Comparison of Diverse Differential Plating Methods to Enrich Bovine Spermatogonial Cells

Autor(es)

Giassetti, M. I. ^[1] ; Goissis, M. D. ^[1] ; de Barros, F. R. O. ^{[1, 2]} ; Bruno, A. H. ^[1] ; Assumpcao, M. E. O. A. ^[1] ; Visintin, J. A. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Sch Vet Med & Anim Sci, Dept Anim Reprod, Av Prof Dr Orlando Marques de Pavia 87, BR-05508270 Sao Paulo, SP - Brazil

^[2] Hosp Sick Children, 555 Univ Ave, Toronto, ON M5G 1X8 - Canada

• Tipo de documento: Artigo Científico
• Fonte: REPRODUCTION IN DOMESTIC ANIMALS; v. 51, n. 1, p. 26-32, FEB 2016.
• Citações Web of Science: 4

Resumo

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 degrees 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. (AU)

• Processo FAPESP: 12/05474-8 - Genes alvo de YY2 como marcadores de SSC
• Beneficiário: Mariana Ianello Giassetti
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado
• Processo FAPESP: 13/03495-0 - Identificação e cultivo de células-tronco espermatogoniais indiferenciadas em bovinos
• Beneficiário: José Antonio Visintin
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 11/20115-1 - A expressão de marcadores moleculares para células-tronco espermatogoniais bovinas difere entre animais pré-púberes e adultos?
• Beneficiário: Mariana Ianello Giassetti
• Modalidade de apoio: Bolsas no Brasil - Doutorado