Establishment of a culture condition for strong proliferation and enrichment of chicken spermatogonial stem cells in vitro

Document Type : Research Articles

Authors

1 Ferdowsi University of Mashhad

2 Hakim Sabzevari University

3 Gonbad Kavous University

Abstract

Poultry spermatogonial stem cells (SSCs) have the potential to serve as a model for studying the basic biology of SSC and they can also be used for biotechnological purposes. However, the small number of SSCs and the presence of the testicular somatic cells with SSCs have limited their applications. Therefore, this study was undertaken for the first time to investigate the effect of a serum-free medium supplemented with a combination of specific growth factors and B27 on the proliferation and enrichment of newborn chicken SSCs in vitro. Newborn chicken testicular cells were cultured in a serum-free DMEM, supplemented with GDNF, bFGF, LIF, and EGF growth factors and also B27 as an alternative for FBS. Presence and maintenance of the SSCs in the enriched cultures were evaluated by detection of alkaline phosphatase (AP) activity and ASZ1, POU5F1, CVH and GPR125 gene expression. A small number of clusters and colonies were emerged in testicular cell cultures before treatment with the enriched cell culture medium. Enrichment of the DMEM with the above indicated factors strongly promoted the proliferation of the chicken SSCs. Moreover, this culture condition declined attachment and maintenance of the testicular somatic cells and thus they decreased gradually in the cultures. The enriched SSCs were positive for AP activity and with detectable levels of ASZ1, POU5F1, CVH and GPR125 gene expression. This study shows that serum-free medium supplemented with a combination of B27 and the above indicated growth factors induces proliferation and enrichment of chicken SSCs in vitro in a short period of time.

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References

Brewer, G.J. (1995) Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. Journal of Neuroscience Research, 42, 674683.
Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Barres, B.A. & Hell, J.W. (2008) NS21: re-defined and modified supplement B27 for neuronal cultures. Journal of Neuroscience Methods, 171, 239-247.
Eildermann, K., Gromoll, J. & Behr, R. (2012) Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture. Human Reproduction, 27, 1754-1767.
Hamra, F.K., Chapman, K.M., Nguyen, D.M., Williams-Stephens, A.A., Hammer, R.E. & Garbers, D.L. (2005) Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proceedings of the National Academy of Sciences, 102, 17430-17435.
He, S., Nakada, D. & Morrison, S.J. (2009) Mechanisms of stem cell self-renewal. Annual Review of Cell and Developmental Biology, 25, 377-406.
He, Z., Kokkinaki, M., Jiang, J., Dobrinski, I. & Dym, M. (2010) Isolation, characterization, and culture of human spermatogonia. Biolgy of Reproduction, 82, 363-372.
Huang, Y.H., Chin, C.C., Ho, H.N., Chou, C.K., Shen, C.N., Kuo, H.C., Wu, T.J., Wu, Y.C., Hung, Y.C., Chang, C.C. & Ling, T.Y. (2009) Pluripotency of mouse spermatogonial stem cells maintained by IGF-1- dependent pathway. FASEB Journal, 23, 2076-2087.
Jung, J.G., Lee, Y.M., Kim, J.N., Kim, T.M., Shin, J.H., Kim, T.H., Lim, J.M. & Han, J.Y. (2010) The reversible developmental unipotency of germ cells in chicken. Reproduction, 139, 113-119.
Jung, J.G., Lee, Y.M., Park, T.S., Park, S.H., Lim, J.M. & Han, J.Y. (2007) Identification, culture, and characterization of germline stem cell-like cells in chicken testes. Biology of Reproduction, 76, 173-182.
Kanatsu-Shinohara, M., Inoue, K., Miki, H., Ogonuki, N., Takehashi, M., Morimoto, T., Ogura, A. & Shinohara, T. (2006) Clonal origin of germ cell colonies after spermatogonial transplantation in mice. Biology of Reproduction, 75, 68-74.
Kanatsu-Shinohara, M., Miki, H., Inoue, K., Ogonuki, N., Toyokuni, S., Ogura, A. & Shinohara, T. (2005) Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions. Biology of Reproduction, 72, 985-991.
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. Biology of Reproduction, 69, 612-616.
Krishnan, S., Lakshmanan, S., Iyer, G.K., UmaMaheswari, K. & Krishnakumar, S. (2010) The role of signaling pathways in the expansion of corneal epithelial cells in serum-free B27 supplemented medium. Molecular Vision, 16, 1169-1177.
Lavial, F., Acloque, H., Bachelard, E., Nieto, M.A., Samarut, J. & Pain, B. (2009) Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate. Developmental Biology, 330, 73-82.
Li, B., Sun, G., Sun, H., Xu, Q., Gao, B., Zhou, G., Zhao, W., Wu, X., Bao, W., Yu, F., Wang, K. & Chen, G. (2008) Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection. Science China Life Sciences, 51, 734-742.
Li, J.J. & Lu, L.Z. (2010) Recent progress on technologies and applications of transgenic poultry. African Journal of Biotechnology, 9, 3481-3488.
Momeni-Moghaddam, M., Matin, M.M., Boozarpour, S., Sisakhtnezhad, S., Mehrjerdi, H.K., Farshchian, M., Dastpak, M. & Bahrami, A.R. (2014) A simple method for isolation, culture and in vitro maintenance of chicken spermatogonial stem cells. In Vitro Cellular & Developmental Biology - Animal, 50, 155-161.
Oatley, J.M., Avarbock, M.R., Telaranta, A.I., Fearon, D.T. & Brinster, R.L. (2006) Identifying genes important for spermatogonial stem cell self-renewal and survival. Proceedings of the National Academy of Sciences, 103, 9524-9529.
Oatley, J.M. & Brinster, R.L. (2008) Regulation of spermatogonial stem cell self-renewal in mammals. Annual Review of Cell and Developmental Biology, 24, 263-286.
Seandel, M., James, D., Shmelkov, S.V., Falciatori, I., Kim, J., Chavala, S., Scherr, D.S., Zhang, F., Torres, R., Gale, N.W., Yancopoulos, G.D., Murphy, A., Valenzuela, D.M., Hobbs, R.M., Pandolfi, P.P. & Rafii, S. (2007) Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature, 449, 346-350.
Sisakhtnezhad, S., Bahrami, A.R., Matin, M.M., Dehghani, H., Momeni-Moghaddam, M., Boozarpour, S., Farshchian, M. & Dastpak, M. (2015) The molecular signature and spermatogenesis potential of newborn chicken spermatogonial stem cells in vitro. In Vitro Cellular & Developmental Biology - Animal, 51, 415-425.
Yan, W., Rajkovic, A., Viveiros, M.M., Burns, K.H., Eppig, J.J. & Matzuk, M.M. (2002) Identification of Gasz, an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper. Molecular Endocrinology (Baltimore, Md.), 16, 1168-1184.
Yu, F., Ding, L.J., Sun, G.B., Sun, P.X., He, X.H., Ni, L.G. & Li, B.C. (2010) Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells. Molecular Reproduction and Development, 77, 340-347.
Zohni, K., Zhang, X., Tan, S.L., Chan, P. & Nagano, M.C. (2012) The efficiency of male fertility restoration is dependent on the recovery kinetics of spermatogonial stem cells after cytotoxic treatment with busulfan in mice. Human Reproduction, 27, 44-53.
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  • Receive Date: 21 December 2014
  • Accept Date: 21 December 2014

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