In vitro differentiation of fertile sperm from cryopreserved spermatogonia of the endangered endemic cyprinid honmoroko (Gnathopogon caerulescens)

Shogo Higaki,Yasuhiro Fujioka,Takaaki Todo,Toshihiro Kawasaki,Kazuaki Kawamoto,Ikuo Tooyama,Tatsuyuki Takada,Noriyoshi Sakai,Manami Shimada

doi:10.1038/srep42852

Abstract

Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials.

Highlights

Many endemic fish species are threatened with extinction
Intense Vasa signal was observed in the spermatogonial cytoplasm; Vasa signal was present in primary spermatocytes, but no signal was detected in secondary spermatocytes, spermatids, or spermatozoa
This study demonstrates that in the critically endangered endemic cyprinid G. caerulescens[28], spermatogonia from juvenile fish and from non-spawning adults can be cryopreserved by vitrification, and fertile sperm can be differentiated in vitro

Summary

Introduction

Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must be investigated. We demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. In vitro production of sperm from spermatogonia has been reported for three fish species, Japanese eel (Anguilla japonica)[12], medaka[13], and zebrafish[14,15] Such sperm has only been confirmed to be fertile for zebrafish, using sperm differentiated from freshly prepared spermatogonia[14] or using spermatogonial stem cells cultured for up to 1 month[15]. We describe the establishment of culture and cryopreservation conditions for spermatogonia of the critically endangered species Gnathopogon caerulescens and demonstrate the differentiation of fertile sperm from cryopreserved spermatogonia using juveniles and non-spawning adults

Methods

Results

Conclusion