Identification of differentially expressed mRNAs and miRNAs in spermatozoa of bulls of varying fertility

Bulls used in artificial insemination, with apparently normal semen quality, can vary significantly in their field fertility. This study aimed to characterize the transcriptome of spermatozoa from high (HF) and low (LF) fertility bulls at the mRNA and miRNA level in order to identify potential novel markers of fertility. Holstein-Friesian bulls were assigned to either the HF or LF group (n = 10 per group) based on an adjusted national fertility index from a minimum of 500 inseminations. Total RNA was extracted from a pool of frozen-thawed spermatozoa from three different ejaculates per bull, following which mRNA-seq and miRNA-seq were performed. Six mRNAs and 13 miRNAs were found differentially expressed (P < 0.05, FC > 1.5) between HF and LF bulls. Of particular interest, the gene pathways targeted by the 13 differentially expressed miRNAs were related to embryonic development and gene expression regulation. Previous studies reported that disruptions to protamine 1 mRNA (PRM1) had deleterious consequences for sperm chromatin structure and fertilizing ability. Notably, PRM1 exhibited a higher expression in spermatozoa from LF than HF bulls. In contrast, Western Blot analysis revealed a decrease in PRM1 protein abundance for spermatozoa from LF bulls; this was not associated with increased protamine deficiency (measured by the degree of chromatin compaction) or DNA fragmentation, as assessed by flow cytometry analyses. However, protamine deficiency was positively and moderately correlated with the percentage of spermatozoa with DNA fragmentation, irrespective of fertility group. This study has identified potential biomarkers that could be used for improving semen quality assessments of bull fertility.

[1]  B. Lazzari,et al.  A Combined Flow Cytometric Semen Analysis and miRNA Profiling as a Tool to Discriminate Between High- and Low-Fertility Bulls , 2021, Frontiers in Veterinary Science.

[2]  P. Lonergan,et al.  Comprehensive functional analysis reveals that acrosome integrity and viability are key variables distinguishing artificial insemination bulls of varying fertility. , 2021, Journal of dairy science.

[3]  A. Kumaresan,et al.  Comparative Transcriptomic Analysis of Spermatozoa From High- and Low-Fertile Crossbred Bulls: Implications for Fertility Prediction , 2021, Frontiers in Cell and Developmental Biology.

[4]  P. Lonergan,et al.  Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle , 2020, Scientific Reports.

[5]  J. Morrell,et al.  RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa , 2020, Animal reproduction science.

[6]  Frøydis D Myromslien,et al.  Differences in sperm functionality and intracellular metabolites in Norwegian Red bulls of contrasting fertility. , 2020, Theriogenology.

[7]  F. Peñagaricano,et al.  Integration of whole-genome DNA methylation data with RNA sequencing data to identify markers for bull fertility. , 2020, Animal genetics.

[8]  L. Jouneau,et al.  A comprehensive overview of bull sperm-borne small non-coding RNAs and their diversity across breeds , 2020, Epigenetics & Chromatin.

[9]  Xin Wang,et al.  MicroRNA expression profile analysis in sperm reveals hsa-mir-191 as an auspicious omen of in vitro fertilization , 2020, BMC Genomics.

[10]  E. Menezes,et al.  Sperm miR‐15a and miR‐29b are associated with bull fertility , 2020, Andrologia.

[11]  F. Peñagaricano,et al.  Characterization and functional roles of paternal RNAs in 2–4 cell bovine embryos , 2019, Scientific Reports.

[12]  M. I. Sousa,et al.  The mTOR Pathway in Reproduction: From Gonadal Function to Developmental Coordination. , 2019, Reproduction.

[13]  M. E. Assumpção,et al.  Sperm DNA fragmentation: causes and identification , 2019, Zygote.

[14]  C. Celik-Ozenci,et al.  FoxO transcription factors 1 regulate mouse preimplantation embryo development , 2019, Journal of Assisted Reproduction and Genetics.

[15]  Ana Kozomara,et al.  miRBase: from microRNA sequences to function , 2018, Nucleic Acids Res..

[16]  N. Yang,et al.  Identification and differential expression of microRNAs in the testis of chicken with high and low sperm motility. , 2018, Theriogenology.

[17]  S. Eggener,et al.  Region-specific microRNA signatures in the human epididymis , 2018, Asian journal of andrology.

