Vladimir N. Parfenov

Karyosphere in oogenesis and intranuclear morphogenesis

Publisher Summary This chapter highlights karyosphere in the phenomena of oogenesis and intranuclear morphogenesis. Karyosphere is formed as the result of all chromosomes of the gametocyte joining in a limited nuclear volume with final formation of a single complex chromatin structure—a type of nucleus inside the germinal vesicle. Karyosphere formation is the result of relative inactivity of chromosomes during RNA synthesis because it is formed either in nutrimental oogenesis or after a long period of lampbrush-chromosome activity. Formation of the karyosphere is accompanied by intensive nuclear cytoplasmic exchange that results in the appearance of numerous protein granules and bodies in the karyosphere and their subsequent transfer toward the nuclear periphery. Karyosphere is quite common in gametogenesis—in particular, oogenesis. The stage of gametogenesis in which the karyosphere is formed is always a longterm diplotene of meiosis. In vertebrates, karyosphere formation is paralleled by the appearance around the chromosomes of newly formed capsule-shaped structures. A characteristic trait of karyosphere in some species is that it is surrounded by a multilayered capsule. The capsule usually appears in large germinal vesicles rather than in small nuclei.

Universal nuclear domains of somatic and germ cells: some lessons from oocyte interchromatin granule cluster and Cajal body structure and molecular composition

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.

Nuclear distribution of Oct-4 transcription factor in transcriptionally active and inactive mouse oocytes and its relation to RNA polymerase II and splicing factors

The intranuclear distribution of the transcription factor Oct‐4, which is specifically expressed in totipotent mice stem and germ line cells, was studied in mouse oocytes using immunogold labeling/electron microscopy and immunofluorescence/confocal laser scanning microcopy. The localization of Oct‐4 was studied in transcriptionally active (uni/bilaminar follicles) and inactive (antral follicles) oocytes. Additionally, the Oct‐4 distribution was examined relative to that of the unphosphorylated form of RNA polymerase II (Pol II) and splicing factor (SC 35) in the intranuclear entities such as perichromatin fibrils (PFs), perichromatin granules (PGs), interchromatin granule clusters (IGCs), Cajal bodies (CBs), and nucleolus‐like bodies (NLBs). It was shown that: (i) Oct‐4 is localized in PFs, IGCs, and in the dense fibrillar component (DFC) of the nucleolus at the transcriptionally active stage of the oocyte nucleus; (ii) Oct‐4 present in PFs and IGCs colocalizes with Pol II and SC 35 at the transcriptionally active stage; (iii) Oct‐4 accumulates in NLBs, CBs, and PGs at the inert stage of the oocyte. The results confirm the previous suggestion that PFs represent the major nucleoplasmic structural domain involved in active pre‐mRNA transcription/processing. The colocalization of Oct‐4 with Pol II in both IGCs and PFs in active oocytes (uni/bilaminar follicles) suggests that Oct‐4 is intimately associated with the Pol II holoenzyme before and during transcription. The colocalization of Oct‐4, Pol II, and SC 35 with coilin‐containing structures such as NLBs and CBs at the inert stage (antral follicles) suggests that the latter may represent storage sites for the transcription/splicing machinery during the decline of transcription. J. Cell. Biochem. 89: 720–732, 2003.

An immunoelectron study of karyosphere and nuclear bodies in oocytes of mealworm beetle, Tenebrio molitor (Coleoptera: polyphaga).

