The potentialities of transplanted early gastrula nuclei ofDrosophila melanogaster. Production of their imago descendants by germ-line transplantation

Summary Wild-type nuclei, taken out of cells from five regions of early gastrula embryos, were implanted singly into unfertilizedy w sn ³lz^50e eggs ofDrosophila melanogaster. The different types of nuclei initiated development with nearly equal frequencies of about 60%. 2.9% of the 1073 nuclear transfers developed as far as one of the three larval instars, and one reached the pupal stage.All individuals showed stage-specific patterns of defect. Most of these abnormalities were probably due to some inevitable damage caused by the implantation procedure such as disarrangement of the internal egg morphology and loss of peripheral egg substance. The proportions of individuals arrested at different embryonic and larval stages were similar for the five nuclear groups.Fertile imagos, descendants of all five types of donor nuclei, were produced via germ-line mosaics in two ways: (1) Pole cells of nuclear-transplant blastoderm stages were implanted into the pole cell region of host blastoderm eggs. (2) Gonads were taken from nuclear-transplant larvae and implanted into host larvae. In both cases gametes developed from the transplants as could be recognized from the genotypes of their progeny. By means of suitable crosses it was possible to get clones of flies whose large chromosomes were descended from the chromosomes of only one transplanted nucleus, that is, each clone was the descendant of one somatic nucleus. The data presented show that the nuclei remain omnipotent until the early gastrula stage.     

Isolation of 125I-concanavalin A-labeled plasma membrane from unfertilized mouse eggs

A procedure was developed for isolation of plasma membrane (PM) preparations from unfertilized mouse eggs. Zona-free mouse eggs prepared by the method of Boldt and Wolf (Gamete Res 13:213–222, 1986) were labeled with ¹²⁵I-concanavalin A (ConA) prior to sonication and fractionation on iso-osmotic self-generated Percoll density gradients. Experiments using the ConA-specific sugar α-methylmannoside (αMM) indicated that ¹²⁵I-ConA bound specifically to the egg PM. Greater than 95% of ¹²⁵I-ConA binding to zona-free eggs was blocked in the presence of 0.1 M αMM, and incubation of eggs in αMM after ¹²⁵I-ConA labeling caused release of 85–90% of bound label. Fractionation of ¹²⁵I-ConA-labeled eggs by Percoll density gradient centrifugation yielded a single radioactive peak at density = 1.025, corresponding to egg PM material. Prolonged incubation of ¹²⁵I-ConA-labeled eggs or egg sonicates prior to fractionation did not alter the location of the radioactive peak, indicating that ¹²⁵I-ConA did not label other organelles. As a control, human erythrocytes were labeled with ¹²⁵I-ConA and fractionated under identical experimental conditions and yielded a single radioactive peak at density (1.020) comparable to that observed for ¹²⁵I-ConA-labeled eggs. These results indicate that ¹²⁵I-ConA can be used as a specific marker to support PM isolation from small numbers of zona-free mouse eggs.     

The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Exploring the foraging environment of a natural enemy of Callosobruchus maculatus: Spatial egg distribution in stored cowpea

Knowledge of the spatial distribution of stored product insects may reduce the dependency on chemicals for control of these insects. Biological control, for instance, could be improved based on such knowledge. In this paper we describe the three-dimensional spatial oviposition pattern of Callosobruchus maculatus in stored cowpea. Individual C. maculatus females oviposited in clusters of 70±15 (SD) eggs. These clusters were variable in shape. In any cluster 90 to 95% of the eggs fitted into a volume of 19.1±3.5 cm³. The egg density was highest (0.6 eggs bean^–1) at the center of a cluster and decreased towards the periphery. A statistically significant relationship existed between the number of eggs n in a cluster and the cluster volume, V(cm³): V=11.5+0.11n. We also investigated the spatial egg distribution of beetles which emerged from egg clusters such as those produced by individual females. Their oviposition was not confined to one specific area but was scattered throughout the bean mass. A point pattern analysis showed that the density of the `parent' cluster had no effect on the spatial egg pattern. These results give insight into the foraging environment which the egg parasitoid Uscana lariophaga, a promising candidate for biological control of C. maculatus, is facing. We argue that the probability p of encountering at least one other bean with eggs after a parasitization is a function of the number n of beans that are visited: p=1–0.42 (0.37)^(n–1).     

