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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 -
PLoS Genetics Mar 2024Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and...
Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In Drosophila melanogaster, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated in vitro, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation.
Topics: Animals; Female; Drosophila; Drosophila melanogaster; Fatty Acids; Calcium; Fertilization; Oocytes; Pheromones
PubMed: 38483976
DOI: 10.1371/journal.pgen.1011186 -
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 -
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 -
BioRxiv : the Preprint Server For... Apr 2024Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in . We have...
Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in . We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, , by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO's role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5'-TAACNGT-3' OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA Polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (), egg activation (), translational regulation (, , ), and vitelline membrane formation (, , ). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.
PubMed: 38076814
DOI: 10.1101/2023.11.01.565184 -
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 -
Journal of Veterinary Science Sep 2023Hystricomorpha rodents display a similar placentation model to humans. The present study was carried out considering the scarcity of information concerning the placental...
BACKGROUND
Hystricomorpha rodents display a similar placentation model to humans. The present study was carried out considering the scarcity of information concerning the placental development in agouti.
OBJECTIVE
Describe the microscopy of the placenta, subplacenta and yolk sac of agoutis in early pregnancy and report on the inversion of the yolk sac.
METHODS
Fifteen females between the 14-32 day of gestation were used following euthanasia. Gestational buttons were collected, fixed, processed, stained to optical microscopy or immunohistochemistry.
RESULTS
Chorioallantoic placenta (CP) ranged from conical to a half-sphere, as follows: from the 14 to 17 day, the CP displays an inverted "V" shape, predominantly formed by cytotrophoblasts; from 20 to 22 days, formed almost entirely by cytotrophoblasts; at 28 days, a half sphere, with distinct lobes and interlobular area, numerous maternal gaps delimited by syncytiotrophoblasts and trophoblast giant cells; at 32 days, globose and undergoing the maturation process. Subplacenta, located between decidua and CP, initially presents septa consisting of simple columnar epithelium and after 17 days, comprising stratified epithelium. Visceral yolk sac (VYS) is attached to two CP projections between 14 and 17 days, formed by a simple cubic epithelium and inverted. Between 20 and 22 days, the epithelium displays apical villous projections with cytoplasmic vacuoles and a vascularized mesoderm. After the 24 day, the VYS near the placenta is pleated, very vascularized and villous, with decreased villi sizes further away from the placenta.
CONCLUSION
The agouti CP displays similar characteristics to other hystricomorpha, including placenta lobulation, a subplacenta and an inverted vitelline placenta.
Topics: Pregnancy; Female; Animals; Humans; Placentation; Placenta; Dasyproctidae; Rodentia; Yolk Sac
PubMed: 38031643
DOI: 10.4142/jvs.22323 -
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 -
Changes in physicochemical parameters of duck eggs and extra-embryonic structures during incubation.Animal : An International Journal of... Dec 2023Duckling embryogenesis should be deepened due to the hatching technology and its modification possibilities. Many changes occur in incubated eggs, which expose the...
