Isolation, in vitro study, and stem cell markers for type A spermatogonia in a Characiformes species

Autor(es)

Batlouni, Sergio R. ^[1] ; Cassel, Monica ^[2] ; Camargo, Marilia P. ^[3]

Afiliação do(s) autor(es)

^[1] Sao Paulo State Univ UNESP, Sao Paulo State Univ CAUNESP, Aquaculture Ctr, Campus Jaboticabal, Jaboticabal, SP - Brazil

^[2] Mato Grosso Fed Inst Educ Sci & Technol, Dept Educ, Sci Anim Sci, Campus Alta Floresta, Floresta, Mato Grosso - Brazil

^[3] Univ Sao Paulo, Inst Biomed Sci, Dept Cell & Dev Biol, Fish Endocrinol Lab, Ave Prof Lineu Prestes 1524, Lab 426, BR-05508000 Sao Paulo, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Molecular Reproduction and Development; v. 87, n. 7 JUN 2020.
• Citações Web of Science: 0

Resumo

The objective of this study was to establish a protocol for the characterization, isolation, and culture of type A spermatogonia using specific molecular markers for these cells in fish. To this end, adultProchilodus lineatustestes were collected and digested enzymatically and the resulting testicular suspension was separated using a discontinuous Percoll gradient, followed by differential plating. The cell cultures obtained were monitored for 15 days and analyzed using the immunofluorescence method with anti-Vasa, anti-GFR alpha 1, and anti-OCT4 antibodies. Spermatogonial enrichment was also performed using flow cytometry. Although discontinuous Percoll gradient centrifugation followed by differential plating enabled the removal of differentiated germ cells and somatic cells, enriching the pool of type A spermatogonia, the enrichment of type A spermatogonia through flow cytometry of samples without Percoll proved to be more efficient. Prominent cell agglomerates that were characterized according to different stem cell markers as type A spermatogonia were observed during the 15 days of the cell culture. The use of immunoperoxidase and western blot analysis methods confirmed the specificity of the markers for type A spermatogonia ofP. lineatus. When combined with specific cell culture conditions, the positive characterization of these molecular markers clarified certain aspects of spermatogonial regulation, such as survival and proliferation. Finally, understanding the regulation of the in vitro germ cell maintenance process may contribute to the enhancement of in vivo and in vitro reproduction techniques of endangered or aquaculture fish species. (AU)

• Processo FAPESP: 10/01899-9 - CARACTERIZAÇÃO, ISOLAMENTO E CULTURA DE ESPERMATOGÔNIAS PRIMÁRIAS DE CURIMBATÁ, Prochilodus lineatus (Valencienes, 1847).
• Beneficiário: Gisele Cristiane de Melo Dias
• Modalidade de apoio: Bolsas no Brasil - Doutorado