冷冻保存对正常和不育症精子PH-20表达和凋亡的影响

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(P0.05);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(P0.05)。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(P0.05)。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(P0.05),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。

Effect of cryopreservation on the expression of sperm surface protein PH-20 and ratio of apoptosis in normal birth and infertility

Objective To investigate the effect of cryopreservation on the expression of sperm surface protein PH-20 and the sperm apoptosis. Methods Semen samples were obtained from fertile men (n=14, group A) and infertile men (n=20, group B). Western blotting was used to detect the PH-20 protein expression in human spermatozoa.The localization of this protein on human spermatozoa was determined by indirect immunofluorescent staining using PH-20 antibody. The sperm apoptosis was examined by terminal deoxynucleotidyl transferase(TdT) mediated deoxyuridine triphophate-biotin nick end labeling(TUNEL). Results After cryopreservation, the level of PH-20 protein expression was significantly lower in both group A and B than that of fresh sperm (P<0.05). The percentage of PH-20 positive rate was significantly lower in both group A and B than that of fresh sperm (P<0.05). The ratio of sperm apoptosis was not significantly different in thawed group A than that of fresh sperm (P>0.05). The ratio of sperm apoptosis was significantly lower in thawed group B than that of fresh sperm (P<0.05). Conclusion Cryopreservation caused significant reduction of PH-20 protein expression, the percentage of PH-20 positive rate in human sperm. But the cryopreservation had no significant influence on normal fertility of sperm apoptosis.