FINE-STRUCTURAL CYTOCHEMICAL AND IMMUNOCYTOCHEMICAL ANALYSIS OF NUCLEIC-ACIDS AND RIBONUCLEOPROTEIN DISTRIBUTION IN NUCLEI OF PIG OOCYTES AND EARLY PREIMPLANTATION EMBRYOS

The fine structure of pig oocytes at the germinal vesicle (GV) stage a nd early preimplantation embryos (one to four blastomeres) isolated at slaughter was investigated by cytochemical and immunocytochemical met hods. The distribution of nucleic acids and ribonucleoproteins (RNPs) in ''compact nucleoli'' [denominated nucleolus-like bodies (NLB) in oo cytes and nucleolus precursor bodies (NPB) in early embryos] and in in tranuclear bodies or granules was investigated by staining methods pre ferential for nuclear RNPs or using the osmium ammine or ethidium brom ide-phosphotungstic acid (EB-PTA) reactions for nucleic acids. The dis tributions of the Sm antigen of nucleoplasmic small nuclear RNPs (snRN Ps), the methyl-3 guanosine (m(3)G) cap of snRNAs and the splicing fac tor SC-35 were detected by immunoelectron microscopy using specific an tibodies. The RNP nature of both NLBs and NPBs, and of nuclear granule s 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 th e enzymatic removal of RNA, or by osmium-ammine staining without previ ous acid hydrolysis, while the absence of DNA in NLBs was established by Feulgen-like osmium-ammine staining. In addition, autoradiography d emonstrated the absence of [6-H-3]thymidine incorporation into NPBs. O ther autoradiographic evidence attested the accumulation of RNA in NLB s of oocytes after a 60 min in vitro pulse of [5-H-3]uridine. Immunoel ectron 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 m(3)G-cap st ructure. Another spliceosomal component, the protein SC-35 was also de tected in NLBs. Among the numerous and variable intranuclear granules occurring mostly in aggregates, the Sm antigen was clearly detected on ly in the in terchromatin 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-m(3)G-cap antibody . The aggregates consisting of large granules and a finely fibrillar c omponent were intensely immunolabeled by the anti-SC-35 splicing facto r 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 cont aining nonnucleolar, spliceosomal components.