Yolk Layer Research Articles

Physical Quality of Native Chicken Eggs in Laying Phase and Fed with Different Shrimp Flours

This study aimed to determine the physical quality of native chicken eggs fed with diets containing different shrimp waste flour. The sample used was 16 layer native chickens. Chickens were reared out intensively and eggs and eggs were collected every afternoon. Feeding was done twice a day, 7:00 in the morning and 14:00 in the afternoon, while drinking water was given ad libitum. The design of this study uses a completely randomized design (CRD). The shrimp waste flour treatment in native chicken feed is P0 (feed without shrimp waste flour), P1 (feed + 3% shrimp waste flour), P2 (feed + 6% shrimp waste flour) and P3 (feed + 9% flour waste shrimp). The parameters observed in this study were egg weight, egg width, egg length, yolk color, yolk width, albumen width, yolk height, albumen height, and eggshell thickness. The results showed thad the addition of shrimp waste flour in the ration to a level of 9% had no significant effect (p> 0.05) on egg weight, egg width, egg length, yolk width, albumen width, yolk height, and albumen height, but had a significant effect (p < 0.05) to the color of the yolk layer of layered chicken eggs.

Composition control in biphasic silicate microspheres on stimulating new bone regeneration and repair of osteoporotic femoral bone defect.

Application of bioactive materials as synthetic bone graft substitutes in regenerative medicine has seen great evolution over the past decades in treating challengeable bone defects. However, balancing the preparation conditions and biological performances of inorganic biomaterials remain a great challenge, especially when there is lack of biomaterial design on how to control component distribution and how pathological bone responds to the biomaterial stimulations and osteogenesis. Here, our objective is to develop yolk-shell Ca-silicate microspheres and to investigate the potential biological performances to overcome the limitations in repair of osteoporotic bone defects. The introduction of β-calcium silicate (CaSiO3 ) or mesoporous bioactive glass (MBG) into self-curing β-dicalcium silicate (Ca2 SiO4 ) cement shell to form spherical granules (CaSiO3 @Ca2 SiO4 , MBG@Ca2 SiO4 ) was to retain the physicochemical property and/or microstructure of each component for optimizing bioactive ion release that could maximize osteostimulation in osteoporosis. We report a scalable shape-controlled mild fabrication protocol to yield the yolk-shell granules, endowing to different phases in yolk layer and interconnected macropore networks in the closely packed granule scaffolds. This unique heterostructure preparation is governed by coaxially aligned bilayer nozzle, inorganic powders and biocompatible binders. Extensive in vitro and in vivo evaluation showed that the CaSiO3 @Ca2 SiO4 and MBG@Ca2 SiO4 granules exhibited many superior properties such as controllable ion release, improved biodegradation and enhanced osteogenic capability in comparison with the pure Ca2 SiO4 @Ca2 SiO4 , thereby opening new mild-condition approach in fabricating osteogenesis-tailored silicate biomaterials for bone regenerative medicine, especially for efficient reconstruction of challenging pathological bone defects.

Yolk-porous shell biphasic bioceramic granules enhancing bone regeneration and repair beyond homogenous hybrid

Bioactive stimulation and spatiotemporal evolution of porous scaffolds with time are crucial for bone regeneration rate in bone repair process. Granule-type bioceramic scaffolds have attracted significant interest in biomedical applications in recent years. However, the major limitation of such porous architecture is that the low initial porosity is disadvantageous for enhancing new bone tissue ingrowth. Here we reported that the yolk-shell-structured biphasic bioceramic granules with adjustable shell microstructures were favorable for controllable ion release in vitro, superior to the granules with the conventional homogenous hybrid structures. Also, we illustrated a significant difference in biodegradation of the granules in vivo, and especially the porous-shell granules exhibited appreciable new bone tissue ingrowth with time. The underlying fundamental mechanisms governing the new bone tissue ingrowth behavior of the yolk-shell granule scaffolds were elucidated based on microCT analyses and histological observation. It was underscored that during biodegradation in vivo, the highly bioactive ions in yolk layer were continuously released due to the porous structures of the sparingly dissolvable shell layer, thereby resulting in hollow shell and rapid new bone tissue ingrowth. Hence, these results demonstrate that the slight tailoring in microstructure and component distribution of biphasic composites is beneficial for adjusting the bone regeneration, and may help us to precisely control bone repair efficiency for a variety of clinical conditions.

