Melatonin is a clock- hormone produced at night and responsible for the synchronization of biological rhythms, including reproduction. However, its exact production sites, targets and function in fish are still unclear. This study aimed to ascertain melatonin presence in Senegalese sole (Solea senegalensis ) seminal plasma, its effects on sperm quality, mechanisms of action in spermatozoa, and its origin. For this, wild and F1 broodstocks were sampled along three reproductive seasons to collect sperm and blood at mid-light (ML) and mid-dark (MD) daytimes. Firstly , using CASA software, the effects of endogenous melatonin on sperm motility and velocity were assessed. In the second year, melatonin was quantified in blood and seminal plasmas through radioimmunoassay (RIA), and the concentration of sex steroids (T and 11KT ) and antioxidant status (TAS) were evaluated in seminal plasma with commercial kits. The mechanism of melatonin entrance in the cell was studied using FITC-melatonin and a confocal microscope . Finally, the expression of genes involved in melatonin biosynthesis (tph1a, tph2, hiomt1, aanat1a, aanat1b, and aanat2) and melatonin receptors ( mel1, mel1c and mel2 ) was evaluated in the brain, eye and testis, by quantitative real-time PCR.
Blood plasma melatonin was higher at MD, together with s perm m otility parameters , likely to be enhanced by the endogenous melatonin found to be present in seminal plasma , with daily oscillations, and contributing to sperm TAS (Fig 1A). Also, it was demonstrated that melatonin easily crosses the plasmatic membrane of spermatozoa, spreading inside the nucleus and mitochondria. Gene expression of receptors mel1 and mel2 , and the enzymes tph1a, aanat1a, aanat2, and hiomt1 , was detected in male gonads , indicating testes as a peripherical location for melatonin production (Fig 1B). Throughout the study, wild and F1 males showed distinct patterns, giving new information on the dysregulation of the circadian system in F1 animals.
This study described the melatonin synthesis , pathway and action in the fish reproductive system for the first time , revealing that Senegalese sole testes are an extra-pineal production site of melatonin. Also, the dysregulation in the hypothalamus-pituitary-gonad axis in F1 sole males was highlighted . These findings can enable the aquaculture industry to obtain better sperm quality from F1 males in a non-invasive way (collecting sperm at night ) and improve Senegalese sole reproduction success.
This study was funded by FCT through SFRH/BD/148280/2019 and COVID/BD/153473/2023 grants to FF, contract DL 57/2016/CP1361/CT0007 to CCVO, and CCMAR Strategic Project (UIDB/04326/2020, UIDP/04326/2020 and doi.org/10.54499/LA/P/0101/2020); and by “Programa Crescimento Azul (#4)” through BREEDFLAT (PT-INNOVATION-0080) project.