Loss of the seipin gene perturbs eggshell formation in Caenorhabditiselegans.

Seipin, an evolutionary conserved protein, plays pivotal roles during lipid droplet (LD) biogenesis and is associated with various human diseases with unclear mechanisms. Here, we analyzed Caenorhabditis elegans mutants deleted of the sole SEIPIN gene, seip-1 Homozygous seip-1 mutants displayed penetrant embryonic lethality, which is caused by the disruption of the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell. In C. elegans oocytes and embryos, SEIP-1 is associated with LDs and is crucial for controlling LD size and lipid homeostasis. The seip-1 deletion mutants reduced the ratio of polyunsaturated fatty acids (PUFAs) in their embryonic fatty acid pool. Interestingly, dietary supplementation of selected n-6 PUFAs rescued the embryonic lethality and defective permeability barrier. Accordingly, we propose that SEIP-1 may maternally regulate LD biogenesis and lipid homeostasis to orchestrate the formation of the permeability barrier for eggshell synthesis during embryogenesis. A lipodystrophy allele of seip-1 resulted in embryonic lethality as well and could be rescued by PUFA supplementation. These experiments support a great potential for using C. elegans to model SEIPIN-associated human diseases.

Expression of follistatin is associated with egg formation in the oviduct of laying hens.

The objective of this study was to examine the expression profiles of follistatin (FST) and its associated molecules (MSTN, INHA, INHBB, INHBA, ACVR2A, and ACVR2B) in the oviduct of laying hens at 3 hr and 20 hr post-ovulation (p.o., n = 5; 35 weeks old), molting (n = 5; 60 weeks old), and non-laying (n = 4; 35-60 weeks old) hens and also to localize the FST by using immunohistochemistry assay. Expression of FST was significantly higher (p < .05), and MSTN was lower in the uterus of laying hens around 15-20 hr p.o. (during eggshell formation), however, their expressions in the magnum remain unchanged across different physiological stages of hens. FST was mainly expressed in the luminal and glandular epithelium of the uterine tissues, and their expression intensity was highest in laying hens during the eggshell mineralization. There was a relatively increased expression of INHA in the magnum of laying hens around 3 hr p.o. as compared to non-laying and molting hens. At the same time (3 hr p.o.), there was a significant (p < .05) decrease in the expression of the INHBB, ACVR2A, and ACV2B. These results indicate that follistatin may regulate the differentiation of uterine luminal and glandular epithelium during eggshell biomineralization.

Comparison of different brown and white layer hybrid embryonic development and uptake of nutrients in the egg.

The objective of this study was to evaluate the comparison of different brown and white layers in embryonic development and uptake of nutrients in the egg. A total of 360 fertilized eggs obtained from two brown (Atak-S and Brown Nick) and two white (Atabey and Nick) layer breeders at 28 wk old. Hatching eggs from each genotype were examined on the day of setting for fresh egg analysis and then at the beginning of the embryonic day (E19) and embryonic day (E21) for egg, embryo and villus analysis. Differences in egg weight, shell percentages, relative weight of yolk and albumen, relative weight and length of embryo, villus height, some values of shell, yolk and albumen and relative chick weight in examined hybrids were significant. Yolk sac utilization of embryos during the incubation in the white layer hybrids was greater than that in the brown layer hybrids. Villus heights in the duodenum, jejenum and ileum of embryos in the brown layer hybrids was greater than that in the white layer hybrids. Genotype is important parameter to determine the egg composition at the same age and in animals being fed the same diet. It was observed that the consumption of yolk and shell nutrients from the embryos during the incubation was not related to whether embryos were from the brown or white layer hybrids. Only uptake of the yolk sac and villus height in the embryo among examined variables varied depending on whether the embryos were from the brown or white layer hybrids.

Effect of heat stress on the gene expression of ion transporters/channels in the uterus of laying hens during eggshell formation.

