Estimation of semen qualities in light of dynamic balance between spermatogenesis and destruction in testes

1.用TUNEL法观察人精子DNA的断裂情况：为获得DNA断裂的阴性对照，用99%Percoll密度梯度离心法纯化精子，然后上游。DNA断裂引起的DNA缺口通过TDT反应用FITC标记，PI反染色。用高灵敏度的CCD摄像机观察荧光。按WHO标准对129例精液质量进行分类时，少弱精子症患者TUNEL阳性率(25.9±16.0%，n=24)高于正常精子症患者(9.4±6.2%，n=73)。彗星细胞电泳法观察到DNA双链断裂。精子固定在0.7%的琼脂中，用蛋白酶K和洗涤剂树枝状排列。在5.0V下电泳40min后，用赛伯金对精子DNA进行染色。带有拉长彗星图像的精子估计为双链断裂，而没有图像的精子则无法检测到。当将彗星阳性率与精液质量进行比较时，彗星阳性率与精子密度和精子动力的相关性较差。血浆和精浆中含有腺苷。在前者中，ATP占50%以上，cAMP漏检。另一方面，在后者中，腺苷占57%，三磷酸腺苷不到1.0%。两种体液中腺嘌呤的组成有很大不同。

1. DNA fragmentation in human sperm was observed by means of TUNEL method : To obtain negative control for DNA fragmentation, sperm was purified by the density gradient centrifugation in 99% Percoll and subsequent swim up. DNA nicks caused by DNA fragmentation were labeled with FITC by TdT reaction and counter stain with PI. Fluorescence was observed by the aid of high sensitive CCD camera. When the qualities of human semen (n=129) were classified according to the criteria of WHO, The TUNEL positive rate was higher in oligo-asthenozoospermia (25.9±16.0%, n=24) than normozoospermia(9.4 ±6.2%, n=73).2. Double strand break in DNA was observed by means of Comet cell electrophoresis. Sperm was mounted in 0.7% agar and treeated with proteinase K and detergent. After electrophoresis at 5.0V for 40 min, the sperm DNA was stained with cyber gold. The sperm with elongated comet image was estimated to be with double strand break and those without the image being under detectable. When the comet positive rate was compared with semen qualities, the rate gave poor correlations with sperm density and motility.3. The blood plasma and seminal plasma involved adenyl purins. In the former, ATP occupied more than 50% and cAMP was under-detectable. On the other hand, adenosine occupied 57% in the latter and ATP was found to be less than 1.0%. The composition of adeny purines in two body fluids were quite different each other.