title: Comparison of Diverse Differential Plating Methods to Enrich Bovine Spermatogonial Cells.
abstract: Spermatogonial stem cells (SSC) have important applications in domestic animal reproduction and advanced biotechnologies. Because differential plating is one of the most common methods used for SSC enrichment, the goal of this study was to compare three differential plating methods for the enrichment of bovine SSC. To achieve this goal, testicular parenchyma from pre-pubertal calves was minced and single cells were obtained after two enzymatic digestions. We compared three coating methods for differential plating: laminin (20 ng/ml), BSA (0.05 mg/ml) and PBS. Cells were incubated at 37°C, 5% CO2 in air for 15 min onto laminin-coated dishes or 2 h onto BSA- or PBS-coated dishes. Cell viability was assessed by trypan blue exclusion method. Recovered cells were analysed for the expression of SSC molecular markers by quantitative RT-PCR (GFRA1, CXCR4, ITGA6, THY1) and flow cytometry (GFRA1, CXCR4 and ITGA6). Cells at time 0, adherent cells on laminin and non-adherent cells from BSA and PBS groups had the same cell viability (p = 0.0655). GFRA1, CXCR4 and THY1 relative gene expression was higher (p = 0.0402, p = 0.0007, p = 0.0117, respectively) for non-adherent cells selected in PBS group. Flow cytometry analysis revealed that the presence of GFRA-positive (GFRA+) cells was higher in non-adherent cells from BSA and PBS groups (p < 0.001). However, laminin-adherent cells had higher number of ITGA6+ cells (p < 0.001) and lower presence of CXCR4+ cells (p = 0.0012). In conclusion, differential plating is an effective method for the enrichment of bovine undifferentiated spermatogonia and higher expression of SSC markers is obtained without laminin or BSA coating.
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: M I
authors.initials: MI
authors.lastname: Giassetti
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: M D
authors.initials: MD
authors.lastname: Goissis
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: Fro
authors.initials: F
authors.lastname: de Barros
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: A H
authors.initials: AH
authors.lastname: Bruno
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: Meoa
authors.initials: M
authors.lastname: Assumpção
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: J A
authors.initials: JA
authors.lastname: Visintin
mesh_terms: D000072956:Adult Germline Stem Cells / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002417:Cattle; D018929:Cell Culture Techniques / Q000662:veterinary*; D003470:Culture Media; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000662:veterinary; D015870:Gene Expression; D039503:Integrin alpha6 / Q000032:analysis / Q000235:genetics; D007797:Laminin; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D019718:Receptors, CXCR4 / Q000032:analysis / Q000235:genetics; D012710:Serum Albumin, Bovine; D012741:Sexual Maturation; D013737:Testis / Q000166:cytology
chemical_list: D015415:Biomarkers; D003470:Culture Media; D039503:Integrin alpha6; D007797:Laminin; D019718:Receptors, CXCR4; D012710:Serum Albumin, Bovine; D004247:DNA