Balbiani body of basal insects is potentially involved in multiplication and selective elimination of mitochondria.

Oocytes of both vertebrates and invertebrates often contain an intricate organelle assemblage, termed the Balbiani body (Bb). It has previously been suggested that this assemblage is involved in the delivery of organelles and macromolecules to the germ plasm, formation of oocyte reserve materials, and transfer of mitochondria to the next generation. To gain further insight into the function of the Bb, we performed a series of analyses and experiments, including computer-aided 3-dimensional reconstructions, detection of DNA (mtDNA) synthesis as well as immunolocalization studies. We showed that in orthopteran Meconema meridionale, the Bb comprises a network of mitochondria and perinuclear nuage aggregations. As oogenesis progresses, the network expands filling almost entire ooplasm, then partitions into several smaller entities, termed micro-networks, and ultimately into individual mitochondria. As in somatic cells, this process involves microfilaments and elements of endoplasmic reticulum. We showed also that at least some of the individual mitochondria are surrounded by phagophores and eliminated via mitophagy. These findings support the idea that the Bb is implicated in the multiplication and selective elimination of (defective) mitochondria and therefore may participate in the transfer of undamaged (healthy) mitochondria to the next generation.

Morphology and ultrastructure of the Balbiani body in the oocytes of closely related bush cricket species. Shared features reveal important aspect of functioning.

Balbiani bodies (Bbs) are female germline-specific organelle assemblages usually composed of mitochondria, Golgi complexes, elements of endoplasmic reticulum and accumulations of fine granular material, termed the nuage. Here we present results of morphological and ultrastructural analysis of the Bb of four bush crickets nested in four subfamilies of the family Tettigonidae. This study has revealed that Bbs of closely related species (belonging to the defined evolutionary line) are morphologically rather different. In two species (Meconema meridionale and Pholidoptera griseoaptera) the Bb has the form of a hollow hemisphere that covers a part of the germinal vesicle surface. In contrast, the Bb of Conocephalus fuscus and Leptophyes albovittata is less distinct and surrounds the whole or the majority of the germinal vesicle surface. Aside from this difference, the Bbs of all four studied species are built of identical sets of organelles and, most importantly, share one significant feature: close association of mitochondria and nuage accumulations. We show additionally that mitochondria remaining in direct contact with the nuage are characterized by distinct morphologies e.g. elongated, dumbbell shaped or bifurcated. In the light of our results and literature survey, the ancestral function of the Bb is discussed.

Flutamide treatment reveals a relationship between steroidogenic activity of Leydig cells and ultrastructure of their mitochondria.

Our present knowledge on interrelation between morphology/ultrastructure of mitochondria of the Leydig cell and its steroidogenic function is far from satisfactory and needs additional studies. Here, we analyzed the effects of blockade of androgen receptor, triggered by exposure to flutamide, on the expression of steroidogenic proteins (1) and ultrastructure of Leydig cells' constituents (2). We demonstrated that increase in the expression level of steroidogenic (StAR, CYP11A1, 3ß-HSD, and CYP19A1) proteins (and respective mRNAs) in rat testicular tissue as well as elevation of intratesticular sex steroid hormone (testosterone and estradiol) levels observed in treated animals correspond well to morphological alterations of the Leydig cell ultrastructure. Most importantly, up-regulation of steroidogenic proteins' expression apparently correlates with considerable multiplication of Leydig cell mitochondria and subsequent formation of local mitochondrial networks. Interestingly, we showed also that the above-mentioned processes were associated with elevated transcription of Drp1 and Mfn2 genes, encoding proteins implicated in mitochondrial dynamics. Collectively, our studies emphasize the importance of mitochondrial homeostasis to the steroidogenic function of Leydig cells.

Morphogenesis of the Balbiani body in developing oocytes of an orthopteran, Metrioptera brachyptera, and multiplication of female germline mitochondria.

Balbiani body (Bb) is a female germline specific organelle complex. Although the morphology and morphogenesis of the Bb have been analyzed in numerous vertebrate and invertebrate species, the role and ultimate fate of this organelle assemblage are still under debate. As a result, various functions have been attributed to the Bb in given animal lineages or even species. Our analyses showed that in the bush cricket, Metrioptera brachyptera, the Bb is an elaborate and highly dynamic structure positioned at one side of the oocyte nucleus. It forms in early previtellogenic oocytes and consists of two compartments: perinuclear and cytoplasmic. In the cytoplasmic compartment, characteristic complexes of nuage and polymorphous mitochondria are present. Computer-aided 3D reconstructions revealed that mitochondria clustered around neighboring nuage accumulations remain in a physical contact and form an extensive, though dispersed network. As oogenesis progresses, nuage/mitochondria complexes are partitioned into progressively smaller entities that become separated from each other. Concurrently, the mitochondrial network splits into small individual mitochondria populating the whole ooplasm. Immunohistochemical analysis showed that the latter process involves dynamin-related protein 1 (Drp1). Collectively, our findings suggest that in basal insect species, the Bb might be responsible for the selection as well as multiplication of the oocyte mitochondria.

