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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

Structural annotation of 19, 257 mapped RNA sequence tags (AC≥1):(a) Distribution of the tags in structural annotation categories by ERANGE; (b) Comparison of annotated genes by GOanna (human genome) and ERANGE (horse genome).
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pone-0056535-g005: Structural annotation of 19, 257 mapped RNA sequence tags (AC≥1):(a) Distribution of the tags in structural annotation categories by ERANGE; (b) Comparison of annotated genes by GOanna (human genome) and ERANGE (horse genome).

Mentions: Structural and GO annotations of the 19,257 mapped RNA-seq tags with AC≥1 were conducted by alignment to the equine reference sequence (EcuCab2; UCSC Genome Browser; http://genome.ucsc.edu/) using Enhanced Read Analysis of Gene Expression (ERANGE) software packages [47], as well as by homology-based approach against the human genome in GOanna (AgBase; http://www.agbase.msstate. edu/cgi-bin/tools/GOanna.cgi) pipeline. A total of 5,903 (∼30%) of all mapped tags, aligned with annotated genes in the horse genome and were classified by ERANGE as expressed sequence tags (5,268), mRNAs (495) and micro RNAs (140) (Fig. 5a). Since the structural annotation of the equine genome is as yet incomplete, we used a permissive ±20 kb parameter to identify additional untranscribed regions (UTRs), new external exons, and to discriminate best candidates for novel genes. Among the 5,903 annotated transcripts, ∼17% entirely fell within the boundaries of annotated genes, 83% partially aligned with known genes, and 0.03% localized within the extended gene boundaries (see Materials and Methods). Only 1,378 annotations uniquely corresponded to individual equine genes. Similarly, 34% (6,606) of all mapped RNA-seq tags aligned with annotated sequences in the human genome identifying 3,262 unique genes. Because the horse (ERANGE) and human (GOanna) annotations shared only 136 genes in common (Table S6), stallion sperm transcripts as observed by RNA-seq analysis corresponded to a total of 4,504 annotated genes (Fig. 5b).


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)

Structural annotation of 19, 257 mapped RNA sequence tags (AC≥1):(a) Distribution of the tags in structural annotation categories by ERANGE; (b) Comparison of annotated genes by GOanna (human genome) and ERANGE (horse genome).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056535-g005: Structural annotation of 19, 257 mapped RNA sequence tags (AC≥1):(a) Distribution of the tags in structural annotation categories by ERANGE; (b) Comparison of annotated genes by GOanna (human genome) and ERANGE (horse genome).
Mentions: Structural and GO annotations of the 19,257 mapped RNA-seq tags with AC≥1 were conducted by alignment to the equine reference sequence (EcuCab2; UCSC Genome Browser; http://genome.ucsc.edu/) using Enhanced Read Analysis of Gene Expression (ERANGE) software packages [47], as well as by homology-based approach against the human genome in GOanna (AgBase; http://www.agbase.msstate. edu/cgi-bin/tools/GOanna.cgi) pipeline. A total of 5,903 (∼30%) of all mapped tags, aligned with annotated genes in the horse genome and were classified by ERANGE as expressed sequence tags (5,268), mRNAs (495) and micro RNAs (140) (Fig. 5a). Since the structural annotation of the equine genome is as yet incomplete, we used a permissive ±20 kb parameter to identify additional untranscribed regions (UTRs), new external exons, and to discriminate best candidates for novel genes. Among the 5,903 annotated transcripts, ∼17% entirely fell within the boundaries of annotated genes, 83% partially aligned with known genes, and 0.03% localized within the extended gene boundaries (see Materials and Methods). Only 1,378 annotations uniquely corresponded to individual equine genes. Similarly, 34% (6,606) of all mapped RNA-seq tags aligned with annotated sequences in the human genome identifying 3,262 unique genes. Because the horse (ERANGE) and human (GOanna) annotations shared only 136 genes in common (Table S6), stallion sperm transcripts as observed by RNA-seq analysis corresponded to a total of 4,504 annotated genes (Fig. 5b).

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