Practical method of estimating fresh mass of Adélie penguin eggs

Fresh egg mass (FEM) is an important measure as it relates to many allometric relationships. Adélie penguin (Pygoscelis adeliae) eggs, as other bird eggs, begin to lose weight as soon as they are laid but their linear dimensions remain constant. The FEM of an Adélie penguin’s egg can be estimated within 1% from the relationship FEM = 0.564 × LW ², where L is the length and W is the width. The constant 0.564 was calculated by weighing the eggs after replacing the air cell with water. The calculated mean egg mass agrees well with that obtained by weighing eggs after replacement of the air cell with water (r ² = 0.996). This method of estimating FEM based on linear measurements is easily applicable in the field.     

Gamete interaction in the white sturgeon Acipenser transmontanus: a morphological and physiological review

Synopsis Sturgeon gametes differ from those of most fish in that the sperm possess acrosomes that undergo exocytosis and filament formation while the eggs possess numerous micropyles. Acipenser transmontanus eggs are encased by multilayered envelopes that consist of outer adhesive jelly coats and three structured layers interior to the jelly. The glycoprotein jelly layer only becomes adhesive upon exposure to freshwater. The layer interior to the jelly, layer 3, is the other carbohydrate-containing component of the egg envelope. This layer consists of a water-insoluble glycoprotein that, upon freshwater exposure, is hydrolyzed by a trypsinlike protease to yield a water-soluble, lower molecular weight carbohydrate-containing component. This component can be identified in the surrounding medium when unfertilized eggs are incubated in freshwater. This egg water component elicits acrosome reactions only in homologous sperm. The A. transmontanus sperm acrosome reaction is a Ca⁺⁺ and/or Mg⁺⁺ dependent event that includes the formation of a 10 μ long fertilization filament. A. transmontanus fertilization can occur at low sperm per egg ratios; however, crossfertilization of A. transmontanus eggs with lake sturgeon, A. fluvescens, sperm results in a very low number of fertilized eggs, even at high sperm per egg ratios. The morphological, physiological, and biochemical phenomenon reviewed in this paper are related to the environment in which they occur. Also, the possible role of the acrosome and the presence of numerous micropyles are discussed.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

Aster formation in sand dollar eggs by micro-injection of calcium buffers and centriolar complexes isolated from starfish sperm

Centriolar complexes isolated from sperm of the starfish, Asterina pectinifera, could initiate irregular cleavage by injecting them into the fertilized eggs of the sand dollar, Scaphechinus mirabilis. Unfertilized sand dollar eggs could be activated when Ca²⁺ concentration in the egg cytoplasm increased by means of microinjecting calcium buffers in which the concentration of free Ca²⁺ was controlled by mixing calcium salt and calcium chelator at various proportions. When the centriolar complexes were injected into Ca²⁺-activated eggs, aster formation was induced in 25 out of the 55 eggs injected with calcium buffers at an intracellular Ca²⁺ level above 2.9 μM. The number of asters detected in one egg was quite variable, ranging from 2 to 20. The pronucleus in injected eggs was seen to migrate towards the induced asters. Eight of those 25 eggs cleaved.     

