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The undifferentiated spermatogonial population consists of stem and progenitor germ cells which function to provide the foundation for spermatogenesis. The stem cell component, termed spermatogonial stem cells (SSCs), is capable of self-renewal and differentiation. These unique attributes have made them a target for novel technologies to enhance reproductive function in males. With bulls, culture and transplantation of SSCs have the potential to enhance efficiency of cattle production and provide a novel avenue to generate transgenic animals. Isolation of SSCs is an essential component for the development of these techniques. In rodents and non-human primates, undifferentiated spermatogonia and SSCs express the surface marker THY1. The hypothesis tested in this study was that THY1 is a conserved marker of the undifferentiated spermatogonial population in bulls. Flow cytometric analyses showed that the THY1+ cell fraction comprises a rare sub-population in testes of pre-pubertal bulls. Immunocytochemical analyses of the isolated THY1+ fraction for expression of VASA showed that this cell population is comprised mostly of germ cells. Additionally, expression of the undifferentiated spermatogonial specific transcription factor promyelocytic leukemia zinc finger (PLZF, ZBTB16) protein was found to be enriched in the isolated THY1+ testis cell fraction. Lastly, xenogeneic transplantation of bull testis cells into seminiferous tubules of immunodeficient mice resulted in greater than sixfold more colonies from isolated THY1+ cells compared to the unselected total testis cell population indicating SSC enrichment. Collectively, these results demonstrate that THY1 is a marker of undifferentiated spermatogonia in testes of pre-pubertal bulls, and isolation of THY1+ cells results in their enrichment from the total testis cell population.

THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis