Abstract
Gonocytes are long-lived primary germ cells in the lumen of seminiferous tubules, that gives rise to undifferentiated spermatogonia. During spermatogenesis, the undifferentiated spermatogonia give rise to spermatogonial stem cells (SCCs) that can self-renew and differentiate. Therefore, undifferentiated spermatogonia have become an attractive target for novel technology to enhance the quality and efficiency of cattle production. However, the rarity of bovine undifferentiated spermatogonia including SSCs has raised the need for an efficient selection method in order to culture and transplant them. The present research develops an efficient negative selection method to choose and enrich functionally viable bovine undifferentiated spermatogonial population using extracellular matrix (ECM), laminin, fibronectin, collagen type IV, and their derivative, gelatin. The result shows 8-fold increase in undifferentiated spermatogonia purity with combinatorial isolation method using laminin and gelatin. The transplanted PKH26-labeled bovine undifferentiated spermatogonial population were functionally active and proliferated and colonized in the recipient mouse testes. The transduction efficiency (TE) of lentiviral vector was 15.4% for the undifferentiated spermatogonial population including SSCs transduced with an enhanced green fluorescent protein (eGFP) transgene using a lentiviral vector. The selection and enrichment method proposed in this study can be applied to harvest a large number of functionally active bovine undifferentiated spermatogonia to aid in creating transgenic cattle with optimal reproductive rate. This research was supported by the Ministry of Education, Science and Technology/Stem Cell Research Center of the 21st Century Frontier Research Program (SC-5140) and the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (110025-03-1-HD110) and the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (2010-0023848). (poster)
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