title: Factors supporting long-term culture of bovine male germ cells.
abstract: Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte:somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Sahare
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Sung-Min
authors.initials: SM
authors.lastname: Kim
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Ayagi
authors.initials: A
authors.lastname: Otomo
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Komatsu
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Naojiro
authors.initials: N
authors.lastname: Minami
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Masayasu
authors.initials: M
authors.lastname: Yamada
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Imai
mesh_terms: D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003470:Culture Media; D008239:Lysine; D008297:Male; D013093:Spermatogonia / Q000166:cytology*; D013234:Stem Cells
chemical_list: D003470:Culture Media; D008239:Lysine