Transcription profile of boar spermatozoa as revealed by RNA-sequencing

Authors: FEUGANG, J - Mississippi State University; LIAO, S - Mississippi State University; SANDERS, W - Mississippi State University; LU, J - Emory University, School Of Medicine; CRENSHAW, M - Mississippi State University; WILLARD, S - Mississippi State University; RYAN, P - Mississippi State University

Submitted to: Joint Abstracts of the American Dairy Science and Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: 4/21/2015
Publication Date: 6/22/2015
Citation: Feugang, J.M., Liao, S.F., Sanders, W.S., Lu, J., Crenshaw, M.A., Willard, S.T., Ryan, P.L. 2015. Transcription profile of boar spermatozoa as revealed by RNA-sequencing. Joint Abstracts of the American Dairy Science and Society of Animal Science. 108:99.

Interpretive Summary: Spermatozoa are highly specialized cells rich in various RNA transcripts and proteins, which full understanding of their biological roles would improve our comprehension of the genetic make-up that influences (re)production and health performances of adult animals. Indeed, a small fraction of these molecules are known to be transferred to the egg (oocyte) at the moment of fertilization. Here we used the most recent high throughput technology to profile the global population of RNA within mature spermatozoa of high fertile boars. Given the dormant status of mature spermatozoa, generated sperm total RNA transcripts profile will provide novel/more materials for in-depth investigations of their biological functions and contribution to the embryo-fetal development and beyond.

Technical Abstract: High-throughput RNA sequencing (RNA-Seq) overcomes the limitations of the current hybridization-based techniques to detect the actual pool of RNA transcripts in spermatozoa. The application of this technology in livestock can speed the discovery of potential predictors of male fertility. As a first step to identify novel gene products of fertility importance in pigs, we conducted a RNA-sequence analysis of boar spermatozoa. Fresh semen of 8 fertile boars (3 ejaculates/boar) were purchased from a commercial stud and pure motile spermatozoa were obtained through a discontinuous percoll gradient. Total sperm RNA were extracted using commercial kits with an in-column DNase digestion. The purity and integrity of RNA samples were checked and those with high quality parameters were used for deep-RNA sequencing to produce millions of short cDNA reads using Illumina RNA-Seq tec hnology. Resulting reads were aligned to the pig reference genome to produce a genome-scale transcription map that consisted of both the transcript structure and the expression level of each gene (fragments per kilobase of exon per million fragments mapped). Total of 18,357 sequence tags were successfully mapped to all pig chromosomes and mitochondrial genome. Five chromosomes (2, 1, 6, 7, and 13) comprised the highest density of mapped transcripts (42%), while the bottom lowest density (8%) was found in chromosomes 10, 18, 16, 11, and Y. The Y chromosome and mitochondrial genome contained only 0.07% and 0.08% of total mapped sequence tags. Structural annotation revealed a diverse population of sperm transcripts comprising both coding and non-coding RNAs. Approximately 12,355 of sequence tags were annotated with ENSEMBL and rRNAs (e.g., 5s and 7sk), snRNA (e.g., U1 and U6), miRNAs (e.g., mir127 and mir935), and mitochondrial RNA (e.g., ND6 and CO1) constituted the most abundant sequence tags. We further confirmed the presence of selected genes (e.g., AQP11 and AQN-1) through RT-PCR. The findings revealed a large pool of coding and non-coding RNA in mature boar spermatozoa. The full investigation of this RNA population will allow for the identification of those having critical roles during fertilization and early embryogenesis.