Heat stress is a problem in laying hens as it decreases egg quality by decreasing eggshell mineralization. Heat stress alters gene expression, hence our aim was to investigate effects of heat stress on gene expression of ion transport elements involving in uterine mineralization (TRPV6, CALB1, ITPR3, SCNN1G, SLC4A4, KCNJ15, SLC4A9, and CLCN2) by real time quantitative PCR. Forty 23-week-old White Leghorn laying hens were housed in two rooms. The control group (n = 20) was maintained at 21-23 °C, and the heat stress group (n = 20) was exposed to 36-38 °C for 8 weeks. All parameters of egg quality including egg weight, surface area, volume, and eggshell weight, thickness, ash weight, and calcium content were decreased in the heat stress group compared to the control group (by 26.9%, 32.7%, 44.1%, 38.4%, 31.7%, 39.4%, and 11.1%, respectively). Total plasma calcium was decreased by 13.4%. Levels of ITPR3, SLC4A4, and SLC4A9 transcripts in the uterine lining were decreased in the heat stress group compared to the control group (by 61.4%, 66.1%, and 66.1%, respectively). CALB1 transcript level was increased (by 34.2 fold) in the heat stress group of hens compared to controls. TRPV6, SCNN1G, KCNJ15, and CLCN2 transcript levels did not significantly differ between control and heat stress groups of laying hens. It is concluded that the down-expression of ITPR3, SLC4A4, and SLC4A9 genes may impair transportation of Cl-, HCO3-, and Na+ in eggshell mineralization during heat stress. Increased CALB1 gene expression may increase resistance of uterine cells to detrimental effects of heat stress.

Epithelial Patterning, Morphogenesis, and Evolution: Drosophila Eggshell as a Model.

Understanding the mechanisms driving tissue and organ formation requires knowledge across scales. How do signaling pathways specify distinct tissue types? How does the patterning system control morphogenesis? How do these processes evolve? The Drosophila egg chamber, where EGF and BMP signaling intersect to specify unique cell types that construct epithelial tubes for specialized eggshell structures, has provided a tractable system to ask these questions. Work there has elucidated connections between scales of development, including across evolutionary scales, and fostered the development of quantitative modeling tools. These tools and general principles can be applied to the understanding of other developmental processes across organisms.

Pathogeny of Fatigued Walking Condition in Pekin Ducks.

Fatigued walking condition (FWC) in ducks is an important welfare and processing issue during the loading/unloading to the processing plant that can be related to heart and bone development. An experiment was conducted to evaluate the effects of incubation conditions on duck embryo bone and heart development and their subsequent effects on ducks showing FWC at market age. Four groups of 2500 Pekin duck eggs were subjected to combinations of two incubation temperature profiles (elevated [E] and normal [N]) and two eggshell conductance profiles (G) (reduced [GR] and normal [GN]). At hatch 10 ducklings from each treatment combination were sampled for heart, liver, residual yolk, and total body weight as well as relative weights (organ percentage of whole body weight). Femur, tibia, and tarsus length and weight were also obtained, and relative asymmetry (RA) was calculated for each leg section. At 35 days of age during unloading of the truck at the slaughter plant, five hens and five drakes demonstrating normal walking and FWC were sampled. Body, heart, and ventricular weights were obtained along with femur, tibia and tarsus length, weight, and RA. Bone strength was evaluated using a three-point bending test, and tibia ash content was assessed. At hatch duckling bone characteristics and organ weights were found to be primarily affected by GR conditions, while heart development in older ducks was mainly impacted by E incubation temperatures. Tibia and relative weight at 35 days were also increased by GR and E. Fatigued ducks presented heavier tibias with more RA and cortical thickness but lower ash percentage. In conclusion, the changes in bone development during incubation and posthatch life were related to duck FWC presence during transportation to the processing plant.

Temperature during the last week of incubation. I. Effects on hatching pattern and broiler chicken embryonic organ development.

We investigated the effects of an eggshell temperature (EST) of 35.6, 36.7, 37.8, and 38.9°C applied from d of incubation (E) 15, E17, and E19 on hatching pattern and embryonic organ development. A total of 2,850 first-grade eggs of a 43-week-old Ross 308 broiler breeder flock were incubated at an EST of 37.8°C until E15. From E15, E17, or E19 onward, eggs were incubated at an EST of 35.6, 36.7, 37.8, or 38.9°C. Moment of internal pipping (IP), external pipping (EP), and hatch was determined, and organ development was measured at E15, E17, E19, IP, EP, and hatch. A lower EST extended incubation duration compared to a higher EST. The lower incubation duration was mainly caused by the extended time until IP, whereas time between IP and hatch hardly varied between treatments. Relative heart weight was affected by EST already from 2 d after the start of EST treatment on E15, and effects became more pronounced at longer exposure time to various EST treatments. At hatch, the largest difference in relative heart weight was found between an EST of 35.6 and 38.9°C started at E15 (Δ=64.4%). From E17 onward, EST affected yolk-free body mass (YFBM) and relative stomach weight, where a lower EST resulted in a lower YFBM and relative stomach weight before IP and a higher YFBM and relative stomach weight after IP. From E19 onward, a lower EST resulted in a higher relative liver and spleen weight regardless of start time of treatment. Yolk weight and relative intestine weight were not affected by EST before and at E19, but a higher EST resulted in a higher yolk weight and lower relative intestine weight from IP onward. Based on the higher YFBM and higher relative organ growth found at hatch, we concluded that an EST lower than 37.8°C from E15 onward appears to be beneficial for optimal embryo development.

