Source: UNIVERSITY OF RHODE ISLAND submitted to
BOVINE SPERM TRANSCRIPTOME: SEQUENCING AND TRANSLATION IN THE BOVINE EMBRYO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0224187
Grant No.
2011-67016-20073
Project No.
RI002010-03193
Proposal No.
2010-03193
Multistate No.
(N/A)
Program Code
A1211
Project Start Date
Dec 15, 2010
Project End Date
Dec 14, 2013
Grant Year
2011
Project Director
Sartini, B. L.
Recipient Organization
UNIVERSITY OF RHODE ISLAND
19 WOODWARD HALL 9 EAST ALUMNI AVENUE
KINGSTON,RI 02881
Performing Department
Fisheries Animal & Vet Science
Non Technical Summary
Male fertility is a limiting factor to livestock production and current tests to detect subfertile sires are inadequate. In the post-genomic era, research in RNA regulation is needed to further our understanding of livestock spermatozoa function to develop more accurate fertility assays. We hypothesize that bovine spermatozoa retain messenger RNA (mRNA) that can be translated into protein in the oocyte after fertilization. We will test this hypothesis by identifying full length mRNA in bovine spermatozoa, from the round spermatid transcriptome using RNA-Seq, and then characterize the transfer of candidate spermatozoa mRNAs to the bovine oocyte during fertilization. We anticipate to identify candidate full-length mRNA in bull spermatozoa that were retained from round spermatids and demonstrate that these mRNAs are transferred to the oocyte during fertilization then translated into protein demonstrating a potential role in embryogenesis. This foundational research will fill in critical gaps in knowledge of bovine spermatozoa mRNA function for further fertility assay development.
Animal Health Component
(N/A)
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30134101040100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The overarching goal of this research is to determine if bovine spermatozoa RNA has a function in early embryonic development. Previous reports of full-length mRNAs in bovine spermatozoa indicate that these transcripts could be translated into protein in the oocyte after fertilization and have a function in the early embryo. The complete full-length transcriptome of bull spermatozoa and subsequent protein expression of specific spermatozoa transcripts during embryogenesis has not been investigated. The objectives of this project are to 1) map the complete transcriptome in bovine spermatozoa by comparison with the bovine round spermatid transcriptome to identify full-length candidate transcripts and 2) characterize the transfer of candidate sperm mRNAs to the bovine oocyte during fertilization and their subsequent translation in the early embryo. Outputs for this project will include a complete mapped transcriptome of bovine round spermatids and spermatozoa.
Project Methods
This project will be conducted by sequencing the bovine round spermatid and spermatozoa transcriptome by RNA-Sequencing (RNA-Seq). Resulting sequences will be aligned to the bovine genome and novel full length messenger RNAs (mRNA) in round spermatids and spermatozoa will be identified. The presence of these novel mRNAs in oocytes and zygotes will be compared to determine if the sperm RNAs are delivered to the oocyte during fertlization. Protein levels of candidate sperm mRNAs will be determined if mRNA transfer is found.

Progress 12/15/10 to 12/14/13

Outputs
Target Audience: Reproductive biologists, animal scientists, livestock producers and the bovine artificial insemination industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two publications (one published and one in prep), three conference poster presentations, a Ph.D dissertation, and a M.S thesis (training of 2 graduate students) have resulted from this project. Seven undergraduate students have been trained in this project resulting in three undergraduate poster presentations at the University of Rhode Island. How have the results been disseminated to communities of interest? Two publications (one published and one in prep), three conference poster presentations (Society for the Study of Reproduction and National Association of Animal Breeders) have disseminated results from this project to reproductive biologists, animal scientists, livestock producers and the bovine artificial insemination industry. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The complete bovine sperm transcript profile was sequenced using RNA-Seq and published (Card C, Anderson L, Zamberlan J, Kreiger KE, Kaproth M and Sartini BL. 2013. Cryopreserved bovine spermatozoal transcript profile revealed by high-throughput ribonucleic acid sequencing. Biology of Reproduction. 88(2): 1-9). From this population, we chose 24 spermatozoal transcripts, from high FPKM, Gene Ontology, Y chromosome specificity, and previous studies, to determine if full-length transcripts are present in sperm and to characterize transfer of these sperm transcripts to the bovine oocyte during fertilization. Of these sperm transcripts, 16 transcripts were validated as full-length including the 3’UTRs and coding regions by 3'RACE PCR. Alternative polyadenylation sites were identified in four transcripts PSMA6, ATPase β, CHMP5, and DDX3Y. Although full-length sperm transcripts were identified, we were unable to confirm the transfer of full-length spermatozoal transcripts to oocytes and 2-cell stage bovine embryo after extensive PCR analysis of individual pools of bovine oocytes and early stage embryos.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Anderson EJ, Card CJ, Sartini BL. 2013. Full-Length mRNAs in Bos taurus Spermatozoa. Society for the Study of Reproduction. Montreal Canada. Poster Presentation.


