Integrin-mediated attachment of the blastoderm to the vitelline envelope impacts gastrulation of insects

During gastrulation, physical forces reshape the simple embryonic tissue to form a complex body plan of multicellular organisms1. These forces often cause large-scale asymmetric movements of the embryonic tissue2,3. In many embryos, the tissue undergoing gastrulation movements is surrounded by a rigid protective shell4,5. While it is well recognized that gastrulation movements depend on forces generated by tissue-intrinsic contractility6,7, it is not known if interactions between the tissue and the protective shell provide additional forces that impact gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle Tribolium castaneum tightly adheres in a temporally coordinated manner to the vitelline envelope surrounding the embryo. This attachment generates an additional force that counteracts the tissue-intrinsic contractile forces to create asymmetric tissue movements. Furthermore, this localized attachment is mediated by a specific integrin, and its knock-down leads to a gastrulation phenotype consistent with complete loss of attachment. Moreover, analysis of another integrin in the fruit fly Drosophila melanogaster suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline. Together, our findings reveal a conserved mechanism whereby the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable counter-forces that shape gastrulation movements in insects.

Gonads and gametogenesis in Chaetodactylus osmiae (Acariformes: Astigmata: Chaetodactylidae) a parasite of solitary bees

Chaetodactylus osmiae (Dufour, 1839) is a mite parasitizing the solitary bee - Osmia rufa L.- used as a commercial pollinator. In this study we present the anatomy of female and male reproductive systems of this species as well as its gonadal structure and gametogenesis at the ultrastructural level. The reproductive systems are similar to those of other Astigmata. The ovaries are paired and each contains germ-line cells – a giant nutritive ovarian cell connected via funnel-type intercellular bridges to oogonia and previtellogenic oocytes. Germinal cells are embedded in several large somatic stroma cells. Remarkable numerous protrusions of the nutritive ovarian cell penetrate into the stroma cell cytoplasm. Conspicuous ER cisterns run close and parallel to the surface of the germinal cells. Oocytes entering vitellogenesis disassociate with the nutritive cell and a vitelline envelope composed of heterogeneous material appears on their surface. When vitellogenesis is completed, the oocytes are full of lipid droplets and two types of yolk spheres; the vitelline envelope transforms into a thin and homogeneous chorion.Paired testes are located on one side of the body, whereas the opposite side is filled by a male accessory gland. In testis, germinal cells are embedded in a few somatic stroma cells. The earliest spermatogonia form a compact germarium, whereas later stages are dispersed randomly within the testis. Spermatocytes are characterized by a superficial spongy layer, formation of mitochondrial derivatives, loss of nuclear envelope and condensation of chromatin in threads. A single electron-dense lamella appears during the spermatid stage, separating chromatin threads from a large spongy body surrounded by arcuate, double-membrane bounded cisterns. In spermatids, the superficial spongy layer is absent. The testicular central cell in the germarium and structures related to meiotic division were not observed in the testes. Spermatozoa are multiform cells (approx. 4x11µm) containing electron-dense lamella (ca. 45 nm thick) surrounded by mitochondrial derivatives which separate chromatin threads 45-50 nm thick from remnants of the spongy body i.e. arcuate cistern profiles. Spermatozoa deposited in female spermatheca are more electron dense; the electron-dense lamella is deeply folded several times, whereas chromatin threads are present in the center of the spermatozoon and are either flanked by lamella folds or located more peripherally under the plasmalemma. Remnants of the spongy body are not discernible.

The effector of Hippo signaling, Taz, is required for formation of the micropyle and fertilization in zebrafish

The mechanisms that ensure fertilization of eggs by a single sperm are not fully understood. In all teleosts, a channel called the ‘micropyle’ is the only route of entry for sperm to enter and fertilize the egg. The micropyle forms by penetration of the developing vitelline envelope by a single specialized follicle cell, the micropylar cell, which subsequently degenerates. The mechanisms underlying micropylar cell specification and micropyle formation are poorly understood. Here, we show that an effector of the Hippo signaling pathway, the Transcriptional co-activator with a PDZ-binding domain (Taz), plays crucial roles in micropyle formation and fertilization in zebrafish. Genome editing mutants affectingtazcan grow to adults, however, eggs from homozygoustazfemales are not fertilized even though oocytes in mutant females are histologically normal with intact animal-vegetal polarity, complete meiosis and proper ovulation. However,tazmutant eggs have no micropyle. We show that Taz protein is specifically enriched from mid-oogenesis onwards in two follicle cells located at the animal pole of the oocyte, and co-localizes with the actin and tubulin cytoskeleton. Taz protein and micropylar cell are not detected intazmutant ovaries. Our work identifies a novel role for the Hippo/Taz pathway in micropylar cell specification in zebrafish, and uncovers the molecular basis of micropyle formation in teleosts.

