Purification of human sperm by a discontinuous Percoll density gradient with an innercolumn

Abstract

Human sperm were highly purified through the use of a discontinuous Percoll density gradient placed in an inner column of a centrifuge tube. Six ml of 80% Percoll solution were poured into a centrifuge tube with an inner column containing successive 1.0-ml layers of 70, 60, and 40% Percoll solutions. Diluted semen was placed on top of the gradient, and the tube was centrifuged at 600 X g for 30 min using a swing-out rotor. After centrifugation, the majority of the progressive motile sperm were isolated in the sediment; they had a mean motility of 93 +/- 4.1% (n = 10). Other cellular components, including bacteria, remaining in the inner column. The level of bacterial contamination in the purified sperm fraction was below detection for most of the species quantified. The purified sperm were found to be more than 92 +/- 3.2% viable, as judged by dye exclusion, and abnormal sperm were reduced to 5.2 +/- 1.4%. Because of the use of the inner column, the contamination by seminal plasma was negligible in the purified sperm, as estimated by residual protein, fructose, and acid phosphatase activity.

Human Seminal Plasma Proteinase Inhibitor: Action Toward Some Trypsinlike Arginine Amidases from Humans

“…Acrosin activity could not be detected directly from the semen because of the presence of proteinase inhibitors [ 141. However, our recent report showed [8] that direct detection of human acrosin activity in a sperm-extracted fraction can be performed when the seminal plasma is completely removed using the Percoll discontinuous gradient method [6].…”

Trypsin-Like Arginine Amidases Including Plasminogen and Plasmin in Human Seminal Plasma by Affinity Adsorption and Elution

“…Human seminal plasma separated from human semen using the method of Kaneko et al [7] was dialyzed against 0.01 M Tris-HC1 buffer at pH 7.0 and was adjusted at conductivity of 1 mS/cm or less after dialysis. The solution was separately applied to DEAE and CM cellulose columns for adsorption of acidic and basic arginine amidases, respectively, and then eluted with the same buffer containing 0.6 M NaCl.…”

Sialic Acid in Purified Human Sperm

“…After 30-min liquefaction at room temperature, sperm density was measured with a hemocytometer and the sperm motility was estimated quantitatively by a bulb-exposure method [18]. In the present study human sperm were prepared by two methods: dilution-washing method and inner column method [13]. In the former, 1 ml of semen was diluted 10-fold with 10 mM N-2-hydroxyethyI-piperadine-N-2-ethane sulfonic acid (HEPES)-buffered Hanks' solution containing 4 m g h l of BSA, pH 7.4 (sperm culture medium), and then centrifuged twice at 600 x g for.…”

“…The present authors have developed a Percoll density gradient centrifugation with an inner column inserted in a centrifuge tube for biochemical analyses of human sperm [13]. It is capable of isolating progressively motile sperm and reducing the seminal plasma to the extent of less than lo-' of the original semen.…”