A comparison of methods for preparing enriched populations of bovine spermatogonia.
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 microg 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 microg 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.
Herrid
Muren
M
CSIRO Food Futures National Research Flagship. muren.herrid@csiro.au
Davey
Rhonda J
RJ
Hutton
Keryn
K
Colditz
Ian G
IG
Hill
Jonathan R
JR
D000818:Animals; D002417:Cattle; D002469:Cell Separation / Q000379:methods / Q000662:veterinary*; D002478:Cells, Cultured; D002498:Centrifugation; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007797:Laminin; D008280:Magnetics; D008297:Male; D037121:Plant Lectins; D011205:Povidone; D012822:Silicon Dioxide; D013094:Spermatozoa / Q000166:cytology* / Q000502:physiology*
C041384:Datura stramonium lectin; D005456:Fluorescent Dyes; D007797:Laminin; D037121:Plant Lectins; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D016650:Fluorescein-5-isothiocyanate