-
PloS One 2024The yellow fever mosquito Aedes aegypti is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for...
The yellow fever mosquito Aedes aegypti is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for the collection, fixation, and preparation of A. aegypti embryos for immunohistochemical and in situ hybridization studies. The processing of A. aegypti embryos for such studies is complicated by the inability to easily remove the vitelline membrane, which prevents the reagents needed for staining from reaching their targets, and which furthermore obscures visualization of the embryo since the membrane is highly sclerotized. Previously described protocols for removal of the vitelline membrane are very low throughput, limiting the capacity of work that can be accomplished in a reasonable timeframe. Our adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to prepare an average of 100-150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and in situ hybridization studies. The protocol has been successfully tested on embryos of ages ranging from 14h after egg laying (AEL) at 27°C through to 55h AEL. Critical to the success of the optimized protocol is the selection, fabrication, and description of the tools required. To this end, a video-demonstrated protocol has been placed at protocols.io to clarify the protocol and provide easy access and training to anyone interested in the preparation of A. aegypti embryos for biological studies.
Topics: Animals; Aedes; In Situ Hybridization; Embryo, Nonmammalian; Tissue Fixation; Immunohistochemistry; Female
PubMed: 38820371
DOI: 10.1371/journal.pone.0304802 -
Poultry Science Apr 2021The aim of the experiment was to evaluate the potential use of citric acid as a modifier of quality changes in table eggs during their storage. About 780 table hen eggs...
The aim of the experiment was to evaluate the potential use of citric acid as a modifier of quality changes in table eggs during their storage. About 780 table hen eggs were collected on the same day. They were numbered individually and placed on trays 30 pcs on each. Control group (CA0) consisted of eggs unmodified with any additional substances. In experimental groups CA10 and CA15, eggshells were sprayed with the aqueous solution of citric acid (10 and 15% concentration, respectively). At the start of the experiment, only quality traits of eggs from the control group were analyzed. The remaining eggs were stored at 14°C and 70% RH (typical storage conditions). Their quality was evaluated after 7, 14, 21, and 28 d. The depth of the air cell, egg weight and specific gravity, traits of shell (permeability, strength, weight, thickness, density), and egg content (pH of yolk and albumen, Haugh units, yolk weight and color) were evaluated each time. The use of citric acid decreased the severity of qualitative changes. Citric acid-treated eggs demonstrated smaller weight loss, shallower air cell, higher structural albumen, less-intensive water diffusion from albumen to yolk indicating the improved resistance of the vitelline membrane. Owing to the fact that citric acid is accepted and recognized as a safe food preservative is a relatively cheap and available substance, it seems that it can be used to inhibit quality changes in table eggs during their storage.
Topics: Animals; Chickens; Citric Acid; Egg Shell; Eggs; Food Handling; Food Preservatives; Vitelline Membrane
PubMed: 33610892
DOI: 10.1016/j.psj.2021.01.018 -
International Journal For Parasitology.... Aug 2019Some pouting caught off the Atlantic coast of Portugal are discarded as unmarketable due to a dark discolouration of the skin and muscle. This study investigates the...
Some pouting caught off the Atlantic coast of Portugal are discarded as unmarketable due to a dark discolouration of the skin and muscle. This study investigates the cause of this condition, describes the new parasite species responsible, and highlights the importance of educating those in charge of premarket inspection of food fish in order to reduce likelihood that consumers will eat infected fish. Macroscopically, infected fish showed considerable heterogeneity in darkening of the skin and hypaxial and epaxial muscles. Microscopical observation revealed bipolar nematode eggs in varying stages of development arranged in a linear pattern along muscle fibers. Histopathology confirmed the presence of eggs of a nematode of the genus Moravec, 1987 as the cause of muscle darkening and established a relationship between infection intensity and consequent darkened appearance of the tissues. The eggs are oval or barrel-shaped, with a smooth surface and polar plugs at opposite ends. The thin outer vitelline membrane is smooth and lacks ornamentation. Under light microscopy, the main eggshell of older eggs exhibits the outermost delicate and smooth vitelline membrane, and a thicker layer, correspondent to chitinous and chondroitin proteoglycan layers. Scanning electron microscopy of eggs confirmed light microscopic studies, namely the presence of a smooth vitelline membrane surrounding the egg. Microscopic and ultrastructural characteristics of eggs, and a new host family in a new geographic area, all suggest that a new species, herein named sp. n. is involved.
