Kopecny V, Biggiogera M, Laurincik J, Pivko J, Grafenau P, Martin T E, Fu X D, Fakan S
Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne, Switzerland.
Chromosoma. 1996 Jun;104(8):561-74. doi: 10.1007/BF00352296.
The fine structure of pig oocytes at the germinal vesicle (GV) stage and early preimplantation embryos (one to four blastomeres) isolated at slaughter was investigated by cytochemical and immunocytochemical methods. The distribution of nucleic acids and ribonucleoproteins (RNPs) in "compact nucleoli" [denominated nucleolus-like bodies (NLB) in oocytes and nucleolus precursor bodies (NPB) in early embryos] and in intranuclear bodies or granules was investigated by staining methods preferential for nuclear RNPs or using the osmium ammine or ethidium bromide-phosphotungstic acid (EB-PTA) reactions for nucleic acids. The distributions of the Sm antigen of nucleoplasmic small nuclear RNPs (snRNPs), the methyl-3 guanosine (m3G) cap of snRNAs and the splicing factor SC-35 were detected by immunoelectron microscopy using specific antibodies. The RNP nature of both NLBs and NPBs, and of nuclear granules in oocytes and embryos, and of fibrillar strands radially projecting from NLBs was revealed. Cytochemical evidence for RNA as a component of NLBs was further provided by EB-PTA staining in combination with the enzymatic removal of RNA, or by osmium-ammine staining without previous acid hydrolysis, while the absence of DNA in NLBs was established by Feulgen-like osmium-ammine staining. In addition, autoradiography demonstrated the absence of [6-3H]thymidine incorporation into NPBs. Other autoradiographic evidence attested the accumulation of RNA in NLBs of oocytes after a 60 min in vitro pulse of [5-3H]uridine. Immunoelectron microscopy using specific antibodies revealed the occurrence of nucleoplasmic snRNPs in both NLBs and NPBs. The presence of snRNA in NLB was confirmed by means of an antibody recognizing the m3G-cap structure. Another spliceosomal component, the protein SC-35 was also detected in NLBs. Among the numerous and variable intranuclear granules occurring mostly in aggregates, the Sm antigen was clearly detected only in the interchromatin granule-type component. Some Sm labeling was occasionally seen in other categories of larger granules. No reaction was detected over any granules when using the anti-m3G-cap antibody. The aggregates consisting of large granules and a finely fibrillar component were intensely immunolabeled by the anti-SC-35 splicing factor probe. Our observations suggest that the compact nucleoli, known to be present before and after fertilization in mammals (NLBs of oocytes and NPBs of early embryos), represent nuclear structural elements containing nonnucleolar, spliceosomal components.
采用细胞化学和免疫细胞化学方法，研究了屠宰时分离出的猪生发泡（GV）期卵母细胞和植入前早期胚胎（1至4个卵裂球）的精细结构。通过对核核糖核蛋白（RNP）有偏好的染色方法，或使用锇胺或溴化乙锭 - 磷钨酸（EB - PTA）核酸反应，研究了核酸和核糖核蛋白（RNP）在“致密核仁”（卵母细胞中称为核仁样体（NLB），早期胚胎中称为核仁前体（NPB））以及核内体或颗粒中的分布。使用特异性抗体通过免疫电子显微镜检测核质小核核糖核蛋白（snRNP）的Sm抗原、snRNA的甲基 - 3鸟苷（m3G）帽和剪接因子SC - 35的分布。揭示了NLB和NPB以及卵母细胞和胚胎中的核颗粒以及从NLB径向突出的纤维状链的RNP性质。通过EB - PTA染色结合RNA的酶促去除，或通过未经酸水解的锇胺染色，进一步提供了RNA作为NLB成分的细胞化学证据，而通过类福尔根锇胺染色确定NLB中不存在DNA。此外，放射自显影显示[6 - 3H]胸苷未掺入NPB。其他放射自显影证据证明，在[5 - 3H]尿苷体外脉冲60分钟后，RNA在卵母细胞的NLB中积累。使用特异性抗体的免疫电子显微镜显示，NLB和NPB中均存在核质snRNP。通过识别m3G帽结构的抗体证实了NLB中存在snRNA。另一种剪接体成分，蛋白质SC - 35也在NLB中被检测到。在大多聚集成团的众多且多样的核内颗粒中，仅在染色质间颗粒类型的成分中清晰检测到Sm抗原。在其他较大颗粒类别中偶尔可见一些Sm标记。使用抗m3G帽抗体时，在任何颗粒上均未检测到反应。由大颗粒和精细纤维状成分组成的聚集体被抗SC - 35剪接因子探针强烈免疫标记。我们的观察结果表明，已知在哺乳动物受精前后存在的致密核仁（卵母细胞的NLB和早期胚胎的NPB）代表含有非核仁剪接体成分的核结构元件。
