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    Preparation of spermatogenic cell populations at specific stages of differentiation in the human. Blanchard Y,Lavault M T,Quernee D,Le Lannou D,Lobel B,Lescoat D Molecular reproduction and development Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon. 10.1002/mrd.1080300316
    Variation of docosahexaenoic acid content in subsets of human spermatozoa at different stages of maturation: implications for sperm lipoperoxidative damage. Ollero M,Powers R D,Alvarez J G Molecular reproduction and development The oxidation of phospholipid-bound docosahexaenoic acid (DHA) has been shown to be one of the major factors that limit the motile life span of sperm in vitro. Sperm samples show high cell-to-cell variability in life span and, consequently, in susceptibility toward lipid peroxidation. Therefore, we postulated that there is also cell-to-cell variability in DHA concentration in human spermatozoa. In this study, the concentration of DHA in subsets of human spermatozoa isolated by a discontinuous Percoll density gradient was determined by gas chromatography. Four subsets of human spermatozoa were isolated using a discontinuous Percoll gradient: fraction 1 was enriched in immature germ cells and immature sperm, fractions 2 and 3 contained, mostly, immature sperm with cytoplasmic droplets, and fraction 4 contained, for the most part, morphologically normal sperm, as determined by histochemical analysis. The results indicated that there were significant differences in DHA content in sperm from all 4 fractions. DHA content in sperm from fraction 1 was 2.5-fold higher than that found in fraction 4. DHA content in mouse sperm obtained from the seminiferous tubules was 3-fold higher than that found in mouse sperm obtained from the epididymis, consistent with the findings observed in ejaculated human sperm. The results of this study indicate (i) there is cell-to-cell variability in the concentration of DHA in human sperm and (ii) that there is a net decrease in DHA content in sperm during the process of sperm maturation. 10.1002/(SICI)1098-2795(200003)55:3<326::AID-MRD11>3.0.CO;2-A