In oocyte nuclei of the scorpionfly, Panorpa communis, we have recently defined a population of nuclear bodies (NBs) that contain some components of Cajal bodies (CBs). In the present study, we used several criteria [presence of coilin, U7 snRNA, RNA polymerase II (pol II) and specific ultrastructure] to identify these NBs as CBs. The essential evidence for CB identification came from experiments with microinjection of fluorescein-tagged U7 snRNA. Consistent with the U7 data, we found pol II and pre-mRNA splicing factor, SC35, in Panorpa oocyte CBs. We show here that the dynamics of CBs differs from that in somatic cells and correlates with the level of oocyte chromosome condensation. We also found that the significant increase of CB size is accompanied by condensation of the chromosomes in the karyosphere, which is indicative of a decline in transcription. Using immunogold microscopy we determined that pol II and coilin are shared by CBs and the granular material associated with condensed chromosomes in the Panorpa karyosphere. The colocalization of pol II, U7 snRNA and splicing factors with CBs at the inactive stage of late oogenesis suggests that the latter may serve as storage domains for components that were earlier engaged in RNA transcription and processing.

It is now clear that two prominent nuclear domains, interchromatin granule clusters (IGCs) and Cajal bodies (CBs), contribute to the highly ordered organization of the extrachromosomal space of the cell nucleus. These functional domains represent structurally stable but highly dynamic nuclear organelles enriched in factors that are required for different nuclear activities, especially RNA biogenesis. IGCs are considered to be the main sites for storage, assembly, and/or recycling of the essential spliceosome components. CBs are involved in the biogenesis of several classes of small RNPs as well as the modification of newly assembled small nuclear RNA. We have summarized data on the molecular composition, structure, and functional roles of IGCs and CBs in the nuclei of mammalian somatic cells and oocytes of some animals with a special focus on insects. We have focused on similarities and differences between the IGCs and CBs of oocytes and the well-studied CBs and IGCs of cultured mammalian somatic cells. We have shown the heterogeneous character of oocyte IGCs and CBs, both in structure and molecular content. We have also demonstrated the unique capacity of oocytes to form close structural interactions between IGC and CB components. We proposed to consider these joint structures as integrated entities, sharing the features of both IGCs and CBs.

Most of the human genome is non-coding. However, some of the non-coding part is transcriptionally active. In humans, the tandemly repeated (TR) pericentromeric non-coding DNA—human satellites 2 and 3 (HS2, HS3)—are transcribed in somatic cells. These transcripts are also found in pre- and post-implantation embryos. The aim of this study was to analyze HS2/HS3 transcription and cellular localization of transcripts in human maturating oocytes. The maternal HS2/HS3 TR transcripts transcribed from both strands were accumulated in the ooplasm in GV-MI oocytes as shown by DNA–RNA FISH (fluorescence in-situ hybridization). The transcripts’ content was higher in GV oocytes than in somatic cumulus cells according to real-time PCR. Using bioinformatics analysis, we demonstrated the presence of polyadenylated HS2 and HS3 RNAs in datasets of GV and MII oocyte transcriptomes. The transcripts shared a high degree of homology with HS2, HS3 transcripts previously observed in cancer cells. The HS2/HS3 transcripts were revealed by a combination of FISH and immunocytochemical staining within membraneless RNP structures that contained DEAD-box helicases DDX5 and DDX4. The RNP structures were closely associated with mitochondria, and are therefore similar to membraneless bodies described previously only in oogonia. These membraneless structures may be a site for spatial sequestration of RNAs and proteins in both maturating oocytes and cancer cells.

The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis.

We performed immunofluorescent analysis of DNA hydroxymethylation and methylation in human testicular spermatogenic cells from azoospermic patients and ejaculated spermatozoa from sperm donors and patients from infertile couples. In contrast to methylation which was present throughout spermatogenesis, hydroxymethylation was either high or almost undetectable in both spermatogenic cells and ejaculated spermatozoa. On testicular cytogenetic preparations, 5-hydroxymethylcytosine was undetectable in mitotic and meiotic chromosomes, and was present exclusively in interphase spermatogonia Ad and in a minor spermatid population. The proportions of hydroxymethylated and non-hydroxymethylated diploid and haploid nuclei were similar among samples, suggesting that the observed alterations of 5-hydroxymethylcytosine patterns in differentiating spermatogenic cells are programmed. In ejaculates, a few spermatozoa had high 5-hydroxymethylcytosine level, while in the other ones hydroxymethylation was almost undetectable. The percentage of highly hydroxymethylated (5-hydroxymethylcytosine-positive) spermatozoa varied strongly among individuals. In patients from infertile couples, it was higher than in sperm donors (P<0.0001) and varied in a wider range: 0.12-21.24% versus 0.02-0.46%. The percentage of highly hydroxymethylated spermatozoa correlated strongly negatively with the indicators of good semen quality – normal morphology (r=-0.567, P<0.0001) and normal head morphology (r=-0.609, P<0.0001) – and strongly positively with the indicator of poor semen quality: sperm DNA fragmentation (r=0.46, P=0.001). Thus, the immunocytochemically detected increase of 5hmC in individual spermatozoa is associated with infertility in a couple and with deterioration of sperm parameters. We hypothesize that this increase is not programmed, but represents an induced abnormality and, therefore, it can be potentially used as a novel indicator of semen quality.