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PloS One 2023The eggshell of the fruit fly Drosophila melanogaster is a useful model for understanding the synthesis of a complex extracellular matrix. The eggshell is synthesized...
The eggshell of the fruit fly Drosophila melanogaster is a useful model for understanding the synthesis of a complex extracellular matrix. The eggshell is synthesized during mid-to-late oogenesis by the somatic follicle cells that surround the developing oocyte. We previously reported that female flies mutant for the gene drop-dead (drd) are sterile, but the underlying cause of the sterility remained unknown. In this study, we examined the role of drd in eggshell synthesis. We show that eggs laid by drd mutant females are fertilized but arrest early in embryogenesis, and that the innermost layer of the eggshell, the vitelline membrane, is abnormally permeable to dye in these eggs. In addition, the major vitelline membrane proteins fail to become crosslinked by nonreducible bonds, a process that normally occurs during egg activation following ovulation, as evidenced by their solubility and detection by Western blot in laid eggs. In contrast, the Cp36 protein, which is found in the outer chorion layers of the eggshell, becomes crosslinked normally. To link the drd expression pattern with these phenotypes, we show that drd is expressed in the ovarian follicle cells beginning in mid-oogenesis, and, importantly, that all drd mutant eggshell phenotypes could be recapitulated by selective knockdown of drd expression in the follicle cells. To determine whether drd expression was required for the crosslinking itself, we performed in vitro activation and crosslinking experiments. The vitelline membranes of control egg chambers could become crosslinked either by incubation in hyperosmotic medium, which activates the egg chambers, or by exogenous peroxidase and hydrogen peroxide. In contrast, neither treatment resulted in the crosslinking of the vitelline membrane in drd mutant egg chambers. These results indicate that drd expression in the follicle cells is necessary for vitelline membrane proteins to serve as substrates for peroxidase-mediated cross-linking at the end of oogenesis.
Topics: Animals; Female; Drosophila; Drosophila melanogaster; Egg Shell; Oogenesis; Peroxidases; Drosophila Proteins
PubMed: 38051756
DOI: 10.1371/journal.pone.0295412 -
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 -
Animals : An Open Access Journal From... Feb 2023This study evaluates the effect of housing environment on the egg quality characteristics of brown egg layers as many different environments are currently used in the...
This study evaluates the effect of housing environment on the egg quality characteristics of brown egg layers as many different environments are currently used in the industry. Battery cages, barren colony cages, enriched colony cages, cage-free, and free-range environments were evaluated. Overall, all egg quality measurements were affected by housing environment ( < 0.01) except for vitelline membrane strength, elasticity, and egg solids. Eggshells and yolks were lightest in barren colony cages and darkest from free-range hens ( < 0.0001). Free-range eggs were heavier than eggs from all other environments ( < 0.0001). Cage-free eggs had lower albumen height and Haugh units than other environments ( < 0.0001). Lastly, cage-free and free-range eggs had stronger eggshells than the other environments ( < 0.0001), and free-range eggs had more elastic eggshells than eggs from conventional battery cages and barren colony cages ( < 0.01). Access to the range seemed to give free-range hens different nutritional advantages, which allowed for the darker yolks and shells. Furthermore, eggs from barren colony cages seemed to exhibit more negative characteristics. Simply adding enrichments to colony cages did not improve or detract from egg quality. From this research, it appears that, as the industry moves toward extensive environments, the egg quality of brown egg layers will improve.
PubMed: 36830504
DOI: 10.3390/ani13040716 -
Molecular Reproduction and Development Mar 2020Fertilization by more than one sperm causes polyploidy, a condition that is generally lethal to the embryo in the majority of animal species. To prevent this occurrence,... (Review)
Review
Fertilization by more than one sperm causes polyploidy, a condition that is generally lethal to the embryo in the majority of animal species. To prevent this occurrence, eggs have developed a series of mechanisms that block polyspermy at the level of the plasma membrane or their extracellular coat. In this review, we first introduce the mammalian egg coat, the zona pellucida (ZP), and summarize what is currently known about its composition, structure, and biological functions. We then describe how this specialized extracellular matrix is modified by the contents of cortical granules (CG), secretory organelles that are exocytosed by the egg after gamete fusion. This process releases proteases, glycosidases, lectins and zinc onto the ZP, resulting in a series of changes in the properties of the egg coat that are collectively referred to as hardening. By drawing parallels with comparable modifications of the vitelline envelope of nonmammalian eggs, we discuss how CG-dependent modifications of the ZP are thought to contribute to the block to polyspermy. Moreover, we argue for the importance of obtaining more information on the architecture of the ZP, as well as systematically investigating the many facets of ZP hardening.