Laboratory methods to predict the cleaning behaviour of egg yolk layers in a flow channel

Cleaning-in-place (CIP) is an established technique for the cleaning of industrial food processing plants. To design CIP protocols, laboratory methods are helpful to predict the cleaning behaviour and to reduce the number of costly in situ cleaning investigations. In this work, laboratory methods (differential scanning calorimetry, rheology, optical swelling measurements and micromanipulation) were used to analyse different aspects and stages of the cleaning process (swelling, chemical and mechanical actions, phase transition and mass transfer). The suitability of these methods to predict the cleaning behaviour was tested by using a representative egg yolk deposit. Temperature (24–60°C) and NaOH concentration (0–1.5%) of the cleaning fluid served as independent factors, and the time necessary to remove the deposit in a flow channel was evaluated using an optical method. Cleaning time decreased with increasing temperature and also depended on NaOH concentration. Within the examined limits, the pushing energy measured by the micromanipulation method showed the best correlation with cleaning time, whereas swelling, diffusion and rheological measurements only reflected tendencies of the cleaning behaviour.

Distribution and concentration of maternal progesterone in the yolk of Greater Rhea eggs (Rhea americana).

Progesterone is the most concentrated maternal yolk steroid characterized to date in birds; however, no information about it is available in ratite eggs. We collected freshly laid eggs from zoo-housed Greater Rhea females (Rhea americana) bred under similar rearing conditions during two breeding seasons to characterize concentration and distribution of maternal yolk progesterone. After high-performance liquid chromatography analysis, yolk hormone was measured using a commercial electrochemiluminescence immunoassay. Progesterone concentrations were found to vary significantly among the yolk layers, supporting a follicular origin for this steroid in Greater Rhea eggs. Additionally, highly similar mean absolute yolk progesterone concentrations were detected between 2013 and 2015 breeding seasons (1,332.98 ± 82.59 and 1,313.59 ± 85.19 ng/g, respectively). These values are also comparable to those found in some domestic carinate species. Findings suggest that at population level, when rearing conditions are similar, mean absolute yolk maternal progesterone concentrations also appear bounded. Future research on the factors and mechanisms that regulate progesterone deposition in Greater Rhea eggs is needed to better understand whether its levels depend on different rearing conditions.

Swelling and hydration studies on egg yolk samples via scanning fluid dynamic gauge and gravimetric tests

Hydration and swelling in initially dry protein-based samples represent the first stage in their cleaning from hard surfaces. These phenomena have been studied in technical egg yolk stains via scanning Fluid Dynamic Gauge (sFDG) and gravimetric tests. Temperature (30 °C to 55 °C) and pH (9.5 to 11.5) were investigated as factors influencing the process. The kinetics did not appear to be significantly different as 95% of the equilibrium swelling was reached at approximately 90 min in all tests. No removal of the egg yolk layer was observed in most cases, except at high alkaline conditions (pH 11.5), where a lift-up followed by a partial removal of the protein network was seen when an external shear stress was applied. The process mimicked creep behaviour of plastic materials. Gravimetric data on the hydration of the sample suggested a Fickian diffusion transport (Case I), where solvent diffusion is the rate limiting stage. The initial hydration was proved to be linear. Two diffusion theories of increasing complexity were applied to estimate effective diffusion coefficients: Fick's second law (with moving boundaries) and a non-linear poroelasticity theory. The temperature dependence of different diffusion coefficients assuming an Arrhenius equation gave an activation energy in the range of 16.4 (±6.7) KJ/mol to 18.4 (±9.0) KJ/mol.