Temperature and CO2 during the hatching phase. I. Effects on chick quality and organ development.

The objective of this study was to investigate the effect of eggshell temperature (EST) and carbon dioxide (CO2) concentration during only the hatching phase on embryonic development and chick quality. Three batches of eggs were incubated at an EST of 37.8°C until d of incubation (E) 19. From E19, embryos were incubated at low (36.7°C), normal (37.8°C), or high (38.9°C) EST and at low (0.2%) or high (1%) CO2 concentration. Organ growth and embryo and chick quality were measured at E19, internal pipping (IP), hatch, and 12 h after hatch. A few interactions between EST and CO2 were found at IP, hatch, and 12 h after hatch, but all of these interactions were temporary and in most cases weak. High EST resulted in a lower relative heart weight compared with low ( = 0.05) and normal EST ( = 0.06) at IP, compared with low ( = 0.11) and normal EST ( = 0.08) at hatch, and compared with low ( = 0.11) and normal EST ( = 0.08) at 12 h after hatch. At hatch, high EST resulted in a lower YFBM compared with low EST ( = 0.65). At 12 h after hatch, high EST resulted in a lower relative liver weight compared with low EST ( = 0.12). At low EST, greater relative intestinal weight was found compared with normal ( = 0.41) and high EST ( = 0.37). The effect of CO2 solely was found at 12 h after hatch at which a higher relative heart weight ( = 0.05) and a higher relative lung weight ( = 0.0542) was found at high CO2 compared with low CO2. High EST during only the hatching phase negatively affected chick development, mainly expressed by the lower relative heart weight at IP, hatch, and 12 h after hatch and lower YFBM at hatch. The resolving effect of CO2 demonstrates that CO2 only seem to have a temporary effect during the hatching phase.

Temperature and CO2 during the hatching phase. II. Effects on chicken embryo physiology.

The objective of this study was to investigate the effect of eggshell temperature (EST) and carbon dioxide concentration during only the hatching phase on physiological characteristics of embryos and chicks. Three groups of eggs were incubated at an EST of 37.8°C until d 19 of incubation (E19). From E19, embryos were incubated at a low (36.7°C), normal (37.8°C), or high (38.9°C) EST and at a low (0.2%) or high (1.0%) CO2 concentration. For E19, internal pipping (IP), hatch, and 12 h after hatch, blood parameters were analyzed and hepatic glycogen was determined. At IP, hatch, and 12 h after hatch, interactions were found between EST and CO2, but all these interactions were temporary and in most cases weak. High EST resulted in a lower hepatic glycogen concentration compared with low ( = 21.1) and normal EST ( = 14.43) at IP, and a lower hepatic glycogen concentration compared with low EST ( = 6.24) at hatch. At hatch, high EST resulted in lower hematocrit value ( = 2.4) and higher potassium ( = 0.5) compared with low EST. At 12 h after hatch, high EST resulted in a higher lactate concentration compared with low ( = 0.77) and normal EST ( = 0.65). And high EST resulted in higher potassium compared with low ( = 0.4) and normal EST ( = 0.3). An effect of CO2 solely was only found at IP, at which high CO2 resulted in a lower pH ( = 0.03) and a lower hepatic glycogen concentration ( = 7.27) compared with low CO2. High EST during only the hatching phase affected embryo and chick physiology, indicated by the lower hepatic glycogen levels at IP and hatch. High CO2 affected pH and hepatic glycogen at IP. Effects of CO2 were only found at low EST, which emphasizes the large effect of EST during the hatching phase.

The effect of developmental stage on eggshell thickness variation in endangered falcons.

