Testis

Establishment of a Short-Term In Vitro Assay for Mouse Spermatogonial Stem Cells

Abstract
Spermatogonial stem cells (SSCs) are responsible for life-long, daily production of male gametes and for transmission of genetic information to the next generation. An unequivocal detection of SSCs has relied on spermatogonial transplantation, in which functional SSCs are analyzed qualitatively and quantitatively based on their regenerative capacity. However, this technique involves some significant limitations. For example, it is a time-consuming procedure, as data acquisition requires at least 8 weeks after transplantation. It is also laborious, requiring microinjection of target cells into the seminiferous tubules of individual testes. Donor-recipient immunocompatibility for successful transplantation and large variance in data obtained represent further limitations of the technique. In this study, we provide the evidence that a recently developed SSC culture system can be employed as a reliable, short-term in vitro assay for SSCs. In this system, donor cells generate three dimensional structures of aggregated germ cells in vitro, termed "clusters," within 6 days. We show that each cluster originates from a single cell; thus, by counting clusters, "cluster-forming cells" can be quantified. We also observe a strong linear correlation between the numbers of clusters and SSCs over extended culture periods; therefore, cluster numbers faithfully reflect SSC numbers. These results indicate that by simply counting the number of clusters, functional SSCs can readily be detected within 1 week in a semiquantitative manner. The faithfulness of this in vitro assay to the transplantation assay was further confirmed under two experimental situations. This in vitro "cluster-formation assay" provides a reliable short-term technique to detect SSCs.

Key words: Testis • Growth factors • Spermatogenesis • Stem cells

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				D. G. de Rooij and S. C. Mizrak Deriving multipotent stem cells from mouse spermatogonial stem cells: a new tool for developmental and clinical research Development, July 1, 2008; 135(13): 2207 - 2213. [Abstract] [Full Text] [PDF]