PubMed: 31245277
DOI: 10.1016/j.ijppaw.2019.05.010 -
Cells Sep 2022In sea urchins, the sequence of the cellular and molecular events characterizing the fertilization process has been intensively studied. We have learned that to activate...
In sea urchins, the sequence of the cellular and molecular events characterizing the fertilization process has been intensively studied. We have learned that to activate the egg, the fertilizing sperm must undergo morphological modifications (the acrosome reaction, AR) upon reaching the outer gelatinous layer enveloping the egg (egg jelly), which triggers the polymerization of F-actin on the sperm head to form the acrosomal process. The AR exposes bindin, an adhesive sperm protein essential for the species-specific interaction with the cognate receptor on the egg vitelline layer. To investigate the specific roles of the egg jelly and vitelline layer at fertilization of sea urchin eggs, eggs were incubated in acidic seawater, which removes the egg jelly, i.e., experimental conditions that should prevent the occurrence of the AR, and inseminated in the same medium. At variance with the prevailing view, our results have shown that these dejellied eggs can still interact with sperm in acidic seawater, albeit with altered fertilization responses. In particular, the eggs deprived of the vitelline layer reacted with multiple sperm but with altered Ca signals. The results have provided experimental evidence that the plasma membrane, and not the vitelline layer, is where the specific recognition between gametes occurs. The vitelline layer works in unfertilized eggs to prevent polyspermy.
Topics: Actins; Animals; Fertilization; Male; Ovum; Sea Urchins; Semen; Sperm-Ovum Interactions
PubMed: 36230946
DOI: 10.3390/cells11192984 -
Developmental Cell Jan 2024During morphogenesis, mechanical forces induce large-scale deformations; yet, how forces emerge from cellular contractility and adhesion is unclear. In Drosophila...
During morphogenesis, mechanical forces induce large-scale deformations; yet, how forces emerge from cellular contractility and adhesion is unclear. In Drosophila embryos, a tissue-scale wave of actomyosin contractility coupled with adhesion to the surrounding vitelline membrane drives polarized tissue invagination. We show that this process emerges subcellularly from the mechanical coupling between myosin II activation and sequential adhesion/de-adhesion to the vitelline membrane. At the wavefront, integrin clusters anchor the actin cortex to the vitelline membrane and promote activation of myosin II, which in turn enhances adhesion in a positive feedback. Following cell detachment, cortex contraction and advective flow amplify myosin II. Prolonged contact with the vitelline membrane prolongs the integrin-myosin II feedback, increases integrin adhesion, and thus slows down cell detachment and wave propagation. The angle of cell detachment depends on adhesion strength and sets the tensile forces required for detachment. Thus, we document how the interplay between subcellular mechanochemical feedback and geometry drives tissue morphogenesis.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Actomyosin; Myosin Type II; Integrins; Morphogenesis
PubMed: 38103554
DOI: 10.1016/j.devcel.2023.11.022 -
Biomolecules Nov 2023Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma...
Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca response at fertilization and the subsequent embryonic development.
Topics: Animals; Female; Male; Starfish; Dithiothreitol; Actins; Semen; Oocytes; Fertilization
PubMed: 38002342
DOI: 10.3390/biom13111659 -
Nature Aug 2019Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by...
Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by controlling actomyosin contractility. Here we report two modes of control over Rho1 and myosin II (MyoII) activation in the Drosophila endoderm. First, Rho1-MyoII are induced in a spatially restricted primordium via localized transcription of the G-protein-coupled receptor ligand Fog. Second, a tissue-scale wave of Rho1-MyoII activation and cell invagination progresses anteriorly away from the primordium. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocks Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. We find that MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane mediated by integrins, apical spreading, MyoII activation and invagination in the next row. Endoderm morphogenesis thus emerges from local transcriptional initiation and a mechanically driven cycle of cell deformation.
Topics: Animals; Cell Adhesion; Cell Shape; Drosophila Proteins; Drosophila melanogaster; Endoderm; Integrins; Morphogenesis; Myosin Type II; Transcriptional Activation; Vitelline Membrane; rho GTP-Binding Proteins
PubMed: 31413363
DOI: 10.1038/s41586-019-1492-9 -
Poultry Science Feb 2024The study aimed to analyze the biological value of eggs and extra-embryonic structures affecting pheasant hatchability depending on the eggshell's color. Eggs (1,415)...
