Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis

Abstract

The discovery of a large number of long noncoding RNAs (lncRNAs), and the finding that they may play key roles in different biological processes, have started to provide a new perspective in the understanding of gene regulation. It has been shown that the testes express the highest amount of lncRNAs among different vertebrate tissues. However, although some studies have addressed the characterization of lncRNAs along spermatogenesis, an exhaustive analysis of the differential expression of lncRNAs at its different stages is still lacking. Here, we present the results for lncRNA transcriptome profiling along mouse spermatogenesis, employing highly pure flow sorted spermatogenic stage-specific cell populations, strand-specific RNAseq, and a combination of up-to-date bioinformatic pipelines for analysis. We found that the vast majority of testicular lncRNA genes are expressed at post-meiotic stages (i.e. spermiogenesis), which are characterized by extensive post-transcriptional regulation. LncRNAs at different spermatogenic stages shared common traits in terms of transcript length, exon number, and biotypes. Most lncRNAs were lincRNAs, followed by a high representation of antisense (AS) lncRNAs. Co-expression analyses showed a high correlation along the different spermatogenic stage transitions between the expression patterns of AS lncRNAs and their overlapping protein-coding genes, raising possible clues about lncRNA-related regulatory mechanisms. Interestingly, we observed the colocalization of an AS lncRNA and its host sense mRNA in the chromatoid body, a round spermatidsspecific organelle that has been proposed as a reservoir of RNA-related regulatory machinery. An additional, intriguing observation is the almost complete lack of detectable expression for Y-linked testicular lncRNAs, despite that a high number of lncRNA genes are annotated for this chromosome.

Transcriptomics of Meiosis in the Male Mouse

“…In our laboratory, we have developed a protocol for the purification of testicular cell populations by FACS using Vybrant DyeCycle Green, a non-cytotoxic vital dye with the advantage over Hoechst 33342 that it is excited with a blue laser thus avoiding the need of a UV laser, which in turn minimizes potential damage to nucleic acids caused by UV light exposure ( Rodríguez-Casuriaga et al, 2014 ; Geisinger and Rodríguez-Casuriaga, 2017 ). This protocol allowed the profiling of coding transcripts and lncRNAs along spermatogenesis through RNAseq, using highly pure (>95%) stage-specific cell populations ( da Cruz et al, 2016 ; Trovero et al, 2020 ). These studies included the L/Z cell population, thus enabling for the first time to compare the transcriptomic profiles, as obtained through NGS, between early and medium/late meiotic prophase, and providing information on gene expression fluctuations along meiotic prophase ( da Cruz et al, 2016 ).…”

“…GC, gonocytes; SPG, spermatogonia; PL, preleptotene; L, leptotene; Z, zygotene; PS, pachytene; D, diplotene; M, meiotic divisions; 1–16 represent the different spermatid stages. Adapted from Trovero et al (2020) , under the Creative Commons Attribution License.…”

lncRNA FDNCR promotes apoptosis of granulosa cells by targeting the miR-543-3p/DCN/TGF-β signaling pathway in Hu sheep

“… 6 Recently, numerous lncRNAs have been identified in humans and other organisms that play diverse roles in regulating various phenomenon, such as cell proliferation and apoptosis, 7 , 8 cell development, 9 cell differentiation, 10 and development of certain diseases. 11 , 12 Specifically, lncRNAs are involved in regulating spermatogenesis, 13 , 14 steroidogenesis, 15 embryo implantation 16 and development, 17 follicular development, 18 oocyte maturation, 19 GC apoptosis and proliferation, 20 , 21 and reproductive diseases, 22 , 23 suggesting their importance in reproduction. 24 However, only a few lncRNAs have been identified in domestic animals.…”

Contributions of Flow Cytometry to the Molecular Study of Spermatogenesis in Mammals

“…More recently, we applied our VDG-based sorting protocol [ 49 , 50 ] for RNA seq of four testicular cell populations, including two meiotic prophase I populations (L/Z and P/D) (see Figure 2 B). The high purity of the sorted fractions (>95%), combined with RNAseq technology, enabled accurately establishment of the transcriptome fluctuations along spermatogenesis, both for coding and for long non-coding transcripts (lncRNAs) [ 94 , 95 ]. Moreover, the purification of the L/Z population allowed for the first time the comparison of the RNAseq-derived transcriptomes of early meiotic prophase I (in which essential events such as homologous chromosome alignment and pairing occur) and medium/late meiotic prophase cells (in which crossing over takes place) [ 94 ].…”