Enrichment of CD9+ spermatogonial stem cells from goat (Capra aegagrus hircus) testis using magnetic microbeads


The well documented source for adult multipotent stem cells is spermatogonial stem cells (SSCs) of mammalian testis. It is foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. SSCs isolation from mammalian testis is difficult because of their scarcity and the lack of well characterized cell surface markers. Thus, the isolation of SSCs is of great interest for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. CD9 is a surface marker expressed in mouse and rat male germline stem cells. In this study, CD9 positive SSCs were successfully isolated from the goat testis using enzymatic digestion followed by three step purification: Differential plating, Percoll discontinuous density gradient followed by Magnetic activated cell sorting (MACS). Percoll discontinuous density gradient showed significant differences in the percentage of CD9+ SSCs across individual fraction. The fraction 36% and 40% gave the highest percentage of CD9+ SSCs i.e. 82% ± 1.2 and 9.2% ± 1.3 respectively. Magnetic activated cell sorting of CD9+ cells in the magnetic fraction of goat testes was in the range of 15% - 18% which is upto threefolds. CD9+ SSCs were further recovered with appreciable efficiency after immunomagnetic isolation by using various bead: cells ratio in which 4:1 ratio gave the highest yield of 69.06 × 105 with 18% of CD9+ SSCs. Magnetic activated cell sorting using anti-CD9 antibodies provides an efficient and fast approach as compared to conventional approaches such as differential plating and percoll discontinuous density gradient for enrichment strategy for spermatogonial stem cells from goat testes for undertaking research on basic and applied reproductive biology.

Share and Cite:

Kaul, G. , Kumar, S. and Kumari, S. (2012) Enrichment of CD9+ spermatogonial stem cells from goat (Capra aegagrus hircus) testis using magnetic microbeads. Stem Cell Discovery, 2, 92-99. doi: 10.4236/scd.2012.23014.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Creemers, L.B., den Ouden, K., van Pelt, A.M.M. and de Rooij, D.G. (2002) Maintenance of adult mouse type A spermatogonia in vitro: Influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture. Reproduction, 124, 791-799. doi:10.1530/rep.0.1240791
[2] Creemers, L.B., Meng, X., den Ouden, K., van Pelt, A.M.M., Izadyar, F., Santoro, M., Sariola, H. and de Rooij, D.G. (2002) Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation. Biology of Reproduction, 66, 1579-1584. doi:10.1095/biolreprod66.6.1579
[3] Nagano, M., Ryu, B., Brinster, C.J., Avarbock, M.R. and Brinster, R.L. (2003) Maintenance of mouse male germ line stem cell in vitro. Biology of Reproduction, 68, 2207-2214. doi:10.1095/biolreprod.102.014050
[4] Shinohara, T., Avarbock, M.R. and Brinster, R.L. (1999) beta1- and alpha-6-integrin are surface markers on mouse spermatogonial stem cells. Proceedings of the National Academy of Sciences of the United States of America, 96, 5504-5509. doi:10.1073/pnas.96.10.5504
[5] Kanatsu-Shinohara, M., Toyokuni, S. and Shinohara, T. (2004) CD9 is a surface marker on mouse and rat germ line stem cells. Biology of Reproduction, 70, 70-75. doi:10.1095/biolreprod.103.020867
[6] Oka, M., Tagoku, K., Russell, T.L., Nakano, Y., Hamazaki, T., Meyer, E.M., Yokota, T. and Terada, N. (2002) CD9 is associated with leukemia inhibitory factor-mediated maintenance of embryonic stem cells. Molecular Biology of the Cell, 13, 1274-1281. doi:10.1091/mbc.02-01-0600
[7] Ikeyama, S., Koyama, M., Yamaoko, M., Sasada, R. and Miyake, M. (1993) Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP/CD9) DNA. The Journal of Experimental Medicine, 177, 1231-1237. doi:10.1084/jem.177.5.1231
[8] Masellis-Smith, A. and Shaw, A.R. (1994) CD9-regulated adhesion: Anti-CD9 monoclonal antibody induces pre-B cell adhesion to bone marrow fibroblasts through de novo recognition of fibronectin. Journal of Immunology, 152, 2768-2777.
