Cytochemical studies of oocyte development in Sarcophaga lineatocollis Macq. (Muscidae: Diptera)

The ovary of Sarcophaga lineatocollis is a typical polytrophic ovary. Each of its 25-30 ovarioles is composed of a small terminal filament, a small germarium and a vitellarium consisting of the egg follicle. The tunica propria is a noncellular, PAS-positive membrane. The ovarian follicle contains fifteen trophocytes and one oocyte. RNA is synthesized with the aid of the nuclei in the trophocyte cytoplasm, which are RNA- and PAS-positive. Protein is deposited intensively in the early stages of the trophocytes. The trophocytes of Sarcophaga lineatocollis synthesize RNA and protein more actively than the oocyte. In this fly, protein yolk precursor (PYP) bodies are supplied by the trophocyte cytoplasm to the ooplasm at an advanced stage of development. Nucleolar budding and vacuolation are observed in the trophocytes. RNA, DNA, protein and PYP bodies appear to be transported to the ooplasm from the trophocytes. Pyknotic trophocyte nuclei can be seen entering the ooplasm. The perinuclear Golgi bodies of the trophocytes help in the production and maturation of PYP bodies in the trophocytes before they are organized and passed on to the oocytes. Some RNA is contributed to the oocyte by the follicular epithelium. All these processes leading to maturation and development of the oocyte are discussed and interpreted.     

Ultrastructure of trophocyte nuclei in polytrophic ovarioles of Chrysopa perla (Neuroptera)]

The light and electron microscope and autoradiographic studies (H3-uridin incorporation) were carried out on the trophocyte nuclei of imago polytrophic ovarioles of Chrysopa perla (Neuroptera), from the trophocyte differentiation up to their degeneration. Like the oocytes, one of the seven nurse cells o every ovariole chamber contains extrachromosomal DNA bodies. This nurse cell is formed during differential mitoses in the germarium as one of two prooocytes. In contrast to extrachromosomal DNA of oocytes the trophocyte DNA bodies are less active structures. Several (2--4) complex nucleoli develop in the trophocytes of Chrysopa in the early stages of oogenesis. They consist of three main components: the chromatin mass, fibrillar bodies and granular strands. Such nucleoli grow, through increasing in number of fibrillar bodies and granular strands. They are most developed by the start of the vitellogenesis. At the middle vitellogenesis the general nucleolar structure modify due to the beginning of trophocyte degeneration. The consecutive stages of nuclear degeneration are described. The trophocyte nucleoli synthesize RNA still in germarium. The most intensive RNA synthesis is observed at the beginning of the vitellogenesis to decrease by the beginning of trophocyte degeneration.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). II. The oocyte nucleus of Blaps lethifera and Gnaptor spinimanus. Light optical data]

Changes in the nuclear structures and their participation in RNA synthesis in the growing oocytes were followed in two species of beetles Blaps lethifera and Gnaptor spinimanus. In the oocytes of both the species, the chromosomes join into the karyosphere following the short-term lampbrush stage. A large capsule appears around the karayosphere which consists of the fibrous substance, granules and karyosphere nucleoli. The latter form in the karyosphere and contain RNP but they are not true nucleoli since they do not include 3H-uridine. RNA synthesis on the chromosomes, active at the lampbrush stage, falls markedly following their joining into the karyosphere. The oocyte nuclei of these beetles are, thus, characterized by the absence of RNA synthesizing nucleolar system and, as compared with the trophocytes, by the low level of RNA synthesis on the chromosomes.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). III. The oocyte nucleus. Electron microscopic data]

The fine structural organization of nuclei was studied in the growing oocytes of Blaps lethifera, B. mortisaga and Gnaptor spinimanus. In the beginning of diplotene the nuclei contain primary fibrillar nucleoli and numerous electron dense globules dispersed all over the nucleus; the loose chromosome material (lampbrush chromosomes) is distributed all over the nucleus. With the oocyte growth the chromosomes are spiralized and join into the karyosphere. A capsule of fibrous material forms around the karyosphere. The karyosphere nucleoli appear on the chromosomes and, then, move to the capsule region and outside its limits, to the nuclear envelope. They are fibrillar and non-active with respect to RNA synthesis. The fibrous material of the capsule is represented by strands which consist of bundles of cross-striated filaments. These latter contact directly with the chromosomes in the karyosphere and with the surface of the karyosphere nucleoli. The fibrillar-granular bodies are distributed along the strands in the capsule; they contain both RNA and DNA. The nature of extrachromosomal DNA in the karyosphere capsule and its participation in the formation of the capsule material are discussed. A suggestion is put forward on the similarity of the capsule strands with the modified central elements of synaptinemal complex.     

