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.     

Assembly and breakdown of Cajal bodies in accessory nuclei of Hymenoptera

In some species of insects, oocytes have vesicular organelles, termed accessory nuclei (ANs). The ANs form by budding off from the nuclear envelope of the oocyte and are filled with translucent matrix containing dense inclusions. One type of these inclusions contains coilin and small nuclear ribonucleoproteins (snRNPs) and is homologous to Cajal bodies. We describe the early events in the morphogenesis of Cajal bodies in the ANs (ANCBs) of the common wasp, Vespula germanica, and show that they contain survival of motor neurons (SMN) protein. We present evidence that in the wasp, ANCBs form by the gradual accumulation of aggregates composed of SMN and small nuclear RNAs. We also show that ANCBs break down and disperse within the ANs as the ANs, which initially surround the oocyte nucleus, localize to the oocyte cortex. The components of dispersed ANCBs are retained within ANs until the end of oogenesis, which suggests that their function may be required at the onset of embryonic development. Because the morphology and behavior of ANs and their Cajal body-like inclusions are conserved in two other hymenopteran species, these features might be characteristic of all hymenopterans.     

Structure of the germinal vesicle during oogenesis in leech Glossiphonia heteroclita (Annelida, Hirudinea, Rhynchobdellida)

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.     

Subcortical microtubule network separates the periplasm from the endoplasm and is responsible for maintaining the position of accessory nuclei in hymenopteran oocytes

Oocytes of hymenopterans are equipped with peculiar organelles termed accessory nuclei. These organelles originate from the germinal vesicle (oocyte nucleus) and gather preferentially at the anterior pole. To gain insight into the mechanism of uneven (asymmetrical) distribution of accessory nuclei, the organization of the microtubule cytoskeleton in the oocytes of two hymenopterans Chrysis ignita and Cosmoconus meridionator has been studied. It is shown that during late previtellogenesis two networks of microtubules are present along the contact zone between the oocyte and enveloping follicular epithelium. The external one is associated with belt desmosomes connecting neighbouring follicular cells. The internal network is composed of randomly orientated microtubules and separates transparent, organelle-free periplasm from the endoplasm. All cellular organelles and the germinal vesicle are localized in the endoplasm. Accessory nuclei are accumulated in the anterior endoplasm; they always lie in direct contact with the subcortical network. Treatment with colchicine results in the disappearance of the periplasm as well as in the redistribution of cellular organelles including accessory nuclei. Presented findings suggest that subcortical microtubules play an important role in the positioning of accessory nuclei throughout the ooplasm.     

Accessory nuclei revisited: the translocation of snRNPs from the germinal vesicle to the periphery of the future embryo

Oocytes of certain insects contain peculiar organelles termed accessory nuclei (AN). These organelles originate by budding off from the envelope of the oocyte nucleus and contain 1-2 dense inclusions immersed in a translucent ground substance. We have demonstrated that in the wasp Vespula germanica each inclusion consists of two elements: a spherical body, and a hemispherical structure composed of numerous 20-30 nm particles. Immunoelectron microscopy and whole-mount in situ hybridization have shown that the inclusions contain AgNOR-staining proteins, p80-coilin, Sm proteins, and small nuclear RNAs (snRNAs). These results indicate that the inclusions and hemispherical structures are homologous to Cajal bodies and B-snurposomes of Xenopus germinal vesicles, respectively. During previtellogenesis, AN (together with their Cajal bodies) migrate to the cortical ooplasm of the oocyte where they reside at least until the onset of embryogenesis. We suggest that AN are vehicles for the transport and localization of snRNPs to the periphery of the oocyte, i.e., to the region where the blastoderm of the embryo develops and where there is a requirement for a high concentration of RNA-processing factors.     

Morphological markers of anteroposterior and dorsoventral polarity in developing oocytes of the hymenopteranCosmoconus meridionator (Ichneumonidae)

Summary The progressive establishment of anteroposterior and dorsoventral polarity in developing oocytes ofCosmoconus meridionator is described. In fully grown oocytes, the asymmetrical (polar) organization is apparent in the localization of the oocyte nucleus (germinal vesicle) and oosome, and in the uneven (graded) distribution of lipid droplets, yolk spheres and specific organelles termed accessory nuclei (AN). The latter structures occur preferentially within the anteroventral periplasm. The developmental significance of AN is discussed.     

Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy

Anti-tubulin immunofluorescence and laser-scanning confocal microscopy were used to examine microtubule organization during Xenopus oogenesis (Dumont stages I-VI). Stage I oocytes contained a poorly ordered microtubule array, characterized by concentrations of microtubule in the cortex, surrounding the germinal vesicle, and associated with the mitochondrial mass. No focus of microtubule organization was detectable by optical sectioning or in microtubule regrowth experiments, suggesting that stage I oocytes lack a functional MTOC. The microtubule array becomes progressively more complex and polarized during oogenesis; an extensive array of microtubules and microtubule bundles was apparent in the animal hemisphere of stage VI oocytes, and a less ordered array was observed in the vegetal hemisphere. A dense network of microtubules surrounded the germinal vesicle, apparently extending from a tubulin- and microtubule-rich region of cytoplasm adjacent to the vegetal surface of the GV. The organization of microtubules in normal oocytes, in oocytes recovering from cold-induced microtubule depolymerization, and in enucleated oocytes, suggested that the germinal vesicle serves as an MTOC in stage VI oocytes. Antibodies to acetylated alpha-tubulin revealed numerous acetylated, presumably stable, microtubules in stage I and stage VI oocytes. The array of oocyte microtubules thus might function as a stable framework for the localization of developmentally important molecules and organelles during oogenesis.     

Formation and function of accessory nuclei in the oocytes of the bird louse,Eomenacanthus stramineus (Insecta,Mallophaga)

In the oocytes of Eomenacanthus stramineus accessory nuclei arise by budding from the nuclear envelope. It is suggested that microtubules and the thick layer of the nuclear lamina are involved in this process. Newly formed accessory nuclei contain aggregations of fibrillogranular material. These aggregations are slightly Feulgen positive, RNA negative and stain positively with the AgNOR method. During later developmental stages one dense, RNA-positive inclusion appears in each accessory nucleus. These inclusions consist of on Ag-NOR-positive cortical layer and an Ag-NOR-negative core. The function of accessory nuclei in the species investigated is discussed in the light of these results.     

Early oogenesis in the ascidian Ciona savignyi

Summary

Morphology of the germinal epithelium and the early follicular oocyte in the ascidian Ciona savignyi as examined by electron microscopy. The oogenetic part of the germinal epithelium contains oocytes at two different stages and the dark and clear cells. The smaller oocyte contains synaptonemal complexes. The larger oocyte in the initial phase of growth has a conspicuous nucleolus, electron-dense materials and some mitochondria close to the nuclear envelope. The nucleus of the larger oocyte is round and has the smooth contour. The dark cell contains a relatively large nucleus and is sometimes connected to each other by an intercellular bridge. Therefore, the dark cell, which has been suggested to be the progenitor cell of two kinds of accessory cells, may be also the oogonium. The early follicular oocyte just after migration from the germinal epithelium retains most of cytological features similar to those of the larger oocyte. However, the nuclear contour of the early follicular oocyte is uneven. This difference in the nuclear contour probably indicates that such a follicular oocyte is in the second phase of growth.     

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.     

东方扁虾卵子发生的超微结构

根据卵细胞的形态、内部结构特征及卵母细胞与滤泡细胞之间的关系,东方扁虾的卵子发生可划分为卵原细胞、卵黄发生前卵母细胞、卵黄发生卵母细胞和成熟卵母细胞等四个时期。卵原细胞胞质稀少,胞器以滑面内质网为主。卵黄发生前卵母细胞核明显膨大,特称为生发泡;在靠近核外膜的胞质中可观察到核仁外排物。卵黄发生卵母细胞逐渐为滤泡细胞所包围;卵黄合成旺盛,胞质中因而形成并积累了越来越多的卵黄粒。东方扁虾卵母细胞的卵黄发生是二源的。游离型核糖体率先参与内源性卵黄合成形成无膜卵黄粒。粗面内质网是内源性卵黄形成的主要胞器。滑面内质网、线粒体和溶酶体以多种方式活跃地参与卵黄粒形成。卵周隙内的外源性物质有两个来源:滤泡细胞的合成产物和血淋巴携带、转运的卵黄蛋白前体物。这些外源性物质主要通过质膜的微吞饮作用和微绒毛的吸收作用这两种方式进入卵母细胞,进而形成外源性卵黄。内源性和外源性的卵黄物质共同参与成熟卵母细胞中富含髓样小体的卵黄粒的形成。卵壳的形成和微绒毛的回缩被认为是东方扁虾卵母细胞成熟的形态学标志。       