Abstract. The karyosphere and nuclear bodies (NBs) were studied in Tenebrio molitor oocytes using immunoelectron cytochemistry. During early diplotene (previtellogenic stage), oocyte chromosomes begin to unite in a small nuclear volume forming the karyosphere. In vitellogenic oocyte nuclei, the chromatin undergoes condensation, and the karyosphere acquires a ring-shaped structure. The karyosphere is the only structure containing DNA in the oocyte nucleus. Pre-mRNA splicing factors [small nuclear ribonucleoproteins (snRNPs) and SC35] are not found in the karyosphere itself. In previtellogenic oocyte nuclei, these factors are present in NBs and in a fibrogranular substance surrounding the chromosomes in the early stages of karyosphere formation. At this stage, larger fibrillar NBs contain the non-snRNP splicing factor SC35. Smaller roundish NBs were shown to contain snRNPs. Some NBs with the same morphology contain neither snRNPs nor SC35. In the vitellogenic oocyte, there are fibrogranular NBs containing both snRNPs and SC35 splicing factors, fibrillar NBs containing snRNPs only, and complex NBs containing both. Complex NBs are often connected with the ring-shaped karyosphere. Based on the obtained immunoelectron data, we suggest that T. molitor oocyte NBs containing both snRNPs and the non-snRNP splicing factor SC35 are homologs of the well-characterized B-snurposomes in amphibian germinal vesicles and clusters of interchromatin granules in mammalian oocyte nuclei. Other NBs containing only snRNPs are suggested to represent a special class of insect oocyte snurposomes. The nuclear organelles mentioned seem to play a role as storage domains for pre-mRNA splicing factors during T. molitor oogenesis.

Nuclear distribution of RNA polymerase II in human oocytes from antral follicles: dynamics relative to the transcriptional state and association with splicing factors.

The intranuclear distribution of two (unphosphorylated and hyperphosphorylated) forms of RNA polymerase II (Pol II) was studied in human oocytes from antral follicles using immunogold labeling/electron microscopy. The distribution of Pol II was analyzed relative to the transcriptional state of the oocyte as well as to the distribution of two splicing factors (snRNPs and SC‐35) in the intranuclear entities, namely, interchromatin granule clusters (IGCs), nucleolus‐like bodies (NLBs), and perichromatin fibrils (PFs). The results showed that (1) antibodies directed against two forms of Pol II have similar pattern of intranuclear distribution, (2) both Pol II and splicing factors progressively accumulate in IGCs with decrease in the transcriptional activity of the oocyte nucleus, (3) both Pol II and splicing factors localize to PFs, and (4) Pol II is present in the NLBs at all transcriptional states of the oocyte nucleus. These studies confirm earlier proposals that PFs represent a nuclear domain in which RNA transcription/processing are spatially coupled. The accumulation of Pol II and splicing factors in IGCs concomitant with a decrease in the transcriptional activity suggests a coordinated mechanism for the movement of both Pol II and splicing factors from the sites of action to the sites of storage. J. Cell. Biochem. 77:654–665, 2000.

Chapter 2 Structure of the Insect Oocyte Nucleus with Special Reference to Interchromatin Granule Clusters and Cajal Bodies

The nuclear structure of insect oocyte is described with reference to interchromatin granule clusters and Cajal bodies. It is suggested that the intranuclear structure is determined by the ovary organization and reflects the sources of oocyte RNA. Inactivated oocyte chromosomes in meroistic ovaries form a karyosphere, and their features are discussed. In insects with panoistic ovaries, the organization and molecular composition of complicated oocyte Cajal bodies are discussed, and the homology between a structure located inside Cajal bodies and interchromatin granule clusters is proved. Along with revealing some essential Cajal body components and factors involved in RNA polymerase II transcription, we analyze the absence of RNAP II itself from oocyte Cajal bodies of Acheta. In insects with meroistic ovaries, Cajal bodies of Drosophila and homology of Panorpa nuclear bodies to Cajal bodies and their peculiarities are described, because the nuclear bodies may share some features of both Cajal bodies and interchromatin granule clusters to represent a single nuclear domain.

Dynamics of distribution of splicing components relative to the transcriptional state of human oocytes from antral follicles

The distribution of two splicing components (snRNP and SC‐35) and coilin were studied by immunogold/electron microscopy in human oocytes from antral follicles at different levels of transcriptional activity (i.e., active, intermediate, and inactive). The results showed a decrease of snRNPs and SC‐35 in the karyoplasm as the oocytes progress from a transcriptionally active to the inactive state. The main areas of accumulation of both these splicing components in all stages of oocytes appeared to be the interchromatin granule clusters (IGCs). Within the IGCs, the two splicing components seemed to be spatially segregated, with the snRNPs predominantly bound to the fibrillar region, whereas the SC‐35 factors are being enriched in the granular zone. The p80 coilin was found only in the nucleolus‐like body (NLB), which is present in all three stages of oocytes; no coiled bodies were evident. These data are consistent with the notion that splicing occurs in the karyoplasm and that the splicing components are mobilized from a storage site (IGCs) to the site of action. J. Cell. Biochem. 69:72–80, 1998.

