Reproduction Abstracts

Introduction: Spermatogenesis is a complex process in which spermatogonial stem cells (SSCs) self-renew, develop to differentiated spermatogonia, and then give rise to meiotic spermatocytes. Functional sperm are ultimately differentiated from postmeiotic haploid spermatids. Cell culture methods representing this process will facilitate analyzing molecular function and imaging molecules. Here we describe consecutive two culture methods by which zebrafish SSCs enable to differentiate to functional sperm in vitro.

Materials and methods: We used sox17 promoter–EGFP transgenic zebrafish that express GFP in the early stage of spermatogonia. Cells of the hypertrophied testis were cultured under effective long-term culture conditions for propagating zebrafish SSCs. After SSCs were maintained for 1 month, they were transferred and cultured on feeder layers of the Sertoli cell line in the medium that induced differentiation of spermatogonia.

Results and discussion: After 1 month of culture for SSCs, most germ cells became SSCs in morphology and expressed GFP. After plating SSCs on the feeders, meiotic spermatocytes appeared for 10 days. Expression of a meiotic marker, Sycp3, was also detected in spermatocytes. After 24 days of plating on the feeders, fertilized embryos were obtained by artificial insemination with differentiated sperm in vitro. They grew up normally, suggesting that normal haploid sperm were produced under this culture condition. Interestingly, sperm production continued for 1 month and fertilized embryos were obtained every week. These results indicate that a whole spermatogenic process in zebrafish is represented in our culture conditions.