Structure and ultrastructure of oogenic stage in short mackerel Rastrelliger brachysoma (Teleostei: Scombidae)

Introduction: Structure, ultrastructural features and degeneration of oogenesis were first investigated in female Rastrelliger brachysoma as new candidate species for aquaculture by transmission electron microscopy. Materials and Methods: Specimens were naturally collected during the breeding season from Samut Songkhram Province, Thailand. Results: The ultrastructure of female oogenesis was principally divided into five stages based on the nuclear characterization and cytoplasmic organelles; (i) oogonium; (ii) previtellogenic; (iii) lipid and cortical alveolar; (iv) early and (v) late vitellogenic stages. Initially, oogonium was present within cell nest in the epithelial compartment. Its cell was supported by prefollicular cells. The multiple nucleoli in previtellogenic stage, referring to primary growth stage were the first to appear and they were scattered around the peripheral of nuclear membrane with the increasing number of cytoplasmic organelles. Some microvilli of granulosa cell initially protruded into the vitelline envelope. A simple layer of flattened granulosa and theca cells was also observed. The lipid and cortical alveolar stage under secondary growth oocyte was accumulated with two inclusions; the lipid droplets and cortical alveoli in the ooplasm. Another characterization, the increasing of numerous microvilli was also detected in the vitelline envelope. Finally, in the vitellogenic stage, a massive uptake and processing of proteins into yolk platelets due to embedding of the numerous microvilli in the largest vitelline envelope was observed. Oocyte degeneration in R. brachysoma was novel found especially oogonial and previtellogenic stages. Conclusion: Five oogenic stages of this fish are found with the changing of the arrangement of nucleus, cytoplasmic organelles and follicular complex, which will be applied to further studies.

On the embryonic cell division beyond the contractile ring mechanism: experimental and computational investigation of effects of vitelline confinement, temperature and egg size

Embryonic cell division is a mechanical process which is predominantly driven by contraction of the cleavage furrow and response of the remaining cellular matter. While most previous studies focused on contractile ring mechanisms of cytokinesis, effects of environmental factors such as pericellular vitelline membrane and temperature on the mechanics of dividing cells were rarely studied. Here, we apply a model-based analysis to the time-lapse imaging data of two species (Saccoglossus kowalevskiiandXenopus laevis) with relatively large eggs, with the goal of revealing the effects of temperature and vitelline envelope on the mechanics of the first embryonic cell division. We constructed a numerical model of cytokinesis to estimate the effects of vitelline confinement on cellular deformation and to predict deformation of cellular contours. We used the deviations of our computational predictions from experimentally observed cell elongation to adjust variable parameters of the contractile ring model and to quantify the contribution of other factors (constitutive cell properties, spindle polarization) that may influence the mechanics and shape of dividing cells. We find that temperature affects the size and rate of dilatation of the vitelline membrane surrounding fertilized eggs and show that in native (not artificially devitellinized) egg cells the effects of temperature and vitelline envelope on mechanics of cell division are tightly interlinked. In particular, our results support the view that vitelline membrane fulfills an important role of micromechanical environment around the early embryo the absence or improper function of which under moderately elevated temperature impairs normal development. Furthermore, our findings suggest the existence of scale-dependent mechanisms that contribute to cytokinesis in species with different egg size, and challenge the view of mechanics of embryonic cell division as a scale-independent phenomenon.

Calcium waves occur as Drosophila oocytes activate

Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female’s reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components.

Shotgun proteomics of rainbow trout ovarian fluid

In the present study we used a shotgun proteomic approach to identify 54 proteins of rainbow trout ovarian fluid. The study has unravelled the identity of several proteins not previously reported in fish ovarian fluid. The proteome of trout ovarian fluid consists of diverse proteins participating in lipid binding and metabolism, carbohydrate and ion transport, innate immunity, maturation and ovulation processes. Most trout ovarian fluid proteins correspond to follicular fluid proteins of higher vertebrates, but 15% of the proteins were found to be different, such as those related to the immune system (precerebellin-like protein), proteolysis (myeloid cell lineage chitinase), carbohydrate and lipid binding and metabolism (vitellogenins), cell structure and shape (vitelline envelope protein gamma) and a protein with unknown functions (UPF0762 protein C6orf58 homologue). The present study could help in the decoding of the biological function of these proteins and in the discovery of potential biomarkers of oocyte quality.