Fig 1.
Aggregation of glycerophospholipids into sub-μm-sized micelles.
(A) Images of sperm and GPL micelles in a confocal microscope with visible light. Precipitated ultra-sonicated sub-μm-sized GPL micelles (thin arrow) with human sperm (thick arrow) after a 6-h incubation with GPL (left), or human sperm after a similar incubation in media without GPL (right). (B) Precipitated GPL micelles seen in a Scanning Electron Microscope. The left image shows the micelles as protruding images from a thin-layered film (thin arrow). In other regions, isolated micelles can also be seen (middle image, thin arrow). The control image without GPL micelles is shown to the right. (C) GPL micelles observed in a transmission electron microscope with negative staining. To the left single GPL micelles (thin arrow) can be seen, and some micelles colliding and forming larger micelles can be observed. A background image without GPL micelles is shown to the right.
Fig 2.
Increase in size of the sperm heads after incubation with sub-μm-sized GPL micelles.
(A) Histogram and normal distribution of sperm head size after incubation in control and in 1% GPL mixture. (B) Sperm Head Area and GPL mixture concentration.
Fig 3.
Incorporation of GPL from sub-μm-sized micelles into sperm membranes can be observed with fluorescent GPL crosslinked with Rhodamine 123.
Human sperm were incubated with sub-μm-sized micelles made of GPL crosslinked with Rhodamine 123. Images of stained sperm are shown merged with trans-illumination (upper) or in fluorescence mode alone (lower). Note that more than one-half of the sperm are strongly fluorescent.
Fig 4.
Dose response curves of overall and fastest sperm motility versus concentration of GPL.
Both types of velocities are increased by incubation of sperm with sub-μm-sized micelles prepared from GPL. The solid line represents the best fit of a Hill equation. The effect is more pronounced (IC50 of approximately 0.5%) for the fastest sperm (right) compared with 0.15% for the overall motile sperm (left).
Fig 5.
Analysis of motility of mature human sperm from adult males incubated under different experimental conditions (control, GPL, H2O2, GPL plus H2O2).
For each experimental condition (listed on the top of each graph set) the percent overall motility (grey bar) and immotile sperm (dashed white bar) are shown. Below these plots are shown the curvilinear velocity (VCL), the straight line velocity (VSL) and the average path velocity (VAP) in μm/s, for each type of sperm motile pattern (average, slow, medium and fast motility)(n = 8 experiments). (A) control (B) 0.1% GPL (C) 300 μM H2O2 (D) 300 μM H2O2 plus 0.1% GPL.
Fig 6.
Dose response curves of overall motility versus hydrogen peroxide concentration.
The data correspond to the average motility obtained after incubation in the presence of varying concentrations of H2O2 (solid circles) or in the presence of varying concentrations of H2O2 with 0.1% GPL added (open triangles) (n = 8 experiments). The solid lines represent the best fit of a Hill equation to the data.
Fig 7.
Dot plots from flow cytometry of sperm loaded with JC-1, a mitochondrial reporter, under different experimental conditions.
(A) Results obtained after control incubation of sperm. (B) Incubation of sperm with 0.1% GPL. (C) Incubation of sperm with 300 μM H2O2. (D) Incubation of sperm with 300 μM H2O2 with the addition of 0.1% GPL. (E) Control obtained with unstained sperm (without JC-1). (F) Cells stained with JC-1 and exposed to the toxic molecule PI.
Fig 8.
Histograms of red and green fluorescence intensity of sperm stained with JC-1.
(A) Histograms of fluorescence of sperm red (left panels) and green (right panels) fluorescence intensities obtained after the control incubation (semi-log scale). (B) Fluorescence histograms of sperm obtained after incubation with 0.1% GPL. (C) Fluorescence histograms of sperm obtained after incubation in 300 μM H2O2. (D) Fluorescence histograms of sperm after exposure to 300 μM H2O2 in the presence of 0.1% GPL. (E) Fluorescence histograms of sperm cells without JC-1. (F) Fluorescence histograms of sperm in the presence of propidium iodide (PI).
Fig 9.
Confocal images of human sperm stained with Rhodamine 123 under different experimental conditions.
Segment A has been inserted in a sperm sector enriched in mitochondria, whereas segment B has been placed in a sector devoid of mitochondria. Fluorescence intensity plots from segments A and B are shown to the right. (A) A typical spermatozoon stained with Rhodamine 123 under control conditions. (B) A sperm stained with Rhodamine 123 in medium containing 0.1% GPL. (C) A typical spermatozoon stained with Rhodamine 123 in medium containing 300 μM H2O2. (D) A sperm stained with Rhodamine 123 in 300 μM H2O2 with 0.1% GPL.
Fig 10.
Confocal images of human sperm stained with JC-1 under different experimental conditions.
(A) Live imaging of sperm incubated in control medium. (B) The same experiment as in A, but in the presence of 0.1% GPL. (C) Sperm incubated in 300 μM H2O2. (D) Sperm co-incubated with 300 μM H2O2 and 0.1% GPL.