Chemotactic behavior of spermatozoa captured using a microfluidic chip

Abstract

Chemotaxis, as a mechanism for sperm guidance in vivo, is an enigma which has been difficult to demonstrate. To address this issue, various devices have been designed to study sperm chemotaxis in vitro. Limitations of traditional chemotaxis devices were related to the inability to maintain a stable concentration gradient as well as track single sperm over long times. Microfluidics technology, which provides superior control over fluid flow, has been recently used to generate stable concentration gradients for investigating the chemotactic behavior of several cell types including spermatozoa. However, the chemotactic behavior of sperm has not been unequivocally demonstrated even in these studies due to the inability to distinguish it from rheotaxis, thermotaxis, and chemokinesis. For instance, the presence of fluid flow in the microchannels not only destabilizes the concentration gradient but also elicits a rheotactic response from sperm. In this work, we have designed a microfluidic device which can be used to establish both, a uniform concentration and a uniform concentration gradient in a stationary fluid. By facilitating measurement of sperm response in ascending, descending ,and uniform chemoattractant concentration, the assay could isolate sperm chemotactic response from rheotaxis and chemokinesis. The device was validated using acetylcholine, a known chemoattractant and further tested with rat oviductal fluid from the estrus phase.

FIG. 1.

Microfluidic device design and dimensions. A model of the basic device geometry drawn in COMSOL Multiphysics 5.2, which consists mainly of two inlets, chemoattractant input (I₁), and diluent input (I₂); two outlets, Outlet₁ (O₁) and Outlet₂ (O₂); a small contact area termed as the “contact zone”; and the cell reservoir from which the sperm enter the main channels. The dimensions of the device are mentioned for each section in μm. A close-up view of the test zone (magnified as an inset) consisting of two main channels that join to form the transverse channels. The angular measurements of the corners are specified in degrees and dimensions of the channels are listed in μm.

FIG. 2.

Experimental set-up for the chemotaxis assay. Schematic representation of the experimental set up with an inset image of the PDMS device bonded on a glass slide with tubing connections to inlets (I₁ and I₂) and outlets (O₁ and O₂); and a port in the center for the sperm reservoir. This microfluidic device set-up is positioned on a microscope stage and the fluidic connections for the inlets and outlets are made as shown, with flow controlled using a syringe pump. The video frames of sperm moving in transverse channels are recorded using a sCMOS camera and stored on a computer hard drive.

FIG. 3.

Uniform concentration gradient in the microfluidic device. Simulation results in 3-D for gradient formed in the microfluidic device; concentration in I₂ (Blue) = 0 mol/m³, I₁ (Red) = 1 mol/m³; Velocities at both inlets: 1 × 10⁻³ m/s Pressure at outlet: 0 Pa, transverse channel width: 55 μm and length: 1000 μm. Concentration profile (Non-dimensionalized with the maximum concentration) obtained after COMSOL simulation in the entire device (A). A mixing zone between the contact area and the test zone allowed mixing of the inlet streams. Enlarged 3-D view of the transverse channels (Test zone); the scale bar on the right represents the concentration range of the gradient (B). Concentration gradient profile in the transverse channels after COMSOL simulation (C). LSCM imaging of gradient generated in the transverse channels of the device using 0.5 mM propidium iodide (D). Experimental results obtained using 0.5 mM propidium iodide shows a linear and stable gradient over time (E).

FIG. 4.

The directionality of sperm in the transverse channels in the presence of media or chemoattractant. Sperm tracks with schematic illustration of sperm (denoted by an arrow) swimming from left to right (blue track, increasing concentration) and right to left (green track, decreasing concentration) in the transverse channels (A). The fraction of sperm entering the transverse channels from the left end in a linear gradient of ACH (ACh-G), uniform concentration of ACh (ACh-N) and media (M) was determined (B). The assay was performed using 2.76 mM and 5.51 mM of ACh in DMEM. The results are cumulative for 20 experiments in total with different combinations as described in Sec. II. Each column represents mean values (±SEM) for the individual conditions. Sperm traveling in a gradient of ACh concentration (2.76 M/m and 5.51 M/m) are preferentially directed along increasing ACh concentration as compared to those in other conditions. Statistical significance was determined by one sample t-test. “*”: p < 0.05.

FIG. 5.

Straight line velocities (VSLs) of sperm exposed to uniform or gradient concentrations of ACh. VSL was determined in the transverse channels for sperm exposed to either a gradient of ACh (ACh-G); a uniform concentration of ACh (ACh-N), at 2.76 and 5.51 mM ACh and the mean VSL was calculated for each group. Sperm exposed to media (M) devoid of ACh served as the control. The bar graph shows the mean VSL (±SEM) for sperm moving from Left to Right (L - R) and Right to Left (R - L) in response to the different conditions tested (A). Sperm VSLs are significantly higher when sperm move along the increasing concentration gradient of ACh compared to those moving along decreasing concentration (2.76 and 5.51 M/m) and the respective controls. The frequency distribution profile of velocity for sperm moving in media, increasing ACh Gradient (G L-R), decreasing ACh Gradient (G R-L), and uniform concentrations of ACh (N) 2.76 mM (B) and 5.51 mM (C). Sperm VSL distribution pattern showed a shift in the curve for 2.76 M/m gradient L-R and a plateau effect for 5.51 M/m gradient L-R, as compared to their respective controls with increased distribution towards higher VSL. A comparison of the percent sperm (±SEM) with VSL ≥ 85 μm/s in the different groups (D) showed a modest increase in the percentage of sperm exposed to a gradient of 5.51 M/m. The results are cumulative from 20 experiments in total with different combinations as described in Sec. II. “*”: p < 0.05.

FIG. 6.

Sperm response to Oviductal Fluid (OF). Directionality of sperm in the transverse channels in the presence of media, uniform OF concentration or OF gradient (A), sperm VSL in response to the aforementioned conditions (B), distribution profile of sperm VSL (C), and percentage of sperm with VSL ≥ 85 μm/s under each condition (D). OF-G: gradient of OF; OF-N: uniform concentration of OF; and M: media control. The assay was performed using a single oviductal fluid protein concentration of (∼0.38 g/l) in DMEM. The results are cumulative from 6 experiments in total as detailed in Sec. II. Each column represents mean ± SEM for individual conditions tested. Sperm exhibit a tendency to travel towards higher OF concentration with increased velocities when compared to media and non-gradient conditions. In the case of Fig. “6(B),” as there was no difference in the VSLs for sperm moving in either direction, the VSL values for both the directions were combined in the case of ‘M’ and ‘OF-N’ groups, ∼7% of the sperm population demonstrated chemotactic response to OF. “*” p < 0.05; “****” p < 0.00005.