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RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa.

ABSTRACT: Sperm, which are believed to be transcriptionally and translationally inactive, synthesize RNA and proteins before there is gradual disappearance of the ribosome during chromatin compaction. Sperm transfer several functionally relevant transcripts to the oocyte, controlling maternal-zygotic transition and embryonic development. The present study was undertaken to profile and analyze sperm transcripts comprehensively using Next Generation Ribonucleic acid sequencing technology in Holstein Friesian x Tharparkar crossbred bulls. The results from global transcriptomic profiling revealed transcripts for 13,814 genes; of which 431 transcripts were expressed with >1 FPKM and 13,383 transcripts were expressed with >0 or <1 FPKM. The abundant mRNA transcripts of crossbred bull sperm were PRM1 and HMGB4. Gene ontology of transcripts with>1 FPKM revealed there was a major involvement in the structural constituent of ribosomes and translation. Results from pathway enrichment indicated the connection between ribosome, oxidative phosphorylation and spliceosome pathways and the transcripts of crossbred bull spermatozoa. The transcriptional abundance of selected genes, validated using RT-qPCR, indicated significant variations between bulls. Collectively, it may be inferred that the transcripts in crossbred bull sperm were heavily implicated in functions such as the structural constituent of ribosomes and translation, and pathways such as ribosome, oxidative phosphorylation and spliceosome. Further studies using larger sample sizes are required to understand the possible implications of transcriptomic variations on semen quality and fertility.

SUBMITTER: Prakash MA

PROVIDER: S-EPMC7607363 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa.

Prakash Mani Arul MA Kumaresan Arumugam A Sinha Manish Kumar MK Kamaraj Elango E Mohanty Tushar Kumar TK Datta Tirtha Kumar TK Morrell Jane M JM

Animal reproduction science 20201010

Sperm, which are believed to be transcriptionally and translationally inactive, synthesize RNA and proteins before there is gradual disappearance of the ribosome during chromatin compaction. Sperm transfer several functionally relevant transcripts to the oocyte, controlling maternal-zygotic transition and embryonic development. The present study was undertaken to profile and analyze sperm transcripts comprehensively using Next Generation Ribonucleic acid sequencing technology in Holstein Friesia ...[more]

PMID: 33069132

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Project description:Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analyzing sperm and testes RNA on a 21,000-element equine whole-genome oligoarray and by RNA-seq. Microarray analysis revealed 6,761 transcripts in the sperm, of which 165 were sperm-enriched, and 155 were differentially expressed between the sperm and testes. Next, 70 million raw reads were generated by RNA-seq of which 50% could be aligned with the horse reference genome. A total of 19,257 sequence tags were mapped to all horse chromosomes and the mitochondrial genome. The highest density of mapped transcripts was in gene-rich ECA11, 12 and 13, and the lowest in gene-poor ECA9 and X; 7 gene transcripts originated from ECAY. Structural annotation aligned sperm transcripts with 4,504 known horse and/or human genes, rRNAs and 82 miRNAs, whereas 13,354 sequence tags remained anonymous. The data were aligned with selected equine gene models to identify additional exons and splice variants. Gene Ontology annotations showed that sperm transcripts were associated with molecular processes (chemoattractant-activated signal transduction, ion transport) and cellular components (membranes and vesicles) related to known sperm functions at fertilization, while some messenger and micro RNAs might be critical for early development. The findings suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs.

Project description:BackgroundRecent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data (H3K4me3, H3K36me3 and Pol II) to detect signatures of active transcription for reported lncRNAs.ResultsWe have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the poly(A)-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a poly(A) tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24% of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not.ConclusionsIn this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets (93 sets in total) from multiple sources (lncRNAs, RNA-seq and ChIP-seq). The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.

Dataset Information

RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa.

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RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa.

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