Pittoggi C, Renzi L, Zaccagnini G, Cimini D, Degrassi F, Giordano R, Magnano A R, Lorenzini R, Lavia P, Spadafora C
Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, Rome, Italy.
J Cell Sci. 1999 Oct;112 ( Pt 20):3537-48. doi: 10.1242/jcs.112.20.3537.
We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of 'active' chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.
我们已对来自小鼠精子的核酸酶超敏染色质组分进行了表征。通过适当刺激可在小鼠附睾精子中诱导内源性核酸酶活性，并导致染色体DNA的局部降解。基于这些观察结果，我们从沉淀精子细胞上清液中的精子释放物中分离出核酸酶超敏染色质区域，并对DNA进行了克隆和表征。末端标记的释放DNA片段的凝胶电泳显示出典型的核小体分布。通过免疫荧光在精子细胞核中观察到外周分布的核组蛋白，并且通过蛋白质印迹在精子染色质中鉴定出组蛋白。此外，释放的DNA富含来自各种家族的逆转座子DNA。荧光原位杂交（FISH）和免疫荧光分析表明，逆转座子DNA和核组蛋白染色质共定位，并且都在外周分布于精子细胞核中。相比之下，用于对照的主要卫星DNA探针与精子细胞核中心区域高度浓缩的染色质共定位。核Ran和RCC1蛋白也在精子细胞核的背缘被观察到，并且与超敏染色质组分一起大量释放。总之，这些结果表明，具有“活性”染色质典型特征的核组蛋白染色质组分存在于小鼠精子中，对核酸酶切割超敏，富含逆转座子DNA并组织成核小体结构域。这些观察结果表明，核组蛋白结构域确定了精子基因组的一部分，其在早期胚胎发育过程中可能具有功能。
