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

Autor: Dias GCM; Fish Endocrinology Laboratory, Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil., Batlouni SR; Aquaculture Center of São Paulo State University (CAUNESP), São Paulo State University (UNESP), Campus Jaboticabal, Jaboticabal, São Paulo, Brazil., Cassel M; Department of Education - Bachelor of Science in Animal Science, Mato Grosso Federal Institute of Education, Science, and Technology, Campus Alta Floresta, Alta Floresta, Mato Grosso, Brazil., Chehade C; Fish Endocrinology Laboratory, Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil., De Jesus LWO; Laboratory of Applied Animal Morphophysiology, Department of Histology and Embryology, Institute of Biological Sciences and Health, Federal University of Alagoas, Campus A. C. Simões, Maceió, Alagoas, Brazil., Branco GS; Fish Endocrinology Laboratory, Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil., Camargo MP; Fish Endocrinology Laboratory, Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil., Borella MI; Fish Endocrinology Laboratory, Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil.
Jazyk: angličtina
Zdroj: Molecular reproduction and development [Mol Reprod Dev] 2020 Jul; Vol. 87 (7), pp. 783-799. Date of Electronic Publication: 2020 Jun 18.
DOI: 10.1002/mrd.23394
Abstrakt: 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, adult Prochilodus lineatus testes 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-GFRα1, 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 of P. 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.
(© 2020 Wiley Periodicals LLC.)
Databáze: MEDLINE
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