The study aimed to analyze the biological value of eggs and extra-embryonic structures affecting pheasant hatchability depending on the eggshell's color. Eggs (1,415) from 62-wk-old pheasants were used. The quality of fresh blue (BL), brown (BR), and green (G) eggs were analyzed. Incubation lasted for 25 d. Thick albumen (d 0, 1, 7, 14), amniotic fluid (d 14, 18), and the yolk (d 0-14) were collected. The pH, viscosity, lysozyme activity, crude protein (CP) content in albumen and amnion, pH, vitelline membrane strength, and fatty acids (FA) content in the yolk were performed. The lowest hatchability was in the BL group, and the highest was in the G group. BL group showed lower eggshell thickness and strength and higher egg weight. In thick albumen and amniotic fluid, the pH decreased with the incubation. In the yolk, there was an increasing trend (P = 0.015), with a decrease on d 18 (P < 0.001). The vitelline membrane strength decreased after 1 d of incubation, excluding BR eggs (P < 0.001). Thick albumen viscosity was higher on d 14 in the G group than in other dates and groups, the lowest in amniotic fluid, and slightly higher in BL and BR eggs. On d 18, amniotic fluid viscosity increased (P < 0.001). The lowest viscosity was indicated in BL eggs (P < 0.001). The lysozyme activity in thick albumen on d 14 was the highest (uniquely in BR and G groups), and the lowest values were found in amniotic fluid on d 14; after four d, the activity increased (P < 0.001). The CP content was higher in the BL group on d 14. In amnion, on d 14, the CP content was the lowest (<1%) and increased on d 18 (P < 0.001). There was a higher FA content (especially UFA) in the G group and a decrease in FA content after d 14 (P < 0.001). It was found that eggs with green eggshells have the highest biological value, and blue eggs are the least useful for incubation.
Topics: Animals; Chickens; Egg Shell; Muramidase; Ovum; Meat; Albumins; Quail; Fatty Acids; Eggs; Egg Yolk
PubMed: 38134460
DOI: 10.1016/j.psj.2023.103338 -
Poultry Science Jun 2023The study aimed to assess various quality characteristics (physical, morphologic, mechanical) of hatching eggs during the early-mid incubation period. Hatching eggs...
The study aimed to assess various quality characteristics (physical, morphologic, mechanical) of hatching eggs during the early-mid incubation period. Hatching eggs (1,200) were bought from a broiler Ross 308 breeder flock. Before incubation, 20 eggs were analyzed for dimensions and morphologic composition. Eggs (1,176) were incubated for 21 d. Hatchability was analyzed. On d 1, 2, 4, 6, 8, 10, and 12, eggs were collected (n = 20). The eggshell surface temperature and water loss were measured. The eggshell strength and thickness and the vitelline membrane strength were analyzed. The pH of thick albumen, amniotic fluid, and yolk were determined. The viscosity and lysozyme activity were studied for the thick albumen and amniotic fluid. Water loss was proportional and significantly different between incubation days. The yolk vitelline membrane strength highly depended on incubation days, decreasing steadily within the first 2 d (R = 0.9643). The albumen pH decreased from d 4 till d 12 of incubation, whereas the yolk pH first increased from d 0 to d 2 before a decline on d 4. Albumen viscosity was highest on d 6. There was a strong dependence of viscosity decrease with increasing shear rate (R = 0.7976). On the first day of incubation, the highest lysozyme hydrolytic activity was demonstrated (33,790 U/mL) compared to the activity from the amniotic fluid (8-12 d). From d 6, lysozyme activity decreased to 70 U/mL (d 10). On d 12, amniotic fluid lysozyme activity increased by over 6,000 U/mL compared to d 10. The lysozyme hydrolytic activity was lower in the amniotic fluid (d 8-12) compared to the thick albumen (0-6 d) (P < 0.001). The embryo's protective barriers are changed, and the fractions are hydrated during incubation. It could be concluded that the lysozyme is transferred from the albumen to the amniotic fluid due to its activity.
Topics: Animals; Chickens; Muramidase; Ovum; Albumins; Egg Shell
PubMed: 37116284
DOI: 10.1016/j.psj.2023.102689 -
Cells Dec 2021The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays...
The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg's fertilization response.
Topics: Actins; Animals; Calcium; Dithiothreitol; Embryonic Development; Fertilization; Ovum; Sea Urchins
PubMed: 34944081
DOI: 10.3390/cells10123573