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Culture conditions for maintaining the survival and mitotic activity of rainbow trout transplantable type A spermatogonia
Author(s) -
Shikina Shinya,
Ihara Shoko,
Yoshizaki Goro
Publication year - 2008
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20771
Subject(s) - biology , rainbow trout , mitosis , andrology , transplantation , sperm , somatic cell , cell culture , microbiology and biotechnology , fetal bovine serum , broodstock , gametogenesis , immunology , embryo , medicine , genetics , embryogenesis , aquaculture , gene , fishery , fish <actinopterygii>
Abstract Germ–cell transplantation is a powerful tool for studying gametogenesis in many species. We previously showed that spermatogonia transplanted into the peritoneal cavity of trout hatchlings were able to colonize recipient gonads, and produced fully functional sperm and eggs in synchrony with the germ cells of the recipient. An in vitro‐culture system enabling spermatogonia to expand, when combined with transplantation, would be valuable in both basic and applied biology. To this end, we optimized culture conditions for type A spermatogonia in the present study using immature rainbow trout at 8–10 month of age. Spermatogonial survival and mitotic activity were improved during culture in Leibovitz's L‐15 medium (pH 7.8) supplemented with 10% fetal bovine serum at 10°C compared with culture under standard conditions for salmonids (Hank's MEM (pH 7.3) supplemented with 25 mM HEPES and 5% FBS, and culture at 20°C). Elimination of testicular somatic cells promoted spermatogonial mitotic activity. In addition, insulin, trout embryonic extract, and basic fibroblast growth factor promoted the mitosis of purified spermatogonia in an additive manner. Mitotic activity increased nearly sevenfold over 19 days of culture compared with growth factor‐free conditions and was maintained for >1 month. Furthermore, the cultured spermatogonia could colonize and proliferate in recipient gonads following transplantation. This study represents the first step towards establishing a cell line that can be transplanted for use in surrogate broodstock technology and cell‐mediated gene‐transfer systems. Mol. Reprod. Dev. 75: 529–537, 2008. © 2007 Wiley‐Liss, Inc.