Melatonin May Improve Post-Thaw Sperm Motility in Epinephelus fuscoguttatus by Potentially Regulating Mitochondrial mPTP via the MT2/PI3K/GSK-3β Pathway: First Evidence in Teleosts

Melatonin, a well-known antioxidant, has been widely used in sperm cryopreservation of various animals, but its regulatory mechanism in fish remains unclear. This first study on teleosts suggests a potential molecular mechanism by which melatonin may improve post-thaw sperm quality of Epinephelus fuscoguttatus via targeting mitochondrial function. Compared with the melatonin group, the MT1 receptor-inhibited group showed slightly higher sperm motility (77.09 ± 3.41% vs. 76.50 ± 1.10%), significantly inhibited mitochondrial permeability transition pore (mPTP) opening (12.64 ± 1.05% vs. 18.29 ± 1.38%), and maintained higher mitochondrial membrane potential (MMP; 85.86 ± 0.18% vs. 81.81 ± 0.69%), with both groups performing better than the control. In contrast, the MT2-inhibited and MT1/2 dual-inhibited groups exhibited reduced sperm quality compared with the MT group, suggesting that MT2 may serve as the core receptor for melatonin to regulate mitochondrial homeostasis in teleosts. Mechanistically, melatonin-activated MT2 potentially inhibits mPTP opening via the PI3K/Akt/GSK-3β pathway, and this protective effect was abrogated by the PI3K and GSK-3β inhibitors. This receptor-mediated process synergized with melatonin’s direct antioxidant effect, as ROS levels in all melatonin-treated groups were significantly lower than the control. This study is the first to find pharmacological evidence for the melatonin–MT2/PI3K/GSK-3β axis in maintaining teleost sperm mitochondrial function; it also reveals potential mechanistic differences between teleosts and mammals and fills a critical knowledge gap regarding this signaling cascade in teleost reproductive biology.