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First published online October 19, 2007
Journal of Experimental Biology 210, 3805-3820 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005439
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Assessment of sperm chemokinesis with exposure to jelly coats of sea urchin eggs and resact: a microfluidic experiment and numerical study

Munish V. Inamdar1, Taeyong Kim1, Yao-Kuang Chung2, Alex M. Was1, Xinran Xiang2, Chia-Wei Wang1, Shuichi Takayama2, Christian M. Lastoskie2,3, Florence I. M. Thomas5 and Ann Marie Sastry1,2,4,*

1 Department of Mechanical Engineering, University of Michigan, Ann Arbor, 48109 MI, USA
2 Department of Biomedical Engineering, University of Michigan, Ann Arbor, 48109 MI, USA
3 Department of Civil and Environmental Engineering and University of Michigan, Ann Arbor, 48109 MI, USA
4 Department of Materials Science and Engineering, University of Michigan, Ann Arbor, 48109 MI, USA
5 Hawaii Institute of Marine Biology, University of Hawaii at Manoa, HI, USA


Figure 1
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  		Fig. 1. The PDMS diffusion device, with dimensions, showing the chemochamber (reservoir), the migration channel and the sperm chamber (reservoir). Boundary and initial conditions used to solve the 1D diffusion equation (see Materials and methods) are also shown.

 

Figure 2
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  		Fig. 2. Schematic of highly motile (A) and bulk (B) sperm in the migration channel. (A) The situation immediately after sperm injection in the sperm chamber. After ~1.5–3 min, the bulk of spermatozoa appear in the migration channel (B). The highly motile sperm shown in A move forward and go out of the focus view by the time bulk sperm appear in the migration channel.

 

Figure 3
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  		Fig. 3. An illustration of the methodology employed to estimate the diffusion coefficient values of bulk sperm (D value). The processed image showing diffused bulk sperm is divided into small rectangular zones (gray rectangles) of 29.5–31.5 µm width. The average light intensity of each rectangular zone (I value) and the location of its center along the length of the channel (x value) are recorded. The I values are normalized (called In values and shown in the figure) using the intensity value of the corresponding sample (Io value). The x values as X coordinates and In values as Y coordinates are supplied to the MATLAB curvefitting toolbox. The D value is obtained by curve-fitting Eqn 6 to the In–x data. Not all In and x values are shown for the sake of clarity. erf, error function.

 

Figure 4
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  		Fig. 4. Schematic of egg–sperm collision simulations. The egg is pinned at the center of the domain, and spermatozoa are distributed randomly at the beginning of the simulation. Then sperm are moved within the domain until they collide (penetrate) the jelly coat.

 

Figure 5
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  		Fig. 5. The captured image (A) and processed image (B) of sperm diffusing in ASW. The sperm sample was prepared by mixing 10 µl dry sperm in 200 µl ASW. (C) The sperm concentration profile along the length of the channel and the D value estimation by curve-fitting Eqn 6. Scale bars, 500 µm.

 

Figure 6
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  		Fig. 6. The captured (A) and processed (B) image of sperm diffusing in 10:250 dilution jelly coat solution. The sperm sample was prepared by mixing 10 µl dry sperm with 200 µl 10:250 dilution jelly coat solution. (C) The sperm concentration profile along the length of the channel and D value estimation by curve-fitting Eqn 6. Scale bars, 500 µm.

 

Figure 7
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  		Fig. 7. The captured image (A) and processed image (B) of sperm diffusing in 250 nmol l–1 resact solution. The sperm sample was prepared by mixing 10 µl dry sperm with 200 µl 250 nmol l–1 resact solution. (C) The sperm concentration profile along the length of the channel and the D value estimation by curve-fitting Eqn 6. Scale bars, 500 µm.

 

Figure 8
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  		Fig. 8. The captured image (A) and processed image (B) of sperm diffusing in 25 nmol l–1 resact solution. The sperm sample was prepared by mixing 10 µl dry sperm with 200 µl 25 nmol l–1 resact solution. (C) The sperm concentration profile along the length of the channel and the D value estimation by curve-fitting Eqn 6. Scale bars, 500 µm.

 

Figure 9
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  		Fig. 9. Presentation of the sperm–egg collision data using dimensionless variables. (A) The highly motile sperm–egg collision data. The normalized sperm diffusion coefficient values are obtained using Formula 10, where tsim is the simulation time (1800 s) and {phi}egg is the egg diameter. The normalized collision times are the ratio of time required for 10/30 highly motile sperm to collide the egg, to the simulation time. The values along y-axis are the average collision time values. (B) The bulk sperm–egg collision data. The normalized sperm diffusion coefficient values are obtained using Formula 10, where tsim is the simulation time (1800 s) and {phi}egg is the egg diameter. The normalized number of collision values are obtained by dividing the number of bulk sperm hitting the egg in 1800 s by the initial number of sperm. The values along y-axis are average values of number of collisions. Lines joining data points show trends only.

 

Figure 10
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  		Fig. 10. Images of sperm treated with 250 nmol l–1 resact (A) and 10:250 dilution jelly coat (B). The resact sample shows clusters of sperm, which are absent in jelly coat sample. These images were taken immediately after filling the microfluidic device with respective samples. Scale bars, 500 µm.

 

Figure 11
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  		Fig. B1. The schematic of PDMS poured on the silanized wafer with channel features.

 

Figure 12
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  		Fig. C1. A typical image of from the sperm life experiments showing immobile sperm among mobile ones. (A) The image captured using the CCD camera and SimplePCI. (B) The image processed by ImageJ. The sperm dilution for this image was 40x and hence the sperm clusters are visible in both the images.

 

Figure 13
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  		Fig. C2. The fraction of immobile sperm shown as a function of time. (A–C) These three graphs were obtained for sperm from three sea urchins (shown separately in A, B and C). The fraction of immobile sperm is shown for both the sperm dilutions, 40x and 400x. The data for all sperm samples studied for the sperm life experiments are shown in these figures.

 

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© The Company of Biologists Ltd 2007