Transmission of Functional, Wild-Type Mitochondria and the Fittest mtDNA to the Next Generation: Bottleneck Phenomenon, Balbiani Body, and Mitophagy.

The most important role of mitochondria is to supply cells with metabolic energy in the form of adenosine triphosphate (ATP). As synthesis of ATP molecules is accompanied by the generation of reactive oxygen species (ROS), mitochondrial DNA (mtDNA) is highly vulnerable to impairment and, consequently, accumulation of deleterious mutations. In most animals, mitochondria are transmitted to the next generation maternally, i.e., exclusively from female germline cells (oocytes and eggs). It has been suggested, in this context, that a specialized mechanism must operate in the developing oocytes enabling escape from the impairment and subsequent transmission of accurate (devoid of mutations) mtDNA from one generation to the next. Literature survey suggest that two distinct and irreplaceable pathways of mitochondria transmission may be operational in various animal lineages. In some taxa, the mitochondria are apparently selected: functional mitochondria with high inner membrane potential are transferred to the cells of the embryo, whereas those with low membrane potential (overloaded with mutations in mtDNA) are eliminated by mitophagy. In other species, the respiratory activity of germline mitochondria is suppressed and ROS production alleviated leading to the same final effect, i.e., transmission of undamaged mitochondria to offspring, via an entirely different route.

Morphogenesis of the ovarian follicular epithelium during initial stages of embryogenesis of the viviparous earwig, Hemimerus talpoides.

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended "postfertilization" activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.

Evolutionary origin and functioning of pregenital abdominal outgrowths in a viviparous insect, Arixenia esau.

Although pregenital abdominal outgrowths occur only rarely in pterygote insects, they are interesting from the evolutionary viewpoint because of their potential homology to wings. Our previous studies of early development of an epizoic dermapteran, Arixenia esau revealed that abdominal segments of the advanced embryos and larvae, growing inside a mother's uterus, are equipped with paired serial outgrowths. Here, we focus on the origin and functioning of these outgrowths. We demonstrate that they bud from the lateral parts of the abdominal nota, persist till the end of intrauterine development, and remain in contact with the uterus wall. We also show that the bundles of muscle fibers associated with the abdominal outgrowths may facilitate flow of the haemolymph from the outgrowths' lumen to the larval body cavity. Following completion of the intrauterine development, abdominal outgrowths are shed together with the larval cuticle during the first molt after the larva birth. Using immunohistochemical and biochemical approaches, we demonstrate that the Arixenia abdominal outgrowths represent an evolutionary novelty, presumably related to intrauterine development, and suggest that they are not related to serial wing homologs.

Viviparity in Two Closely Related Epizoic Dermapterans Relies on Disparate Modifications of Reproductive Systems and Embryogenesis.

Nutritional modes operating during embryonic/larval development of viviparous species range from "pure" lecitothrophy in which embryos rely solely on reserve materials (yolk spheres, lipid droplets, and glycogen particles) accumulated in the egg cytoplasm to matrotrophy in which embryos are continuously supplied with nutrients from a parental organism. Interestingly, a wide spectrum of diverse "mixed" modes employed in the embryo nourishment have also been described among viviparous species. Here, we summarize results of histochemical, ultrastructural, and biochemical analyses of reproductive systems as well as developing embryos of two closely related viviparous species of earwigs (Dermaptera), Hemimerus talpoides and Arixenia esau. These analyses clearly indicate that morphological as well as physiological modifications (adaptations) supporting viviparity and matrotrophy in Hemimerus and Arixenia, with the exception of a complex biphasic respiration, are markedly different. Most importantly, Hemimerus embryos complete their development inside terminal (largest) ovarian follicles, whereas Arixenia embryos, after initial developmental stages, are transferred to highly modified lateral oviducts, that is the uterus, where they develop until the release (birth) of larvae. The obtained results strongly suggest that viviparity in hemimerids and arixeniids had evolved independently and might therefore serve as an example of evolutionary parallelism as well as remarkable functional plasticity of insect reproduction and embryonic development.

