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目的探讨冷冻保存对人精子膜蛋白PH-20表达和精子凋亡的影响。方法 14例正常生育力精液标本(A组)和20例不育症精液标本(B组)行冷冻保存。Western blotting检测PH-20蛋白在人精子中的表达,免疫荧光用来观察PH-20蛋白在人精子上的定位,应用末端脱氧核苷酸转移酶(Td T)介导的原位末端标记(TUNEL)法检测精子凋亡情况。结果解冻后正常生育组和不育组的PH-20/β-actin平均吸光度与冷冻前比较均有显著性下降(P<0.05);解冻后正常生育组和不育组的PH-20阳性率与冷冻前比较均有显著性下降(P<0.05)。解冻后正常生育组的精子凋亡率与冷冻前的比较差异无显著性意义(P>0.05)。而解冻后不育组的精子凋亡率与冷冻前的比较均显著性下降(P<0.05),且不育组的降低程度大于正常生育组。结论冷冻-解冻过程可引起精子PH-20蛋白表达减少和精子PH-20阳性率降低,但冷冻保存对正常生育者的精子凋亡率无显著影响。
Objective To investigate the effect of cryopreservation on the expression of human sperm membrane protein PH-20 and sperm apoptosis. Methods Fourteen normal fertility semen samples (group A) and 20 infertility semen samples (group B) were cryopreserved. Western blotting was used to detect the expression of PH-20 protein in human spermatozoa. Immunofluorescence was used to observe the localization of PH-20 protein in human spermatozoa. Terminal deoxynucleotidyl transferase (TdT) TUNEL) method to detect sperm apoptosis. Results After thawing, the average absorbance of PH-20 / β-actin in normal and infertile groups was significantly decreased compared with those before freezing (P <0.05). After thawing, the positive rate of PH-20 in normal and infertile groups Compared with that before freezing, there was a significant decrease (P <0.05). After thawing, the sperm apoptosis rate in normal fertility group was not significantly different from that before freezing (P> 0.05). After thawing, the sperm apoptosis rate in sterile group decreased significantly compared with that before freezing (P <0.05), and the decrease in infertility group was greater than that in normal fertility group. Conclusion Freezing-thawing process can reduce the expression of sperm PH-20 protein and decrease the positive rate of sperm PH-20, but cryopreservation has no significant effect on the sperm apoptosis rate of normal fertility.