Browse > Article
http://dx.doi.org/10.12749/RDB.2013.37.3.143

Establishment of Spermatogonial Stem Cells using Total Testicular Cell Culture System in Mouse  

Lee, Won Young (Department of Animal & Food Bioscience, College of Biomedical & Health Science, Konkuk University)
Kim, Hee Chan (Department of Animal & Food Bioscience, College of Biomedical & Health Science, Konkuk University)
Kim, Dong Hoon (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Chung, Hak Jae (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Park, Jin Ki (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Song, Hyuk (Department of Animal & Food Bioscience, College of Biomedical & Health Science, Konkuk University)
Publication Information
Reproductive and Developmental Biology / v.37, no.3, 2013 , pp. 143-148 More about this Journal
Abstract
Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-$34^{TM}$ cell culture media at $37^{\circ}C$. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin ${\alpha}6$ and ${\beta}1$, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.
Keywords
Mouse spermatogonial stem cells (mSSCs); Alkaline phosphatase; Oct4; Vasa; Dazl; PLZF;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Aponte PM, van Bragt MP, de Rooij DG, van Pelt AM (2005): Spermatogonial stem cells: characteristics and experimental possibilities. Apmis 113:727- 742.   DOI
2 Brinster RL, Zimmermann JW (1994): Spermatoge- nesis following male germ-cell transplantation. Proc Natl Acad Sci USA 91:11298-11302.   DOI
3 Clermont Y (1972): Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 52:198-236.   DOI
4 Dann CT, Alvarado AL, Molyneux LA, Denard BS, Garbers DL, Porteus MH (2008): Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells 26:2928-2937.   DOI
5 Fujihara M, Kim SM, Minami N, Yamada M, Imai H (2011): Characterization and in vitro culture of male germ cells from developing bovine testis. J Reprod Dev 57:355-364.   DOI
6 Goel S, Sugimoto M, Minami N, Yamada M, Kume S, Imai H (2007): Identification, isolation, and in vitro culture of porcine gonocytes. Biol Reprod 77:127- 137.   DOI
7 Kanatsu-Shinohara M, Inoue K, Ogonuki N, Morimoto H, Ogura A, Shinohara T (2011) Serum- and feeder-free culture of mouse germline stem cells. Biol Reprod 84:97-105.   DOI
8 Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T (2003): Long- term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69:612-616.   DOI
9 Kim J, Seandel M, Falciatori I, Wen D, Rafii S (2008): CD34+ testicular stromal cells support long- term expansion of embryonic and adult stem and progenitor cells. Stem Cells 26:2516-2522.   DOI
10 Kobayashi H, Nagao K, Nakajima K, Miura K, Ishii N (2009): Thy-1+ cells isolated from adult human testicular tissues express human embryonic stem cell genes OCT3/4 and NANOG and may include spermatogonial stem cells. Reprod Med Biol 8:71-77.   DOI
11 Liu S, Tang Z, Xiong T, Tang W (2011): Isolation and characterization of human spermatogonial stem cells. Reprod Biol Endocrinol 9:141.   DOI
12 Meistrich ML (1993): Effects of chemotherapy and radiotherapy on spermatogenesis. Eur Urol 23:136- 141.   DOI
13 Nagano M, Avarbock MR, Leonida EB, Brinster CJ, Brinster RL (1998): Culture of mouse spermatogonial stem cells. Tissue Cell 30:389-397.   DOI
14 Nagano M, Brinster CJ, Orwig KE, Ryu BY, Avarbock MR, Brinster RL (2001): Transgenic mice produced by retroviral transduction of male germ-line stem cells. Proc Natl Acad Sci USA 98:13090-13095.   DOI
15 Oakberg EF (1956): A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am J Anat 99:391-413.   DOI
16 Pesce M, Wang X, Wolgemuth DJ, Scholer H (1998): Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech Dev 71:89-98.   DOI
17 Phillips BT, Gassei K, Orwig KE (2010): Spermatogonial stem cell regulation and spermatogenesis. Phil Trans R Soc B 3651663-1678.
18 Sato T, Katagiri K, Yokonishi T, Kubota Y, Inoue K, Ogonuki N, Matoba S, Ogura A, Ogawa T (2011): In vitro production of fertile sperm from murine spermatogonial stem cell lines. Nat Commun 2:472.   DOI
19 Reding SC, Stepnoski AL, Cloninger EW, Oatley JM (2010): THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis. Reproduction 139:893-903.   DOI
20 Ryu BY, Orwig KE, Oatley JM, Lin CC, Chang LJ, Avarbock MR, Brinster RL (2007): Efficient generation of transgenic rats through the male germline using lentiviral transduction and transplantation of spermatogonial stem cells. J Androl 28:353- 360.
21 Tadokoro Y, Yomogida K, Ohta H, Tohda A, Nishimune Y (2002): Homeostatic regulation of germinal stem cell proliferation by the GDNF/FSH pathway. Mech Dev 113:29-39.   DOI
22 van Pelt AM, Morena AR, van Dissel-Emiliani FM, Boitani C, Gaemers IC, de Rooij DG, Stefanini M (1996): Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes. Biol Reprod 55:439-444.   DOI
23 Xu FC, Wu SH, Guo Y, Mei XM (2006): Proliferation and undifferentiation of spermatogonial stem cells in vitro. Sichuan Da Xue Xue Bao Yi Xue Ban 37:625-628.