Submitted May 9, 2003
Returned for revision May 27, 2003
Accepted July 10, 2003
Testis
Genetic Analysis of the Clonal Origin of Regenerating
Mouse Spermatogenesis Following Transplantation
Xiangfan Zhang ,
Kevin T. Ebata ,
and
Makoto C. Nagano *
* To whom correspondence should be addressed. E-mail: makoto.nagano{at}muhc.mcgill.ca.
Abstract
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/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.
Key words:
Testis •
Spermatogenesis
