comparison of methods for preparing enriched populations of bovine spermatogonia

• Published in: Reproduction fertility and development Vol. 21; no. 3; pp. 393 - 399
• Main Authors: Herrid, Muren, Davey, Rhonda J, Hutton, Keryn, Colditz, Ian G, Hill, Jonathan R
• Format: Journal Article
• Language: English
• Published: Australia Collingwood, Victoria: CSIRO Publishing 01.01.2009
• Subjects: Animals; Cattle; Cell Separation - methods; Cell Separation - veterinary; Cells, Cultured; Centrifugation; enrichment; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; isolation; Laminin; Magnetics; Male; Plant Lectins; Povidone; Silicon Dioxide; Spermatozoa - cytology; Spermatozoa - physiology; stem cells
• ISSN: 1031-3613
• DOI: 10.1071/RD08129

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 x 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.