Topics: Animals; Exocytosis; Female; Glycosylation; Humans; Lectins; Male; Mice; Oocytes; Polyploidy; Sperm-Ovum Interactions; Spermatozoa; Zinc; Zona Pellucida; Zona Pellucida Glycoproteins
PubMed: 32003503
DOI: 10.1002/mrd.23320 -
Frontiers in Cell and Developmental... 2021Fertilization is one of the most important events in living organisms to generate a new life with a mixed genetic background. To achieve successful fertilization, sperm... (Review)
Review
Fertilization is one of the most important events in living organisms to generate a new life with a mixed genetic background. To achieve successful fertilization, sperm and eggs must undergo complex processes in a sequential order. Fertilization of marine invertebrate type has been studied for more than a hundred years. Ascidian sperm are attracted by chemoattractants from eggs and bind to the vitelline coat. Subsequently, sperm penetrate through the vitelline coat proteolytically and finally fuse with the egg plasma membrane. Here, we summarize the fertilization mechanisms of ascidians, particularly from sperm-egg interactions to sperm penetration of the egg coat. Since ascidians are hermaphrodites, inbreeding depression is a serious problem. To avoid self-fertilization, ascidians possess a self-incompatibility system. In this review, we also describe the molecular mechanisms of the self-incompatibility system in type A governed by three allelic gene pairs of and .
PubMed: 35186958
DOI: 10.3389/fcell.2021.827214 -
Membranes Jul 2022oocytes are commonly used in many fundamental biological studies. One of the major limitations of oocytes is their short storage lifespan with most defolliculated...
oocytes are commonly used in many fundamental biological studies. One of the major limitations of oocytes is their short storage lifespan with most defolliculated oocytes physically deteriorating in 10 days or less. Herein, we identified a 3D Cultrex-based storage media that incorporates extracellular membrane-based hydrogels to maintain oocyte integrity. Under these treatments, the lifespan of the oocytes increased to more than 20 days compared to standard conditions. The treatment preserved the oocytes membrane integrity and did not interfere with mRNA- or cDNA-derived protein expression.
PubMed: 36005669
DOI: 10.3390/membranes12080754 -
Developmental Biology May 2019Stereotyped left-right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the neurula rotation in the...
Stereotyped left-right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the neurula rotation in the ascidian, Halocynthia roretzi. Neurula embryos rotate along the anterior-posterior axis in a counterclockwise direction, and the rotation stops when the left side of the embryo is oriented downwards, resulting in contact of the left-side epidermis with the vitelline membrane at the bottom of perivitelline space. Then, such contact induces the expression of nodal and its downstream Pitx2 gene in the left-side epidermis. Vitelline membrane is required for the promotion of nodal expression. Here, we showed that a chemical signal from the vitelline membrane promotes nodal gene expression, but mechanical stimulus at the point of contact is unnecessary since the treatment of devitellinated neurulae with an extract of the vitelline membrane promoted nodal expression on both sides. The signal molecules are already present in the vitelline membranes of unfertilized eggs. These signal molecules are proteins but not sugars. Specific fractions in gel filtration chromatography had the nodal promoting activity. By mass spectrometry, we selected 48 candidate proteins. Proteins that contain both a zona pellucida (ZP) domain and epidermal growth factor (EGF) repeats were enriched in the candidates of the nodal inducing molecules. Six of the ZP proteins had multiple EGF repeats that are only found in ascidian ZP proteins. These were considered to be the most viable candidates of the nodal-inducing molecules. Signal molecules are anchored to the entire vitelline membrane, and contact sites of signal-receiving cells are spatially and mechanically controlled by the neurula rotation. In this context, ascidians are unusual with respect to mechanisms for specification of the left-right axis. By suppressing formation of epidermis monocilia, we also showed that epidermal cilia drive the neurula rotation but are dispensable for sensing the signal from the vitelline membrane.