Viviparity in the dermapteran Arixenia esau: respiration inside mother's body requires both maternal and larval contribution.

Earwigs (Dermaptera) use different strategies to increase their reproductive success. Most species lay eggs; however, viviparity of the matrotrophic type has been reported in two groups: Hemimeridae and Arixeniidae. In Arixeniidae, offspring develop in two separate places: inside an ovary (the intraovarian phase) and within a uterus (the intrauterine phase). Both morphological and physiological aspects of viviparity in Arixeniidae have begun to be unraveled only recently. Here, we characterize how the first instar larvae of Arixenia esau, developing inside the mother's reproductive system, manage respiration and gas exchange. Using modern light and electron microscopy techniques as well as immunological approach, we provide a detailed account of the maternal and larval tissue interactions during the intrauterine development. We demonstrate that respiration in the Arixenia first instar larvae relies on the extensive tracheal system of the mother as well as a respiratory pigment (hemocyanin) present within the body cavity of the larvae. Our results indicate that the larval fat body tissue is the likely place of the hemocyanin synthesis. Our study shows that characteristic cone-shaped lobes of the outgrowths located on the larval abdomen are a part of a placenta-like organ and mediate the gas exchange between the maternal and larval organisms. Based on the obtained results, we propose that Arixenia esau evolved a unique biphasic system supporting respiration of the first instar larvae during their development inside the mother's reproductive tract.

Organelle assemblages implicated in the transfer of oocyte components to the embryo: an insect perspective.

Besides reserve materials (yolk spheres, lipid droplets), ribosomes and various mRNA species, insect oocytes contain large easily morphologically recognizable organelle assemblages: the Balbiani body and the oosome (pole plasm). These assemblages are implicated in the transfer of oocyte components (mitochondria, polar granules) to the embryo that is to offspring. Here, we review present knowledge of morphology, morphogenesis, molecular composition and function/s of these assemblages. We discuss also the morphogenesis and presumed function of unconventional organelle assemblages, dormant stacks of endoplasmic reticulum, recently described in the oocytes and early embryos of a viviparous dermapteran, Hemimerus talpoides.

Morphogenesis of serial abdominal outgrowths during development of the viviparous dermapteran, Arixenia esau (Insecta, Dermaptera).

The embryos and first instar larvae of the epizoic earwig, Arixenia esau, develop sequentially in two different compartments of the female reproductive system, that is ovarian follicles and the lateral oviducts (the uterus). Here we show that the second (intrauterine) phase of development consists of three physiologically disparate stages: early embryos (before dorsal closure, surrounded by an egg envelope), late embryos (after dorsal closure, surrounded by an egg envelope) and the first instar larvae (after "hatching" from an egg envelope). Early and late embryos float in the fluid filling the uterus, whereas the first instar larvae develop attached to the uterus wall. Our analyses revealed also that in Arixenia serial multilobed outgrowths develop on dorso-lateral aspects of all abdominal segments. At the onset of the third developmental stage and after liberation from an egg envelope, these outgrowths (or more precisely their lobes) adhere to the epithelium lining the uterus, forming a series of small contact sites, where the mother and embryo tissues are separated only by a thin, presumably permeable, embryonic cuticle. We suggest that all these contact sites collectively constitute a dispersed placenta-like organ involved in the nourishment of the embryo.

Excretion in the mother's body: modifications of the larval excretory system in the viviparous dermapteran, Arixenia esau.

The vast majority of Dermaptera are free-living and oviparous, i.e., females lay eggs within which embryonic development occurs until the larva hatches. In contrast, in the epizoic dermapteran Arixenia esau, eggs are retained within mother's body and the embryos and first instar larvae develop inside her reproductive system. Such a reproductive strategy poses many physiological challenges for a mother, one of which is the removal of metabolic waste generated by the developing offspring. Here, we examine how the Arixenia females cope with this challenge by analyzing features of the developing larval excretory system. Our comparative analyses of the early and late first instar larvae revealed characteristic modifications in the cellular architecture of the Malpighian tubules, indicating that these organs are functional. The results of the electron probe microanalyses suggest additionally that the larval Malpighian tubules are mainly involved in maintaining ion homeostasis. We also found that the lumen of the larval alimentary track is occluded by a cellular diaphragm at the midgut-hindgut junction and that cells of the diaphragm accumulate metabolic compounds. Such an organization of the larval gut apparently prevents fouling of the mother's organism with the offspring metabolic waste and therefore can be regarded as an adaptation for viviparity.

