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Scientific Reports Nov 2023The specific functions and essentiality of type II vitellogenin (Vtg2) in early zebrafish development were investigated in this study. A vtg2-mutant zebrafish line was...
Genomic disturbance of vitellogenin 2 (vtg2) leads to vitellin membrane deficiencies and significant mortalities at early stages of embryonic development in zebrafish (Danio rerio).
The specific functions and essentiality of type II vitellogenin (Vtg2) in early zebrafish development were investigated in this study. A vtg2-mutant zebrafish line was produced and effects of genomic disturbance were observed in F2 females and F3 offspring. No change in vtg2 transcript has been detected, however, Vtg2 abundance in F2 female liver was 5×, and in 1 hpf F3 vtg2-mutant embryos was 3.8× less than Wt (p < 0.05). Fecundity was unaffected while fertilization rate was more than halved in F2 vtg2-mutant females (p < 0.05). Hatching rate was significantly higher in F3 vtg2-mutant embryos in comparison to Wt embryos. Survival rate declined drastically to 29% and 18% at 24 hpf and 20 dpf, respectively, in F3 vtg2-mutant embryos. The introduced mutation caused vitelline membrane deficiencies, significant mortalities at early embryonic stages, and morphological abnormalities in the surviving F3 vtg2-mutant larvae. Overrepresentation of histones, zona pellucida proteins, lectins, and protein degradation related proteins in F3 vtg2-mutant embryos provide evidence to impaired mechanisms involved in vitellin membrane formation. Overall findings imply a potential function of Vtg2 in acquisition of vitellin membrane integrity, among other reproductive functions, and therefore, its essentiality in early zebrafish embryo development.
Topics: Animals; Female; Embryo, Nonmammalian; Embryonic Development; Genomics; Larva; Vitellins; Vitellogenins; Zebrafish
PubMed: 37914813
DOI: 10.1038/s41598-023-46148-2 -
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 -
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 -
Nature Communications Jun 2023Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the...
Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that the chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. Experimental relaxation of the VM early in development impairs blastoderm expansion, while maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, failure of neural tube closure, and axis rupture. Biochemical and structural analysis shows that VM weakening is associated with the reduction of outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO release from the egg. Our results identify a previously unrecognized potential cause of body axis defects through mis-regulation of extraembryonic tissue tension.
Topics: Animals; Down-Regulation; Chickens; Blastoderm; Embryonic Development
PubMed: 37277340
DOI: 10.1038/s41467-023-38988-3 -
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 -
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 -
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 -
International Journal of Molecular... Mar 2023(1) Hematological malignancies are characterized by an immortalization, uncontrolled proliferation of blood cells and their differentiation block, followed by the loss...
(1) Hematological malignancies are characterized by an immortalization, uncontrolled proliferation of blood cells and their differentiation block, followed by the loss of function. The primary goal in the treatment of leukemias is the elimination of rapidly proliferating leukemic cells (named blasts). However, chemotherapy, which removes proliferating blasts, also prevents the remaining immune cells from being activated. Acute leukemias affect elderly people, who are often not fit to survive aggressive chemotherapy. Therefore, there is a need of milder treatment, named differentiation therapy, which might simulate the immune system of the patient. 1,25-Dihydroxyvitamin D, or low-calcemic analogs of this compound, were proposed as supporting therapy in acute leukemias. (2) Bone marrow blasts from patients with hematological malignancies, and leukocytes from healthy volunteers were ex vivo exposed to 1,25-dihydroxyvitamin D, and then their genomes and transcriptomes were investigated. (3) Our analysis indicates that 1,25-dihydroxyvitamin D regulates in blood cells predominantly genes involved in immune response, such as (cathelicidin antimicrobial peptide), (ceruloplasmin), (C-X-C motif chemokine ligand 9), (CD14 molecule) or (vitelline membrane outer layer 1 homolog). This concerns blood cells from healthy people, as well as blasts from patients with hematological malignancies. In addition, in one patient, 1,25-dihydroxyvitamin D significantly downregulated transcription of genes responsible for cell division and immortalization. (4) In conclusion, the data presented in this paper suggest that addition of 1,25-dihydroxyvitamin D to the currently available treatments would stimulate immune system, inhibit proliferation and reduce immortal potential of blasts.
Topics: Humans; Aged; Leukemia, Myeloid, Acute; Leukocytes; Blood Cells; Cell Differentiation; Dihydroxycholecalciferols; Hematologic Neoplasms
PubMed: 37047477
DOI: 10.3390/ijms24076504 -
Nanotoxicology Feb 2023Despite the great potential of using positively charged gold nanoparticles (AuNPs) in nanomedicine, no systematic studies have been reported on their synthesis...
Polyethyleneimine/polyethylene glycol-conjugated gold nanoparticles as nanoscale positive/negative controls in nanotoxicology: testing in frog embryo teratogenesis assay- and mammalian tissue culture system.
Despite the great potential of using positively charged gold nanoparticles (AuNPs) in nanomedicine, no systematic studies have been reported on their synthesis optimization or colloidal stability under physiological conditions until a group at the National Institute of Standards and Technology recently succeeded in producing remarkably stable polyethyleneimine (PEI)-coated AuNPs (Au-PEI). This improved version of Au-PEI (Au-PEI25kB) has increased the demand for toxicity and teratogenicity information for applications in nanomedicine and nanotoxicology. In vitro assays for Au-PEI25kB in various cell lines showed substantial active cytotoxicity. For advanced toxicity research, the frog embryo teratogenesis assay- (FETAX) method was employed in this study. We observed that positively-charged Au-PEI25kB exhibited significant toxicity and teratogenicity, whereas polyethylene glycol conjugated AuNPs (Au-PEG) used as comparable negative controls did not. There is a characteristic avidity of Au-PEI25kB for the jelly coat, the chorionic envelope (also known as vitelline membrane) and the cytoplasmic membrane, as well as a barrier effect of the chorionic envelope observed with Au-PEG. To circumvent these characteristics, an injection-mediated FETAX approach was utilized. Like treatment with the FETAX method, the injection of Au-PEI25kB severely impaired embryo development. Notably, the survival/concentration curve that was steep when the standard FETAX approach was employed became gradual in the injection-mediated FETAX. These results suggest that Au-PEI25kB may be a good candidate as a nanoscale positive control material for nanoparticle analysis in toxicology and teratology.
Topics: Animals; Teratogenesis; Gold; Polyethyleneimine; Polyethylene Glycols; Xenopus laevis; Metal Nanoparticles; Embryo, Nonmammalian; Teratogens; Mammals
PubMed: 36919473
DOI: 10.1080/17435390.2023.2187322