Bianchi P G, Manicardi G C, Bizzaro D, Bianchi U, Sakkas D
Department of Obstetrics and Gynecology, Hôpital Cantonal Universitaire de Genève, Switzerland.
Biol Reprod. 1993 Nov;49(5):1083-8. doi: 10.1095/biolreprod49.5.1083.
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.
精子发生过程中，鱼精蛋白取代组蛋白是增强精子染色质稳定性的一个主要事件。在本研究中，我们展示的结果表明，放线菌素A3（CMA3）可用于显示精子染色质中鱼精蛋白的缺乏。成熟小鼠精子的固定染色质在用溴化乙锭（EB）处理后显示出高荧光，但在用CMA3处理后完全未被染色。同样的染色质被发现对原位切口平移具有高度抗性。相比之下，相当一部分人类精子对CMA3呈阳性反应。如果玻片预先用鱼精蛋白进行原位处理，CMA3对人类精子DNA的可及性就会消除。这种处理并不影响EB对染色质的可及性。个体人类精子样本显示，具有内源性切口的精子频率相当高，且发现该频率与对CMA3染色呈阳性反应的精子频率相同。用鱼精蛋白处理制剂可防止内源性切口的识别。总体而言，这些数据表明CMA3可能是检测精子染色质中鱼精蛋白缺乏的有用工具。此外，对与切口平移敏感性和CMA3阳性相关的实验进行确认，可能会实现对有切口和部分变性DNA的间接原位可视化，这可能与某些形式的男性因素不育有关。
