Spermatogonia are increasingly used in biotechnological innovation related to transplantation in endangered or commercially cultured fish species and to cryopreserve genetic material. In some species, spermatogonia enrichment and in vitro cultivation are crucial steps to obtain enough of these germ cells. Senegalese sole (Solea senegalensis) is a flatfish species farmed in the South of Europe with a high market price, but that presents a reproductive disorder which does not allow to close the live cycle in captivity, making this species propitious to carry out spermatogonial biotechnological applications. While a protocol using strainers has been recently implemented to enrich Senegalese sole spermatogonia, the parameters required for the in vitro culture of spermatogonia are still unknown for this species. In this context, the aim of this study was to determine the growth factors needed for the in vitro cultivation of Senegalese sole spermatogonia.
For this purpose, testes from juvenile fish (~1 year old; 35-50 g) were pooled (3 pools; 12 testes per pool) and processed for spermatogonia (SPG) enrichment. Briefly, testes were cut into 1 mm3 fragments, dissociated using a mechanical and enzymatic treatment, and passed through a 100 µm-strainer to remove fragments. Cells present in the flowthrough were passed sequentially through two 5 µm-strainers to enrich cell suspension in SPG (> 5 µm). Cells were seeded in a 6-well gelatine-coated plate at 2x05 cells/well density in triplicates for each experimental group. Experimental groups were: control (without growth factors), GF (a cocktail of recombinant murine growth factors: GDNF, IGF-1, and bFGF) and S (Senegalese sole serum). Cells were maintained at 18 ºC and the number of spermatogonia was determined at 24- and 48-hours post-seeding (hps) from 6 images taken using an inverted microscope equipped with phase contrast.
While the number of SPG was significantly higher in GF (40 ± 24 cells; P = 0.03) than in Control (14 ± 7 cells) at 24-hps, the number of SPG in S (27 ± 13 cells) was not different from Control or GF. No differences were observed among the experimental groups at 48-hps. Interestingly, the number of SPG increased in all groups from 24 to 48-hps (Control: 97 ± 45; GF: 103 ± 45; S: 92 ± 39). This suggests that the presence of growth factors in the media is important until 24-hps but not at 48-hps. However, the use of growth factors, including serum, boosted spermatogonia proliferation faster than the control group after 24 hours of SPG culture. The initial seeding density seems to be very important for SPG proliferation. More studies are needed to validate these conditions and test other growth factors to finally describe a protocol for spermatogonia cultivation in Senegalese sole.
This study was funded by Portuguese national funds (FCT) through projects GERMROS (EXPL/CVT-CVT/0305/2021), UIDB/04326/2020, UIDP/04326/2020 and LA/P/0101/2020, and by European funds through project BREEDFLAT (PT-INNOVATION-0080-EEA grants). Contract 2020.04181.CEECIND was awarded to E. Fatsini. European Union’s Horizon 2020 research and innovation programme under grant agreement No. 871108 (AQUAEXCEL3.0).