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Open Biology Aug 2021Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation...
Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In , egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. Here we use physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in mature oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. We show that the swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na channels. Furthermore, through pharmacological and genetic disruption, we reveal a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. Our data establish osmotic pressure as a mechanism that initiates egg activation in and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants.
Topics: Animals; Calcium; Calcium Signaling; Drosophila Proteins; Drosophila melanogaster; Embryonic Development; Female; Fertilization; Oocytes; Osmolar Concentration
PubMed: 34343463
DOI: 10.1098/rsob.210067 -
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 -
Developmental and Comparative Immunology Jul 2022Semigranular cells (SGCs) and granular cells (GCs) are two dominant groups of circulating hemocytes in crayfish Cherax quadricarinatus. Molecular markers are required...
Semigranular cells (SGCs) and granular cells (GCs) are two dominant groups of circulating hemocytes in crayfish Cherax quadricarinatus. Molecular markers are required for the clear classification of the hemocytes and the research of their function and differentiation. In this study, we compared the protein content of GCs and SGCs by using two workflows: one-dimensional gel electrophoresis followed by LC-MS/MS and in-solution digestion of cell lysate followed by LC-MS/MS. Cell type-specific proteins were identified, and their expression in SGCs and GCs was further investigated by RT-PCR, Western blotting, and immunofluorescence analysis. Three molecular markers for GCs (peroxinectin, a mannose-binding protein, and prophenoloxidase-activating enzyme 2a) and three molecular markers for SGCs (a vitelline membrane outer layer protein I-like protein, a C-type lectin, and a peptidase) were identified. The application of some of the markers in Eriocheir sinensis was also analyzed. These molecular markers are useful tools for the research of crustaceans hemocytes.
Topics: Animals; Astacoidea; Cell Differentiation; Chromatography, Liquid; Hemocytes; Tandem Mass Spectrometry
PubMed: 35364134
DOI: 10.1016/j.dci.2022.104407 -
Journal of Food Science Apr 2021In this study, after washing, changes in the quality characteristics of chicken eggs coated with apricot, almond, and sour cherry tree resins were examined during two...
In this study, after washing, changes in the quality characteristics of chicken eggs coated with apricot, almond, and sour cherry tree resins were examined during two different temperatures (4 °C and 22 °C) storage for 60 days. While air cell height, weight loss, albumen and yolk pH and a* (redness) values increased in all samples during storage, Haugh unit, albumen and yolk index, shell fracture and vitelline membrane strength, albumen and yolk L* (lightness) and b* (yellowness) values decreased (P < 0.05). The lowest weight loss (0.54 g) and air cell height (2.89 mm), highest Haugh unit (73.95 HU), albumen index (8.81%), and yolk index (40.37%) were found in the samples coated with sour cherry wood resin stored at 4 °C. The shell breakage and vitelline membrane strength of the coated samples were determined to be higher than the control samples and the samples stored after washing. Higher weight loss, air cell height, and pH values, while lower Haugh unit, Albumen and yolk index were found in samples stored at 22 °C (P < 0.05). At the end of storage, the maximum increase in the counts of total aerobic mesophilic and psychrophilic bacteria was found in the albumin and egg yolk of washed samples stored at ambient temperature. As a result, the coating materials prepared with the resin of apricot, almond, and sour cherry trees were suitable for eggshell's shelf life extension. PRACTICAL APPLICATION: The consumers demand the eggs be in their freshest condition, but the currently available storage conditions are not sufficient to maintain freshness in many regions of Turkey. The physical, chemical and, microbiological qualities of the eggs coated with wood resins were determined to be superior compared to other samples. Because resins have good barrier properties, it is recommended to conduct extensive studies on their applicability in different products.
