Dynein-ATPase is the intracellular motor for sperm motility. In the present work we assayed the dynein-ATPase activity in an axoneme-containing fraction of human sperm, free of plasma membranes, in normozoospermic and asthenozoospermic donors. Axonemecontaining fractions were isolated from semen samples obtained from healthy donors with either normozoospermia or asthenozoospermia, as indicated by a sperm motility lower than 50% (WHO grade a + b). The dynein-ATPase activity was assayed and partially characterized. The dynein-ATPase activity in the axoneme-containing fractions was identified as Mg2+-dependent ATPase activity inhibited by 10 μM vanadate. This inhibition was not seen when the assay was done in the presence of 1 mM norepinephrine. The dynein-ATPase activity is Mg2+-dependent, Li+-sensitive, and insensitive to 2 mM ouabain, 1 μM oligomycin, and 1 μM thapsigargin. The dynein-ATPase activity was significantly lower (p < 0.001) for asthenozoospermic donors as compared to normozoospermic donors. This is a straightforward dynein-ATPase assay that can be used to obtain data of functional interest in clinical or experimental settings

Human sperm has several mechanisms to control its ionic milieu, such as the Na,K-ATPase (NKA), the Ca-ATPase of the plasma membrane (PMCA), the Na C /Ca 2C -exchanger (NCX) and the NaOn the other hand, the dynein-ATPase is the intracellular motor for sperm motility. In this work, we evaluated NKA, PMCA, NHE, NCX and dynein-ATPase activities in human sperm and investigated their correlation with sperm motility. Sperm motility was measured by Computer Assisted Semen Analysis. It was found that the NKA activity is inhibited by ouabain with two K i (7.9!10 K9 and 9.8!10 K5 M), which is consistent with the presence of two isoforms of a subunit of the NKA in the sperm plasma membranes (a1 and a4), being a4 more sensitive to ouabain. The decrease in NKA activity is associated with a reduction in sperm motility. In addition, sperm motility was evaluated in the presence of known inhibitors of NHE, PMCA and NCX, such as amiloride, eosin, and KB-R7943, respectively, as well as in the presence of nigericin after incubation with ouabain. Amiloride, eosin and KB-R7943 significantly reduced sperm motility. Nigericin reversed the effect of ouabain and amiloride on sperm motility. Dynein-ATPase activity was inhibited by acidic pH and micromolar concentrations of Ca 2C. We explain our results in terms of inhibition of the dynein-ATPase in the presence of higher cytosolic H C and Ca 2C, and therefore inhibition of sperm motility.

Fluorescence microscopy of cells loaded with fluorescent, Ca 2+ -sensitive dyes is used for measurement of spatial and temporal aspects of Ca 2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca 2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca 2+ -reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca 2+ response as % change in fluorescence is obtained. Video LinkThe video component of this article can be found at http://www.jove.com/video/1996/ ProtocolSperm from healthy fertile males, with a normal semen analysis, are normally prepared for imaging as follows. .8 Na lactate) supplemented with 0.3% charcoal de-lipidated/fatty acid free Fraction V BSA (quality of the BSA is crucial for successful capacitation of sperm). 1 ml of sEBBS is pipetted into each of a series of 5 ml tubes and gently underlayered with 0.3 ml of semen. After incubation for 1 hour (37°C; 6% CO 2 ) the top 0.7 ml is gently removed from each tube and pooled. 10 μl of the sperm suspension is diluted with 90 μl of 1% (v/v) formalin to immobilize the cells, then sperm are counted in a Neubauer chamber. Cell density in the suspension is then adjusted (with sEBSS) to 6 million cells/ml. 2. The sample is then divided into aliquots of 200 μl in loosely-capped tubes and incubated (37°C; 6% CO 2 ) in for 5-6 h to allow capacitation. 3. Coverslips (22x50 mm) have previously been treated with poly-D-lysine. 10 μl of poly-D-lysine solution (10% w/v) is applied as a number of small drops to the centre of the coverslip. The poly-D-lysine is then allowed to air dry. This can be on a heated stage and should be to complete dryness. A coverslip is attached with vacuum grease to an enclosed, purpose-built, perfusable, polycarbonate imaging chamber (dimensions 35 mm x 20 mm x 5 mm; capacity ≈ 180 μl) similar to the Warner RC20 chamber .The poly-D-lysine-coated coverslip forms the base ...

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