title: Proliferation and differentiation of bovine type A spermatogonia during long-term culture.
abstract: The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2-4 wk at 32 degrees C or 37 degrees C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37 degrees C than at 32 degrees C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.
authors.affiliation: Department of Endocrinology, Faculty of Biology, University Medical Center Utrecht, 3548 CH Utrecht, The Netherlands.
authors.forename: Fariborz
authors.initials: F
authors.lastname: Izadyar
authors.forename: Krista
authors.initials: K
authors.lastname: Den Ouden
authors.forename: Laura B
authors.initials: LB
authors.lastname: Creemers
authors.forename: George
authors.initials: G
authors.lastname: Posthuma
authors.forename: Martti
authors.initials: M
authors.lastname: Parvinen
authors.forename: Dirk G
authors.initials: DG
authors.lastname: De Rooij
mesh_terms: D000818:Animals; D015415:Biomarkers; D002417:Cattle; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D003470:Culture Media; D016895:Culture Media, Serum-Free; D018507:Gene Expression Regulation, Developmental; D008297:Male; D008540:Meiosis; D008854:Microscopy, Electron; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D013091:Spermatogenesis; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013696:Temperature
chemical_list: D015415:Biomarkers; D003470:Culture Media; D016895:Culture Media, Serum-Free; D019009:Proto-Oncogene Proteins c-kit