Maternal corticosterone is transferred into the egg yolk

Hormones deposited in the avian egg are considered in many studies to influence or to adjust offspring phenotype to prevailing conditions in an adaptive way. Several studies demonstrated an effect of corticosterone, the main glucocorticoid in birds, injected into the egg on the developing chick, but the injection of steroids in the egg is far from mimicking the natural distribution of the hormone in the egg. Other studies applied a stressor or corticosterone to the mother. However it is still debated whether an increase of circulating corticosterone in the mother translates into higher concentrations of corticosterone in the egg. Therefore, we investigated in captive barn owls Tyto alba whether circulating corticosterone in egg-laying females elevated within a physiological range, resulted in the deposition of corticosterone in eggs. We found that an increase in circulating corticosterone in the mother within the naturally occurring range translated into elevated concentrations of corticosterone in the yolk of subsequently laid eggs, indicating a specific time frame and yolk layer of corticosterone deposition. We conclude that increasing maternal plasma corticosterone within a naturally occurring range is an efficient tool to increase corticosterone concentration in the egg and to manipulate conditions for the developing embryo.

Change in Body Mass Can Overrule the Effects of Maternal Testosterone on Primary Offspring Sex Ratio of First Eggs in Homing Pigeons

The phenomenon of primary offspring sex ratio adjustment is being extensively studied, yet knowledge of the underlying proximate mechanism is still mainly hypothetical. Female birds are the heterogametic sex, thus potentially controlling the sex of the gamete to be fertilized. In several bird species, independent studies showed effects of maternal plasma testosterone, corticosterone, or condition on primary offspring sex ratio. Our objective was to investigate the causal relation between these two maternal hormones, body condition, and offspring sex ratio in homing pigeons (Columba livia domestica). Following our earlier study, we again implanted females with testosterone and determined embryo sex of first eggs. To identify the pathway of sex ratio adjustment, we repeatedly measured not only maternal plasma testosterone and mass but also plasma corticosterone, cholesterol, and glucose, all indicators of body condition. We also calculated the temporal change in these parameters, which has been proposed to be a more accurate predictor of offspring sex ratio compared with the absolute values. Furthermore, we analyzed testosterone concentrations in outer yolk layers, which potentially influence the first meiotic division of the gamete. We found no relation between plasma parameter and embryo sex of first eggs; testosterone treatment did not affect any of the measured parameters. However, females that increased in mass produced more male embryos, irrespective of treatment group. Outer yolk layer testosterone concentrations did not differ between treatment groups or between male and female eggs. We propose that not only the absolute values but also the complex interactions between maternal hormones and body condition ultimately affect the mechanism of primary offspring sex manipulation.

The embryonic development of the flatworm Macrostomum sp.

Macrostomid flatworms represent a group of basal bilaterians with primitive developmental and morphological characteristics. The species Macrostomum sp., raised under laboratory conditions, has a short generation time of about 2-3 weeks and produces a large number of eggs year round. Using live observation, histology, electron microscopy and immunohistochemistry we have carried out a developmental analysis of Macrostomum sp. Cleavage (stages 1-2) of this species follows a modified spiral pattern and results in a solid embryonic primordium surrounded by an external yolk layer. During stage 3, cells at the anterior and lateral periphery of the embryo evolve into the somatic primordium which gives rise to the body wall and nervous system. Cells in the center form the large yolk-rich gut primordium. During stage 4, the brain primordium and the pharynx primordium appear as symmetric densities anterior-ventrally within the somatic primordium. Organ differentiation commences during stage 5 when the neurons of the brain primordium extend axons that form a central neuropile, and the outer cell layer of the somatic primordium turns into a ciliated epidermal epithelium. Cilia also appear in the lumen of the pharynx primordium, in the protonephridial system and, slightly later, in the lumen of the gut. Ultrastructurally, these differentiating cells show the hallmarks of platyhelminth epithelia, with a pronounced apical assembly of microfilaments (terminal web) inserting at the zonula adherens, and a wide band of septate junctions underneath the zonula. Terminal web and zonula adherens are particularly well observed in the epidermis. During stage 6, the somatic primordium extends around the surface dorsally and ventrally to form a complete body wall. Muscle precursors extend myofilaments that are organized into a highly regular orthogonal network of circular, diagonal and longitudinal fibers. Neurons of the brain primordium differentiate a commissural neuropile that extends a single pair of ventro-lateral nerve trunks (the main longitudinal cords) posteriorly. The primordial pharynx lumen fuses with the ventral epidermis anteriorly and the gut posteriorly, thereby generating a continuous digestive tract. The embryo adopts its final shape during stages 7 and 8, characterized by the morphallactic lengthening of the body into a U-shaped form and the condensation of the nervous system.

Distribution and origin of steroid hormones in the yolk of Japanese quail eggs (Coturnix coturnix japonica).

The yolk of avian eggs contains steroid hormones, which may influence the development and behaviour of hatched birds. The aim of the present study was to investigate the concentration as well as the distribution of various gonadal steroids in the yolk spheres of quail eggs. Steroid concentrations of dissected yolk layers were analysed after alcoholic extraction using enzyme immunoassays (EIAs) for progesterone, androstenedione and testosterone. To monitor the uptake of testosterone into the yolk, radioactive testosterone was injected i.m. into six female quails. The radioactivity of yolk layers of subsequently laid eggs was measured by liquid scintillation counting. Progesterone concentrations were highest in the outer layer (median: 2265 nmol/kg). Androstenedione (median: 453 nmol/kg), as the major androgen, and testosterone (median: 99 nmol/kg) reached their highest concentrations in interior layers, whereas in the centre the concentration of all three hormones was low. No significant variation of steroid levels in yolk layers of subsequently laid eggs was found. The highest radioactivity was detected in the outer yolk layer in those eggs laid 1 day after injection and in subsequently laid eggs was measured nearer to the centre. These results indicated local origin of the steroid hormones especially because of the result that only 0.1% of the radioactivity entered the yolk. We conclude that steroid concentrations in the yolk layers reflected progesterone and androgen production of the cells of the follicular wall at the time.

Effect of gravity on the interaction between the avian germ and neighbouring ooplasm in inverted egg yolk balls.

The developmental capacities of an avian germ (from before symmetrization to the moment of laying) are strongly diminished after inversion of its egg yolk ball followed by culture in egg white. Our present experiments show that even when the avian germ is completely horizontally inverted (without an upper or lower border) below its egg yolk ball before symmetrization, symmetrization and gastrulation phenomena take place. The germ grows slower and becomes smaller than after normal incubation. After culture of inverted unincubated germs, localized on freshly laid eggs, the closure of the neural tube is impaired and it remains open over a long distance. Although a primitive streak (PS) develops, mesoderm migration (mainly from the lateral part of the area pellucida) is also impaired. On sections through the germinal disc one can see the abnormal upward migration into the depth of the ooplasm and yolk of cells from the germ wall and the development of large cellular extensions encircling the yolk globules. Most prominent is the loss of contact between the superficial cell layers and the deep layer elements (junctional endoblast and yolk endoblast in the area opaca). Large areas without deep layer elements (even visible on surface micrographs) develop in the area vasculosa and area vitellina interna. The margin of overgrowth grows and extends normally over the egg yolk ball. An autoradiographic study after labelling of the yolk layers in inverted egg yolks reveals that mainly compression of the peripheral subgerminal and perigerminal ooplasm takes place. This suggests that the compression by the neighbouring yolk and upwards growth of cells are at the origin of the impaired development. After return to the normal upward orientation of the germ on the topmost part of the egg yolk ball, a more or less pronounced restoration to normal development takes place (depending on the duration of the inversion period and the age of the germ).

Concentrations of Steroid Hormones in Layers and Biopsies of Chelonian Egg Yolks

The actions of circulating hormones, although relatively well understood for adults, are largely unknown for their developing embryos. Transfer of maternal hormones to the egg is known to occur in oviparous species, and recently the presence of hormonally heterogeneous yolk layers has been described in two avian species. To investigate the possibility of a similar phenomenon occurring in chelonian species, egg yolk layers were analyzed in the painted turtle (Chrysemys picta marginata) and the red-eared slider turtle (Trachemys scripta elegans), two species that exhibit temperature-dependent sex determination. There was a similar pattern of hormonally heterogeneous yolk layers in both species: concentrations of progesterone and testosterone were significantly higher in the external yolk layer while concentrations of 17β-estradiol were significantly higher in the intermediate and internal layers. This pattern of hormone deposition concurs with previously published studies of plasma hormone profiles from females of temperate-zone turtle species. Yolks of freshly laid eggs were also sampled using a biopsy technique to examine the concordance of early yolk hormone concentrations and offspring sex. No relationship was found between yolk hormone concentrations and individual offspring sex. Previous work showing that maternally derived yolk estradiol concentrations are correlated with female-biased sex ratios was, however, replicated. These findings suggest that offspring sex is influenced, in part, by the maternal hormone environment.

