The zebrafish serves as a classical vertebrate model widely used in studying gametogenesis and gonadal differentiation. However, commonly used laboratory strains, such as AB and Tübingen (TU), have lost their genetic sex-determining regions. In these strains, gonadal development in both sexes goes through a "juvenile ovary" stage, and defects in germ cell development often lead to female-to-male sex reversal. In the published literature, numerous studies have mistakenly identified genes affecting zebrafish oogenesis as those influencing sex differentiation.

The Juji (Gobiocypris rarus), a small freshwater cyprinid fish native to streams in Sichuan, China, has been the focus of extensive research. Since the 1990s, a team led by Academician CAO Wenxuan from the Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences has detailed the biological characteristics of this species and conducted systematic research toward its establishment as an experimental animal. It is now widely used in research areas such as molecular toxicology and fish pathology.

Recently, a team led by Prof. SUN Yonghua from IHB constructed a transgenic Juji line, Tg(ddx4:EGFP-UTRddx4), which enables continuous labeling of the germ cell lineage across all developmental stages. Their work demonstrates that the Juji is an excellent model for studying germ cell development and gonadal differentiation. The related research paper has been published in Communications Biology.

The researchers first cloned the regulatory element of the germ cell-specific gene ddx4 in Juji to create the transgenic line Tg(ddx4:EGFP-UTRddx4), allowing continuous tracing of the germ cell lineage from the gastrula stage to sexual maturity. They found that, unlike in zebrafish, the number of early primordial germ cells (PGCs) in Juji does not affect subsequent sex differentiation. Instead, the fluorescence intensity in the gonads of the transgenic line at 20 days post-fertilization (dpf) directly predicts subsequent ovarian or testicular development.

The team further detailed the germ cell developmental trajectories in both sexes: in ovaries, germ cells undergo rapid proliferation and initiate meiosis around 25 dpf; in testes, germ cells remain in a proliferating spermatogonial state from 20 to 60 dpf, after which spermatogenesis begins rapidly. Ultimately, males reach sexual maturity and produce spermatozoa around 90 dpf, while females mature and produce oocytes around 120 dpf.

To identify regulatory genes at critical gonadal differentiation stage, the researchers selected 20 dpf juveniles where germ cells showed no differentiation. They performed RNA-seq analysis on primitive ovaries and testes, identifying a total of 123 sexually dimorphic genes (SDGs). Techniques such as single-molecule fluorescence in situ hybridization (smFISH) revealed differential expression of typical SDGs, including oogenesis‑related genes (e.g., foxl2l) and spermatogenesis‑related genes (e.g., dmrt1, amh), indicating that distinct gene‑expression programs in female and male gonads emerge even before germ cell differentiation.

The researchers further constructed dmrt1 knockout Juji lines and found that all mutants developed into fertile females, confirming that dmrt1 is essential for male development. Moreover, through germline stem cell transplantation experiments, they demonstrated that the loss of dmrt1 function in host gonadal somatic cells is sufficient to drive gonadal feminization and direct the differentiation of donor testis-derived germ stem cells into oocytes. In other words, the fate reversal of somatic cells can reprogram the fate of germ cells.

In conclusion, this study successfully established a Juji line with continuous labeling of the germ cell lineage across all developmental stages and revealed the significant advantages of Juji as a fish model for studying gonadal differentiation and gametogenesis.

(Editor: MA Yun)