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Stallion sperm transcriptome comprises functionally coherent coding and regulatory RNAs as revealed by microarray analysis and RNA-seq.

Das PJ, McCarthy F, Vishnoi M, Paria N, Gresham C, Li G, Kachroo P, Sudderth AK, Teague S, Love CC, Varner DD, Chowdhary BP, Raudsepp T - PLoS ONE (2013)

Bottom Line: 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.The data were aligned with selected equine gene models to identify additional exons and splice variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
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.

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Related in: MedlinePlus

Comparison of RNA-seq data with current equine gene models:(a) PKM2 showing 9 in silico prediction sites, of which two are positioned 5′ upstream to exon 1; (b) CRISP3 with 3 in silico prediction sites, all located 5′ upstream to exon 1; (c) PRM1 and TNP2 cluster (the protamine cluster) with 12 in silico prediction sites of which only two align with PRM1 and TNP2 exons. Black boxes with numbers –exons in current gene models; blue boxes –very highly expressed tags (AC≥100); red boxes–highly expressed tags (10<AC<100); green boxes–tags with medium expression (1≤AC≤100). Exact start and end sites of all mapped tags are presented in Additional file 7.
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pone-0056535-g006: Comparison of RNA-seq data with current equine gene models:(a) PKM2 showing 9 in silico prediction sites, of which two are positioned 5′ upstream to exon 1; (b) CRISP3 with 3 in silico prediction sites, all located 5′ upstream to exon 1; (c) PRM1 and TNP2 cluster (the protamine cluster) with 12 in silico prediction sites of which only two align with PRM1 and TNP2 exons. Black boxes with numbers –exons in current gene models; blue boxes –very highly expressed tags (AC≥100); red boxes–highly expressed tags (10<AC<100); green boxes–tags with medium expression (1≤AC≤100). Exact start and end sites of all mapped tags are presented in Additional file 7.

Mentions: Of the 9 tags that mapped to PKM2, each corresponded to two different NCBI accessions (Table S8) suggesting the presence of two splice variants in stallion sperm. Based on the AC values, the variant comprising of exons 1, 3, 4, 5, and 6 was more abundant than a variant where exons 2 and 9 were included; no tags aligned with exons 7, 8, 10, and 11. However, a relatively abundant (AC = 94.16) sequence tag aligned with a 5′ upstream region of the gene indicating likely presence of an additional exon (Fig. 6a).


Stallion sperm transcriptome comprises functionally coherent coding and regulatory RNAs as revealed by microarray analysis and RNA-seq.

Das PJ, McCarthy F, Vishnoi M, Paria N, Gresham C, Li G, Kachroo P, Sudderth AK, Teague S, Love CC, Varner DD, Chowdhary BP, Raudsepp T - PLoS ONE (2013)

Comparison of RNA-seq data with current equine gene models:(a) PKM2 showing 9 in silico prediction sites, of which two are positioned 5′ upstream to exon 1; (b) CRISP3 with 3 in silico prediction sites, all located 5′ upstream to exon 1; (c) PRM1 and TNP2 cluster (the protamine cluster) with 12 in silico prediction sites of which only two align with PRM1 and TNP2 exons. Black boxes with numbers –exons in current gene models; blue boxes –very highly expressed tags (AC≥100); red boxes–highly expressed tags (10<AC<100); green boxes–tags with medium expression (1≤AC≤100). Exact start and end sites of all mapped tags are presented in Additional file 7.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569414&req=5

pone-0056535-g006: Comparison of RNA-seq data with current equine gene models:(a) PKM2 showing 9 in silico prediction sites, of which two are positioned 5′ upstream to exon 1; (b) CRISP3 with 3 in silico prediction sites, all located 5′ upstream to exon 1; (c) PRM1 and TNP2 cluster (the protamine cluster) with 12 in silico prediction sites of which only two align with PRM1 and TNP2 exons. Black boxes with numbers –exons in current gene models; blue boxes –very highly expressed tags (AC≥100); red boxes–highly expressed tags (10<AC<100); green boxes–tags with medium expression (1≤AC≤100). Exact start and end sites of all mapped tags are presented in Additional file 7.
Mentions: Of the 9 tags that mapped to PKM2, each corresponded to two different NCBI accessions (Table S8) suggesting the presence of two splice variants in stallion sperm. Based on the AC values, the variant comprising of exons 1, 3, 4, 5, and 6 was more abundant than a variant where exons 2 and 9 were included; no tags aligned with exons 7, 8, 10, and 11. However, a relatively abundant (AC = 94.16) sequence tag aligned with a 5′ upstream region of the gene indicating likely presence of an additional exon (Fig. 6a).

Bottom Line: 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.The data were aligned with selected equine gene models to identify additional exons and splice variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus