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. 2019 Aug 13;17(8):e3000398. doi: 10.1371/journal.pbio.3000398

Fig 3. TLR7/8 ligands suppressed progressive motility the sperm bearing the X chromosome.

Fig 3

(A) Percentages of swim-up sperm when sperm were cultured in HTF medium with R848, a TLR7/8 ligand, or R837, a TLR7-specific ligand. Sperm were collected from the mouse epididymis and cultured in HTF medium with several doses of R848 or R837 for 1 hr. The numbers of sperm in the upper layer and the number of total sperm were counted at 1 hr. The percent of swim-up sperm was then calculated. The experiment was repeated four biological replicates. Values represent the mean ± SEM of four replicates. Data associated with this figure can be found in the supplemental data file (S1 Data). (B) Percentages of swim-up sperm when sperm were cultured in HTF medium with 0.3 μM R848 or R837. Sperm were collected from the mouse epididymis and cultured in HTF medium with/without 0.3 μM R848 or R837 for a maximum of 240 min. The numbers of sperm in the upper layer and total sperm were counted at each time point. The percent of swim-up sperm was then calculated. The experiment was repeated three biological replicates. Values represent the mean ± SEM of three replicates. Data associated with this figure can be found in the supplemental data file (S1 Data). (C) Tracks of sperm incubated with/without 0.3 μM R848 or 0.3 μM R837 for 1 hr, determined using the CASA system. White arrows indicated the progressive sperm (VAP > 70 μm/sec). Yellow arrows indicated the slow sperm (VAP < 30 μm/sec). (D, E) Variability of average-path velocity (VAP) of sperm after R848 or R837 treatment (D) and the number of the sperm exhibiting each VAP, such as 0–50, 50–100, 100–150, and over 150 μm/s€(E). Sperm were incubated with 0.3 μM R848 or R837 and then analyzed by CASA technology. The experiment was repeated three biological replicates (more than 200 individual trajectories in each treatment group were analyzed per mouse). Values represent the mean ± SEM. *Denotes significant differences compared with the control. Data associated with this figure can be found in the supplemental data file (S1 Data). (F) Effect of R848 or R837 on sex selection of mouse sperm. Sperm were incubated with 0.3 μM R848 or R837 for 60 min, and then upper layer was collected. After centrifuging, the pellet was used for DNA extraction, and real-time PCR using the specific primer recognized X chromosome or Y chromosome. The experiment was repeated three biological replicates. Values represent the mean ± SEM. *Denotes significant differences compared with the control. Data associated with this figure can be found in the supplemental data file (S1 Data). CASA, computer-assisted sperm analysis; HTF, human tubal fluid; R837, Imiquimod, selective agonist for TLR7; R848, Resiquimod; TLR7/8, Toll-like receptor 7/Toll-like receptor 8; VAP, average-path velocity.