McLay D W, Clarke H J
Department of Biology, McGill University, Montreal, Canada.
Dev Biol. 1997 Jun 1;186(1):73-84. doi: 10.1006/dbio.1997.8581.
Following fertilization of meiotically mature eggs, the chromatin of the sperm becomes biochemically and structurally remodeled within the egg cytoplasm. Despite the essential role of the paternal genome during embryogenesis, little is known of when the activities that regulate this chromatin remodeling appear during oogenesis. To determine whether these activities were acquired during meiotic maturation, we inseminated maturing oocytes of mice shortly after germinal vesicle breakdown. As previously shown, insemination at this stage did not activate the maturing oocytes, which became arrested at metaphase II. Immunofluorescent analysis revealed that at 1 hr postinsemination the sperm chromatin was dispersed and contained protamines but was devoid of core histones H2B and H3. At 4 hr postinsemination, both protamine and core histones were detectable on the sperm chromatin. By 8 hr postinsemination protamines were absent, and histones stained maximally. The appearance of immunoreactive histones was correlated with a morphological transition of the sperm chromatin from the dispersed to a condensed state, which suggests that the assembly of the histones reflected modification of the chromatin to a somatic-like state in which it was competent to respond to the metaphase-promoting factor activity of the oocyte. Both the assembly of histones and chromatin condensation were reversibly blocked when protein synthesis was inhibited, indicating that the remodeling process required proteins synthesized during maturation. Injection of core histones into protein synthesis-inhibited oocytes failed to induce condensation of the sperm chromatin, which implies that correct remodeling requires synthesis during maturation of nonhistone proteins. To test the functional capacity of remodeled sperm chromatin, maturing oocytes were inseminated, allowed to continue maturation for 17 hr and then parthenogenetically activated. Following activation, the sperm-derived chromatin as well as that of the oocyte became decondensed within pronuclei and underwent DNA replication, indicating that sperm chromatin remodeled in maturing oocyte cytoplasm was functionally normal. When the postinsemination incubation time was reduced to 11 hr; however, neither the female nor the male pronuclei underwent DNA replication, implying that factors synthesized late during maturation are required for DNA replication after activation. Taken together, these results indicate that the ability to organize sperm DNA into functional somatic-like chromatin develops in oocytes during meiotic maturation, requires proteins synthesized during maturation, and can be expressed independently of activation.
减数分裂成熟的卵子受精后，精子的染色质在卵细胞质内发生生化和结构重塑。尽管父本基因组在胚胎发育过程中起着至关重要的作用，但对于调控这种染色质重塑的活动在卵子发生过程中何时出现却知之甚少。为了确定这些活动是否在减数分裂成熟过程中获得，我们在生发泡破裂后不久对成熟的小鼠卵母细胞进行授精。如先前所示，在此阶段授精并未激活成熟的卵母细胞，这些卵母细胞停滞在减数第二次分裂中期。免疫荧光分析显示，授精后1小时，精子染色质分散，含有鱼精蛋白，但缺乏核心组蛋白H2B和H3。授精后4小时，在精子染色质上可检测到鱼精蛋白和核心组蛋白。到授精后8小时，鱼精蛋白消失，组蛋白染色最强。免疫反应性组蛋白的出现与精子染色质从分散状态到浓缩状态的形态转变相关，这表明组蛋白的组装反映了染色质向体细胞样状态的修饰，使其能够对卵母细胞的促成熟因子活性作出反应。当蛋白质合成受到抑制时，组蛋白的组装和染色质浓缩均被可逆性阻断，这表明重塑过程需要在成熟过程中合成的蛋白质。将核心组蛋白注射到蛋白质合成受抑制的卵母细胞中未能诱导精子染色质浓缩，这意味着正确的重塑需要在成熟过程中合成非组蛋白。为了测试重塑的精子染色质的功能能力，对成熟的卵母细胞进行授精，使其继续成熟17小时，然后进行孤雌激活。激活后，精子来源的染色质以及卵母细胞的染色质在原核内解聚并进行DNA复制，这表明在成熟卵母细胞细胞质中重塑的精子染色质功能正常。然而，当授精后的孵育时间缩短至11小时时，雌原核和雄原核均未进行DNA复制，这意味着成熟后期合成的因子是激活后DNA复制所必需的。综上所述，这些结果表明，在减数分裂成熟过程中，卵子具备将精子DNA组织成功能性体细胞样染色质的能力，这一过程需要在成熟过程中合成的蛋白质，并且可以独立于激活而表达。