Unusual morphological adaptations and processes associated with viviparity in an epizoic dermapteran.

Matrotrophic viviparity is a reproductive pattern in which offspring develop inside a female's body which provides gas exchange and nutrients necessary for development. Besides placental mammals, structural and physiological aspects of matrotrophic viviparity are poorly characterized. In insects, the majority of species is oviparous, i.e. lay eggs, and viviparous reproduction has been reported only in 11 out of 44 orders, including earwigs (Dermaptera). Among dermapterans, matrotrophic viviparity has been reported in two epizoic subgroups: Arixeniidae and Hemimeridae. Here, we provide morphological evidence for distinct adaptations for this mode of viviparity in embryonic and maternal tissues in a representative of the latter subgroup, Hemimerus talpoides. Our study reveals a novel mechanism of maternal contribution to embryonic development which operates during oogenesis and involves characteristic modification of endoplasmic reticulum cisternae. Conspicuous and apparently inactive para-crystalline stacks of the endoplasmic reticulum are deposited in the oocyte cytoplasm and become activated during early embryonic development. Our analyses indicate additionally that in Hemimerus, transformed follicular/ovarian cells (on the mother's side) and an evagination of the dorsal vessel (on the embryo's side) converge to form a cephalic vesicle, structure analogous to a placenta. The cellular architecture of this unusual "cephalic placenta" points to its participation in an exchange of low molecular weight substances between a mother and developing embryo.

Ovaries and oogenesis in an epizoic dermapteran, Hemimerus talpoides (Dermaptera, Hemimeridae): Structural and functional adaptations to viviparity and matrotrophy.

The Dermaptera are traditionally classified in three taxa: the free living Forficulina and two viviparous (matrotrophic) groups, the Hemimerina and Arixeniina. Recent molecular and histological analyses suggest that both matrotrophic groups should be nested among the most derived taxon of the Forficulina, the Eudermaptera. We present results of ultrastructural analyses of ovary/ovariole morphology and oogenesis in a representative of the Hemimerina, Hemimerus talpoides (Walker, 1871). Our results strongly reinforce the idea that the Hemimerina should be classified within the Eudermaptera. We show additionally that the ovaries of the studied species are characterized by two peculiar modifications, i.e. the presence of numerous tracheoles in contact with the basement lamina covering the ovarioles, and an unusual development of the ovariole stalks. We believe that both characters are related to viviparity and unconventional "intra-ovariolar" embryo development. Finally, our study also indicates that the oocytes of H. talpoides reveal characters apparently associated with a matrotrophic type of embryo nourishment. They are completely yolkless and devoid of the typical, multilayered egg envelopes; instead, they comprise unconventional organelles (para-crystalline stacks of endoplasmic reticulum cisternae and translucent vacuoles) that seem to function after initiation of embryonic development. Thus, the ovaries as well as the oocytes of H. talpoides are characterized by an exceptional mixture of features shared with derived dermapterans and adaptations to matrotrophy.

The Pole (Germ) Plasm in Insect Oocytes.

Animal germline cells are specified either through zygotic induction or cytoplasmic inheritance. Zygotic induction takes place in mid- or late embryogenesis and requires cell-to-cell signaling leading to the acquisition of germline fate de novo. In contrast, cytoplasmic inheritance involves formation of a specific, asymmetrically localized oocyte region, termed the germ (pole) plasm. This region contains maternally provided germline determinants (mRNAs, proteins) that are capable of inducing germline fate in a subset of embryonic cells. Recent data indicate that among insects, the zygotic induction represents an ancestral condition, while the cytoplasmic inheritance evolved at the base of Holometabola or in the last common ancestor of Holometabola and its sister taxon, Paraneoptera.In this chapter, we first describe subsequent stages of morphogenesis of the pole plasm and polar granules in the model organism, Drosophila melanogaster. Then, we present an overview of morphology and cytoarchitecture of the pole plasm in various holometabolan and paraneopteran insect species. Finally, we focus on phylogenetic hypotheses explaining the known distribution of two different strategies of germline specification among insects.

Selection of mitochondria in female germline cells: is Balbiani body implicated in this process?

Early oocytes of nearly all animal species contain a transient organelle assemblage termed the Balbiani body. Structure and composition of this assemblage may vary even between closely related species. Despite this variability, the Balbiani body always comprises of numerous tightly clustered mitochondria and accumulations of nuage material. It has been suggested that the Balbiani body is an evolutionarily ancestral structure, which plays a role in various processes such as the localization of organelles and macromolecules to the germ plasm, lipidogenesis, as well as the selection/elimination of dysfunctional mitochondria from female germline cells. We suggest that the selection/elimination of mitochondria is a primary and evolutionarily ancestral function of Balbiani body, and that the other functions are secondary, evolutionarily derived additions. We propose a simple model explaining the role of the Balbiani body in the selection of mitochondria, i.e., in the mitochondrial DNA (mtDNA) bottleneck phenomenon.

