Development of commercial aquaculture for many marine flatfish species such as southern flounder is severely limited by poor male reproductive performance, especially reductions in sperm motility and fertility, but the underlying causes of these reductions and the mechanisms regulating sperm motility are largely unknown. Our previous studies have shown that acute treatment of southern flounder and Atlantic croaker sperm in vitro with two progestins, a teleost progestogen hormone (20β-S) and a specific agonist (Org-OD-02-0), causes rapid induction of sperm hypermotility and increased fertility through activation of progestin membrane receptor alpha (mPRα) coupled to a stimulatory G protein and increases in cAMP/Acy (adenylyl cyclase) activity. Progestin-induced hypermotility is also mediated through Egfr (epidermal growth factor receptor)/Mapk (mitogen-activated protein kinase)/Erk1/2 (extracellular-regulated kinase 1/2), and Pi3 (phosphatidylinositol-3-kinase)/Akt (protein kinase B)/PDE (phosphodiesterase) signaling pathways as well as increases in calcium levels in croaker sperm, but additional studies are required to determine whether they are involved in the motility response in flounder sperm.
In the present study the role of the Egfr/Mapk/Erk1/2 signaling pathways in progestin stimulation of flounder sperm hypermotility was investigated using specific pharmacological tools. Aliquots of freshly collected sperm, diluted in physiological saline, were preincubated with two EGFR inhibitors, AG1478, and AG825, and an Erk inhibitor U0126, for 30 min. prior to treatment with progestins for 1 min. and motility activation with a hyperosmotic medium. Sperm motility was observed under a microscope and recorded for 1 min. with a video camera and swimming speed was calculated using motion analysis software. Whereas treatment with the three inhibitors did not alter basal sperm motility, all of them significantly attenuated the hypermotility response to the progestins. Interestingly, 1 min. in vitro treatment with 100 nM human recombinant Egf, the Egfr agonist, mimicked the stimulatory effects of progestins on sperm motility, calcium levels, and fertility. The results suggest Egfr/Mapk/Erk signaling is involved in the hypermotility and fertility response to progestins in flounder sperm. The cAMP/Acy signaling pathway has been implicated in progestin upregulation of flounder sperm motility which is accompanied by a rapid increase in cAMP levels. The Acy activator, forskolin, mimicked the stimulatory effects of progestins on cAMP levels but its effects on sperm motility were not investigated. The present results show that direct in vitro treatment with 10µM forskolin for 1 min. mimics the stimulatory action of progestins on flounder sperm motility, calcium levels, and fertility.
These results demonstrate that flounder sperm motility and fertility can be directly stimulated by recombinant Egfr and forskolin and suggest they could potentially be used as pharmacological agents to enhance reproductive performance of flounder broodstock. This research was supported by USDA NIFA grant 2018-67015-27574 to PT.