[18]  Vimlesh Kumar,et al.  Transcriptional abundance of type-1 endocannabinoid receptor (CB1) and fatty acid amide hydrolase (FAAH) in bull spermatozoa: Relationship with field fertility. , 2018, Theriogenology.

[19]  L. Jouneau,et al.  A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific features , 2018, BMC Genomics.

[20]  K. Boekelheide,et al.  High‐quality human and rat spermatozoal RNA isolation for functional genomic studies , 2018, Andrology.

[21]  F. Morel,et al.  Sperm RNA preparation for transcriptomic analysis: Review of the techniques and personal experience , 2017, Andrologia.

[22]  R. Aitken.  Reactive oxygen species as mediators of sperm capacitation and pathological damage , 2017, Molecular reproduction and development.

[23]  H. Khatib,et al.  MicroRNA Signaling in Embryo Development , 2017, Biology.

[24]  J. Ravindra,et al.  Spermatozoal transcripts expression levels are predictive of semen quality and conception rate in bulls (Bos taurus). , 2017, Theriogenology.

[25]  M. Palladino,et al.  MicroRNAs downregulated following immune activation of rat testis , 2017, American journal of reproductive immunology.

[26]  Shujun Zhang,et al.  Analysis of Bos taurus and Sus scrofa X and Y chromosome transcriptome highlights reproductive driver genes. , 2017, Oncotarget.

[27]  J. Ravindra,et al.  Occurrence and functional significance of the transcriptome in bovine (Bos taurus) spermatozoa , 2017, Scientific Reports.

[28]  M. Kaproth,et al.  Oligo-dT selected spermatozoal transcript profiles differ among higher and lower fertility dairy sires. , 2017, Animal reproduction science.

[29]  J. Kastelic,et al.  Associations of hypoosmotic swelling test, relative sperm volume shift, aquaporin7 mRNA abundance and bull fertility estimates. , 2017, Theriogenology.

[30]  A. Stella,et al.  Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between High- and Low-motile sperm populations , 2017, BMC Genomics.

[31]  B. Nixon,et al.  Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome , 2016, Scientific Reports.

[32]  Wei Yan,et al.  Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development , 2016, Development.

[33]  L. Chevrier,et al.  Use of combinations of in vitro quality assessments to predict fertility of bovine semen. , 2015, Theriogenology.

[34]  J. Ravindra,et al.  Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus). , 2015, Analytical biochemistry.

[35]  K. Klinga-Levan,et al.  Expression of miRNAs in Bull Spermatozoa Correlates with Fertility Rates. , 2015, Reproduction in domestic animals = Zuchthygiene.

[36]  D. Bartel,et al.  Predicting effective microRNA target sites in mammalian mRNAs , 2015, eLife.

[37]  A. Kaya,et al.  Sperm Protamine-Status Correlates to the Fertility of Breeding Bulls1 , 2015, Biology of reproduction.

[38]  A. Giwercman,et al.  Serum miR-155 as a potential biomarker of male fertility. , 2015, Human reproduction.

[39]  K. Aston,et al.  Paternal influence of sperm DNA integrity on early embryonic development. , 2014, Human reproduction.

[40]  Md. Munir Hossain,et al.  The Expression Pattern of microRNAs in Granulosa Cells of Subordinate and Dominant Follicles during the Early Luteal Phase of the Bovine Estrous Cycle , 2014, PloS one.

[41]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[42]  J. Mercader,et al.  New insights into the expression profile and function of micro-ribonucleic acid in human spermatozoa. , 2014, Fertility and sterility.

[43]  N. Corbet,et al.  Sperm protamine deficiency correlates with sperm DNA damage in Bos indicus bulls , 2014, Andrology.

[44]  Björn Usadel,et al.  Trimmomatic: a flexible trimmer for Illumina sequence data , 2014, Bioinform..

[45]  Zongfu Cao,et al.  MiR‐98 is involved in rat embryo implantation by targeting Bcl‐xl , 2014, FEBS letters.

[46]  U. Singh,et al.  Differential expression of protamine 1 and 2 genes in mature spermatozoa of normal and motility impaired semen producing crossbred Frieswal (HF×Sahiwal) bulls. , 2013, Research in veterinary science.

[47]  J. Kastelic,et al.  Associations of adiponectin and fertility estimates in Holstein bulls. , 2013, Theriogenology.

