title: Male infertility and copy number variants (CNVs) in the dog: a two-pronged approach using Computer Assisted Sperm Analysis (CASA) and Fluorescent In Situ Hybridization (FISH).
abstract: BACKGROUND
  Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors.
  
  
  RESULTS
  We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development.
  
  
  CONCLUSIONS
  We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species.
authors.forename: Daniele
authors.initials: D
authors.lastname: Cassatella
authors.forename: Nicola Antonio
authors.initials: NA
authors.lastname: Martino
authors.forename: Luisa
authors.initials: L
authors.lastname: Valentini
authors.forename: Antonio Ciro
authors.initials: AC
authors.lastname: Guaricci
authors.forename: Maria Francesca
authors.initials: MF
authors.lastname: Cardone
authors.forename: Flavia
authors.initials: F
authors.lastname: Pizzi
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Dell'Aquila
authors.affiliation: Dipartimento di Biologia, Università degli Studi di Bari "Aldo Moro", Bari, Italy. mario.ventura@uniba.it.
authors.forename: Mario
authors.initials: M
authors.lastname: Ventura
mesh_terms: D000818:Animals; D002874:Chromosome Mapping; D056915:DNA Copy Number Variations / Q000235:genetics*; D004285:Dogs; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence; D007248:Infertility, Male / Q000235:genetics* / Q000473:pathology; D008297:Male; D013091:Spermatogenesis / Q000235:genetics; D013094:Spermatozoa / Q000473:pathology*