Progress 12/15/11 to 12/14/12

Outputs
OUTPUTS: Further analysis of RNA-Seq data was completed. RNA-Seq transcript levels (n=9) were highly correlated with qPCR copy number (r2=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 (FPKM>100) and expression of the 5'and 3' ends for some transcripts were confirmed. 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 top 368 spermatozoal transcripts revealed that translation was the most predominant biological process represented. PARTICIPANTS: BL Sartini, Principal Investigator, lead the study design and data analysis. CJ Card, PhD Student, participated in study design and executed experiments and data analysis. L. Anderson, MS student, participated in data analysis J. Zamberlan, Undergraduate, conducted experiments E. Aparicio, Undergraduate, conducted experiments TARGET AUDIENCES: Reproductive biologists, animal scientists, livestock producers and the bovine artificial insemination industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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. Characterization of the cryopreserved bovine sperm transcript profile allows for further investigation of idiopathic bull subfertility based on gamete gene expression. By identifying factors that contribute to variation in sire fertility, more accurate fertility estimates could increase cow pregnancy rates and therefore improve the sustainability of the dairy and beef industries in the United States.

Publications

  • Card C, Anderson L, Zamberlan J, Kreiger KE, Kaproth M and Sartini BL. 2013. Cryopreserved bovine spermatozoal transcript profile revealed by high-throughput ribonucleic acid sequencing. Biology of Reproduction. 88(2): 1-9. PMID 23303677
  • Card C and Sartini BL. 2012. Spermatozoal transcriptome in bull fertility. Proceedings of the National Association of Animal Breeders (NAAB) 24rd Biennial Technical Conference on Artificial Insemination and Reproduction. Milwaukee, WI.
  • Card C, Anderson L, Zamberlan J, Kreiger KE, Kaproth M and Sartini BL. 2012. Cryopreserved bovine spermatozoal transcriptome as revealed by RNA-Seq. Society for the Study of Reproduction, Pennsylvania State University. Abstract No. 445. Poster Presentation.


Progress 12/15/10 to 12/14/11

Outputs
OUTPUTS: Ejaculated bovine spermatozoa retain a pool of RNAs that may have a function during early embryogenesis and could also serve as a male fertility assay. The bovine sperm transcriptome remains incomplete because previous studies have relied on hybridization based microarray techniques, which evaluate a limited pool of transcripts and cannot provide information about full-length transcripts. The goal of this study was to sequence the complete cryopreserved bovine sperm transcriptome using Illumina RNA-Seq. Cryopreserved bovine sperm RNA was converted to cDNA using SMARTer (Clontech) to enrich full-length mRNAs. Sperm cDNA was pooled from nine bulls with CR scores ranging from -2.9 to 3.5 and was confirmed to be free of contamination from genomic DNA and somatic cell mRNA. Over 37 million 100-bp pair-end reads were generated and 5543 transcripts were identified when these reads were aligned to the bovine genome (UMD 3.1/bosTau6). We are currently validating the expression levels and gene ontology of the cryopreserved bovine sperm transcriptome. PARTICIPANTS: BL Sartini, Principal Investigator, lead the study design, and data analysis. Three students have received research training on this project: C Card, Ph.D. student, participated in study design, executed experiments and data analysis. L. Baird, M.S. student, executed experiments and data analysis J. Zamberlan, undergraduate student, executed experiments and data analysis. TARGET AUDIENCES: Target audiences include reproductive biologists, animal scientist, livestock producers and the bovine artificial insemination industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This is the first complete report of the spermatozoa transcriptome in any species. Characterization of the cryopreserved bovine sperm transcriptome will allow for further investigation of idiopathic bull subfertility based on gamete gene expression. By identifying factors that contribute to variation in sire fertility, more accurate fertility estimates could increase cow pregnancy rates and therefore improve the economic sustainability of the dairy and beef industries in the United States.

Publications

  • No publications reported this period