Different Migration Patterns of Sea Urchin and Mouse Sperm Revealed by a Microfluidic Chemotaxis Device

<div><p>Chemotaxis refers to a process whereby cells move up or down a chemical gradient. Sperm chemotaxis is known to be a strategy exploited by marine invertebrates such as sea urchins to reach eggs efficiently in moving water. Less is understood about how or whether chemotaxis is used by mammalian sperm to reach eggs, where fertilization takes place within the confinement of a reproductive tract. In this report, we quantitatively assessed sea urchin and mouse sperm chemotaxis using a recently developed microfluidic model and high-speed imaging. Results demonstrated that sea urchin <i>Arbacia punctulata</i> sperm were chemotactic toward the peptide resact with high chemotactic sensitivity, with an average velocity <i>V_x</i> up the chemical gradient as high as 20% of its average speed (238 μm/s), while mouse sperm displayed no statistically significant chemotactic behavior in progesterone gradients, which had been proposed to guide mammalian sperm toward eggs. This work demonstrates the validity of a microfluidic model for quantitative sperm chemotaxis studies, and reveals a biological insight that chemotaxis up a progesterone gradient may not be a universal strategy for mammalian sperm to reach eggs.</p></div

Differential chemotactic behavior of sea urchin and mouse sperm.

<p>Trajectories of sea urchin sperm (25 sperm in each plot) when the resact concentration in the source channel is 0 (A); 100 pM (C); 10 nM (E). Trajectories of mouse sperm (39 sperm in each plot) when the progesterone concentration in the source channel is 0 (B); 2.5 µM (D); 250 µM (F). We placed the starting point of each of the trajectories at the (0, 0) coordinate. Each colored line is a cell trajectory that is 2 s long and starts at t = 0.</p

Quantitative analysis of sea urchin and mouse sperm migration pattern.

<p>Scatter plot of the speed of sea urchin (A) and mouse (B) sperm. Scatter plot of the velocity up the chemical gradient for sea urchin (C) and mouse (D) sperm. Scatter plot of the persistence length for sea urchin (E) and mouse (F) sperm. Cell numbers that contribute to the scatter plot are indicated. The duration of the cell track length ranges from 0.24 s to 38.0 s for sea urchin, and from 0.12 s to 13.76 s for mouse sperm.</p

Differential morphology and motility of sea urchin versus mouse sperm.

<p>An illustration of sea urchin (A) and mouse (B) sperm drawn to scale. Both sea urchin and mouse sperm use flagella having axonemes of 9 outer microtubule doublets and a single central pair of microtubules in order to move. Sea urchin sperm has a typical length scale of 50 µm, and mouse sperm 100 µm. Drawing credit: C. Rose Gottlieb. Trajectories of sea urchin (C) and mouse (D) sperm swimming in a microfluidic channel in the absence of putative chemoattractant. Each colored line represents a trajectory, and each trajectory is 2 s long and starts at t = 0.</p

Microfluidic device setup, operation principle and calibration.

<p>(A) Device setup. Four devices were patterned on a 1 mm thick agarose gel membrane, which was sandwiched between a Plexiglas manifold and a stainless steel supporting frame (Drawing credit: Andrew Darling). (B) Device layout. Each device contained three parallel channels that were 400 µm wide and 167 µm deep, and spaced 250 µm apart. Sperm are not shown to scale. This drawing is reproduced from Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060587#pone.0060587-Cheng1" target="_blank">[45]</a> by permission of The Royal Society of Chemistry. Chemical/buffer were flowed through two side channels and a chemical gradient formed in the center channel via molecular diffusion through the agarose ridges between the center and the side channels. (C) Device characterization. Time evolution of fluorescence intensity profile across all three channels when flowing 4 kDa FITC-dextran/buffer along the source and sink channels respectively. Time (t)  = 0 is defined as the time when the chemoattractant was flowed into the source channel.</p

Chemoinvasive and chemokinetic behavior of tumor cells to linear SDF-1α gradients.

<p><b>A.</b> Average cell velocity along the SDF-1α gradient as a function of the SDF-1α gradient . Solid line is a fit to the ligand – receptor binding kinetics . <b>B.</b> Average cell speed as a function of the SDF-1α concentration gradient. <b>C.</b> Average persistence length along the gradient direction as a function of SDF-1α concentration gradient. <b>D.</b> Average persistence length as a function of SDF-1α concentration gradient. The stars were obtained using a nonparametric <i>t</i>-test compared to the control group (Mann-Whitney test with * for 0.01<<i>p</i><0.05, ** for 0.001<<i>p</i><0.01, and *** for <i>p</i><0.001).</p

Cooperative roles of EGF and SDF-1α in tumor cell chemoinvasion.

<p>Average cell velocity (<b>A</b>) and speed (<b>B</b>) in the presence of a SDF-1α gradient of 111 nM/mm and a uniform EGF concentration of 0, 0.25 or 8.33 nM. Control conditions were without SDF-1α and EGF. <b>C.</b> Average cell speed under indicated conditions. The stars were obtained using a nonparametric <i>t</i>-test compared to the control group (Mann-Whitney test with * for 0.01<<i>p</i><0.05, ** for 0.001<<i>p</i><0.01, and *** for <i>p</i><0.001).</p

Plasticity and heterogeneity of tumor cell morphology and motility behavior.

<p><b>A–B</b>. Bright field images of MDA-MB-231 cells embedded in 3D collagen matrices within the microfluidic channel at t = 0 and 8 h. Here, t = 0 is defined as the time when buffer and 100 nM SDF-1α solution were introduced into the two side channels. Cells were pre-incubated for 24 hours after seeding before the introduction of the gradients. <b>C.</b> Graphical description of cell speed , cell velocity along the gradient direction , persistence length , and gradient-directed persistence length . <b>D.</b> Graphical description of aspect ratio. Distribution of cell aspect ratios at t = 0 and 8 h. <b>E.</b> Distribution of cell speed of elongated cells (aspect ratio larger than 3) and amoeboid-like cells (aspect ratio less than 3).</p

Tumor cells display no chemoinvasion but mild chemokinesis in linear EGF gradients.

<p>Average cell velocity along the EGF gradient (<b>A</b>), average cell speed (<b>B</b>), average persistence length along the EGF concentration gradient (<b>C</b>) and average persistence length (<b>D</b>) as a function of EGF gradients. The stars were obtained using a nonparametric <i>t</i>-test compared to the control group (Mann-Whitney test with * for 0.01<<i>p</i><0.05, ** for 0.001<<i>p</i><0.01, and *** for <i>p</i><0.001).</p

Microfluidic device setup and data acquisition.

<p><b>A.</b> An image of the microfluidic device on the microscope stage. A penny is placed on the side for scale. <b>B.</b> Schematic illustration of the microfluidic device. Three parallel channels are patterned on a 1-mm thick agarose gel membrane. A stable linear gradient is generated across the center channel by flowing solutions of chemokine and buffer through the source and the sink channels respectively. A mixture of cells (1 million cells/ml) embedded in type I collagen (1.5 mg/ml) is seeded in the center channel. All three channels are 400 µm wide and 250 µm deep, and the ridges between the channels are 250 µm wide. <b>C.</b> 3D reconstruction of a z-stack of 65 images (5 µm each) of the cell-embedded collagen matrix viewed from x-y plane (top view) and the x-z plane (side view); scale bar, 50 µm. <b>D.</b> Cell trajectory plots (60 cells each) from the four conditions indicated. In the last panel, the uniform 0.25 nM EGF is generated by supplying 0.25 nM EGF solutions along the two side channels. Each colored line represents one cell trajectory tracked in 16 h.</p