Identification of specific molecular markers for spermatogonial stem cells in teleost is crucial for enhancing the efficacy of reproductive biotechnologies in aquaculture, such as transplantation and surrogate production in fishes. Since it is not yet possible to distinguish spermatogonial stem cells of European eel (Anguilla anguilla) using specific molecular markers, we isolated spermatogonial cells from immature European eels to find these potential markers. We attempted this by studying three candidate genes: vasa, nanos2, and dnd1. Two vasa (vasa1 and vasa2) genes, nanos2, and dnd1 were identified, characterized, and studied in the muscle, testis, and isolated spermatogonia. Our results showed that vasa1 and vasa2 had the highest levels of expression when measured by qPCR. In situ hybridization and immunochemistry assays showed that the four genes were localized explicitly in type A spermatogonia. However, vasa1 and vasa2 exhibited stronger signals in the immature testicular tissue than the other two potential markers. According to this, vasa1 and vasa2 were found to be the most effective markers for spermatogonial cells in the European eel.

Heat shock factors (HSFs) are transcription factors that regulate responses to environmental changes and play roles in physiological mechanisms like spermatogenesis. This study analyzed the HSF gene family and their expression profiles in the European eel under different environmental conditions and during testis maturation. Six HSF genes were identified in the studied vertebrates, in which the eel presents two HSF1 paralogs (HSF1a and HSF1b), likely resulting from the teleost whole-genome duplication event, while only one paralog is present for the other HSF (HSF2, HSF4, and HSF5). All five HSF genes were highly expressed in the testis, but some were also detected in the brain, intestine, and gills. Our findings showed that HSF1 changed their expression in response to different temperature and salinity conditions, suggesting that these may support males in perceiving the temperature and salinity changes possibly found during reproductive migration. During hCGrec-induced spermatogenesis, HSF genes presented a decreasing expression profile throughout testis maturation (with significant differences in HSF1a and HSF4), except HSF5, which showed the highest levels after 4 weeks of hormonal treatment. Our study indicates that HSF genes are potentially implicated in the response to environmental changes perception and during gonadal maturation.