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.通过TUNEL法观察人类精子中的DNA断裂：为了获得DNA断裂的阴性对照，在99％Percoll中通过密度梯度离心纯化精子，随后用FITC标记由DNA断裂引起的游动DNA切口。根据WHO标准对人类精液（n=129）进行TdT反应和PI复染，观察荧光。少弱精子症TUNEL阳性率(25.9±16.0%,n=24)高于正常精子症(9.4±6.2%,n=73)。2.彗星细胞电泳观察到精子DNA双链断裂。封固于 0.7% 琼脂中，并用蛋白酶 K 和去垢剂进行树化，5.0V 电泳 40 秒后。分钟，精子DNA被赛博金染色，有拉长彗星图像的精子估计有双链断裂，而没有彗星图像的精子则检测不到。当将彗星阳性率与精液质量进行比较时，该比率显示出较差的相关性。 3.血浆和精浆中腺嘌呤含量占50%以上，而cAMP含量较低。后者为57%，而ATP含量低于1.0%。两种体液中腺嘌呤的组成差异很大。