Topics: Animals; Body Patterning; Cell Extracts; Cilia; Egg Proteins; Embryo, Nonmammalian; Epidermis; Gene Expression Regulation, Developmental; Glycosylation; Nodal Protein; Protein Domains; Quinazolinones; Rotation; Signal Transduction; Sugars; Urochordata; Vitelline Membrane
PubMed: 30710513
DOI: 10.1016/j.ydbio.2019.01.016 -
Parasites & Vectors Aug 2023Stephanofilaria stilesi is a vector-borne filarioid nematode of cattle in North America that is transmitted via the hematophagous horn fly (Haematobia irritans)...
BACKGROUND
Stephanofilaria stilesi is a vector-borne filarioid nematode of cattle in North America that is transmitted via the hematophagous horn fly (Haematobia irritans) intermediate host. Despite being relatively common, little attention has been given to a thorough description of S. stilesi lesions and the potential integration of pathological and molecular diagnostic findings to confirm infection.
METHODS
To characterize the cutaneous lesions caused by S. stilesi in cattle (Bos taurus taurus and Bos taurus indicus), skin of the ventral abdominal midline was collected from 22 animals during postmortem examination. Skin samples were processed for histology, transmission electron microscopy (TEM), DNA extraction, PCR, and Sanger sequencing targeting molecular markers cytochrome oxidase c subunit 1 (cox1), 12S, 18S rDNA, and 28S rDNA.
RESULTS
Macroscopically, lesions ranged from 5 × 4 cm to 36 × 10 cm, consisting of one large single lesion, or two to four ovoid areas at the ventral abdominal midline, surrounding the umbilicus. Each lesion presented as ulcerative dermatitis with dry, serocellular crusts, or alopecic and lichenified areas. Histologically, eosinophilic, neutrophilic, and ulcerative dermatitis with furunculosis, folliculitis, and epidermal hyperplasia was observed. Cross sections of adult nematodes were identified in ~ 60% of the cases (n = 13) within intact follicles, sebaceous ducts, crusts, and areas of furunculosis. Stephanofilaria first-stage larvae (L1) were observed in five cases within "vitelline membranes" in the superficial dermis and crusts. Ultrastructurally, the L1 cross sections were compounded of smooth multilayered cuticle and somatic cells. The "vitelline membrane" is a tri-layered membrane where L1 are suspended in a matrix. Stephanofilaria stilesi DNA was found in 5 out of the 13 cases in which adults or L1 were histologically observed (38%) and in 1 out of the 9 cases without adults or L1 present (11%). Phylogenetic analyses suggest a closer relationship of the genus Stephanofilaria with Thelazioidea, instead of the family Filariidae (Filarioidea), in which it has been historically allocated.
CONCLUSIONS
Our study improved the characterization of lesions and described ultrastructural findings of S. stilesi and highlights that molecular tools should be utilized in combination with histology for improved diagnostic resolution.
Topics: Animals; Cattle; Phylogeny; Furunculosis; Filarioidea; Dermatitis; Muscidae; DNA, Ribosomal
PubMed: 37573424
DOI: 10.1186/s13071-023-05905-y -
Animals : An Open Access Journal From... Mar 2021The yolk is the principal part of the egg that contains vitamins, minerals, lipids, and proteins which are essential for embryo development and hatching. The egg yolk...