We compared eggshell thickness of hatched eggs with that of non-developed eggs in endangered falcon taxa to explore the effect of embryo development on eggshell thinning. To our knowledge, this has never been examined before in falcons, despite the fact that eggshell thinning due to pollutants and environmental contamination is often considered the most common cause of egg failure in falcons. Because of the endangered nature of these birds, and the difficulty in gaining access to the nests and their eggs, there is a large gap in our knowledge regarding eggshell thickness variation and the factors affecting it. We used a linear mixed-effects (LME) model to explore the variation in eggshell thickness (n=335 eggs) in relation to the developmental stage of the eggs, but also in relation to the falcon taxa, the laying sequence and the study zone. Female identity (n=69) and clutch identity (n=98) were also included in the LME model. Our results are consistent with the prediction that eggshell thickness decreases during incubation because of the important effect of calcium uptake by the embryo during development. Our results also show that eggs laid later in the sequence had significantly thinner eggshells. In this study, we provide the first quantitative data on eggshell thickness variation of hatched eggs in different falcon taxa that were not subjected to contamination or food limitation (i.e., bred under captive conditions). Because eggshell thickness strongly influences survival and because the species examined in this study are endangered, our data represent a valuable control for future studies on the effects of pollution on eggshells from wild populations and thus are an important contribution to the conservation of falcons.

The eggshell is required for meiotic fidelity, polar-body extrusion and polarization of the C. elegans embryo.

BACKGROUND: Fertilization restores the diploid state and begins the process by which the single-cell oocyte is converted into a polarized, multicellular organism. In the nematode, Caenorhabditis elegans, two of the earliest events following fertilization are secretion of the chitinous eggshell and completion of meiosis, and in this report we demonstrate that the eggshell is essential for multiple developmental events at the one-cell stage. RESULTS: We show that the GLD (Germline differentiation abnormal)-1-regulated hexosamine pathway enzyme, glucosamine-6-phosphate N-acetyltransferase (GNA)-2, is required for synthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the substrate for eggshell chitin synthesis by chitin synthase-1 (CHS-1). Furthermore, while chs-1(RNAi) or combined RNAi with the chitin-binding proteins, CEJ-1 and B0280.5, does not interfere with normal meiotic timing, lagging chromosomes are observed at meiosis, and polar-body extrusion fails. We also demonstrate that chitin, and either CEJ-1 or B0280.5, are essential for the osmotic/permeability barrier and for movement of the sperm pronucleus/centrosome complex to the cortex, which is associated with the initiation of polarization. CONCLUSION: Our results indicate that the eggshell is required in single-cell C. elegans development, playing an essential role in multiple actin-dependent early events. Furthermore, the earliest meiotic roles precede osmotic barrier formation, indicating that the role of the eggshell is not limited to generation of the osmotic barrier.

Minor proteins and enzymes of the Drosophila eggshell matrix.

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.

Histology and ultrastructure of the chorioallantoic membrane of the mallard duck (Anas platyrhynchos).

The histology and fine structure of the chorioallantoic membrane of the mallard duck (Anas platyrhynchos), and the density of vessels per millimeter of membrane were assessed between days 12 and 24 of incubation. Light and transmission electron microscopy of the chorioallantoic membrane of the mallard duck after various days of incubation was carried out. Blood vessels within the mesoderm were counted per millimeter of membrane by light microscopy (40x). The chorioallantoic membrane had three distinct layers from day 12 to 24 of incubation, the chorionic epithelium, the mesoderm, and the allantoic epithelium. After day 12, chorionic epithelium consisted of two layers of flattened, elongated epithelial cells interfaced by numerous desmosomes, and separated from the underlying mesoderm by a basement membrane. At this stage, the allantoic epithelium consisted of a single layer of flattened, overlapping cells. Blood capillaries were observed in the mesoderm close to the chorionic epithelium on days 12 and 13; by day 14, these capillaries were located within the chorionic epithelium, forming a capillary sinus. Between days 14 and 16, the chorion underwent cellular and cytological differentiation into three cell types: capillary covering cells, villus cavity cells, and less differentiated basal cells. The mesoderm was composed of a loose matrix of mesenchymal cells and collagen fibrils through which coursed blood and lymphatic vessels. The vascular density in the mesoderm increased rapidly from 4.2+/-0.6 vessels per mm (n = 12) on day 12 to a maximum of 9.4+/-0.3 vessels per mm (n = 15) by day 16. From day 16, the allantoic epithelium had two to three layers of elongated and overlapping cells. The luminal layer of allantoic epithelial cells had microvillus projections and varying numbers of membrane-bound dense vesicles at all stages from day 12 onward. The histologic and ultrastructural features of mallard duck chorioallantoic membrane from day 12 to 24 of incubation were very similar to those described in the chorioallantoic membrane of the chicken (Gallus gallus) from day 8 to 20 of incubation. Much of the information available concerning the CAM of the chicken also may apply to the CAM of the mallard, with timing adjusted to match the developmental time-frame recorded here.

