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a Departments of Endocrinology, Faculty of Biology,
b Cell Biology, University Medical Center Utrecht, 3548 CH Utrecht, The Netherlands
c Department of Anatomy, Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland
The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2–4 wk at 32°C or 37°C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37°C than at 32°C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.
2 Correspondence: D.G. de Rooij, Department of Endocrinology, Faculty of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. FAX: 0031 30 2532837; d.g.derooij{at}bio.uu.nl
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