Egg Yolk Layers Vary in the Concentration of Steroid Hormones in Two Avian Species

Maternally derived steroid hormones are known to be present in the yolks of avian eggs; however, the physiological mechanisms involved in their deposition remain largely unexplored. Investigations of steroid production by avian follicles have demonstrated temporal differences in the concentrations of progesterone, 17β-estradiol, and testosterone during yolk formation. Because yolk is deposited peripherally in concentric spheres as the oocyte develops, differences in the production of follicular hormones during yolk formation should be manifested in differences in the localization of steroids within layers of the yolk. To investigate this hypothesis we analyzed steroid hormone concentrations in layers of individual eggs of the dark-eyed junco (Junco hyemalis) and the red-winged blackbird (Agelaius phoeniceus). We found that in the dark-eyed junco the concentration of progesterone is significantly greater at the periphery of the yolk, while the concentration of 17β-estradiol is significantly greater near the center of the yolk. We also found in both the dark-eyed junco and the red-winged blackbird that the concentration of testosterone remains constant from the interior to the intermediate layers of the yolk and then drops sharply between the intermediate and exterior layers. The patterns of hormone localization that we found agree with those predicted by studies of temporal changes in steroidogenesis in the maturing follicle of the chicken, thus suggesting that within-yolk variation in yolk steroid concentrations in the dark-eyed junco and the red-winged blackbird reflects temporal differences in the pattern of follicular steroidogenesis. Variation in the concentration of hormones among yolk layers presents a methodological concern for studies that involve the removal of yolk samples from viable eggs for subsequent hormonal analysis. This variation also has implications for the timing of embryonic exposure to steroid hormones.

The in vitro development from egg to prepupa of Campoletis sonorensis (Hymenoptera: Ichneumonidae) in an artificial medium: importance of physical factors

A further attempt to improve the in vitro rearing of the solitary larval endoparasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae) was made after third instars were obtained in previous studies. Respiration was an important factor for the parasitoid's continued development: both small diet volumes that exposed the larval cuticle to air and O 2 greatly improved growth and development of C. sonorensis. Fifth instars were obtained when the parasitoid larvae were reared in medium volumes of 0.25 ml and 0.1 ml per well. Increasing the O 2 tension of the air resulted in larger larvae and prepupae. The agar layer under the nutritional medium was designed to slowly remove water from the diet. The results indicated that a drier environment was important for the parasitoid at the terminal stage. One percent and 0.8% agar gels, which removed water from the diet more rapidly, provided a drier environment and resulted in improved larval growth compared to the 0.6% agar gel. The detrimental effect of higher concentration of chicken egg yolk mixed in the diet was eliminated by using a dry egg yolk layer covered by an agar layer. This sandwich technique resulted in more parasitoid larvae molting to the final larval instar (5th) and the formation of many prepupae. However, the development of prepupae was premature and none of the fifth instar larvae successfully formed a normal cocoon.

Establishment and movement of egg regions revealed by the size class of yolk platelets in Xenopus laevis.

Sizes of yolk platelets were measured in sections of oocytes and embryos in Xenopus. It was found that the average size of the largest group of platelets in cells differed between germ layers of neurulae. It was small (3 to 5 μm) in the ectoderm, medium-sized (5 to 8 µm) in the mesoderm, and large (over 8 μm) in the endoderm. Platelets of these size classes formed layers in egg, the yolk gradient, by the end of oocyte maturation. The yolk gradient contained products of the mitochondrial cloud and a part of the germinal vesicle material at certain positions. The layers of small, medium and large platelets in the egg changed their locations to distribute to the ectoderm, mesoderm and endoderm of neurulae, respectively. The yolk layers in the egg thus represented different prospective fates, and a figure describing the locations of these layers could be regarded as a fate map for the one-cell stage. Most of the marginal blastomeres of embryos at cleavage stages consisted of a few parts with different prospective fates. Results were discussed with reference to available fate maps for cleavage stage embryos.

