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Bioengineering (Basel, Switzerland) May 2023The lipid layer surrounding the vitelline membrane of insect eggs has a critical role in the waterproofing and desiccation resistance of embryos. However, this lipid...
The lipid layer surrounding the vitelline membrane of insect eggs has a critical role in the waterproofing and desiccation resistance of embryos. However, this lipid layer also prevents the flux of chemicals into the embryos, such as cryoprotectants, which are required for successful cryopreservation. The permeabilization studies of silkworm embryos remain insufficient. Therefore, in this study, we developed a permeabilization method to remove the lipid layer in the silkworm, , and examined factors affecting the viability of dechorionated embryos, including the types and exposure times of chemicals and embryonic stages. Among the chemicals used, hexane and heptane were effective for permeabilization, whereas Triton X-100 and Tween-80 were less effective. Regarding the embryonic stages, there were significant differences between 160 and 166 h after egg laying (AEL) at 25 °C. Consequently, we found that the treatment of 160 AEL embryos with hexane for 30 s was the best condition for the permeability and viability of embryos, in which over 62% of the permeabilized embryos grew up to the second larval instar and their moths could lay fertilized eggs. Our method can be used for various purposes, including permeability investigations using other chemicals and embryonic cryopreservation.
PubMed: 37237633
DOI: 10.3390/bioengineering10050563 -
International Journal of Molecular... Aug 2022Vitelline membrane proteins (VMPs) are the main proteins that form the inner shell (vitelline membrane layer) of insect eggs and are an integral part of egg formation...
Vitelline membrane proteins (VMPs) are the main proteins that form the inner shell (vitelline membrane layer) of insect eggs and are an integral part of egg formation and embryo development. Here, we characterized the molecular structure and expression patterns of the gene and analyzed its reproductive functions in diamondback moth, (L.), a worldwide migratory pest of cruciferous plants. The gene was shown to be a single exon gene that contained an open reading frame of 852 base pairs (bp) encoding 283 amino acids. Both qPCR and western blot analyses showed that VMP26 was specifically expressed in female adults and was significantly highly expressed in the ovary. Further anatomical analysis indicated that the expression level of in the ovarian tube with an incomplete yolk was significantly higher than that in the ovarian tube with a complete yolk. CRISPR/Cas9-induced knockout successfully created two homozygous strains with 8- and 46-bp frameshift mutations. The expression deficiency of the VMP26 protein was detected in the mutant strains using immunofluorescence and western blot. No significant difference was found in the number of eggs laid within three days between wild and mutant individuals, but there was a lower egg hatchability. The loss of the gene changed the mean egg size, damaged the structure of the vitelline membrane, and increased the proportion of abnormal eggs due to water loss, resulting in egg collapse. This first analysis of the roles of the gene in the oocyte formation and embryonic development of , using CRISPR/Cas9 technology, provides a basis for screening new genetic control targets of .
Topics: Animals; CRISPR-Cas Systems; Egg Proteins; Female; Moths; Mutagenesis; Vitelline Membrane
PubMed: 36076934
DOI: 10.3390/ijms23179538 -
Scientific Reports Nov 2020In this study, we aimed to perform structural and proteomic analysis of the vitelline membrane (VM) of two species birds belonging to the family Turdidae: blackbird...
In this study, we aimed to perform structural and proteomic analysis of the vitelline membrane (VM) of two species birds belonging to the family Turdidae: blackbird (Turdus merula) and song thrush (Turdus philomelos). We performed structural analyses using scanning electron microscopy. The VM proteins were identified and compared to the best-known chicken VM proteins. According to our results, VM of both species has a typical three-layered structure: the outer layer, inner layer, and the continuous membrane between them. An unusual observation was the finding of "convexity" formed by the inner layer in blackbird. The role of these convex structures is not known, but they can be typical for the species and can be used in their identification. In addition, we identified two proteins in the VM of both species of birds, of which U3KEZ1 FICAL was not previously identified in any other bird species, and the U3JXV8 FICAL protein was confirmed only once in cockatiel parrot VM. The function of these proteins is not exactly known, but their structure shows similarities to the SERPIN proteins that are involved in microbiological defense, i.e., they are immune proteins. This study contributes to the current knowledge about the structure and composition of proteins of VM, especially because similar analyses have never been performed for Turdidae family. Knowledge of the structure and specific proteins of blackbird and song thrush VM can be beneficial in research on ecology and bird biology and also helpful in developing noninvasive and nongenetic identification methods.
Topics: Animals; Chickens; Egg Proteins; Egg Yolk; Female; Immune System; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Proteome; Songbirds; Species Specificity; Vitelline Membrane
PubMed: 33168893
DOI: 10.1038/s41598-020-76559-4 -
BioRxiv : the Preprint Server For... May 2023The eggshell of the fruit fly is a useful model for understanding the synthesis of a complex extracellular matrix. The eggshell is synthesized during mid-to-late...
The eggshell of the fruit fly 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 ( ) are sterile, but the underlying cause of the sterility remained unknown. In this study, we examined the role of in eggshell synthesis. We show that eggs laid by 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 expression pattern with these phenotypes, we show that is expressed in the ovarian follicle cells beginning in mid-oogenesis, and, importantly, that all mutant eggshell phenotypes could be recapitulated by selective knockdown of expression in the follicle cells. To determine whether 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 mutant egg chambers. These results indicate that 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.
PubMed: 37163052
DOI: 10.1101/2023.04.25.538335 -
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 -
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