Nucleoli and perinuclear bodies in trophocytes of Panorpa communis contain factors of pre-mRNA splicing

The distribution of pre-mRNA splicing factors and protein coilin was examined in trophocyte nuclei (TN) in polytrophic ovarioles of Panorpa communis. In situ hybridization, using antisense U1 and U6 snRNA 3H-riboprobes, showed that TN were labeled evenly. Immunostaining at light and electron microscopic levels revealed in some TN nucleolar structures containing small nuclear RNP (snRNP) and protein coilin characteristic of the Cajal bodies/coiled bodies (CB). No free CBs were found in TN. These data showed that CB in TN are present only in the nucleoli. One of characteristic features of P. communis trophocytes is the presence of several types of perinuclear bodies (PB) in the cytoplasm. We distinguish between three types of PBs. PB-1 consist of spherical bodies (10-20 microns) with vacuoles composed of closely packed fibrils. PB-2 are irregularly shaped bodies (0.3-2.0 microns) consisting of a fibro-granular material. PB-2 are located near the nuclear envelope and contact the nucleoplasm material through nuclear pores. PB-1 and PB-2 join together to form a complex PB of the third type. All types of PB are not surrounded with a membrane and sometimes have mitochondria on their surface. The immunogold technique at the ultrastructural level revealed snRNP in PB-2. These results have enabled us to make a conclusion that PB-2 may be storage sites of snRNPs required for a future development of the embryo.     

Karyosphere and extrachromosomal nuclear bodies in oocytes of the scorpionfly, Panorpa communis

Oocyte nuclear structures were studied for the scorpionfly Panorpa communis at different stages of oocyte growth, from pachytene to the first meiotic division. Using immunofluorescent and immunogold microscopy, we analyzed the nuclear distribution of RNA polymerase II, splicing factors and coilin. These factors were revealed in close association with perichromatin fibrils and, later, with some elements of the karyosphere and extrachromosomal nuclear bodies (NBs). Besides, it was shown that large amounts of P. communis oocyte NBs represent Cajal bodies (CBs) and contain CB marker protein, coilin, as well as RNA polymerase II, and in some cases an essential splicing factor, SC35. The presence of SC35 is commonly not characteristic of CBs in somatic cells. CB dynamics was traced in inactivated oocyte nuclei, during a gradual condensation of chromosomes and their final assembling into the karyosphere. It has been shown that coilin, RNA polymerase II and SC35 protein are common compounds shared by CBs and some granular material associated with these condensed chromosomes. CB remnants were demonstrated in the ooplasm after the breakdown of nuclear envelope before the first meiotic division. In inactivated oocyte nuclei, CBs serve presumably as storage compartments for some inactive components essential for gene expression.     

Characteristics of karyosphere formation in the lake frog. Light microscopy data

Morphological peculiarities of the oocyte nuclear organization were examined in R. ridibunda during winter and spring (February-March). Numerous nucleoli were seen to be assembled around regressive lampbrush chromosomes in the centre of the nucleus, and a central body was formed to which the chromosomes were attached. As result, a structural complex is constituted that involves a karyosphere and a capsule. Nucleoli are characterized by segregation and intensive fragmentation of their material. In result, a considerable part of nucleolar DNA is eliminated in the form of ring and polymorphous structures (micronucleoli). Besides the membranous component of nucleoli (nucleolar threads or tails) is lost. Towards the end of this period, nucleoli with complicated morphology become spherical again. The formation of the central body is started from the appearance of some small optically-light protein structures 5-20 nm in diameter (central body precursors-CBP). CBP are closely surrounded with ring micronucleoli to make intimate contact with the chromosomes and nucleolar threads. CBP commonly lie in one region of the nucleus not far from each other. The formation of a definitive central body obviously occurs due to a fusion of some small CBP. A conclusion is made of the nucleolar origin of the ring and polymorphous structures and of their essential role in the central body formation. The participation of chromosomal and eliminated nucleolar DNA in this process is discussed.     

Intranuclear lipids in the late oocytes of the common frog

The complex of chromosomes and nucleoli, constituting the karyosphere with a capsule, was removed micro-surgically from the late oocyte nuclei of Rana temporaria. Lipids of nuclei and of karyosphere were investigated using biochemical and autoradiographical methods in hormone-stimulated maturing oocytes in vitro. Neutral lipids (triglyceride, diglyceride, cholesterol ester) were found in the karyosphere substance by thin-layer chromatography. During oocyte maturation the incorporation of a precursor (3H-glycerol) into triglyceride was seen to increase much more than into lecithin. The autoradiography on the sectioned oocytes showed that the intranuclear level of 3H-glycerol was more densely distributed in the nucleolar zone over the material of a fibrous component of the karyosphere capsule. The level was also detected over the central part of the karyosphere in close proximity to the chromosomes. The involvement of lipids in organization of the complicated intranuclear complex of the karyosphere with a capsule is discussed. It is suggested that lipid accumulation in the area of the karyosphere fibrous component may reflect their functional relation with the oocyte nuclear matrix.     

