Researchers have recently developed an expanded germline stem cell (eGSC) technology that enables the stable expansion and preservation of donor germline stem cells (GSCs), overcoming a major bottleneck in zebrafish surrogate reproduction technology.

The study, led by Prof. SUN Yonghua from the Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences, was published in Communications Biology.

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 difficulty of obtaining a stable and sufficient supply of donor 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.

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 their 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 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," SUN said.

The team also 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 eGSC transplantation platform, the team achieved rapid genetic reconstruction of the maternal-zygotic mutant (MZnanog) in a single generation. Compared with conventional sperm cryopreservation-based strategies, this approach significantly shortened the time required for functional line recovery, highlighting its potential for preserving and rapidly restoring complex genetic resources.

According to the researchers, these findings establish a robust platform for the large-scale in vivo expansion and cryopreservation of donor GSCs, providing 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, they said.