Nuclear bodies in the oocyte nucleus of ground beetles are enriched in snRNPs

Within the oocyte nucleus of many insect species, a variable number of intensely stained spherical bodies occur. These nuclear bodies differ significantly from nucleoli and their precise role in nuclei has not been elucidated yet. I have examined some of the histochemical properties as well as the molecular composition of these structures in a representative of ground (carabid) beetles. I demonstrate, using molecular markers, that the nuclear bodies are composed of small nuclear RNAs and associated proteins, including p80 coilin. Hence, they correspond to Cajal bodies (= coiled bodies) described in somatic cell nuclei as well as oocyte germinal vesicles in plant and animal organisms. It is suggested that Cajal bodies in the carabid germinal vesicle serve as a storage site for splicing factors.     

Structure in the amphibian germinal vesicle

The germinal vesicle (GV) of Xenopus laevis is an enormous nucleus that contains 18 giant lampbrush chromosomes and thousands of inclusions. The inclusions are primarily of three types: approximately 1500 extrachromosomal nucleoli, 50-100 Cajal bodies, and several thousand B-snurposomes, which correspond to speckles or interchromatin granule clusters in other nuclei. The large size and abundance of the GV organelles, as well as the ease with which they can be studied both in vivo and in vitro, make the GV an ideal object for analysis of nuclear structure and function.     

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.     

Previtellogenic oocytes of South African largemouth bass Micropterus salmoides Lacépède 1802 (Actinopterygii,Perciformes) - the Balbiani body,cortical alveoli and developing eggshell

The ovaries of the largemouth bass Micropterus salmoides, an alien and invasive species in South Africa, contain a germinal epithelium which consists of germline and somatic cells, as well as previtellogenic and late vitellogenic ovarian follicles. The ovarian follicle consists of an oocyte surrounded by follicular cells and a basal lamina; thecal cells adjacent to this lamina are covered by an extracellular matrix. In this article, we describe the Balbiani body and the polarization and ultrastructure of the cytoplasm (ooplasm) in previtellogenic oocytes. The nucleoplasm in all examined oocytes contains lampbrush chromosomes, nuclear bodies and several nucleoli near the nuclear envelope. The ultrastructure of the nucleoli is described. Numerous nuage aggregations are present in the perinuclear cytoplasm in germline cells as well as in the ooplasm. Possible roles of these aggregations are discussed. The ooplasm contains the Balbiani body, which defines the future vegetal region in early previtellogenic oocytes. It is comprised of nuage aggregations, rough endoplasmic reticulum, Golgi apparatus, mitochondria, complexes of mitochondria with nuage-like material, and lysosome-like organelles. In mid-previtellogenic oocytes, the Balbiani body surrounds the nucleus and later disperses in the ooplasm. The lysosome-like organelles fuse and transform into vesicles containing material which is highly electron dense. As a result of the fusion of the vesicles of Golgi and rough endoplasmic reticulum, the cortical alveoli arise and distribute uniformly throughout the ooplasm of late previtellogenic oocytes. During this stage, the deposition of the eggshell (zona radiata) begins. The eggshell is penetrated by canals containing microvilli and consists of the following: the internal and the external egg envelope. In the external envelope three sublayers can be distinguished.     

Oogenesis in Actinoposthia beklemischevi (Platyhelminthes, Acoela): an ultrastructural and cytochemical study

The oogenesis of the acoel Actinoposthia beklemischevi can be divided into a previtellogenic and a vitellogenic stage. Maturing oocytes are surrounded by accessory cells (a.c.) that produce electrondense granules, the content of which is released into the space between the oocyte and a.c. and gives rise to a thin primary egg envelope. The a.c. may also contribute to yolk synthesis by transferring low molecular weight precursors to the oocyte. Two types of inclusion are produced in maturing oocytes. Type I inclusions are small, roundish granules produced by the Golgi complex. They have a proteinaceous non-polyphenolic content which is discharged in the intercellular space and produce a thicker secondary egg envelope. Type I inclusions represent eggshell-forming granules (EFGs). Type II inclusions are variably sized globules progressively changing their shape from round to crescent. They appear to be produced by the ER, contain glycoproteins and remain scattered throughout the cytoplasm in large oocytes. Type II inclusions represent yolk. The main features of oogenesis in Actinoposthia are: (a) EFGs have a non-polyphenolic composition; (b) the egg envelope has a double origin and is not sclerotinized; (c) yolk production appears to be autosynthetic. The present ultrastructural findings are compared with those from other Acoelomorpha and Turbellaria.     

