Characterization of the migratory pattern of Primordial Germ Cells in three neotropical fish species aiming genetic conservation actions

Abstract

Abstract Primordial germ cells (PGCs) are embryonic pluripotent cells that can differentiate into spermatogonia and oogonia, making them a genetic source for germplasm conservation through cryobanking and the generation of germline chimeras. Therefore, PGC studies should be conducted for each species, given possible divergences in migratory patterns of PGCs. In this work, the PGCs of 3 neotropical species (Pseudopimelodus mangurus, Astyanax altiparanae, and Prochilodus lineatus) were characterized by microinjection into zygotes of mRNA synthesized from the ddx4 3'UTR sequence of P. mangurus. Visualization of labeled PGCs was achieved by fluorescence microscopy during embryonic development. In addition, ddx4 and dnd1 expressions were evaluated during embryonic development, larvae, and adult tissues of P. mangurus, to validate their use as a PGC marker. As a result, the effective identification of PGCs was obtained. DsRed-positive PGC of P. mangurus was observed in the hatching stage, GFP-positive PGC of A. altiparanae in the gastrula stage, and GFP-positive PGCs from P. lineatus were identified at the segmentation stage, with representative labeling percentages of 29% and 16% in A. altiparanae and P. lineatus respectively. The expression of ddx4 and dnd1 of P. mangurus confirmed the specificity of these genes in germ cells. These results point to the functionality of the P. mangurus ddx4 3'UTR sequence as a PGC marker, demonstrating that PGC labeling was more efficient in A. altiparanae and P. lineatus. The procedures used to identify PGCs in P. mangurus consolidate the first step for generating germinal chimeras as a conservation action of P. mangurus.