Osteogenic protein-1, a bone morphogenetic protein, induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor-beta1.

Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-betas themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF-beta superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein-1 (OP-1; BMP-7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP-1 at both ortho- and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF-beta1. The single application of relatively high doses of TGF-beta1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP-1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP-1) dose-dependent angiogenic response. The binary application of a relatively low dose of OP-1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF-beta1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis.

Ontogeny of the extraembryonic membranes of the oviparous lizard, Eumeces fasciatus (Squamata: scincidae).

Oviposited eggs of Eumeces fasciatus contain embryos in the limb bud stage. Amniogenesis is complete and two yolk sac membranes, vascular trilaminar omphalopleure (choriovitelline membrane) and bilaminar omphalopleure, enclose the yolk vesicle. A small allantoic vesicle contacts the chorion. The choriovitelline membrane is the primary vascular system. Blood islands, sites of hematopoiesis, are associated with omphalomesenteric vessels of the choriovitelline membrane. The bilaminar omphalopleure, which contacts the eggshell over the abembryonic hemisphere of the egg, lies external to an isolated yolk mass and yolk cleft and is not vascularized. The definitive yolk sac (splanchnopleure) is formed when the extraembryonic coelom and allantoic vesicle intrude into the choriovitelline membrane. Omphalomesenteric vessels are retained with the yolk sac splanchnopleure and the associated hematopoietic sites are present throughout incubation. The chorioallantoic membrane reaches the equator of the egg, entirely supplanting the choriovitelline membrane, after 25% of incubation is completed. Further growth of the allantois is stalled until 65% of incubation is completed when rapid expansion of the allantoic vesicle, in conjunction with resorption of the isolated yolk mass, supplants the bilaminar omphalopleure. As a result, the chorioallantoic membrane completely envelopes the egg for the final 35% of incubation. This developmental event is coincident with published reports for the timing of increased growth and metabolism of embryos. As the isolated yolk mass regresses, intravitelline cells associated with the yolk cleft invade and resorb the yolk to form a large cavity. The wall of this cavity is a germinal epithelium that produces cells that fill the cavity. This structure appears to be a site of hematopoiesis previously undescribed in vertebrates.

Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases.

The homeless (hls) gene of Drosophila is required for anteroposterior and dorsoventral axis formation during oogenesis. At a low frequency, females homozygous for mutations in hls generate early egg chambers in which the oocyte is positioned incorrectly within the cyst. At a high frequency, late-stage egg chambers exhibit a ventralized chorion. Sequence analysis of the hls cDNA predicts a protein with amino-terminal homology to members of the DE-H family of RNA-dependent ATPases and putative helicases. Similarity of 51% in the amino-terminal third of the protein was found to two yeast splicing factors, PRP2 and PRP16, and to Drosophila Maleless, which is required for dosage compensation. To analyze Hls function, RNA localization patterns were determined for seven different transcripts in hls mutant ovaries. Previtellogenic transport to the oocyte was unaffected for all transcripts examined. Transport and localization of bicoid and oskar messages during vitellogenic stages were strongly disrupted, and the distribution and/or quantity of gurken, orb, and fs(1)K10 mRNAs were also affected, but to a lesser degree. In contrast, hu-li tai shao and Bicaudal-D transcripts were transported and localized normally in hls mutants. In addition, Kinesin heavy chain:beta-Galactosidase fusion protein failed to localize correctly to the posterior of the oocyte in vitellogenic egg chambers. Examination of the microtubule structure with anti-alpha-Tubulin antibodies revealed aberrant microtubule organizing center movement and an abnormally dense cytoplasmic microtubule meshwork. We discuss potential roles for Hls in organizing a cytoskeletal framework essential for localizing specific RNAs.