RHO/ROCK信号通路在精子抗冷冻损伤中的作用

Rho/ROCK signaling pathway and anti-cryodamage ability of human sperm

目的:探讨RHO/ROCK信号通路在人精子抗冷冻损伤中的作用,为高效精液冷冻保护剂的研制提供理论依据。方法:选取健康精液25份,每份精液分为新鲜组、对照组与RHO通路抑制剂组(抑制剂组)。检测冷冻前后各组精液精子活力、精子存活率、精子膜完整率、正常形态精子百分率、精子DNA碎片指数(DFI)、精子顶体酶活性及精子线粒体膜电位变化;免疫荧光染色检测RHOa/ROCK蛋白在精子中的表达。结果:抑制剂组精液冷冻复苏后精子活力[(57.50±6.83)%vs(51.20±7.70)%,P=0.002]、精子存活率[(60.24±5.53)%vs(52.87±5.07)%,P=0.001]、精子膜完整率[(67.10±4.43)%vs(59.78±5.56)%,P=0.001]、正常形态精子百分率[(7.46±1.28)%vs(4.83±1.11)%,P=0.001]、精子DFI[(18.87±4.07)%vs(27.64±6.64)%,P=0.001]、精子线粒体膜电位(63.11±2.97 vs 56.30±4.28,P=0.001)指标均明显优于对照组;抑制剂组冷冻后精子顶体酶活性与对照组差异无统计学意义(98.30±11.33 vs 97.65±9.31,P>0.05)。免疫荧光染色显示RHOa/ROCK蛋白在精子头、颈部广泛表达。结论:RHO/ROCK信号通路在精子冷冻损伤中具有一定作用,抑制其通路活性可明显提高精子抗冷冻损伤的能力。

Objective:To investigate the influence of the Rho/ROCK signaling pathway on the anti-cryodamage ability of human sperm and provide some theoretical evidence for the development of high-efficiency semen cryoprotectants.Methods:We collected semen samples from 25 healthy males,each divided into a fresh,a normal cryopreservation control and an Rho-inhibition group.Before and after freezing,we detected sperm motility,viability,membrane integrity,morphology,DNA fragmentation index(DFI),acrosomal enzyme activity(AEA)and mitochondrial membrane potential(MMP)and determined the expressions of RhoA and ROCK proteins in the sperm by immunofluorescence staining.Results:Compared with the normal cryopreservation control,the frozen-thawed sperm of the Rho-inhibition group showed significantly increased sperm motility([51.20±7.70]%vs[57.50±6.83]%,P=0.002),survival rate([52.87±5.07]%vs[60.24±5.53]%,P=0.001),membrane integrity([59.78±5.56]%vs[67.10±4.43]%,P=0.001),percentage of morphologically normal sperm([4.83±1.11]%vs[7.46±1.28],P=0.001)and MMP(56.30±4.28 vs 63.11±2.97,P=0.001),but decreased DFI([27.64±6.64]%vs[18.87±4.07]%,P=0.001).There was no statistically significant difference in the AEA of the frozen-thawed sperm between the control and Rho-inhibition groups(97.65±9.31 vs 98.30±11.33,P>0.05).Immunofluorescence staining revealed extensive expressions of RhoA and ROCK proteins in the head and neck of the sperm.Conclusion:The Rho/ROCK signaling pathway plays a role in the cryodamage to human sperm,and inhibiting the activity of Rho/ROCK can significantly improve the ability of sperm to resist cryodamage.