[48]  M. Kaproth,et al.  Cryopreserved Bovine Spermatozoal Transcript Profile as Revealed by High-Throughput Ribonucleic Acid Sequencing1 , 2013, Biology of reproduction.

[49]  S. Krawetz,et al.  Isolating mRNA and small noncoding RNAs from human sperm. , 2013, Methods in molecular biology.

[50]  Cole Trapnell,et al.  TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions , 2013, Genome Biology.

[51]  J. Kastelic,et al.  Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls. , 2012, Theriogenology.

[52]  J. Padbury,et al.  Dynamics of microRNAs in bull spermatozoa , 2012, Reproductive Biology and Endocrinology.

[53]  Jian Ye,et al.  Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction , 2012, BMC Bioinformatics.

[54]  J. Dejarnette,et al.  Impact of genomic selection of AI dairy sires on their likely utilization and methods to estimate fertility: a paradigm shift. , 2012, Theriogenology.

[55]  P. Jagodziński,et al.  Impact of protamine transcripts and their proteins on the quality and fertilization ability of sperm and the development of preimplantation embryos. , 2012, Reproductive biology.

[56]  Sebastian D. Mackowiak,et al.  miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades , 2011, Nucleic acids research.

[57]  X. Chen,et al.  Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility. , 2011, Clinical chemistry.

[58]  R. Kasimanickam,et al.  Association of CRISP2, CCT8, PEBP1 mRNA abundance in sperm and sire conception rate in Holstein bulls. , 2011, Theriogenology.

[59]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[60]  S. M. Chuva de Sousa Lopes,et al.  The different shades of mammalian pluripotent stem cells. , 2011, Human reproduction update.

[61]  L. O’Neill,et al.  MicroRNAs: the fine-tuners of Toll-like receptor signalling , 2011, Nature Reviews Immunology.

[62]  D. Berry,et al.  Evaluation of bull fertility in dairy and beef cattle using cow field data. , 2011, Theriogenology.

[63]  Antoine H. F. M. Peters,et al.  Genetic vasectomy—Overexpression of Prm1‐EGFP fusion protein in elongating spermatids causes dominant male sterility in mice , 2010, Genesis.

[64]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[65]  M. Goddard,et al.  Invited review: Genomic selection in dairy cattle: progress and challenges. , 2009, Journal of dairy science.

[66]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[67]  S. Krawetz,et al.  The Preparation of Human Spermatozoal RNA for Clinical Analysis , 2007, Archives of andrology.

[68]  P. Jagodziński,et al.  Evaluation of protamines 1 and 2 transcript contents in spermatozoa from asthenozoospermic men. , 2007, Folia histochemica et cytobiologica.

[69]  C. Peterson,et al.  Sperm protamine 1/protamine 2 ratios are related to in vitro fertilization pregnancy rates and predictive of fertilization ability. , 2006, Fertility and sterility.

[70]  D. Carrell,et al.  A novel mechanism of protamine expression deregulation highlighted by abnormal protamine transcript retention in infertile human males with sperm protamine deficiency. , 2006, Molecular human reproduction.

[71]  S. Lambard,et al.  Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors: relationship to sperm motility and capacitation. , 2004, Molecular human reproduction.

[72]  M. Virro,et al.  Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. , 2004, Fertility and sterility.

[73]  M. Nasr-Esfahani,et al.  Chromomycin A3 Staining as a Useful Tool for Evaluation of Male Fertility , 2004, Journal of Assisted Reproduction and Genetics.

[74]  P. Khatri,et al.  Spermatozoal RNA profiles of normal fertile men , 2002, The Lancet.

[75]  D. Miller,et al.  Can relative spermatozoal galactosyltransferase activity be predictive of dairy bull fertility? , 2000, Journal of dairy science.

[76]  D. Evenson,et al.  Sperm chromatin structure assay is useful for fertility assessment. , 2000, Methods in cell science : an official journal of the Society for In Vitro Biology.

[77]  Susumu Goto,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 2000, Nucleic Acids Res..

[78]  Hiroyuki Ogata,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 1999, Nucleic Acids Res..

[79]  I. Georgiou,et al.  Chromomycin A3-staining as an indicator of protamine deficiency and fertilization. , 1996, International journal of andrology.

[80]  R. Oliva,et al.  Rapid analysis of mammalian sperm nuclear proteins. , 1993, Analytical biochemistry.