Bizzaro D, Manicardi G C, Bianchi P G, Bianchi U, Mariethoz E, Sakkas D
Department of Animal Biology, University of Modena, Italy.
Mol Hum Reprod. 1998 Feb;4(2):127-32. doi: 10.1093/molehr/4.2.127.
In this study we investigated the relationship between the presence of bound protamine on mouse and human sperm DNA and the level of chromomycin A3 (CMA3) and 4'6-diamidino-2-phenylindole (DAPI) fluorescence. This was accomplished by performing a competition assay between salmon protamine and fluorochromes on decondensed spermatozoa that had their nuclear proteins extracted and were fixed on slides. Various concentrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon protamine were added to either the CMA3 or DAPI staining solutions. Fluorescence emission measurements of stained sperm nuclei were then performed using a microfluorometer. When the treated decondensed sperm heads were stained with either CMA3 or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA3 fluorescence, while the intensity of DAPI staining was decreased to approximately 50% at the highest concentrations of protamine. The addition of increasing amounts of salmon protamine also induced the sperm nuclei to regain their initial condensed appearance. This study shows that protamine retains a strong affinity for sperm DNA in situ and that CMA3 fluorescence is a strong indicator of the protamination state of spermatozoa.
在本研究中，我们调查了小鼠和人类精子DNA上结合鱼精蛋白的存在与嗜铬霉素A3（CMA3）和4',6-二脒基-2-苯基吲哚（DAPI）荧光水平之间的关系。这是通过在已提取核蛋白并固定在载玻片上的去浓缩精子上进行鲑鱼精蛋白与荧光染料之间的竞争试验来实现的。将各种浓度（0、0.005、0.0225、0.05、0.225、0.5和5 mg/ml）的鲑鱼精蛋白添加到CMA3或DAPI染色溶液中。然后使用微量荧光计对染色的精子细胞核进行荧光发射测量。当用CMA3或DAPI对处理过的去浓缩精子头部进行染色时，发现所有精子都发出强烈荧光。向精子中添加鱼精蛋白会导致CMA3荧光消失，而在鱼精蛋白最高浓度下，DAPI染色强度降低至约50%。添加越来越多的鲑鱼精蛋白还会诱导精子细胞核恢复其初始的浓缩外观。这项研究表明，鱼精蛋白在原位对精子DNA保持着很强的亲和力，并且CMA3荧光是精子鱼精蛋白化状态的有力指标。
