EFFECT OF DEOXYRIBONUCLEIC-ACID PROTAMINATION ON FLUOROCHROME STAINING AND IN-SITU NICK-TRANSLATION OF MURINE AND HUMAN MATURE SPERMATOZOA
EFFECT OF DEOXYRIBONUCLEIC-ACID PROTAMINATION ON FLUOROCHROME STAINING AND IN-SITU NICK-TRANSLATION OF MURINE AND HUMAN MATURE SPERMATOZOA
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DOI：
10.1095/biolreprod49.5.1083
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发表时间：
1993-11-01
影响因子：
3.6
通讯作者：
SAKKAS, D
中科院分区：
文献类型：
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作者：
BIANCHI, PG;MANICARDI, GC;SAKKAS, D
提高精子染色质稳定性的一个主要事件是在精子发生过程中鱼精蛋白取代组蛋白。本研究结果表明，色霉素A3（CMA 3）可用于显示精子染色质中的鱼精蛋白缺陷。用溴化乙锭（EB）处理后，成熟小鼠精子的固定染色质显示高荧光，但用CMA 3处理后完全不染色。发现相同的染色质对原位切口平移具有高度抗性。相反，相当一部分人精子对CMA 3呈阳性。如果载玻片预先用鱼精蛋白原位处理，则CMA 3对人类精子DNA的可及性被消除。这种处理不影响EB对染色质的可及性。个体人精子样本显示具有内源性切口的精子的频率相当高，发现其与对CMA 3染色呈阳性反应的精子的频率相同。鱼精蛋白制剂的治疗阻止了内源性刻痕的识别。这些数据作为一个整体表明，CMA 3可以代表一个有用的工具，用于检测精子染色质中的鱼精蛋白缺乏症。此外，确认与切口平移敏感性和CMA 3阳性相关的实验可能允许切口和部分变性DNA的间接原位可视化，这可能与某些形式的男性因素不育相关。
A major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3) Can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA3 to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility.