3-PARAMETER FLOW CYTOMETRIC ANALYSIS OF RAT SPERMATOGENESIS

Mammalian spermatogenesis is a complex process which is not yet fully understood. In this paper we describe the analysis of rat spermatogene sis by means of S-parameter now cytometry. Since the analysis of DNA c ontent only provides sufficient information for the identification of 4 cell populations, additional parameters were combined with propidium iodide (PI) staining. Immunostaining of the intermediate filament vim entin allowed the identification of somatic (vimentin positive) and ge rm (vimentin negative) cells, Utilizing the combination of DNA and vim entin staining, we have been able to quantitate the somatic cells pres ent in a testicular cell suspension and to analyze somatic and germina l cells separately. Furthermore, the addition of mitochondrial stainin g with the fluorochrome nonyl acridine orange (NAO) allowed several ce ll subpopulations within each ploidy group to be distinguished. After 3-color staining and subsequent cell sorting, 11 testicular cell subpo pulations could be identified: somatic cells, and 10 subtypes of germi nal cells. The method described in this paper represents a valuable to ol for the evaluation of spermatogenesis in both normal and perturbed situations. (C) 1997 Wiley-Liss, Inc.