Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout

Yoshiko Iwasaki-Takahashi,Shinya Shikina,Masaru Yagisawa,Kasumi Takahashi,Goro Yoshizaki,Masaya Watanabe,Akira Banba,Ryo Fujihara,Akihiro Yamauchi,Takafumi Okabe,Delgado M Valdez Jr

doi:10.1038/s42003-020-1025-y

Yoshiko Iwasaki-Takahashi, Shinya Shikina + Show 9 more

Open Access

https://doi.org/10.1038/s42003-020-1025-y

Copy DOI

Abstract

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes. However, application of this technique has been limited because endangered species sometimes have small gonads and do not supply enough GSCs to be used for transplantation. This limitation could be overcome by expanding GSCs in vitro, though this has been difficult due to the complexity of reconstructing the gonadal microenvironment that surrounds GSCs. Here, we describe a novel method of in vitro expansion of rainbow trout GSCs using a feeder layer derived from Sertoli cells and a culture medium containing trout plasma. A transplantation assay demonstrated that the in vitro-expanded GSCs exhibited stem cell activity and potency to produce functional eggs, sperm, and eventually healthy offspring. In vitro expansion of GSCs can aid in rescuing fishes that are on the verge of extinction.

Highlights

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes
We previously found that when GSCs harvested from the testes of rainbow trout were transplanted into female recipients, they were incorporated into recipient ovaries and eventually differentiated into fully functional eggs with full developmental potency[11]
Under the optimized culture medium (ERDF medium supplemented with 10 mM HEPES, 0.25% fish serum, and 6% fetal bovine serum (FBS)), Sertoli cells showed stable proliferation over the 7-week test period (Fig. 1q)

Summary

Introduction

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes. Due to the lack of an available method to cryopreserve fish eggs or embryos[3], a combination of cryopreservation of germline stem cells (GSCs) and their subsequent transplantation into recipient fish is a practical alternative for long-term preservation of genetic resources of endangered fish species or strains[4,5]. This method suffers from difficulties, including that the number of GSCs harvested from donor individuals are not sufficient for transplantation. The in vitro-expanded ASGs were confirmed to exhibit stem cell activity and possessed potency to differentiate into functional eggs and sperm in recipient gonads, allowing for production of viable offspring following fertilization

Methods

Results

Conclusion