The yolk is the principal part of the egg that contains vitamins, minerals, lipids, and proteins which are essential for embryo development and hatching. The egg yolk contains significant amounts of lipoproteins, triacylglycerides, and cholesterol, whose dynamics are indistinct during embryogenesis. The effects of cholesterol on the yolk protein abundance, intensity, and function are ill-defined during embryonic development. Using two-dimensional gel electrophoresis, eggs with respective high and low cholesterol protein abundance were investigated after 0, 2, 6, and 13 days of embryogenesis and further analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. The results revealed that the vitellogenin proteins are the most abundant egg yolk protein that showed proximity and a high degree of variation in isoelectric point and molecular weight. The results demonstrated increased expression of vitellogenin-1 and vitellogenin-3 at two days and vitellogenin-2 protein at 13 days of embryogenesis in both egg types. The ovoinhibitor, immunoglobulin lambda light chain precursor, Ig-gamma (clone-36 chicken), and beta-2-glycoprotein-1 precursor proteins were significantly expressed in high cholesterol eggs while haptoglobin protein PIT-54 and vitelline membrane outer layer proteins intensities were significant in low cholesterol eggs at two days of embryogenesis. The high cholesterol eggs showed a modest increase in egg weight, yolk weight, albumen height, yolk color, and egg strength relative to the low cholesterol eggs. The gene ontology enrichment analysis revealed that the differentially expressed proteins such as vitellogenin proteins were involved in lipid transport and lipid localization biological processes and showed nutrient reservoir activity function. The ovotransferrin regulated the biological processes of plasminogen activation and extracellular matrix disassembly and characterized the anchored component of the plasma membrane. The ovoinhibitor protein was involved in response to mineralocorticoid and corticosterone biological processes whereas the vitellin membrane outer layer protein constituted the extracellular exosome, extracellular organelle, and membrane-bounded vesicle cellular components. Collectively, our study revealed yolk protein abundance, molecular function, cellular components, and biological processes and concluded that yolk protein intensities were significantly altered by cholesterol concentration.
PubMed: 33803097
DOI: 10.3390/ani11030744 -
Poultry Science Dec 2023The study aimed to analyze the hatching egg and physiochemical features of eggshells, thick albumen, amniotic fluid, and yolk during the incubation of Ross 308 chicken...
The study aimed to analyze the hatching egg and physiochemical features of eggshells, thick albumen, amniotic fluid, and yolk during the incubation of Ross 308 chicken eggs. Eggs (n = 755) were incubated for 21 d. Quality analysis of fresh eggs was performed. Eggshells, albumen, and yolk were collected from fresh eggs and incubation d 1, 7, and 14. Eggshell thickness and strength, pH, vitelline membrane strength, fatty acid (FA) in the yolk, pH, viscosity, lysozyme activity, and crude protein content in thick albumen and amniotic fluid were analyzed. Hatching parameters were calculated. Egg weight loss was constant (8.04% overall). Lower egg surface temperature was found on d 7 compared to d 4, 14, and 18. A lower thickness of posthatch eggshells was found. The strength of the vitelline membrane significantly decreased within 24 h (by over 58%). During incubation, there was a decrease in thick albumen/amniotic fluid pH; an opposite trend was found in yolk pH. The vitelline membrane strength was negatively correlated with the albumen pH. Lysozyme activity was higher in fresh thick albumen and up to 2 wk of incubation. On d 7, the lowest activity was found in the amniotic fluid. On d 14, lysozyme activity increased in amniotic fluid. The higher viscosity of the thick albumen was demonstrated on d 7 and 14 of incubation. The lowest viscosity in amniotic fluid was found on the same days. Crude protein content was higher in thick albumen (d 7 and 14) and lowest in amniotic fluid on d 7. The FA content changed between d 0 and 14. The results indicate different use of FA, where PUFA decreased. Eggshell is used in the last week of incubation. The thick albumen is reduced, while the biological value of amniotic fluid is increasing. Lysozyme activity, viscosity, and crude protein content may be interdependent. It may indicate the flow of substances and the transfer of functions from the thick albumen to the amniotic fluid during chicken embryogenesis.
Topics: Animals; Chickens; Egg Shell; Muramidase; Amniotic Fluid; Ovum; Albumins; Fatty Acids; Embryonic Development; Egg Yolk; Eggs
PubMed: 37832191
DOI: 10.1016/j.psj.2023.103119