Developmental potencies of nuclei from cleavage,preblastoderm, and syncytial blastoderm transplanted into unfertilized eggs ofDrosophila melanogaster

Summary Wild-type nuclei from eggs ofDrosophila melanogaster at various developmental stages and from different regions of the egg—cleavage nuclei, pole nuclei from preblastoderm, and lateral nuclei from syncytial blastoderm—were singly implanted into unfertilizedy w sn ³ lz _50e eggs to determine their developmental potencies.All three types of transplanted nuclei were almost equally effective in initiating development of unfertilized eggs. Development was arrested in one of five critieal embryonic stages or in one of the three larval instars. The frequency of individuals reaching a distinct stage was approximately the same for all three types of donor nuclei. The stage-specific pattern of defects was independent of the type of nucleus transplanted.The deviations from normal development were broadly similar to those seen in controls developing from fertilized eggs which had only been punctured or into which cytoplasm had been injected. Many defective embryos also occurred in these control experiments. These and other observations indicate that a large proportion of irregularly developed individuals found after nuclear transfer can be ascribed to loss of egg material, disturbances in the internal organization of the egg during nuclear implantation, and the difficulty the implanted nucleus has in adjusting to the autonomous processes within the egg, such as the formation and migration of cytoplasmic islands.Some of the defective embryos and larvae originating from nuclear transfer were implanted into adult hosts. After culture for 14 days the early embryonic stages had formed several larval structures, and the late embryonic and larval stages had developed all larval organs. The proliferated imaginal primordia of thesein vivo cultured embryos and larvae, as well as the imaginal disks of the third instar larva, were then implanted into larval hosts with which they passed through metamorphosis and differentiated into imaginal structures. All three types of donor nuclei were capable of producing all adult structures derivedin situ from imaginal disks. The phenotype of these structures waswild-type, thus demonstrating their origin from the transplanted nuclei.The problem as to why not all transplanted nuclei initiated development, and why development after nuclear transplantation was arrested at the third larval instar, at the latest, is discussed.This article is dedicated to Professor Friedrich Seidel on the occasion of his 75th birthday.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H₂O₂) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca²⁺/Mg²⁺-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca²⁺/Mg²⁺-supplemented medium. In Ca²⁺/Mg²⁺-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 µm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H₂O₂ (84.40±0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 µm) induced apoptosis and pronounced generation of intracellular H₂O₂ (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H₂O₂ level (81.20±1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H₂O₂ in rat eggs.     

THE EFFECT OF SOLUBLE EGG JELLY ON THE FERTILIZABILITY OF ACID-DEJELLIED SEA URCHIN EGGS*

Acid-dejellied Lytechinus pictus eggs bind few sperm and show decreased fertilizability. Addition of solubilized egg jelly increases sperm binding and fertilizability, presumably by increasing the frequency of the acrosome reaction. However, dejellied Strongylocentrotus purpuratus bind more sperm and show increased fertilizability in the complete absence of soluble egg jelly. Addition of soluble egg jelly greatly decreases fertilizability in S. purpuratus. Such species differences may be the basis for the controversy between Lillie and Tyler on the one hand, who believed that egg jelly increased egg fertilizability; while Loeb and Hagström on the other hand, believed jelly had no effect on, or actually decreased egg fertilizability. ¹²⁵I-labeling of dejellied S. purpuratus egg surfaces and immunofluorescent studies show that egg jelly persists on the surfaces of acid-dejellied eggs. Egg jelly appears to be a non-removable component of the vitelline layer of this species.     

The production of subitaneous and diapause eggs: a reproductive strategy for Acartia bifilosa (Copepods: Calanoida) in Southampton Water, UK

Acartia bifilosa from Southampton Water lays two morphologically distinct types of egg which are described for the first time. Eggs with a smooth surface are considered subitaneous, while eggs covered with thick 'spines' are diapause. During the seasonal occurrence of A.bifilosa in Southampton Water, from November/December to June, subitaneous eggs are laid during the first months of this period. The production of diapause eggs is restricted to a 2 month period before A.bifilosa disappears from the water column. There are significant differences between the response of the eggs produced at seasonal field temperatures (5-12C) and those reported for other Acartia species. In particular, the numbers of eggs female^-1 day^-1, both subitaneous and diapause, are lower and typically <4; and the hatching time of subitaneous eggs is longer, at up to 10 days, at optimum temperatures between 15 and 20°C and optimum salinity >20 PSU. Females acclimated to higher laboratory temperature regimes show higher egg production rates at field salinity. A delayed-hatch subitaneous egg is also reported. Results suggest that fecundity in A.bifilosa from Southampton Water might be limited to some degree by temperature and the responses of the eggs produced appear to offer A.bifilosa a reproductive repertoire to ensure its sustained presence in this seasonally influenced environment.      

