Title : Establishment of in vitro spermatogenesis from spermatocytes in chinese black sleeper, Bostrychus sinensis
Spermatogenesis is a series of unique process including mitosis, meiosis and metamorphosis, which is regulated by the interaction between germ cells and somatic cells in the testis. Many of the details of these processes have yet to be elucidated. Cell culture is a suitable system to investigate the process of spermatogenesis. Chinese black sleeper (Bostrychus sinensis), is a commercially important fish that widely distributed in Southeast Asia. However, long term aquaculture practice found that a high percentage of male infertility in Chinese black sleeper. Therefore, in vitro culture of spermatocytes to produce functional sperm is a useful method to improve the aquaculture of this species. Herein, we performed 3D culture of testis tissue in vitro. The spermatocytes formed clusters to proliferate and differentiate during the long period of 3D culture. Immunofluorescence analysis showed that there were many Vasa-positive cells within the cell cluster. EdU assay indicated that the cultured cells had good proliferation activity. Furthermore, at day 9 after culture, numerous mature sperms were found within the cluster and the total number of sperm was rapidly increased along with the proceeding of 3D culture. Moreover, we performed a comparative in vitro fertilization (IVF) analysis by using fresh sperm and sperms from 3D-culture. IVF results indicated that 3D-culture sperm had the ability to fertilize the egg, and the obtained embryos could normally develop and hatch, despite the fertilization rate was lower than that of fresh sperm. However, the mechanism underlying spermatogenesis in 3D culture is remained unclear. Therefore, our present work provided an important method for production of functional sperms, which is not only useful for aquaculture of male farming fish but also a model for investigating spermatogenesis.