Fig 1.
Design and fabrication of the microfluidic device.
(A) Design of the microfluidic device. (B) A photograph of the chip. (C) A three-dimensional illustration of the device. Liquid level in Pool A was higher than that in both Pool B/C (details were listed in Table 1). (D) Fabrication process of the device (pictures were not drawn to scale).
Table 1.
Initial volume of solution and hydrostatic pressure in each loading pool.
Fig 2.
Simulation analysis of fluid flow in the device.
(A) Schematic illustration of the microfluidic device. A and B/C represent the inlets and outlets of the channels, respectively. (B) Representative simulation of pressure distribution in the device. (C) Representative simulation of flow velocity distribution in the device. Blue arrow indicates the direction of fluid flow. (D) Distribution curves of flow velocity along the chip. I: peripheral channel; II: interconnecting grooves; III: central hexagon. Regions that were analyzed were indicated in (C) by a black dashed line. P1, P2 and P3 refers to different loading plans listed in Table 1.
Fig 3.
Characterization of fluid flow using microspheres.
(A) Illustration of characterization experiment. Channels doped with microspheres were marked as yellow. The microsphere solution was added both in the inlet and outlet of the this channel. Other channels as well as the central hexagon were doped with SWM (marked as white). Red square indicated the region where (C) was captured. (B) The speed of microspheres in different region of the device (I: peripheral channel; II: interconnecting grooves; III: central hexagon) at 0, 15 min, 30 min after sample loading. P1, P2 and P3 were corresponded to the loading plans in Table 1. Data are presented as Mean ± SD (n = 5). (C) The distribution of microspheres in the device when liquid level in each pool reached equilibrium in different loading plans (P1, P2 and P3, successively).
Fig 4.
Gradient formation in the microfluidic device.
(A) Fluorescein solution was doped into one channel and concentration gradient was formed in the hexagon (marked as green). Red square showed the area where fluorescence signals were recorded. (B) A representative fluorescence intensity change at 15 min, 30 min, 1 h, 2 h, 4 h and 7 h were displayed in sequence from left to right, top to bottom. (C) Normalized fluorescence intensity profiles in the central hexagon.
Fig 5.
Chemotactic responses of spermatozoa to progesterone gradients.
(A) An overview of concentration gradients generated in the hexagon. Progesterone solution was added in every other channel of the chip (red). Concentration gradients were generated in three regions in the hexagon corresponding to peripheral channels where progesterone solution was loaded. Blue square showed the field where sperm chemotaxis were observed. (B) A microscopic photograph of spermatozoa with several trajectories indicated (18 sperm). Each colored line represented a sperm trajectory within 3 s. (C-E) Comparisons of chemotactic parameters among three groups. Group A, 100 pM progesterone solution was added in peripheral channels; Group B, 1 mM progesterone solution was added; Group C, control. Data are presented as mean ± SD (n = 5). **: p < 0.05.
Fig 6.
Chemokinetic response of spermatozoa to progesterone gradients.
(A-C) Representative trajectories of sperm in Group A, B and C (18 sperm in each plot). Each colored line is a cell trajectory that is 3 s long. Black dots are the endpoints of the trajectories. (D-F) Different chemokinetic parameters were compared among three groups. Group A, 100 pM progesterone solution was added in peripheral channels; Group B, 1 mM progesterone solution was added; Group C, control. Data are presented as mean ± SD (n = 5). **: p < 0.05.