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BOR - Papers in Press, published online ahead of print May 19, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028894
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BIOLOGY OF REPRODUCTION 71, 942–947 (2004)
DOI: 10.1095/biolreprod.104.028894
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Biological Activity of Cryopreserved Bovine Spermatogonial Stem Cells During In Vitro Culture1

Jon M. Oatley, Jerry J. Reeves, and Derek J. McLean2

Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164

Functional roles of spermatogonial stem cells 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 that 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 exogenous glial cell line-derived neurotrophic factor (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 coculture system with bovine germ cells. Bovine spermatogonial stem cell survival and proliferation in vitro were evaluated by xenogeneic transplantation into the seminiferous tubules of immunodeficient mice. Bovine germ cells cocultured for 1 wk resulted in significantly more round cell donor colonies in recipient mouse testes compared to donor cells transplanted just after thawing. Bovine germ cells cocultured for 2 wk had fewer colony-forming cells than the freshly thawed cell suspensions or cells cultured for 1 wk. 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 1 wk of coculture, but enhanced stem cell maintenance at 2 wk compared to cultures without added GDNF. These data indicate that frozen-thawed bovine spermatogonial stem cells survive cryopreservation and can be maintained during coculture with a feeder cell line in which the maintenance is influenced by GDNF.

1 J.M.O. was supported by an Achievement Rewards for Collegiate Scientists (ARCS) Fellowship. J.J.R. was supported by a Baxter Endowed Grant from Washington State University.

2 Correspondence. FAX: 509 335 4246; dmclean{at}wsu.edu






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Copyright © 2004 by the Society for the Study of Reproduction.