Cryopreserved Bovine Spermatozoal Transcript Profile as Revealed by High-Throughput Ribonucleic Acid Sequencing
Cryopreserved Bovine Spermatozoal Transcript Profile as Revealed by High-Throughput Ribonucleic Acid Sequencing
复制标题
DOI：
10.1095/biolreprod.112.103788
复制
发表时间：
2013-02-01
影响因子：
3.6
通讯作者：
Sartini, Becky L.
中科院分区：
文献类型：
--
作者：
Card, Christopher J.;Anderson, Elizabeth J.;Sartini, Becky L.
排出的牛精子保留了一个可能在早期胚胎发生中起作用的RNA库，并可用作男性生育力的预测因子。牛精子的转录本档案仍然是不完整的，因为以前的研究依赖于杂交为基础的技术，评估有限的池的成绩单，不能确定全长成绩单。本研究的目的是使用Illumina RNA测序（RNA-Seq）对完整的冷冻保存的牛精子转录本谱进行测序。从9头受胎率评分为-2.9至3.5的公牛中收集精子RNA，并确认排除了基因组DNA和体细胞mRNA。在选择性扩增poly（A）（+）RNA和高通量测序后，通过与牛基因组（UMD 3.1/bosTau 6）的比对鉴定了6166个转录物。RNA-Seq转录水平（n = 9）与定量PCR拷贝数高度相关（r（2）= 0.9747）。牛精子转录谱是一个异质群体的降解和全长主要是核编码的mRNA。高丰度的精子转录本包括PRM 1、HMGB 4和精子编码的转录本。全长转录物包含前368个转录物的66%（每百万个映射片段的外显子的每个内切酶的片段[FPKM] > 100），并且对于选定的转录物确认全长转录物或5'和3'末端的扩增。除了在精子中发现了以前没有报道过的转录本外，还发现了几种已知的来自不同物种的精子转录本，对FPKM > 100个精子转录本的基因本体论分析表明，翻译是最主要的生物学过程。这是第一次报告的精子转录谱在任何物种中使用高通量测序，支持精子中的mRNA的存在，进一步的功能和生育力的研究。
Ejaculated bovine spermatozoa retain a pool of RNAs that may have a function in early embryogenesis and be used as predictors of male fertility. The bovine spermatozoal transcript profile remains incomplete because previous studies have relied on hybridization-based techniques, which evaluate a limited pool of transcripts and cannot identify full-length transcripts. The goal of this study was to sequence the complete cryopreserved bovine spermatozoal transcript profile using Illumina RNA-Sequencing (RNA-Seq). Spermatozoal RNA was pooled from nine bulls with conception rate scores ranging from -2.9 to 3.5 and confirmed to exclude genomic DNA and somatic cell mRNA. After selective amplification of poly(A)(+) RNA and high-throughput sequencing, 6166 transcripts were identified via alignment to the bovine genome (UMD 3.1/bosTau6). RNA-Seq transcript levels (n = 9) were highly correlated with quantitative PCR copy number (r(2) = 0.9747). The bovine spermatozoal transcript profile is a heterogeneous population of degraded and full-length predominantly nuclear-encoded mRNAs. Highly abundant spermatozoal transcripts included PRM1, HMGB4, and mitochondrial-encoded transcripts. Full-length transcripts comprised 66% of the top 368 transcripts (fragments per kilobase of exon per million fragments mapped [FPKM] > 100) and amplification of the full-length transcript or 5' and 3' ends was confirmed for selected transcripts. In addition to the identification of transcripts not previously reported in spermatozoa, several known spermatozoal transcripts from various species were also found. Gene ontology analysis of the FPKM > 100 spermatozoal transcripts revealed that translation was the most predominant biological process represented. This is the first report of the spermatozoal transcript profile in any species using high-throughput sequencing, supporting the presence of mRNA in spermatozoa for further functional and fertility studies.