SEPARATION OF ROUND SPERMATIDS FROM THE RAT USING AN IMMUNOSELECTION PANNING TECHNIQUE

A method was devised for the isolation of round spermatids from the ra t using a positive immunoselection technique (panning). A testis suspe nsion was prepared from adult rats by enzymatic digestion of seminifer ous tubules with collagenase. Specific mouse monoclonal antibody (97.2 5) was indirectly attached to Petri dishes and used in a panning proto col to purify spermatids from the testis cell suspension. The quantity and purity of cells isolated were determined by cell counts and histo chemical (periodic acid-Schiff stain) or by immunostaining with acroso me-specific antibodies. A mean yield of 1.38 +/- 0.15 x 10(7) cells pe r dish was obtained with a purity of more than 90%. The viability of t he cells was confirmed by epifluorescent microscopy with propidium iod ide/carboxyfluorescein acetate probes. Northern blot analysis of RNA e xtracted directly from the dish indicated good integrity of a spermati d-specific transcript of glyceraldehyde-3-phosphate dehydrogenase (GAP DH). (C) 1995 Wiley-Liss, Inc.