Germline stem cell transplantation (GSCT)-based surrogate reproduction technology, applicable both intra- and inter-specifically, holds significant promise in areas such as accelerated breeding of economic fish, conservation of rare and endangered species, and construction of model fish resources, owing to its ability to shorten sexual maturation cycles and enable efficient genetic resource preservation and germline reconstruction.

However, a major bottleneck limiting the broader application of this technology is the challenge of obtaining a stable and sufficient supply of donor germline stem cells (GSCs) for transplantation. Cryopreservation of GSCs, which is essential for long-term genetic resource banking, is severely hampered by the limited number of donor cells that can be recovered after freeze-thaw procedures, resulting in extremely low surrogate reproduction efficiency.

Recently, a research group led by Prof. SUN Yonghua from the Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences established an expanded Germline Stem Cell (eGSC) technology that effectively solves the problem of stably obtaining donor GSCs for intraspecific surrogate reproduction. This study was published in Communications Biology.

In this study, researchers transplanted donor GSCs into cyp11a2^−/− zebrafish recipients. Previous work by the team had revealed that loss of cyp11a2, a key gene involved in steroid biosynthesis in gonadal somatic cells, induces GSC proliferation and blocks differentiation. Within the gonadal microenvironment of the cyp11a2^−/− recipients, the donor GSCs maintained robust self-renewal capacity and rarely entered a differentiation state, enabling large-scale in vivo expansion and long-term maintenance of stem cell properties.

"We found that the cyp11a2^−/− gonadal microenvironment provides a unique niche for the massive in vivo expansion of donor GSCs, which maintain their stemness and self-renewal capacity over the long term. We designated these in vivo-expanded GSCs as expanded Germline Stem Cells (eGSCs). Compared with conventional GSC transplantation strategies, the number of eGSCs obtained from a single donor after cryopreservation was increased by approximately 300-fold, and transplantation efficiency was dramatically improved," said Prof. SUN Yonghua.

Furthermore, the team established a novel nanos2^−/− recipient strategy as an ideal empty gonadal niche for GSC transplantation. In contrast to dnd1-deficient recipients, which often undergo recipient masculinization and mainly produce donor-derived sperm, the nanos2^−/− recipients enabled the efficient production of both donor-derived sperm and oocytes.

Using this integrated eGSCs transplantation platform, the team successfully achieved rapid genetic reconstruction of the maternal-zygotic mutant (MZnanog) within a single generation. Compared with conventional sperm cryopreservation-based strategies, this approach significantly shortened the time required for functional line recovery, demonstrating its great potential for the preservation and rapid restoration of complex genetic resources.

These findings firstly establish a robust platform for the large-scale in vivo expansion and cryopreservation of donor GSCs, and thereby provide an efficient strategy for rapid genetic resource recovery in zebrafish. Further optimization and development of the eGSC technology and the nanos2^−/− empty gonadal niche recipient strategy may substantially improve the efficiency of interspecific surrogate reproduction in fish.

(Editor: MA Yun)