Identification and in vitro derivation of spermatogonia in beagle testis

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Abstract

Background In vitro culture of spermatogonial stem cells (SSCs) is important for exploration of SSCs self-renewal, differentiation, and manipulation. There are several reports on rodent SSC cultures; however, data on SSC cultures in domestic animals are limited. To provide basic scientific information on canine SSC cultures, we report canine testes development, and the development of spermatogonia-derived colonies (SDCs) for in vitro cultures. Methodology/Principal Findings Testes from 2-, 3-, and 12-month-old beagles were used for histology, immunohistochemistry, in vitro culture, immunocytochemistry, and PCR. Protein gene product 9.5 (PGP9.5)-positive spermatogonia, both single and paired, were found to be abundant in the testes of 2-month-old beagles. stempro-34 and Dulbecco's modified Eagle medium with 5% fetal bovine serum provided as useful substrates for culture of SDCs, and fibroblast growth factor (FGF) played a key role in colony formation. Colonies were positive for alkaline phosphatase and anti-PGP9.5 staining. The early spermatogonia and stem cell markers such as octamer binding protein 4 (Oct4), Nanog homeobox (Nanog), promyelocytic leukemia zinc finger (PLZF), PGP9.5, and GDNF family receptor alpha-1 (GFRα-1) were expressed in the colonies at higher levels than in the testis tissue. Conclusions Testes of the 2-month-old beagles had abundant single and paired spermatogonia, which can be used for derivation of SDCs, and FGF was important for colony formation.

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Lee, K. H., Lee, R., Lee, W. Y., Kim, D. H., Chung, H. J., Kim, J. H., … Song, H. (2014). Identification and in vitro derivation of spermatogonia in beagle testis. PLoS ONE, 9(10). https://doi.org/10.1371/journal.pone.0109963

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