Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

Development. 2016 Feb 15;143(4):566-74. doi: 10.1242/dev.129643. Epub 2015 Dec 30.


Molecular dissection and chemical screening on a complex process such as spermatogenesis could be facilitated by cell culture approaches that allow easy access for experimental manipulation and live imaging of specific molecules; however, technical limitations have thus far prevented the complete reconstruction of spermatogenic events in cell culture. Here, we describe the production of functional sperm from self-renewing spermatogonial stem cells (SSCs) in cell culture conditions, using zebrafish testicular hyperplasia cells that accumulate early stage spermatogonia. By serially transplanting hyperplasias into immunodeficient rag1 mutant zebrafish, we succeeded in long-term maintenance and efficient production of starting material for SSC culture. Through improvements of culture conditions, we achieved efficient propagation of SSCs derived from the hyperplasia. When SSCs that underwent the SSC-propagating step for 1 month were transferred onto Sertoli feeder cells, they differentiated into functional sperm that gave rise to offspring. Oxygen at the concentration of air proved to be detrimental for sperm differentiation from SSCs, but not for propagation of SSCs. These results indicate that the whole spermatogenic process can be represented in cell culture in zebrafish, facilitating analyses of the molecular mechanisms of spermatogenesis in vertebrates.

Keywords: Allogeneic graft; Cell culture; Oxygen; Rag1 mutant zebrafish; Spermatogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Culture Techniques / methods*
  • Cells, Cultured
  • Homeodomain Proteins / genetics
  • Hyperplasia
  • Male
  • Mutation
  • Oxygen / pharmacology
  • Spermatogonia / cytology*
  • Subcutaneous Tissue / pathology
  • Testis / pathology
  • Testis / transplantation
  • Zebrafish / metabolism*


  • Homeodomain Proteins
  • RAG-1 protein
  • Oxygen