Dual origin of yolk nuclei in the lesser spotted dogfish, Scyliorhinus canicula (Chondrichthyes)

AbstractUsing plastic embedding techniques and semithin sections in order to overcome the difficulties encountered in the sectioning of yolky eggs, we made a histological study of the developing blastodisc and early embryo of the oviparous dogfish Scyliorhinus canicula (L.) (Pisces: Chondrichthyes) from Roscoff (Brittany, France). According to our observations, several conclusions based on classical studies on elasmobranch development have to be revised as follows: (i) Cleavage of the germinative disc into blastomeres lags considerably behind the mitotic activity of diploid nuclei derived from amphimixy. As a result, the number of surface blastomeres does not correspond in any way to the actual number of nuclei, even at the start of segmentation. Thus, segmentation is primarily syncytial. (ii) The first set of yolk nuclei corresponded to the deepest nuclei that remained beneath the segmentation cavity after complete cellularization of the blastodisc. They became polyploid, some of them degenerated, and the others multiplied while remaining close to the extraembryonic endoderm during epiboly. (iii) A second set of yolk nuclei was derived from the embryonic endoderm along its boundary with the extraembryonic endoderm. Endoderm cells escaped into the yolk layer nearby. They then give off free nuclei, which also became very large and finally were indistinguishable from the first set. (iv) No yolk nuclei were derived from supernumerary sperm nuclei, even when the latter were able to divide several times in synchrony. Nuclei from this haploid lineage were always smaller than those in the first set, and they were finally located in the deepest layer of the germinative disc, where they degenerated. It is concluded that this chondrichthyan fish generates its yolk syncytium in a very different way from teleosts, in which all yolk nuclei are derived from the blastodisc. © 1993 Wiley‐Liss, Inc.

Cortical contraction and ooplasmic movement in centrifuged or artificially constricted eggs ofCiona intestinalis.

To discover the force causing bipolar ooplasmic segregation just after fertilization in ascidian eggs (Ciona intestinalis), cortical contraction and cytoplasmic movement were examined by centrifugation and by artificial constricting techniques. In the centrifuged eggs, the surface constriction appeared independently of cytoplasmic stratification. The yolk layer and the sub-centripetal layer moved toward the vegetal pole in the peripheral region. In the eggs which were artificially constricted by partially broken chorion and then fertilized, the inner cytoplasm always flowed from the vegetal sphere into the animal sphere during bipolar segregation. The direction of this cytoplasmic movement was independent of sphere size. This shows that the force causing cytoplasmic movement is supplied by the contraction of the vegetal-side cortex. It is suggested that the contracting cortex pushes the inner cytoplasm toward the animal pole and drags the peripheral cytoplasm toward the vegetal pole.

Abnormal oogenesis and embryogenesis resulting from centrifuging Drosophila melanogaster females

Females of Drosophila melanogaster were centrifuged at 2400 g and 4200 g for 3 h in a number of different orientations. The oocytes in various stages of vitellogenesis become separated into three layers; a centrifugal yolk layer, a central cytoplasmic layer, and a centripetal lipid layer. The direction of layering is related to the orientation of the female. The process of recovery of the ovaries was followed and the development of the eggs laid analysed. Many of the eggs laid over the subsequent 3 days die very early in development and fail to produce any differentiated structures. Some hatch into normal larvae and others produce defective embryos. The most common defect being a misarrangement of the segmentation. The differences in the kinds of experimentally inducible pattern aberrations in Drosophila and other diptera is discussed.