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

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.     

Functional organization of the nucleus in the oogenesis of Chrysopa perla L. (Insecta,Neuroptera)

On the basis of light, autoradiographic (uridine-³H incorporation) and electron microscopic investigation changes of nuclear structures were examined during the oogenesis in Chrysopa perla L. — In early meiotic prophase the oocyte nuclei were found to contain a large body of extrachromosomal DNA. In certain cases the latter splits up into several DNA clumps giving rise to a few (4–7) primary nucleoli, 3–5 in diameter. The primary nucleoli consist of densely packed fibrils 50–100 Å thick. They contain no granular component and are inactive in RNA synthesis. — At the beginning of large growth the extrachromosomal DNA bodies disappear and numerous electron-dense clumps, 0,5–1 in diameter, appear in the nucleus. Instead of the primary nucleoli, the nucleus now contains a great number of ring nucleoli about 0,5–1 in diameter with a granular component (granules are 150 Å). The space between them is filled up with nucleolar strands running from the surface of the ring nucleoli. — At the stage ring nucleoli of uridine^–3 H incorporation into the oocyte nucleus begins. — During later previtellogenesis and at the beginning of vitellogenesis the ring nucleoli disappear and the nucleus is filled with the network of nucleolar strands. Among them there are specific complexes. These consist of electron dense masses, of granular clusters (granules 500 Å in diameter) and large fibrillar electron light bodies. At this stage the nucleus takes the most active part in RNA synthesis. — The process of karyosphere capsule formation was studied by electron microscopy. The capsule was found to be of fibrillar nature; its structure is very peculiar and unlike any known membrane components of the cell. On the basis of cytochemical evidences the characteristics of the capsule are given. — The development of a powerful nucleolar apparatus based on the extrachromosomal DNA and a possible role of the synaptonemal complex and extrachromosomal DNA in formation of the karyosphere capsule is discussed.     

Immunomorphological localization of Vasa protein and pre-mRNA splicing factors in Panorpa communis trophocytes and oocytes

The distribution of Vasa protein and splicing factors of pre-mRNA has been studied in oogenesis of Panorpa communis. This distribution was analyzed relative to three types of perinuclear bodies (PBs) in trophocyte cytoplasm, PBs and polar granules (PGs) in oocyte. Immunoelectron labeling using antibody against Vasa protein revealed PBs of the 2nd type of P. communis trophocytes as well as oocyte PBs and PGs to contain Vasa protein. From this evidence emerged proposal that PBs of the 2nd type are homologues to the "nuage" of Drosophila, a marker of germ line cells. Besides, we suggest that in P. communis, both trophocytes and oocytes take part in formation of PGs. Using immunoelectron microscopy, we also show small nuclear RNPs both in trophocyte PBs of the 2nd type and in oocyte PBs. The functional significance of coupling in the same structure of Vasa protein and snRNPs is discussed.     

Cytoarchitecture of the ovarioles in scale insects (Hemiptera,Coccinea)

Telotrophic ovarioles of scale insects are subdivided into tropharia (=trophic chambers) and vitellaria that contain single developing oocytes. Tropharium encloses trophocytes (=nurse cells) and arrested oocytes. The central area of the tropharium, termed the trophic core, is devoid of cells. Both trophocytes and oocytes are connected to the trophic core: trophocytes by cytoplasmic processes, oocytes by means of nutritive cords. The trophic core, processes and nutritive cords are filled with bundles of microtubules. The trophocytes contain large lobated nuclei with giant nucleoli. Fluorescent labelling with DAPI has shown that trophocyte nuclei are characterized by high contents of DNA. In the cortical cytoplasm of trophocytes, numerous microfilaments are present. The developing oocyte is surrounded by a simple follicular epithelium. The cortical cytoplasm of follicular cells contains numerous microtubules and microfilaments.     