Protein synthesis during meiotic maturation of mouse oocytes in vitro: Synthesis and phosphorylation of a protein localized in the germinal vesicle

Isolated fully grown mouse oocytes, arrested in dictyate of the first meiotic prophase, synthesize a protein with an apparent molecular weight of 28,000 which is localized in the germinal vesicle of the oocyte (germinal vesicle-associated protein; GVAP). Analyses of the distribution of GVAP have been carried out on SDS-polyacrylamide gels using oocytes cultured in vitro in the presence of [³⁵S]methionine or [³H]lysine and germinal vesicles isolated individually from these cultured oocytes. The results of such analyses show that GVAP contains only about 2% of the total radiolabel incorporated into mouse oocyte proteins, but as much as 40% of the total radiolabel incorporated into proteins associated with isolated germinal vesicles. These measurements indicate that GVAP is at least 1000-fold more concentrated in the germinal vesicle than in the cytoplasm of the oocyte. Furthermore, the synthesis and phosphorylation of GVAP are apparently terminated at a time which coincides with germinal vesicle breakdown during spontaneous meiotic maturation of mouse oocytes in vitro. Although the exact nature of GVAP is not known as yet, it appears to be an example of a protein that is selectively sequestered in the germinal vesicle of the oocyte during oogenesis and whose synthesis and modification are dependent upon the presence of an intact germinal vesicle.     

Ultrastructural features of the trophonema and oogenesis in the starlet sea anemone, Nematostella vectensis (Edwardsiidae)

Abstract. The starlet sea anemone, Nematostella vectensis Stephenson 1935, is a burrowing, estuarine species that has become a model organism for fundamental studies of cnidarian and metazoan development. During early oogenesis, oocytes appear in the basal region of the gastrodermis in the reproductive mesenteries and gradually bulge into the adjacent connective tissue space (mesoglea) where the majority of oocyte growth and vitellogenesis occurs. However, oocytes do not physically contact the cellular and amorphous matrix of the mesogleal compartment due to a thin, intervening basal lamina. Oocytes retain limited contact with the basal gastrodermal epithelium via groups of ultrastructurally modified gastrodermal cells called trophocytes. Trophocytes are monociliated accessory cells of somatic origin that collectively form a structure called the trophonema, a unique accessory cell/oocyte association not observed outside the Cnidaria. The trophonema consists of 50–60 trophocytes that maintain contact with <1% of the oocyte surface and forms a circular, bowel‐shaped depression on the luminal surface of the gastrodermis as they sink into the mesoglea with the oocyte. The oocyte remains highly polarized throughout oogenesis with the germinal vesicle positioned near the trophonema and presumably representing the future animal pole of the embryo. Contact between the trophonema and the oocyte is restricted to cell junctions connecting peripheral trophocytes and narrow extensions from the oocyte. Previous studies suggest that the trophonema plays a role in transport of extracellular digestive products from the gastrovascular cavity to the oocyte, and the ultrastructural features described in this study are consistent with that view. Vitellogenesis is described for the first time in a sea anemone. Yolk synthesis involves both autosynthetic and heterosynthetic processes including the biosynthetic activity of the Golgi complex and the uptake of extraoocytic yolk precursors via endocytosis, respectively.     

Conservation of RNA polymerase during maturation of the Rana pipiens oocyte.

DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5% of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.     

泥螺卵子发生的超微结构研究

利用透射电镜观察了泥螺卵子发生过程。结果表明 ,泥螺的卵子发生可划分为卵原细胞、卵黄发生早期、卵黄发生中期及卵黄发生后期卵母细胞 4个时期。卵原细胞核大而圆 ,胞质内分布有少量的线粒体和高尔基囊泡 ,细胞表面具微绒毛。卵黄发生早期的卵母细胞 ,胞质中各类细胞器发达 ,并出现数量较多的类朦胧子。卵黄发生中期的卵母细胞胞体迅速增大 ,核伸出伪足状突起 ,卵质中各种细胞器活动活跃 ,并参与形成卵黄粒和脂滴。此期还可观察到卵母细胞与滤泡细胞间的物质交换现象。卵黄发生后期的卵母细胞体积增至最大 ,细胞器数量减少。本文就卵黄发生前后卵母细胞内部构造的变化、意义及滤泡细胞与卵母细胞蛋白来源间的关系作了探讨