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Expression microarray profiling of sperm and testis mRNA of reproductively normal stallions
Animal Reproduction Science 121S (2010) S175
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Abstract
Expression microarray profiling of sperm and testis mRNA of reproductively normal stallions夽 P.J. Das a , M. Vishnoi a , P. Kachroo a , J. Wang a , C.C. Love b , D.D. Varner b , B.P. Chowdhary a , T. Raudsepp a,∗ a b
Department of Veterinary Integrative Biosciences, Texas A & M University, College Station, TX 77843, USA Department of Large Animal Clinical Sciences, Texas A & M University, College Station, TX 77843, USA
1. Introduction Poor fertility of breeding stallions is a recognized concern in the horse industry and makes the development of genomic-based strategies to evaluate male fertility important. In humans, sperm contain messenger RNAs (mRNAs) that reflect the functional status of the testes, and can be used to identify genetic markers for male fertility. To use sperm as an efficient non-invasive measure of testicular function in horses, detailed knowledge of transcriptional profiles of equine sperm is needed. Therefore, the goal of this study was to obtain information about the number, identity, biological functions and transcriptional profiles of mRNAs present in stallion sperm, and compare this with the mRNA profile of stallion testis. 2. Materials and methods Four testes and four semen samples from reproductively normal stallions were used. The semen samples were cleaned free of somatic cells using a 40% silanized silica particle system (EquiPureTM ), and total RNA was isolated from both sperm and testis. RNA quantity and quality were evaluated using a spectrophotometer, bioanalyzer, and reverse transcriptase PCR with sperm/testis-specific PRM2 and somatic cell-specific PTPRC primers. Sperm and testis total RNA was linearly amplified into sense-strand mRNA, converted into cDNA and labeled with Cy3 and Cy5, respectively. Gene expression profiles of sperm and
夽 This paper is part of the supplement entitled “Proceedings of the Tenth International Symposium on Equine Reproduction”, Guest Edited by Margaret J. Evans. ∗ Corresponding author. Tel.: +1 979 862 2879; fax: +1 979 845 9972. E-mail address: [email protected] (T. Raudsepp). 0378-4320/$ – see front matter doi:10.1016/j.anireprosci.2010.04.086
testis mRNA were analyzed by hybridizing differently labeled pairs of sperm and testis mRNA to the Texas A&M equine whole-genome 21,351-element oligoarray. After signal normalization, the microarray hybridization results were analyzed for absolute and differential gene expression, followed by gene ontology analysis. 3. Results and discussion Total numbers of transcripts in sperm and testis were 7,718 and 15,785, respectively (signal-to-noise ratio = 2). Differential expression analysis of the transcripts shared by sperm and testis showed that 1,168 genes were upregulated and 1,454 down-regulated in sperm (signal fold change > 2). Notably, 84 mRNAs were present only in sperm. Gene ontology analysis showed that sperm-specific and sperm up-regulated mRNAs are associated with membranes and involved in molecular functions associated with G-protein coupled receptor (GPCR) and olfactory receptor activities. In contrast, the mRNAs over-represented in testis are localized in all cellular compartments and are involved in diverse molecular functions and biological processes. We conclude that the sperm of reproductively normal stallions contain a rich collection of mRNAs that are associated with a variety of known sperm functions. The results set an important foundation for comprehensive characterization of the sperm transcriptome of reproductively normal stallions, and of stallions with reduced fertility, thus facilitating the development of non-invasive molecular genetic tools for evaluating fertility in stallions. Conflict of interest None.
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Abstract
Expression microarray profiling of sperm and testis mRNA of reproductively normal stallions夽 P.J. Das a , M. Vishnoi a , P. Kachroo a , J. Wang a , C.C. Love b , D.D. Varner b , B.P. Chowdhary a , T. Raudsepp a,∗ a b
Department of Veterinary Integrative Biosciences, Texas A & M University, College Station, TX 77843, USA Department of Large Animal Clinical Sciences, Texas A & M University, College Station, TX 77843, USA
1. Introduction Poor fertility of breeding stallions is a recognized concern in the horse industry and makes the development of genomic-based strategies to evaluate male fertility important. In humans, sperm contain messenger RNAs (mRNAs) that reflect the functional status of the testes, and can be used to identify genetic markers for male fertility. To use sperm as an efficient non-invasive measure of testicular function in horses, detailed knowledge of transcriptional profiles of equine sperm is needed. Therefore, the goal of this study was to obtain information about the number, identity, biological functions and transcriptional profiles of mRNAs present in stallion sperm, and compare this with the mRNA profile of stallion testis. 2. Materials and methods Four testes and four semen samples from reproductively normal stallions were used. The semen samples were cleaned free of somatic cells using a 40% silanized silica particle system (EquiPureTM ), and total RNA was isolated from both sperm and testis. RNA quantity and quality were evaluated using a spectrophotometer, bioanalyzer, and reverse transcriptase PCR with sperm/testis-specific PRM2 and somatic cell-specific PTPRC primers. Sperm and testis total RNA was linearly amplified into sense-strand mRNA, converted into cDNA and labeled with Cy3 and Cy5, respectively. Gene expression profiles of sperm and
夽 This paper is part of the supplement entitled “Proceedings of the Tenth International Symposium on Equine Reproduction”, Guest Edited by Margaret J. Evans. ∗ Corresponding author. Tel.: +1 979 862 2879; fax: +1 979 845 9972. E-mail address: [email protected] (T. Raudsepp). 0378-4320/$ – see front matter doi:10.1016/j.anireprosci.2010.04.086
testis mRNA were analyzed by hybridizing differently labeled pairs of sperm and testis mRNA to the Texas A&M equine whole-genome 21,351-element oligoarray. After signal normalization, the microarray hybridization results were analyzed for absolute and differential gene expression, followed by gene ontology analysis. 3. Results and discussion Total numbers of transcripts in sperm and testis were 7,718 and 15,785, respectively (signal-to-noise ratio = 2). Differential expression analysis of the transcripts shared by sperm and testis showed that 1,168 genes were upregulated and 1,454 down-regulated in sperm (signal fold change > 2). Notably, 84 mRNAs were present only in sperm. Gene ontology analysis showed that sperm-specific and sperm up-regulated mRNAs are associated with membranes and involved in molecular functions associated with G-protein coupled receptor (GPCR) and olfactory receptor activities. In contrast, the mRNAs over-represented in testis are localized in all cellular compartments and are involved in diverse molecular functions and biological processes. We conclude that the sperm of reproductively normal stallions contain a rich collection of mRNAs that are associated with a variety of known sperm functions. The results set an important foundation for comprehensive characterization of the sperm transcriptome of reproductively normal stallions, and of stallions with reduced fertility, thus facilitating the development of non-invasive molecular genetic tools for evaluating fertility in stallions. Conflict of interest None.