Cajal bodies and interchromatin granule clusters in cricket oocytes: Composition, dynamics and interactions

The organization and molecular composition of complicated Cajal bodies (CBs) and interchromatin granule clusters (IGCs) in oocytes of the house cricket, Acheta domesticus, were studied using immunofluorescent/confocal and Immunogold labeling/electron microscopy. In A. domesticus oocytes, the CB consists of the fibrillar matrix and a central cavity containing a predominantly granular body with insertions of tightly packed fibrillar material. The latter structure was identified as an “internal” IGC, since it is enriched with the SC35 protein, a marker for IGCs. The IGCs located outside the CB were also identified. Microinjections of the fluorescein‐tagged U7 snRNA into the ooplasm showed the targeting of the U7 to the matrix of the CB. Some other essential CB components (coilin, snRNPs, fibrillarin) were found to be colocalized in the matrix of the CB. Neither confocal nor Immunogold microscopy revealed significant amounts of RNA polymerase II (pol II) in the CB of A. domesticus oocytes. The splicing factor SC35 was detected in the matrix of the CB. In oocytes treated with DRB, the amount of IGCs in the nucleoplasm increased significantly, granular and fibrillar components of IGCs were segregated, and the fibrillar areas accumulated pol II. Additionally, IG‐like granules were shown to display on the surface of the CB probably due to a shifting from the internal IGC. We believe that in A. domesticus oocytes, CBs are involved in nuclear distribution of splicing factors, but their role in pol II transport is less significant. We also suggest that the formation of complicated CBs is a result of interconnection between two different nuclear domains, CBs and IGCs.

Localization of poly(A)(+) RNA and mRNA export factors in interchromatin granule clusters of two-cell mouse embryos.

Interchromatin granule clusters (IGCs), also known as nuclear speckles, splicing factor compartments, or SC35-domains, are one of the most universal nuclear organelles of the cell. We have used two-cell mouse embryos as an experimental system to study the possible association of poly(A)+ RNA and factors involved in RNA export (Tip-associated protein [TAP] and heterogenous nuclear ribonucleoprotein A/B [hnRNP A/B]) with IGCs. Poly(A)+ RNA was localized by microinjections of 2′-O-Me(U)22 probes conjugated with tetramethylrhodamine. RNA export proteins were detected by immunofluorescence confocal laser microscopy and immunogold-labeling electron microscopy. We found that poly(A)+ RNA was located in IGCs regardless of the transcriptional state of the nuclei. hnRNP A/B was also found to characterize IGCs of the embryo nuclei with various levels of transcription activity. In transcriptionally active embryo nuclei, TAP was detected in the vicinity of IGCs rather than inside them; however, when transcription was inhibited by drugs, TAP was localized to IGCs. Our data support the idea that IGCs not only serve as splicing factor reservoirs, but also take part in mRNA retention and export.

Nucleolus transformation in mouse antral follicles: Distribution of coilin and components of RNA-polymerase I complex

This study is the next step in the examination of the nucleolus transformation in growing oocytes from mouse multilayer follicles (Pochukalina and Parfenov, 2006). Here, we present our results on the structural organization and molecular composition of postnucleoli in oocytes isolated from mouse antral folicules. Using light and electron microscopic immunocytochemistry, we examined the dynamics of the distribution of molecular components of rRNA synthesis and the processing in postnucleoli. Considerable changes were revealed in the RNA polymerase I distribution and its colocalization with coilin on the periphery of postnucleoli. The putative role of coilin in complex formation with ribosomal RNA synthesis/processing components is discussed.