On the Structure of a Hen's Egg Yolk and Yolk Spheres

1. Yolk of a egg was fixed with 10% acrolein-0.05M phoshate buffer (pH 7.4) of LUFT11) by keeping in a cold dark place for 5-7 days and frazen sections of 3-7μ thickness, usually of 5μ, were prepared. Some of the sections were stained for lipid with Sudan IV or Sudan Black B-55% ethanol solution, 1% Nile Blue A solution, or BAKER'S acid hematein. Other sections were post-fixed with the HELLY solution and stained with Azan or extracted with pyridine and submitted to the SCHIFF reaction (acrolein-SCHIFF reaction).Experimental hens were given diet mixed with Sudan IV or Sudan Black B, or given intravenous injection of dye colloid solution, and the time the yolk spheres were formed was presumed from the striations of the dye formed inside the yolk.The shape of striped patteren of the dye in the yolk seemed to suggest that there might be a kind of potential flow in the growing yolk and a test was made with an electrical model. For the circuit, a KOHLRAUSCH bridge used in hydrodynamics was employed. A brass ring was provided with insulators corresponding to the blastodisc and a highresistance part corresponding to the periphery of the blastodisc, and 10% sodium chloride solution was filled inside the brass frame. An electrode was placed in the center of the ring, and a weak alternating current was passed to such a degree that there would not be any heat generation. Resistance in the high-resistance part was varied to obtain isopotential curves.2. White yolk spheres are distributed in latebra, neck of latebra, nucleus of Pander, and yolk surface layer around the blastodisc, and only yellow yolk spheres were observed in other parts. Yolk spheres in the surface layer of the yolk were medium to small yellow yolk spheres except around the blastodisc. Primordial yolk spheres were distributed from the center of latebra to the part adjacent to the blastodisc, becoming smaller as they approached the blastodisc and forming a vacuole in the inclusion and finally formed a mass of foams.3. White yolk spheres were varied in shape; from a primordial yolk sphere of 3-25μ diameter containing one drop-like inclusion to that of around 40μ containing numerous fine droplet inclusions. The inclusion substance tended to become smaller and more numberous with increase in the diameter of the spheres. On the other hand, yellow yolk spheres were 30-150μ in diameter and contained numerous fine granular inclusions, which were sometimes more coarse.There was no difference in the stainability of the inclusions of these two kinds of yolk spheres, and the staining indicated that the main component was a protein, with a small amount of phospholipid. Stainability of the matrix was also similar in the two spheres and indicated the presence of a large amount of fats besides the protein. Numerous fat drolets showing the characteristic of a neutral fat were observed but they were very fluid and their localization could not be clarified.The greatest difference between these two kinds of yolk spheres lies in the concentration of phospholipid in the matrix. While the matrix of white yolk sphere was almost negative or weakly positive to acid hematein, that of yellow yolk sphere was strongly positive to it.4. Layered structure was invariably seen in the latebra in which primordial yolk spheres and slightly large yolk spheres were arranged alternately. As a whole, the structure resembled the form of yellow yolk spheres nearer the outer layer. The layer of yellow yolk spheres in the outer side of the latebra showed layered changes in the degree of stainability but majority of hen eggs showed approximately uniform structure, irrespective of yellow or white yolk layer.5. The continuous phase between yolk spheres was fairly marked in the site where primordial yolk spheres were present and extremely small in the site with the other yolk spheres. Its staining characteristics indicated the presence of a small amount of protein.

Electron microscope studies on eggs of Mytilus edulis

Normal eggs of the bay mussel, Mytilus edulis, centrifuged eggs, eggs treated with sperm extract, plasmolyzed eggs, and homogenates of whole eggs, were surveyed by electron microscopy. Microvilli increase the egg surface area about fifteenfold. They extend through the vitelline membrane, and delicate fibrils extend from their tips into a jelly coat. Hypertonic sea water detaches the microvilli from the egg and they are retained in the vitelline membrane. Sperm extract causes the vitelline membrane, an intercellular substance, and cortical granules of the cortex to become invisible. The endoplasm of the centrifuged egg stratified into (1) a lipid layer, (2) a clear layer, (3) a mitochondrial layer, and (4) a yolk layer. The constituents of these layers and of the cortex are described. Many of these constituents are recognizable in thin sections of pellets obtained by centrifugation of egg homogenates. The findings are discussed.