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

根据睾丸精子生成和破坏之间的动态平衡估计精液质量

基本信息

批准号：
12671633
负责人：
KANEKO Satoru
金额：
$ 2.18万
依托单位：
TOKYO DENTAL COLLEGE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671633/
关键词：
sperm DNA break DNA fragmentation TUNEL method cell electrophoresis adenyl purine 染色体

项目摘要

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.

1.用TUNEL法观察人精子DNA断裂情况：精子经99%Percoll密度梯度离心后，上游法纯化，作为DNA断裂的阴性对照。用FITC标记DNA断裂产生的DNA缺口，经TdT反应和PI复染。借助高灵敏度CCD相机观察荧光。对129例精液质量按WHO标准进行分类，少弱精子症组TUNEL阳性率（25.9± 16.0%，n =24）高于正常精子症组（9.4 ± 6.2%，n =73）.彗星电泳观察DNA双链断裂。精子固定在0.7%琼脂中，用蛋白酶K和去污剂处理。5.0V电泳40 min后，用Cyber Gold染色。彗星像拉长的精子估计为双链断裂，彗星像消失的精子则为未检出。彗星试验阳性率与精液质量相关性较差，与精子密度和精子活动力相关性较差.血浆和精浆中含有腺苷嘌呤。前者ATP占50%以上，cAMP检测不到。另一方面，腺苷在后者中占57%，ATP被发现小于1.0%。两种体液中腺苷嘌呤的组成差异较大。