[9] Hadjiargyrou, M. and Patterson, P.H. (1995) An anti-CD9 monoclonal antibody promotes adhesion and induces proliferation of Schwann cells in vitro. The Journal of Neuroscience, 15, 574-583.
[10] Maecker, H.T., Todd, S.C. and Levy, S. (1997) The tetraspanin superfamily: Molecular facilitators. Federation of American Societies for Experimental Biology, 11, 428-442.
[11] Tachibana, I. and Hemler, M.E. (1999) Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance. The Journal of Cell Biology, 146, 893-904. doi:10.1083/jcb.146.4.893
[12] Avarbock, M., Brinster, C. and Brinster, R. (1996) Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nature Medicine, 2, 693-696. doi:10.1038/nm0696-693
[13] Nagano, M. and Brinster, R.L. (1998) Spermatogonial transplantation and reconstitution of donor cell spermatogenesis in recipient mice. Acta Pathologica Microbiologica et Immunologica Scandinavica, 106, 47-57. doi:10.1111/j.1699-0463.1998.tb01318.x
[14] Honaramooz, A., Megee, S. and Dobrinski, I. (2002) Germ cell transplantation in Pig. Biology of Reproduction, 66, 21-28. doi:10.1095/biolreprod66.1.21
[15] van Pelt, A., Morena, A., van Dissel-Emiliani, F., Boitani, C., Gaemers, I., de Rooij, D. and Stefanini, M. (1996) Isolation of the synchronized A spermatogonia from adult vitamin A: Deficient rat testes. Biology of Reproduction, 55, 439-444. doi:10.1095/biolreprod55.2.439
[16] Morena, A., Boitani, C., Pesce, M., Felici, M. and Stefanini, M. (1996) Isolation of highly purified type A spermatogonia from prepubertal rat testis. Journal of Andrology, 17, 708-717.
[17] Miltenyi, S., Muller, W., Weichel, W. and Radbruch, A. (1990) High gradient magnetic cell separation with MACS. Cytometry, 11, 231-238. doi:10.1002/cyto.990110203
[18] Cristea, I.M., Williams, R., Chait, B.T. and Rout, M.P. (2005) Fluorescent proteins as proteomic probes. Molecular and Cellular Proteomics, 4, 1933-1941. doi:10.1074/mcp.M500227-MCP200
[19] Jamur, M.C., Grodzki, A.C.G., Moreno, A.N., Swaim, W.D., Siraganian, R.P. and Oliver, C. (1997) Immunomagnetic isolation of rat bone marrow-derived and peritoneal mast cells. The Journal of Histochemistry and Cytochemistry, 45, 1715-1722. doi:10.1177/002215549704501215
[20] Bucci, L.R., Brock, W.A., Johnson, T.S. and Meistrich, M.L. (1986) Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes. Biology of Reproduction, 34, 195-206. doi:10.1095/biolreprod34.1.195
[21] Bellve, A.R., Cavicchia, J.C., Millette, C.F., O’Brien, D.A., Bhatnagar, Y.M. and Dym, M. (1977) Spermatogenic cells of the prepuberal mouse: Isolation and morphological characterization. The Journal of Cell Biology, 74, 68-85. doi:10.1083/jcb.74.1.68
[22] Dym, M., Jia, M., Dirami, G., Price, J.M., Rabin, S.J., Mocchetti, I. and Ravindranath, N. (1995) Expression of c-kit receptor and its autophosphorylation in immature rat type A: Spermatogonia. Biology of Reproduction, 52, 8-19. doi:10.1095/biolreprod52.1.8
[23] Cammareri, P., Lombardo, Y., Francipane, M.G., Bonventre, S., Todaro, M. and Stassi, G. (2008) Isolation and culture of colon cancer stem cells. Methods in Cell Biology, 86, 311-324. doi:10.1016/S0091-679X(08)00014-9
[24] Liu, X.L., Yuan, J.Y., Zhang, J.W., Zhang, X.H. and Wang, R.X. (2007) Differential gene expression in human hematopoietic stem cells specified toward erythroid, megakaryocytic, and granulocytic lineage. Journal of Leukocyte Biology, 82,986-1002. doi:10.1189/jlb.0107014
