Prostaglandin E2 synchronizes lunar-regulated beach spawning in grass puffers.

Many organisms living along the coastlines synchronize their reproduction with the lunar cycle. At the time of spring tide, thousands of grass puffers (Takifugu alboplumbeus) aggregate and vigorously tremble their bodies at the water's edge to spawn. To understand the mechanisms underlying this spectacular semilunar beach spawning, we collected the hypothalamus and pituitary from male grass puffers every week for 2 months. RNA sequencing (RNA-seq) analysis identified 125 semilunar genes, including genes crucial for reproduction (e.g., gonadotropin-releasing hormone 1 [gnrh1], luteinizing hormone ß subunit [lhb]) and receptors for pheromone prostaglandin E (PGE). PGE2 is secreted into the seawater during the spawning, and its administration activates olfactory sensory neurons and triggers trembling behavior of surrounding individuals. These results suggest that PGE2 synchronizes lunar-regulated beach-spawning behavior in grass puffers. To further explore the mechanism that regulates the lunar-synchronized transcription of semilunar genes, we searched for semilunar transcription factors. Spatial transcriptomics and multiplex fluorescent in situ hybridization showed co-localization of the semilunar transcription factor CCAAT/enhancer-binding protein δ (cebpd) and gnrh1, and cebpd induced the promoter activity of gnrh1. Taken together, our study demonstrates semilunar genes that mediate lunar-synchronized beach-spawning behavior. VIDEO ABSTRACT.

Modulation of circadian clock by crude drug extracts used in Japanese Kampo medicine.

Circadian rhythm is an approximately 24 h endogenous biological rhythm. Chronic disruption of the circadian clock leads to an increased risk of diabetes, cardiovascular disease, and cancer. Hence, it is important to develop circadian clock modulators. Natural organisms are a good source of several medicines currently in use. Crude drugs used in Japanese traditional Kampo medicine or folk medicines are an excellent source for drug discovery. Furthermore, identifying new functions for existing drugs, known as the drug repositioning approach, is a popular and powerful tool. In this study, we screened 137 crude drug extracts to act as circadian clock modulators in human U2OS cells stably expressing the clock reporter Bmal1-dLuc, and approximately 12% of these modulated the circadian rhythm. We further examined the effects of several crude drugs in Rat-1 fibroblasts stably expressing Per2-luc, explant culture of lung from Per2::Luciferase knockin mice, and zebrafish larvae in vivo. Notably, more than half of the major ingredients of these crude drugs were reported to target AKT and its relevant signaling pathways. As expected, analysis of the major ingredients targeting AKT signaling confirmed the circadian clock-modulating effects. Furthermore, activator and inhibitor of AKT, and triple knockdown of AKT isoforms by siRNA also modulated the circadian rhythm. This study, by employing the drug repositioning approach, shows that Kampo medicines are a useful source for the identification of underlying mechanisms of circadian clock modulators and could potentially be used in the treatment of circadian clock disruption.