Asymmetric Divisions in Oogenesis.

In the majority of animals, the oocyte/egg is structurally, molecularly, and functionally asymmetric. Such asymmetry is a prerequisite for a flawless fertilization and faithful segregation of maternal determinants during subsequent embryonic development. The oocyte asymmetry develops during oogenesis and must be maintained during consecutive and obligatorily asymmetric oogonial divisions, which depending on the species lead to the formation of either oocyte alone or oocyte and nurse cell complex. In the following chapter, we summarize current knowledge on the asymmetric oogonial divisions in invertebrate (insects) and vertebrate (Xenopus) species.

Meiosis, Balbiani body and early asymmetry of Thermobia oocyte.

The meiotic division guarantees maintenance of a genetic diversity; it consists of several stages, with prophase I being the longest and the most complex. We decided to follow the course of initial stages of meiotic division in ovaries of Thermobia domestica using modified techniques of squash preparations, semithin sections, and electron microscopy. We show that germaria contain numerous germline cells that can be classified into three categories: cystoblasts, meiotic oocytes, and growing previtellogenic oocytes. The cystoblasts are located most apically. The meiotic oocytes occupy the middle part of the germarium, and the previtellogenic oocytes can be found in the most basal part, near the vitellarium. Analyses of the semithin sections and squash preparations show that post leptotene meiotic chromosomes gather in one region of the nucleoplasm where they form the so-called bouquet. The telomeres of the bouquet chromosomes are attached to a relatively small area (segment) of the nuclear envelope. Next to this envelope segment, the nucleolar organizers are also located. We show that in concert to sequential changes inside the oocyte nuclei, rearrangement of organelles within the ooplasm (oocyte cytoplasm) takes place. This leads to the formation of the Balbiani body and consequent asymmetry of the ooplasm. These early nuclear and cytoplasmic asymmetries, however, are transient. During diplotene, the chromosome bouquet disappears, while the Balbiani body gradually disperses throughout the ooplasm. Finally, our observations indicate the presence of lampbrush chromosomes in the nuclei of previtellogenic oocytes. In the close vicinity to lampbrush chromosomes, characteristic spherical nuclear bodies are present.

Gametic synapses, nanotubes and sperm RNAs - Redefining the origin of maternal determinants.

The female germline cells, i.e., the oocytes/eggs, contain a subpopulation of unique organelles and molecules (RNA and proteins) collectively called "the maternal determinants" that are indispensable for the determination of cell fate in the developing embryo. Although it has been known for some time that somatic cells deliver low-molecular-weight molecules to the oocyte/egg, the paradigm has been that the larger molecules and organelles are synthesized by the female germline cells without input from the surrounding somatic cells. However, recent discoveries of novel types of intercellular connections such as gametic synapses and tunneling nanotubes, allowing the transfer of large, externally derived molecules to the oocyte/egg, may dismantle the paradigm of the transcriptional/translational self-containment of the female gamete and add novel and unexpected aspects to the origin and identity of maternal determinants. In addition, the discovery that sperm delivers various RNAs to the egg suggests that sperm may not only epigenetically modify the egg genome but also influence or modify information contained in the maternal determinants.

Exclusion of dysfunctional mitochondria from Balbiani body during early oogenesis of Thermobia.

Oocytes of many invertebrate and vertebrate species contain a characteristic organelle complex known as the Balbiani body (Bb). Until now, three principal functions have been ascribed to this complex: delivery of germ cell determinants and localized RNAs to the vegetal cortex/posterior pole of the oocyte, transport of the mitochondria towards the germ plasm, and participation in the formation of lipid droplets. Here, we present the results of a computer-aided 3D reconstruction of the Bb in the growing oocytes of an insect, Thermobia domestica. Our analyses have shown that, in Thermobia, the central part of each fully developed Bb comprises a single intricate mitochondrial network. This "core" network is surrounded by several isolated bean-shaped mitochondrial units that display lowered membrane potential and clear signs of degeneration. In light of the above results and recent theoretical models of mitochondrial quality control, the role of the Bb is discussed. We suggest that, in addition to the aforementioned functions, the Bb is implicated in the selective elimination of dysfunctional mitochondria during oogenesis.