[25] Zhang, J., Duan, X., Zhang, H., Deng, Z., Zhou, Z., Wen, N., Smith, A.J., Zhao, W. and Jin, Y. (2006) Isolation of neural crest-derived stem cells from rat embryonic mandibular processes. Biology of the Cell, 98, 567-75. doi:10.1042/BC20060012
[26] Gangopadhyay, N.N., Shen, H., Landreneau, R., Luketich, J.D. and Schuchert, M.J. (2004) Isolation and tracking of a rare lymphoid progenitor cell which facilitates bone marrow transplantation in mice. Journal of Immunological Methods, 292, 73-81. doi:10.1016/j.jim.2004.06.015
[27] Le Grand, F., Auda-Boucher, G., Levitsky, D., Rouaud, T., Fontaine-Perus, J. and Gardahaut, M.F. (2004) Endothelial cells within embryonic skeletal muscles: A potential source of myogenic progenitors. Experimental Cell Research, 301, 232-241. doi:10.1016/j.yexcr.2004.07.028
[28] Qin, A.L., Zhou, X.Q., Zhang, W., Yu. H. and Xie, Q. (2004) Characterization and enrichment of hepatic progenitor cells in adult rat liver. World Journal of Gastroenterology, 10, 1480-1486.
[29] Buaas, F.W., Kirsh, A.L., Sharma, M., McLean, D.J., Morris, J.L., Griswold, M.D., de Rooij, D.G. and Braun, R.E. ( 2004) PLZF is required in adult male germ cells for stem cell self-renewal. Nature Genetics, 36, 647-652. doi:10.1038/ng1366
[30] Costoya, J.A., Hobbs, R.M., Barna, M., Cattoretti, G., Manova, K., Sukhwani, M., Orwig, K.E., Wolgemuth, D.J. and Pandolfi, P.P. (2004) Essential role of PLZF inmaintenance of spermatogonial stem cells. Nature Genetics, 36, 551-553. doi:10.1038/ng1367
[31] Hermann, B.P., Sukhwani, M., Lin, C.C., Sheng, Y., Tomko, J., Rodriguez, M., Shuttleworth, J.J., McFarland, D., Hobbs, R.M., Pandolfi, P.P., Schatten, G.P. and Orwig, K.E. (2007) Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques. Stem Cells, 25, 2330-2338. doi:10.1634/stemcells.2007-0143
[32] Luo, J., Megee, S. and Dobrinski, I. (2009) Asymmetric distribution of UCH-L1 in spermatogonia is associated with maintenance and differentiation of spermatogonial stem cells. Journal of Cellular Physiology, 220, 460-468. doi:10.1002/jcp.21789
[33] Herrid, M., Davey, R.J. and Hill, J.R. (2007) Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation. Cell and Tissue Research, 330, 321-329. doi:10.1007/s00441-007-0445-z
[34] Frankenhuis, M.T., Kramer, M.F. and de Rooij, D.G. (1982) Spermatogenesis in the boar. The Veterinary Quarterly, 4, 57-61. doi:10.1080/01652176.1982.9693840
[35] Luo, J., Megee, S., Rathi, R. and Dobrinski, I. (2006) Protein gene product 9.5 is a spermatogonia-specific marker in the pig testis: Application to enrichment and culture of porcine spermatogonia. Molecular Reproduction and Development, 73, 1531-1540. doi:10.1002/mrd.20529
[36] Bartholomew, R.A. and Parks, J.E. (2007) Identification, localization, and sequencing of fetal bovine vasa homolog. Animal Reproduction Science, 101, 241-251. doi:10.1016/j.anireprosci.2006.09.017
[37] Lee, G.S., Kim, H.S., Lee, S.H., Kang, M.S., Kim, D.Y., Lee, C.K., Kang, S.K., Lee, B.C. and Hwang, W.S. (2005) Characterization of pig vasa homolog gene and specific expression in germ cell lineage. Molecular Reproduction and Development, 72, 320-328. doi:10.1002/mrd.20320
[38] Toyooka, Y., Tsuenekawa, N., Takahashi, Y., Matsui, Y., Satoh, M. and Noce, T. (2000) Expression and intercellular localization of mouse vasa-homologue protein during germ cell development. Mechanisms of Development, 93, 139-149. doi:10.1016/S0925-4773(00)00283-5
[39] Zou, K., Hou, L., Sun, K., Xie, W. and Wu, J. (2011) Improved efficiency of female germline stem cell purification using fragilis-based magnetic bead sorting. Stem Cells and Development, 20, 2197-2204.
