMITATIONS, REASONS FOR CAUTIONIsolation procedures may alter the physiological state of testicular cells, especially for somatic cells, leading to substantial changes at the transcriptome level. We therefore cross-validated our findings with three previously published transcriptomic analyses of human spermatogenesis. Despite the use of stringent filtration criteria, i.e. expression cut-off of at least three fragments per kilobase of exon model per million reads mapped, fold-change of at least three and false discovery rate adjusted P-values of less than <1%, the possibility of assembly artifacts and false-positive transcripts cannot be fully ruled out.WIDER IMPLICATIONS OF THE FINDINGSFor the first time, this study has led to the identification of a large number of conserved germline-associated lncRNAs that are potentially important for spermatogenesis and sexual reproduction. In addition to further substantiating the basis of the human testicular physiology, our study provides new candidate genes for male infertility of genetic origin. This is likely to be relevant for identifying interesting diagnostic and prognostic biomarkers and also potential novel therapeutic targets for male contraception.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by l’Institut national de la santé et de la recherche médicale (Inserm); l’Université de Rennes 1; l’Ecole des hautes études en santé publique (EHESP); INERIS-STORM to B.J. [N 10028NN]; Rennes Métropole ‘Défis scientifiques émergents’ to F.C (2011) and A.D.R (2013). The authors have no competing financial interests.