Duckling embryogenesis should be deepened due to the hatching technology and its modification possibilities. Many changes occur in incubated eggs, which expose the embryo to hazards. The study aimed to analyse the physicochemical properties of eggshell, yolk, thick albumen (TA), and amniotic fluid (AF) of incubated hatching eggs from 52-week-old Cherry Valley ducks. The morphological features of 18 fresh eggs were analysed. Over 28 days, a total of 800 eggs underwent incubation. Eggshell surface temperature and egg weight loss were measured on days 1, 4, 7, 10, 14, 18, 21, and 25. Eggshell, TA, AF, and yolk were collected from eggs at incubation days 1-21 (every week). TA was collected on days 0, 1, and 7, while AF on days 7, 14, and 21. The analysis covered a range of physicochemical parameters. Eggshell thickness decreased with incubation, reaching its lowest point posthatch (P < 0.001). The highest pH for TA was recorded on day 1, while the lowest was on day 7 when comparing days 0, 1, and 7 (P < 0.001). TA pH was consistently higher than in AF (P < 0.001). However, the pH of TA was the highest on day 1 and the lowest on day 7 (P < 0.001). Yolk pH increased from days 1 to 21 (P < 0.001). There was also a noticeable in egg weight loss (0.34% daily) (P < 0.001). Vitelline membrane strength decreased from day 0 to day 1 (P < 0.001). Lysozyme activity in thick albumen on day 7 was higher than on days 0 and 1 (P < 0.001). Lysozyme activity in AF was higher on day 21 than days 7 and 14 (P < 0.001). TA viscosity was highest on day 0 and lowest on day 1, compared to other days (P < 0.001). AF viscosity and CP content exhibited an increase on day 21 as compared to days 7 and 14 (P < 0.001). The CP content in TA was notably higher on day 7 than on days 0 and 1 (P < 0.001). Polyunsaturated fatty acids declined, while monounsaturated and transfatty acids increased (P < 0.001). Viscosity and lysozyme activity increased on day 7 in TA and day 21 in AF. TA and the amniotic cavity appeared to facilitate the transfer of substances, particularly CP. Viscosity could be an indicator for optimising incubation conditions, as incorrect changes can affect embryo mortality. The results showed the different utilisation of nutrients, such as fatty acids. It could support research on the in-ovo administration of various substances.
Topics: Animals; Ducks; Muramidase; Ovum; Egg Shell; Weight Loss; Eggs; Chickens
PubMed: 37981451
DOI: 10.1016/j.animal.2023.101024 -
Brazilian Journal of Biology = Revista... 2023Specimens of Cnemidocarpa amphora were collected monthly from the Arabian Gulf from September 2017 to August 2018. Parts of their gonads were prepared for histological...
Gonadal proliferation and reproductive cycle of the exotic sea squirt Cnemidocarpa amphora () (Pleurogona, Styelidae) sampled for the first time from the northern coast of Arabian Gulf in Saudi Arabia.
Specimens of Cnemidocarpa amphora were collected monthly from the Arabian Gulf from September 2017 to August 2018. Parts of their gonads were prepared for histological testing. The gonads' diameters varied by month. Each gonad contained many ovarian follicles with different morphologies and was surrounded by several testicular follicles. The ovarian and testicular follicles were separate, although the latter were always present near the former. Repeated measures ANOVA tests were conducted to investigate monthly changes in oocyte stages. In squirts measuring 12-13 cm in length, the gonads measured 30-50 mm from July to August; 20-25 mm from September to October; 15-20 mm from November to February; and 25-40 mm from March to June. Oogonia budded from the germinal epithelium with diameters of 20-30 µm. Previtellogenic oocytes measuring 70-120 µm were characterized by the deposition of small granules of protein around the nucleus, a continuous layer of follicular cuboidal epithelium, and scattered vacuoles in the ooplasm. The measurement of gonads and oocyte diameters were performed by image analysis (Image scope 2.3, Image Line, Inc.) and stage micrometer. The vitellogenic oocytes measured 130-220 µm and the follicular epithelium consisted of flattened and cuboidal layers. Beneath the vitelline membrane, scattered test cells appeared in the ooplasm and different granules of protein and MPS were deposited in the ooplasm. In the later phase, lipid droplets began to appear in the ooplasm. Yolk bodies formed after the impregnation of various granules together and the oocyte was ready to be shed. Before spawning, a yolk membrane appeared above the ooplasm. Post-vitellogenic oocytes, in which the homogeneity of ooplasm was restored, underwent gradual lysis and entered the atretic phase. Different stages of sperm development were present year-round in different follicles of the same squirt; hence, the testes were always mature.
Topics: Animals; Female; Male; Urochordata; Saudi Arabia; Semen; Oocytes; Ovary; Cell Proliferation
PubMed: 37970899
DOI: 10.1590/1519-6984.273666