Topics: Animals; Chickens; Color; Egg White; Egg Yolk; Eggs; Food Packaging; Food Storage; Prunus armeniaca; Prunus avium; Prunus dulcis; Resins, Plant; Temperature
PubMed: 33761140
DOI: 10.1111/1750-3841.15686 -
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 -
Investigative Ophthalmology & Visual... Sep 2014The aim of this study was to explore the possible interactions between vitelline membrane outer layer 1 homolog (VMO1) and other tear proteins and to determine the... (Comparative Study)
Comparative Study
PURPOSE
The aim of this study was to explore the possible interactions between vitelline membrane outer layer 1 homolog (VMO1) and other tear proteins and to determine the function of VMO1 in tear fluid.
METHODS
Interactions between recombinant human VMO1 and several abundant tear proteins were determined by dot blot, His pull-down, immunoprecipitation, and Western blot assays, as well as by computer-assisted prediction and modeling of molecular interactions. Kirby-Bauer antibiotic testing was performed to determine whether VMO1 possesses antimicrobial activity. Tear samples were collected from dry eye patients and from healthy controls. The role of VMO1 in maintaining the stability of tear film was investigated by measurement of contact angles on Teflon, tear break-up time (TBUT) and the time-dependent reduction in tear film integrity in mice.
RESULTS
Vitelline membrane outer layer 1 homolog showed an interaction with lysozyme C (LYSC) in the dot-blot, His pull-down, and immunoprecipitation assays. Vitelline membrane outer layer 1 homolog revealed no zones of growth inhibition of standard strains of Staphylococcus aureus and Escherichia coli. Tears presented smaller contact angles on Teflon surfaces after the addition of VMO1 (P<0.05). Vitelline membrane outer layer 1 homolog-treated mice showed longer TBUTs (P<0.05). Tear films from VMO1-treated mice maintained their integrity for longer periods of time than tear films from the control group, and this effect was dose-dependent.
CONCLUSIONS
Vitelline membrane outer layer 1 homolog interacts with LYSC and has positive effects on the stabilization of tear film.
Topics: Animals; Blotting, Western; Disease Models, Animal; Dry Eye Syndromes; Egg Proteins; Humans; Immunoprecipitation; Mice; Mice, Inbred C57BL; Muramidase; Tears
PubMed: 25257056
DOI: 10.1167/iovs.14-14491 -
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 -
Advances in Experimental Medicine and... 2017Terminally differentiated avian sperm consist of a head which male genetic material locates and flagellum that provides the motive force to propel them towards the...
Terminally differentiated avian sperm consist of a head which male genetic material locates and flagellum that provides the motive force to propel them towards the fertilization site. The apical end of the sperm head accommodates a secretory vesicle, called an acrosome, that undergoes acrosome reaction releasing proteolytic content to penetrate the peri-vitelline membrane of an egg. Transcriptionally and translationally inactive, sperm need to rely on these distinct compartments in which different functions are preassembled, in order to achieve the goal of "fertilization". How are these complex structures with high functionality formed? Spermatogenesis is divided into an early stage in which diploid spermatogonia is proliferated into round spermatids thorough mitotic and meiotic divisions, and a late stage in which round spermatids are transformed into sperm though nuclear condensation and elongation of the sperm head, and formation of accessory structures. Recently, it was reported in aves that morphologically differentiated sperm undergo post-testicular maturation during passage through the male genital tract, suggesting that a similar system to mammals might be involved in the acquisition of fertilizing ability in avian sperm. Investigation for mechanisms underlying how sperm regulate their functions which are necessary to achieve fertilization is important for developing reproductive biotechnology in aves, because cryopreservation of poultry sperm is still not reliable for use in commercial production or for the preservation of genetic resources. In this review, we firstly provide an update on avian spermatogenesis, and then discuss the uniqueness of structure and functions of avian sperm, highlighting differences from mammalian sperm. Lastly, we discuss the molecular mechanism and current techniques of cryopreservation for avian sperm.
Topics: Animals; Chickens; Cryopreservation; Male; Semen Preservation; Spermatozoa
PubMed: 28980229
DOI: 10.1007/978-981-10-3975-1_4