A comparison of methods for preparing enriched populations of bovine spermatogonia
Muren Herrid A B D , Rhonda J. Davey A B , Keryn Hutton A B , Ian G. Colditz B and Jonathan R. Hill A B CA CSIRO Food Futures National Research Flagship.
B CSIRO Livestock Industries, F. D. McMaster Laboratory, Armidale, NSW 2350, Australia.
C Present address: School of Veterinary Science, University of Queensland, St Lucia, Qld 4072, Australia.
D Corresponding author. Email: muren.herrid@csiro.au
Reproduction, Fertility and Development 21(3) 393-399 https://doi.org/10.1071/RD08129
Submitted: 5 June 2008 Accepted: 29 September 2008 Published: 4 March 2009
Abstract
The objective of the present study was to identify an efficient and practical enrichment method for bovine type A spermatogonia. Four different enrichment methods were compared: differential plating on laminin- or Datura stramonium agglutinin (DSA)-coated flasks, percoll-gradient isolation, magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The isolated cells were characterised with Dolichos biflorus agglutinin (DBA) lectin staining for type A spermatogonia and vimentin-antibody staining for Sertoli cells. A 2 × 2 factorial design was used to investigate the enrichment efficiency on laminin and DSA. In the laminin-enrichment groups, 2 h incubation in plates coated with 20 μg mL–1 laminin yielded a 3.3-fold increase in DBA-positive cells in the adherent fraction, while overnight incubation in flasks coated with 20 μg mL–1 DSA produced a 3.6-fold increase in the non-adherent fraction. However, the greatest enrichment (5.3-fold) of DBA-positive cells was obtained after 2 h incubation in control flasks (coated with bovine serum albumin). Percoll-gradient centrifugation yielded a 3-fold increase in DBA-positive cells. MACS results showed a 3.5- to 5-fold enrichment while FACS produced a 4-fold increase in DBA-positive cells. It is concluded that differential plating is a better method of recovering large numbers of type A spermatogonia for germ cell transplantation, while MACS or FACS can provide highly enriched viable type A spermatogonia for in vitro culture. Further, the combination of differential plating and other enrichment techniques may increase the purification efficiency of type A spermatogonia.
Additional keywords: enrichment, isolation, stem cells.
Acknowledgements
We thank Brendan Hatton and Andrew Eichorn for assistance with experimental animal management and funding from the CSIRO Food Future National Research Flagship and CSIRO Livestock Industries. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
Brinster, R. L. , and Avarbock, M. R. (1994). Germline transmission of donor haplotype following spermatogonial transplantation. Proc. Natl. Acad. Sci. USA 91, 11 303–11 307.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Dirami, G. , Ravindranath, N. , Pursel, V. , and Dym, M. (1999). Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM. Biol. Reprod. 61, 225–230.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Dym, M. , Jia, M. C. , 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. Biol. Reprod. 52, 8–19.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Furimsky, A. , Vuong, N. , Xu, H. , Kumarathasan, P. , Xu, M. , Weerachatyanukul, W. , Bou Khalil, M. , Kates, M. , and Tanphaichitr, N. (2005). Percoll gradient-centrifuged capacitated mouse sperm have increased fertilizing ability and higher contents of sulfogalactosylglycerolipid and docosahexaenoic acid-containing phosphatidylcholine compared to washed capacitated mouse sperm. Biol. Reprod. 72, 574–583.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Glattauer, V. , Irving-Rodgers, H. F. , Rodgers, R. J. , Stockwell, S. , Brownlee, A. G. , Werkmeister, J. A. , and Ramshaw, J. A. (2007). Examination of basement membrane components associated with the bovine semineferous tubule basal lamina. Reprod. Fertil. Dev. 19, 473–481.
| Crossref | GoogleScholarGoogle Scholar | CAS | PubMed |
Herrid, M. , Vignarajan, S. , Davey, R. , Dobrinski, I. , and Hill, J. R. (2006). Successful transplantation of bovine testicular cells to heterologous recipients. Reproduction 132, 617–624.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
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 Tissue Res. 330, 321–329.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Honaramooz, A. , Behboodi, E. , Blash, S. , Megee, S. O. , and Dobrinski, I. (2003). Germ cell transplantation in goats. Mol. Reprod. Dev. 64, 422–428.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Izadyar, F. , Spierenberg, G. T. , Creemers, L. B. , den Ouden, K. , and de Rooij, D. G. (2002). Isolation and purification of type A spermatogonia from the bovine testis. Reproduction 124, 85–94.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Izadyar, F. , Den Ouden, K. , Stout, T. A. , Stout, J. , and Coret, J. , et al. (2003). Autologous and homologous transplantation of bovine spermatogonial stem cells. Reproduction 126, 765–774.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Jiang, F. X. , and Short, R. V. (1995). Male germ cell transplantation in rats: apparent synchronization of spermatogenesis between host and donor seminiferous epithelia. Int. J. Androl. 18, 326–330.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Kanatsu-Shinohara, M. , Toyokuni, S. , and Shinohara, T. (2004). CD9 is a surface marker on mouse and rat male germline stem cells. Biol. Reprod. 70, 70–75.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
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. Mol. Reprod. Dev. 73, 1531–1540.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Ogawa, T. , Aréchaga, J. M. , Avarbock, M. R. , and Brinster, R. L. (1997). Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41, 111–122.
| PubMed | CAS |
Orwig, K. E. , Shinohara, T. , Avarbock, M. R. , and Brinster, R. L. (2002). Functional analysis of stem cells in the adult rat testis. Biol. Reprod. 66, 944–949.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Rodriguez-Sosa, J. R. , Dobson, H. , and Hahnel, A. (2006). Isolation and transplantation of spermatogonia in sheep. Theriogenology 66, 2091–2103.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Scarpino, S. , Morena, A. R. , Petersen, C. , Froysa, B. , Soder, O. , and Boitani, C. (1998). A rapid method of Sertoli cell isolation by DSA lectin, allowing mitotic analyses. Mol. Cell. Endocrinol. 146, 121–127.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Shinohara, T. , Avarbock, M. R. , and Brinster, R. L. (1999). β1- and α6-integrin are surface markers on mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 96, 5504–5509.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
van Pelt, A. M. , Morena, A. R. , van Dissel-Emiliani, F. M. , Boitani, C. , Gaemers, I. C. , de Rooij, D. G. , and Stefanini, M. (1996). Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes. Biol. Reprod. 55, 439–444.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
Wrobel, K. H. (2000). Prespermatogenesis and spermatogoniogenesis in the bovine testis. Anat. Embryol. (Berl.) 202, 209–222.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |
