Recombinant flounder Egf increases southern flounder sperm motility and fertility at the end of the reproductive season

Sub-optimal milt production and sperm quality are major causes of relatively poor fertilization and reproductive success of southern flounder (flounder, Paralichthys lethostigma ) in captivity. Previous work showed that progestin improved low motility of sperm and increased fertility of male flounder through membrane progestin receptor-mediated Egf /epidermal growth factor receptor ( Egfr ) signaling and that this effect can be mimicked by acute in vitro treatments with recombinant human Egf. However, knowledge of seasonal changes in southern flounder sperm Egf/Egfr physiology and confirmation that homologous recombinant southern flounder Egf exerts similar effects on flounder sperm is required to develop practical aquaculture methods to increase male broodstock fertility with recombinant Egf . Here, southern flounder Egf and Egfr were cloned and shown by phylogenetic analysis to have extensive sequence homologies with those of Japanese flounder and several other flatfish species. Egf and Egfr mRNAs and proteins were detected in southern flounder testes and sperm by RT-PCR and Western blotting with specific antibodies, respectively. There was a significant decrease in Egf mRNA expression in milt towards the end of the spawning season whereas Egfr mRNA and protein expression were unchanged. Recombinant southern flounder Egf, generated in a yeast expression system, was highly effective in increasing sperm motility and fertilization success. The results suggest that when endogenous Egf levels have decreased towards the end of the spawning season, Egfr on flounder sperm can still be activated with recombinant flounder Egf treatments to enhance the reproductive performance of male southern flounder broodstock in aquaculture farms. • Cloning of southern flounder Egf and Egfr revealed high homology to other flatfish. • Egf and Egfr were expressed in the sperm and testis of flounder. • Recombinant flounder Egf (rsfEgf) activated the flounder Egf receptor. • rsfEgf increased sperm Ca²⁺ concentrations, motility, and fertilization success.