[40] Hofmann, M.C., Braydich-Stolle, L. and Dym, M. (2005) Isolation of male germ-line stem cells; influence of GDNF. Developmental Biology, 279, 114-124. doi:10.1016/j.ydbio.2004.12.006
[41] Kubota, H., Avarbock, M.R. and Brinster, R.L. (2004) Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biology of Reproduction, 71, 722-731. doi:10.1095/biolreprod.104.029207
[42] Borjigin, U., Davey, R., Hutton, K. and Herrid, M. (2010) Expression of promyelocytic leukaemia zinc-finger in ovine testis and its application in evaluating the enrichment efficiency of differential Plating. Reproduction Fertility and Development, 22, 733-742. doi:10.1071/RD09237
[43] Asano, H., Deguchi, Y., Kawamura, S. and Inaba, M. (2011) A simple method for enrichment of polychromatic erythroblasts from rat bone marrow, and their proliferation and maturation in vitro. The Journal of Toxicological Sciences, 435, 435-444. doi:10.2131/jts.36.435
[44] Xu, C., Police, S., Rao, N. and Carpenter M.K. (2002) Characterization & enrichment of cardiomyocytes derived from human embryonic stem cells. Circulation Research, 91, 501-508. doi:10.1161/01.RES.0000035254.80718.91
[45] von Schonfeldt, V., Krishnamurthy, H., Foppiani, L. and Schlatt, S. (1999) Magnetic cell sorting is a fast & effective method of enriching viable spermatogonia from djungarian hamster, mouse, and marmoset monkey testes. Biology of Reproduction, 61, 582-589. doi:10.1095/biolreprod61.3.582
[46] Gassei, K., Ehmcke, J., Dhir, R. and Schlatt, S. (2010) Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRα1-positive subpopulations in men. Journal of Medical Primatology, 39, 83-91. doi:10.1111/j.1600-0684.2009.00397.x
[47] Semple, J.W., Allen, D., Chang, W., Castaldi, P. and Freedman, J. (1993) Rapid separation of CD4+ and CD19+ lymphocyte populations from human peripheral blood by a magnetic activated cell sorter (MACS). Cytometry, 14, 955-960. doi:10.1002/cyto.990140816
[48] Büsch, J., Huber, P., Pflüger, E., Miltenyi, S., Holtz, J. and Radbruch, A. (1994) Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis. Prenatal Diagnosis, 14, 1129-1140. doi:10.1002/pd.1970141206
[49] Schmitz, B., Radbruch, A., Kümmel, T., Wickenhauser, C., Korb, H., Hansmann, M.L., Thiele, J. and Fischer, R. (1994) Magnetic activated cell sorting (MACS)—A new immunomagnetic method for megakaryocytic cell isolation: Comparison of different separation techniques. European Journal of Haematology, 52, 267-275. doi:10.1111/j.1600-0609.1994.tb00095.x

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.