Journal
THERIOGENOLOGY
Volume 76, Issue 2, Pages 241-251Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.theriogenology.2011.01.031
Keywords
Spermatogonial stem cell; Labeo rohita; Spermatogenesis; Magnetic activated cell sorting; Testis; Rohu
Categories
Funding
- Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India
Ask authors/readers for more resources
The in vitro culture system for spermatogonial stem cells (SSCs) is a powerful tool for exploring molecular mechanisms of male gametogenesis and gene manipulation. Very little information is available for fish SSC biology. Our aim was to isolate highly pure SSCs from the testis of commercially important farmed carp, Labeo rohita. The minced testis of L. rohita was dissociated with collagenase. Dissociated cells purified by two-step Ficoll gradient centrifugation followed by magnetic activated cell sorting (MACS) using Thy 1.2 (CD90.2) antibody dramatically heightened recovery rate for spermatogonial cells. The purified cells were cultured in vitro conditions for more than two months in L-15 media containing 10% fetal bovine serum (FBS), 1% carp serum, and other nutrients. The proliferative cells were dividing as validated by 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and formed colonies/clumps with the typical characteristics of SSCs A majority of enriched cell population represented a Vasa(+), Pou5f1/potof1(+), Ssea-1(+), Tra-1-81(+), plzf(+), Gfr alpha 1/gfr alpha l(-), and c-Kit/c-kit(-) as detected by immunocytochemical and/or quantitative real-time polymerase chain reaction (RT-PCR) analyses. Thus, Thyl(+) SSCs were enriched with greater efficiency from the mixed population of testicular cells of L. rohita. A population of enriched spermatogonial cells could be cultured in an undifferentiated state. The isolated SSCs could provide avenue for undertaking research on basic and applied reproductive biology. (C) 2011 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available