ON STAGE SINGLE-CELL IDENTIFICATION OF RAT SPERMATOGENIC CELLS

The study of spermatogenic cell physiology has been hindered by the ab sence of unbiased methods of identification of cells upon which single cell techniques are being applied. In this work, we have used histoch emical techniques, digital videoimaging, quantification of chromatin-b ound DNA probes, and measurements of cell diameter to identify single spermatogenic cells at different periods of development. Our criteria of identification permit the definition of four developmental stages o f spermatogenesis on which to perform single cell analyses: spermatogo nia B/preleptotene spermatocytes, leptotene/zygotene spermatocytes, pa chytene spermatocytes, and round spermatids. The use of voltage-sensit ive dyes and Ca2+-sensitive dyes does not interfere with the estimatio ns of DNA content. The estimations of DNA content of spermatogenic cel ls can be performed both with near-UV excited dyes (H-33342) and long wavelength-excited dyes (ethidium bromide), allowing the use of a wide range of physiological and immunocytochemical fluorescent probes to s tudy the spermatogenic process.