Karyosphere in oogenesis]

The purpose of this review is to draw attention to the peculiar phenomenon during gametogenesis: the formation of the karyosphere. This phenomenon is characterized by concentration of all chromosomes in the limited area of the nucleus and may be considered as alternative of the genome in the state of lumpbrush chromosomes. The formation of the karyosphere is a widely spread phenomenon during oogenesis of different animal classes. The karyosphere can be developed during different stages of oogenesis in different organisms; but as a rule the chromosomes of diploten stage of meiosis take part in its formation. As to functional identity of the karyosphere in different species, special investigations are to be done, but contemporary knowledge of the karyosphere formation reveals some common feature:1) in the karyosphere the chromosomes are in a relatively spiral state as demonstrated by the positive Feulgen reaction; 2) there is a low level of RNA synthesis or the absence of it in the karyosphere; 3) during the karyosphere formation the nucleus is enriched by the acid proteins and a lot of protein granules and structures appearing in a close contact with the karysphere. The more typical examples of the karyosphere formation can be observed in the insect oocytes belonging to the nutrimentary type of oogenesis. In the oocytes of some animals the peculiar protein substances are formed around the chromosome knot and appear as a fibrillar zone. Such karyosphere appears to be a kind of capsule inside the nucleus. The capsules are developed as a result of complex interaction between the main nuclear structures; chromosomes, nucleoli, and nuclear membrane as it is manifested by the analysis of some recent ultrastructural date obtained in some insect and amphibian oocytes. The function of the karyosphere capsule and the role of the nuclear structure (sinaptonemal complex, extrachromosomal DNA, and nuclear membrane) in formation of the capsule, are discussed as well as the ultrastructural and cytochemical similarity between the karyosphere capsule of oocytes and nuclear bodies of somatic cells.     

Oogenesis in phthirapterans (Insecta: Phthiraptera). I. Morphological and histochemical characterization of the oocyte nucleus and its inclusions

We characterize morphological and histochemical changes occurring within the oocyte nucleus (germinal vesicle) during oogenesis in two phthirapteran species: the pig louse, Haematopinus suis (Anoplura) and the pigeon louse, Columbicola columbae (Mallophaga). In previtellogenic oocytes, within the oocyte nucleus the chromatin condenses and forms the karyosome. In contact with the karyosome numerous dense and highly heterogeneous nuclear bodies occur. We demonstrate that these nuclear inclusions have complex structure and contain RNA and argyrophilic proteins of nucleolar organizer (Ag-NOR proteins). Results of immunogold electron microscopy experiments are also presented. The obtained results suggest that the phthirapteran nuclear bodies are assemblages of ribonucleoproteins that are stored in the oocyte nucleus and might be utilized during early stages of embryonic development. In the investigated species, the nuclear envelope of the germinal vesicle is equipped with characteristic protrusions. Ultrastructural analysis revealed striking similarity of these structures to the initial stages of the formation of accessory nuclei. Based on these results, we speculate on the possible evolutionary origin of the accessory nuclei in phthirapterans.     

Nuclear ultrastructure of the oocytes from human antral follicles. Formation of the karyosphere

The organization of the nucleus in the oocytes from human antral follicles was examined at the electron microscopic level. At this time all the chromosomes are aggregated around an inactivated nucleolus forming a karyosphere 5-7 micron in diameter. The nucleolus bears no granular component and consists of densely packed delicate fibrillar material. The peripheral zone resembling a ring 0.5 micron thick is separated in the nucleolus. Nucleolus-like bodies (NLB), consisting of granules 20 nm in diameter embedded in finely fibrillar material, are constantly observed in contact with the chromatin. The eventually formed karyosphere is a complex of intimately interconnecting structures--the nucleolus, chromosomes and NLB. However, the chromatin surrounding the nucleolus does not form a continuous (compact) mass as it is observed at the light microscopic level. It is determined that the human karyosphere is formed during the preovulatory period when the connection between oocyte and follicular cells of cumulus oophorus is lost. The duration of karyosphere existence in the human oocytes, and relation of the karyosphere to the processes of antral follicle atresia are discussed.     

The karyosphere during late oogenesis in Rana ridibunda.

The organization of the nucleus in the oocytes of Rana ridibunda was examined during late diplotene at the light and electron microscopic level. At this stage the chromosomes are relatively condensed and assembled in the centre of the nucleus, constituting a karyosphere. The chromosomes here are associated with the central "protein sphere" (15--20 microns in diameter), obviously at their telomeres. Numerous nucleoli are accumulated around the chromosomes, forming a karyosphere capsule and contain segregated fibrillar and granular components; structures resembling perinucleolar chromatin and fibrillar bodies (spherules) are associated with the nucleoli. Granules 30 to 40 nm in diameter are seen to surround the fibrillar spherules. "Nucleolus-like bodies" consisting of granules 10 to 15 nm in diameter which are embedded in finely fibrillar material are often associated in contact with the chromosomes. The central sphere is an accumulation of annular structures similar to those of the pore complexes of the nuclear envelope. These structures are bound to the chromosome material, the "nucleolus-like bodies" and the fibrillar bodies. A participation of "nucleolus-like bodies" in the formation of the central sphere is suggested. A possible role of the nuclear protein matrix in the construction of the karyosphere elements is discussed.