Annual patterns of ocular melatonin level in the female grass puffer, Takifugu alboplumbeus: possible involvement in seasonal reproductive response.

The aim of this study was to investigate the expression patterns of ocular melatonin in the annual reproductive cycle of the female grass puffer. Spawning season of the female grass puffer is from June to July in Jeju, South Korea. Time-resolved fluoroimmunoassay revealed that levels of ocular melatonin, which show an annual change, peaked in May (spawning season). Additionally, expression of reproductive-related genes also showed annual patterns: GnRH1 peaked in August, GnRH2 peaked in February, GnRH3, Kiss2, and LPXRFa peaked in November. These results suggest that ocular melatonin may be related to the annual reproductive cycle in the grass puffer. To better understand the photic regulation of AANAT1a mRNA in the retina, we observed the nocturnal pattern of ocular melatonin levels daily, which shows a nocturnal pattern in both short photoperiod (SD) and long photoperiod (LD) conditions. In the brain, AANAT2 mRNA also shows a nocturnal pattern in both SD and LD; however, the time of peak expression of AANAT2 mRNA was unchanged in both conditions. Following intraperitoneal injection of melatonin for 2 weeks, expression of GnRH2 and LPXRFa mRNA in the brain significantly increased, while that of Kiss2 mRNA was decreased, suggesting that melatonin has a reproduction-related effect. Furthermore, under SD and LD conditions for 14 weeks, the gonadosomatic index more increased and the maturity of the ovary progressed under LD compared with those under SD, suggesting that the SD photoperiodic signal inactivated ovarian development. These results indicate that the ocular melatonin may have a possible role in the reproductive endocrinology of the grass puffer.

Involvement of melatonin in transducing moon-related signals into the reproductive network of the female honeycomb grouper Epinephelus merra.

Most groupers (genus Epinephelus) inhabiting tropical and subtropical waters exhibit lunar-related reproductive cycles. Their gametes develop synchronously toward and are released around the species-selected moon phase. Periodical changes in cues from the moon are likely used as zeitgeber, and the hypothalamic-pituitary-gonadal (HPG) axis may be activated after cues are perceived by the sensory organ and transduced as internal signals. The objective of this study was to examine weekly changes in mRNA expression profiles of gonadotropin-releasing hormones (gnrh1 and gnrh2) and the ß-subunit of gonadotropins (fshß and lhß) during the spawning season (May to June) of the female honeycomb grouper Epinephelus merra, which spawns around the full moon period. When mature females were collected based on the lunar cycle, the gonadosomatic index peaked around the full moon. Ovarian histology revealed that oocytes laden with yolk developed toward the full moon and, subsequently, ovulatory follicles appeared around the last quarter moon, confirming lunar-related spawning with a full moon preference. Real-time quantitative polymerase chain reaction analyses revealed high abundances of fshß and lhß toward the first quarter moon, whereas concentrations of gnrh1 and gnrh2 increased around the last quarter moon and the first quarter moon, respectively, suggesting that transcription levels of these hormones fluctuate with the lunar cycle. The measurement of melatonin in the eye around the new moon and the full moon revealed that the ocular melatonin content was higher around the new moon than around the full moon, suggesting that the honeycomb grouper can perceive changes in moonlight. In addition, implantation of an osmotic pump containing melatonin into the body cavity of E. merra reduced the transcription levels of gonadotropins, suggesting that melatonin negatively affects hormonal synthesis at the HPG axis. We concluded that melatonin plays an essential role in transducing periodical changes in moonlight and that decreases in melatonin levels from the new moon to the full moon activate the HPG axis for entrainment of gonadal development and spawning.

Daily expression of a clock gene in the brain and pituitary of the Malabar grouper (Epinephelus malabaricus).

Recent studies have revealed that, in addition to regulating the circadian system, clock genes such as cryptochrome (Cry) genes are involved in seasonal and lunar rhythmicity in fish. This study clarified the transcriptional characteristics of a Cry subtype (mgCry2) in the brain of the Malabar grouper, Epinephelus malabaricus, which is an important aquaculture species that spawns around the new moon. The cDNA sequence of mgCry2 showed high identity (97-99%) with fish Cry2 and had an open reading frame encoding a protein with 170 amino acids. Phylogenetic analyses revealed that mgCRY2 had high identity with CRY in other fish species. Real-time quantitative polymerase chain reaction (qPCR) showed the widespread distribution of mgCry2 in neural (brain, pituitary, and retina) and peripheral (heart, liver, kidney, spleen, gill, intestine, and ovary) tissues. When immature Malabar groupers were reared under a light-dark cycle (LD = 12:12) and the amounts of mgCry2 mRNA in the telencephalon and diencephalon were measured at 4-h intervals, the levels increased during photophase and decreased during scotophase. Day-night variation in mgCry2 mRNA abundance was also observed in the pituitary. These daily profiles suggest that mgCry2 is a light-responsive gene in neural tissues. In situ hybridization analyses showed that mgCry2 was strongly transcribed in the nucleus lateralis tuberis of the ventral hypothalamus, peripheral area of the proximal pars distalis, and the pars intermedia of the pituitary. We conclude that clock genes expressed in the pituitary and diencephalon play a role in entraining the endocrine network of the Malabar grouper to periodic changes in external cues.