Enrichment of spermatogonia by density gradient centrifugation for use as a donor of surrogate production of tiger puffer

Abstract

A method of producing functional gametes from cryopreserved testicular germ cells using surrogate broodstock technology is a powerful tool for preserving the aquacultural genetic resources of the tiger puffer (Takifugu rubripes). Among the various testicular cells, only type A spermatogonia (ASGs) can colonize recipient gonads and shift to gametogenesis; therefore, ASG enrichment is an important factor for successful surrogate production. This study examined the possibility of ASG enrichment by density gradient centrifugation for surrogate production, considering its practical use. Through density gradient centrifugation with Percoll media, the highest distribution of ASGs estimated on the cell morphology and vasa expression was counted at a fraction of 20% Percoll layer. These fractionated testicular cells were transplanted intraperitoneally and successfully incorporated into the gonads of the recipient, grass puffer (T. alboplumbeus); however, no colonization was observed in the control recipients transplanted with unfractionated testicular cells. Moreover, these recipients matured normally and produced functional donor-derived gametes from which tiger puffer offspring originated. Thus, the Percoll gradient centrifugation developed in this study enables the enrichment of ASGs from testicular cells, including many differentiated germ cells, and would be useful for efficiently managing broodstock with desirable genetic traits.