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373 CONTENT AND ORGANIZATION OF SPERMATOZOAN DNA

The development of laser-based instrumentation has made possible the determination of individual cell DNA content at rates of 20,000/min and above. Forward light scatter, an index of cell shape and refractive index, can be concurrently measured. Moreover, subpopulations of cells can be sorted based on either of these parameters and collected for additional biochemical or morphological analysis. In order to determine changes occuring during epididymal maturation individual caput and cauda epididyma were obtained from C57BL/6N and AKR mice. Sperm were rinsed free in saline-EDTA, washed, fixed in ethanol and stained with propidiam iodide. A Coulter TPS-1 cell sorter was employed. In some studies freeze-thawing and sonication were employed to obtain a high yield of isolated sperm heads. A discrete homogeneous peak of fluorescent intensity corresponding to the haploid chromosome DNA content was defined and the purity confirmed by collection and examination by flourescent microscopy. Caput sperm heads had lesser Elourescence than those from the cauda. Heating at 100 C for 10 min resulted in a shift toward greater flourescence. These changes indicate altered organization of DNA. Conclusions: The flourescent activated cell sorter can be used to measure DNA content and morphology of individual sperm with a precision and speed not previously possible. Condensation of DNA occurs during epididymal maturation. This new technology is immediately applicable to a number of new areas of developmental, pharmacologic and clinical studies of reproduction in the male.