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Genetic Analysis of the Clonal Origin of Regenerating Mouse Spermatogenesis Following Transplantation¹

Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada H3A 1A1

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.

¹ This research was supported by the Canadian Institutes of Health Research (MOP-49444) and the Canadian Foundation for Innovation (4177). M.N. is a recipient of the MGH/RVH Scholarship. A part of the study was presented at the 28th Annual Meeting of the American Society of Andrology (Phoenix, AZ, 2003).

² Correspondence: Makoto Nagano, Royal Victoria Hospital, Room F3.07, 687 Pine Ave. West, Montreal, Quebec, Canada H3A 1A1. FAX: 514 843 1662; makoto.nagano{at}muhc.mcgill.ca

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