Abstract
BackgroundIn 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 FindingsTestes 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.ConclusionsTestes 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.
Highlights
Spermatogenesis, or mitosis and meiosis of male germ cells, is a dynamic event that occurs only in testicular seminiferous tubules when spermatogonia transform into spermatozoa through the complex biological process of cellular transformation
Testes of the 2-month-old beagles had abundant single and paired spermatogonia, which can be used for derivation of spermatogonia-derived colonies (SDCs), and fibroblast growth factor (FGF) was important for colony formation
Canine testicular development To identify the types of cells in the canine testes during different developmental stages, histological analysis was performed in 2, 3, and 12 MOBT
Summary
Spermatogenesis, or mitosis and meiosis of male germ cells, is a dynamic event that occurs only in testicular seminiferous tubules when spermatogonia transform into spermatozoa through the complex biological process of cellular transformation. The seminiferous epithelium cycle is divided into 12 stages [1]. The canine seminiferous epithelium cycle is divided into 8 stages that are determined by development of the acrosome system; the morphology of developing spermatids; as well as tubular morphology, in which the shape and location of the spermatid nuclei are considered the main aspects [2,3]. Spermatogonia are important for the maintenance of spermatogenesis, and several spermatogonia markers have been reported in rodents [4,5]. Stage-specific spermatogonia markers remain to be identified in neonatal canine testes [9]. 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
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