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Abstract
Spermatogonial stem cell functional roles in
spermatogenesis are self-renewing proliferation and
production of differentiated daughter progeny. The
ability to recapitulate these actions in vitro is
important for investigating their biology and inducing
genetic modification which could potentially lead to an
alternative means of generating transgenic animals. The
objective of this study was to evaluate the survival and
proliferation of frozen-thawed bovine spermatogonial stem
cells in vitro and investigate the effects of exogeneous
GDNF. In order to accomplish this objective we developed
a bovine embryonic fibroblast feeder cell line, termed
BEF, to serve as feeder cells in a co-culture system with
bovine germ cells. Bovine spermatogonial stem cell
survival and proliferation in vitro was evaluated by
xenogeneic transplantation into the seminiferous tubules
of immunodeficient mice. Bovine germ cells co-cultured
for 1wk resulted in significantly more round cell donor
colonies in recipient mouse testes compared to donor
cells transplanted just after thawing. Bovine germ cells
co-cultured for 2wk had fewer colony forming cells than
the freshly thawed cell suspensions or cells cultured for
1wk. Characterization of the feeder cell line revealed
endogenous expression of GDNF mRNA and protein. Addition
of exogenous GDNF to the culture medium decreased the
number of stem cells present at 1wk of co-culture, but
enhanced stem cell maintenance at 2wk compared to
cultures without added GDNF. These data indicate that
frozen-thawed bovine spermatogonial stem cells survive
cryopreservation and can be maintained during co-culture
with a feeder cell line in which the maintenance is
influenced by GDNF.
Key words:
Gamete Biology •
Testis •
Spermatogenesis
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