Spermatogenesis in fish is a complex process consisting of cell proliferation and differentiation that gives rise to different types of spermatogenic cells, from mitotic spermatogonial stem cells (SSCs) to meiotic spermatocytes and finally to spermatozoa. Characterization of different spermatogenic cells and investigation of the underlying cellular events are important not only for fish reproductive research but also for germ cell-based genetic breeding such as surrogate reproduction.   

By far, the identification of spermatogenic cell subtypes in fish is usually based on the morphological characteristics of cells and their nucleus. Lacking molecular evidence, its accuracy and reliability heavily depend on the experience of researchers. It would be more objective and accurate to identify spermatogenic cells based on the expression patterns of marker proteins. However, the lack of antibody set and the inability of existing immunofluorescence techniques to adapt to a new paradigm of high-throughput research driven by big data seriously hinder the development of this research field. 

Recently, a research group led by Prof. SUN Yonghua from the Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences achieved the fine identification of 12 different spermatogenic cells through simultaneous staining of zebrafish Ddx 4, Pcna, Sycp 3 and cell nucleus. The study was published in Frontiers in Endocrinology.  

In this study, the researchers developed a set of antibodies against zebrafish Ddx4, Piwil1, Sycp3, and Pcna and a high-throughput and high-quality integrated immunofluorescence method for testicular samples.    

Through immunofluorescence analysis of zebrafish testes, the researchers found that the expression of Ddx4 gradually decreases during spermatogenesis, Piwil1 is strongly expressed in type A spermatogonia and moderately expressed in type B spermatogonia, and Sycp3 exhibits distinct expression patterns in different subtypes of spermatocytes.   

The researchers further demonstrated the practicality of the antibodies in other fish species, including Chinese rare minnow, common carp, blunt snout bream, rice field eel and grass carp. By studying the expression patterns of Ddx4, Sycp3, Piwil1, PCNA in the testes of these fish species, they proposed an integrated criterion for identifying different types/subtypes of spermatogenic cells.    

The antibody set together with the integrated immunofluorescence method provides a simple, practical, and efficient tool to study spermatogenesis in various fishes. 

Figure Quadra-staining of Ddx4, Sycp3, Pcna and cell nucleus on zebrafish testis. (Image by IHB) 

(Editor: MA Yun)