College of Police Dog Technology, Criminal Investigation Police University of China, Shenyang, 110000, China.
College of Police Dog Technology, Criminal Investigation Police University of China, Shenyang, 110000, China.
Theriogenology. 2025 Jan 1;231:127-132. doi: 10.1016/j.theriogenology.2024.10.015. Epub 2024 Oct 16.
The aim of this study was to analyze the expression pattern of Toll receptor 7/8 (TLR7/8) in canine sperm, and explore the feasibility of using TLR7/8 ligand resiquimod(R848)to separate canine X and Y sperm. In this study, cellular immunofluorescence was used to analyze the expression of TLR7/8 in canine sperm, real-time fluorescence quantitative PCR was used to calculate the proportion of X sperm in the lower layer of the incubation solution with R848 to evaluate the sorting effect of R848 on canine X/Y sperm, and sperm quality detection system was used to analyze the effect of R848 on the motility of canine sperm. The mechanism of effect of R848 on canine sperm motility was analyzed by Western blot. The results showed that TLR8 was not expressed in all canine sperm, while TLR7 was expressed in all canine sperm and was localized in the head and tail of sperm. When 0.4 μM R848 was incubated with canine sperm for 1 h, the total motility, average path velocity (VAP), average straight-line velocity (VSL), and average curved-line velocity (VCL) of canine sperm were significantly decreased(P < 0.05). There was no significant difference between the lower and upper layers of the R848 treatment group and the control group(P > 0.05), and the proportion of X sperm was nearly half. The levels of NF-κB and GSK3α/β phosphorylation of sperm in R848 treatment group were significantly increased compared with control group(P < 0.05). The above results showed that TLR7/8 was not differentially expressed in canine X and Y sperm. R848 could decrease the motility of canine spermatozoa and inhibit sperm motility by the GSK3α/β-hexokinase pathway through the phosphorylation of NFκB and GSK3α/β, while could not separate X and Y spermatozoa. The method of sorting X/Y sperm based on TLR7/8 is not feasible for dogs.
本研究旨在分析犬精子中 Toll 受体 7/8（TLR7/8）的表达模式，并探讨使用 TLR7/8 配体瑞喹莫德（R848）分离犬 X 和 Y 精子的可行性。本研究采用细胞免疫荧光法分析 TLR7/8 在犬精子中的表达，采用实时荧光定量 PCR 计算 R848 孵育下层溶液中 X 精子的比例，以评估 R848 对犬 X/Y 精子的分选效果，采用精子质量检测系统分析 R848 对犬精子运动能力的影响。采用 Western blot 分析 R848 对犬精子运动能力的作用机制。结果显示，TLR8 未在所有犬精子中表达，而 TLR7 在所有犬精子中表达，并定位于精子头部和尾部。当 0.4 μM R848 与犬精子孵育 1 h 时，犬精子的总活力、平均路径速度（VAP）、平均直线速度（VSL）和平均曲线速度（VCL）均显著降低（P < 0.05）。R848 处理组和对照组的下层和上层之间没有显著差异（P > 0.05），且 X 精子的比例接近一半。与对照组相比，R848 处理组精子中 NF-κB 和 GSK3α/β 磷酸化水平显著升高（P < 0.05）。以上结果表明，TLR7/8 在犬的 X 和 Y 精子中没有差异表达。R848 可通过 NFκB 和 GSK3α/β 的磷酸化作用，通过 GSK3α/β-己糖激酶途径降低犬精子的活力并抑制精子的运动能力，而不能分离 X 和 Y 精子。基于 TLR7/8 分离 X/Y 精子的方法不适用于犬。
