Distinct roles of melatonin receptors in spermatogenesis in vitro in four-eyed sleeper (Bostrychus sinensis)

Melatonin is a master neuroendocrine hormone in regulation of circadian rhythm and seasonal breeding behaviors in animal kingdom. Recently, we used the scaffold material to establish the three-dimension (3D) culture method, by which we found that melatonin promotes spermatogenesis in vitro and production of functional sperm in the marine fish four-eyed sleeper ( Bostrychus sinensis ). However, the mechanism how melatonin regulates spermatogenesis in vitro in 3D culture is unclear. In this study, we improved the method by using scaffold-free material spheroid microplate to shorten the time for generation of haploid cells and to yield a higher proportion of haploid sperm, which serves as a helpful tool to study molecular mechanisms underlying spermatogenesis in vitro . In the spheroid microplate, melatonin significantly increased the expression of its membrane receptors ( Mtnr1 , Mtnr2 and Mtnr3 ), suggesting they might play an important role in spermatogenesis. Therefore, we cloned and characterized their expression patterns and found that they were expressed in various types of male germlines in the testis. Knockdown experiments indicated that inhibition of Mtnr1 , Mtnr2 and Mtnr3 resulted in significant reduction of haploid sperms. Whereas, inhibition of Mtnr2 and Mtnr3 rather than Mtnr1 significantly increased and decreased the expression of proliferation‑related genes. Loss-of and gain-of function experiments indicated that they had different effects on downstream testicular development associated signaling. Mtnr1 regulated phosphorylation of ERK1/2, CREB and CAMK2, whereas Mtnr2 affected phosphorylation of CREB and Mtnr3 selectively affected both ERK1/2 and CAMK2, respectively. The present work demonstrated that Mtnrs are important for production of haploid cells and revealed distinct functions in regulation of cell proliferation and downstream signaling, providing insights how melatonin regulates spermatogenesis in vitro and a promising strategy for enhancing the 3D culture system by leveraging the melatonin pathway in turn. • Improving the 3D culture method of fish spermatogenesis in vitro. • Characterizing the expression patterns of four-eyed sleeper Mtnr. • Revealing distinct roles of Mtnr in cell proliferation and meiosis. • Differential regulation of Mtnrs in downstream signaling.