Incorporation and fate of specific sperm plasma membrane components following insemination as revealed by ultrastructural immunocytochemistry

Studies have been carried out to 1) further characterize sperm specific plasma membrane polypeptides (33 and 35 kDa) that are recognized by a monoclonal antibody previously described (Longo, 1989) and 2) follow the incorporation and dispersal of these proteins within plasmalemmae of monospermic and polyspermic sea urchin (Arbacia punctuluta) eggs and oocytes utilizing immunocytochemical methods at the ultrastructural level of observation. Only sperm labeled when incubated with monoclonal antibody to the 33 and 35 kDa proteins followed by colloidal gold-tagged second antibody. Colloidal gold label was observed on the egg plasma membrane immediately after gamete membrane fusion; the amount and extent of label, i.e., the distance from the site of sperm incorporation, increased with time postinsemination. By 20 min postinsemination approximately one hemisphere of the inseminated egg/oocyte was associated with label. The expanding distribution of colloidal gold label on inseminated eggs and oocytes vs. time reflects the free diffusion of 33 and 35 kDa sperm surface proteins among egg/oocyte plasma membrane components. Label was also found in forming endocytotic vesicles, suggesting that sperm plasma membrane proteins may be internalized.     

Self-referent phenotype matching is a poor predictor of egg rejection by American Robins

Many hosts of obligate brood-parasitic birds use variation in the coloration and pattern of eggshells to identify and reject foreign eggs from their nests. However, egg-rejection behavior of several hosts is not tightly predicted by the modeled output of overall avian-perceivable chromatic differences between foreign and host eggs. This demands a re-investigation into the proximate sensory cues and perceptual processes used for egg recognition. One plausible mechanism is that rejection by hosts depends on the relative stimulation of one (or a combination) of their photoreceptors when comparing their own and parasitic eggs (i.e., self-referencing). To test this, we placed dark-blue egg models (with a known ~ 50% rejection rate) in nests of American Robins (Turdus migratorius), which are occasional hosts of Brown-headed Cowbirds (Molothrus ater). We found that relative metrics of individually repeatable size or shape and overall avian-perceivable differences in color between American Robin eggs and model eggs did not explain the probability of an experimental egg being rejected. However, despite a limited R² value, hosts were significantly more likely to reject a model egg when the estimated contrast between their own egg and the model egg was greater for the blue (SWS) photoreceptor. These results are consistent with female American Robins using a simple physiological algorithm that would confer widespread benefits by reducing the likelihood of rejecting their own eggs, while still eliminating parasitic eggs because of their dissimilar novel coloration.     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.     

A cortical granule-specific enzyme,B-1,3-glucanase,in sea urchin eggs

The ultrastructural localization of B-1,3-glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B-1,3-glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B-1,3-glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.     

Hatching from cysts and egg sacs of Heterodera cruciferae and effects of temperature on hatching and development on oilseed rape

Juveniles hatched readily from field cysts and very readily from eggs in egg sacs of Heterodera cruciferae, when exposed to oilseed rape root diffusate. They hatched very poorly, however, from white or brown females from which the above egg sacs had been removed. Some hatching occurred at 8 ^oC but much more occurred at 12 , 16, 20 and 24 ^oC, with most at 16 ^oC. Development of juveniles in roots of oilseed rape occurred throughout the range 8–24 ^oC, and proceeded faster the higher the temperature. The basal development temperature was taken as 5 ^oC and the number of day degrees above this temperature required to reach each stage of development was calculated. From invasion of roots to the hatching of F₁ juveniles required an average of 680 day degrees, but only 210 day degrees were required for the first appearance of egg sacs on adult females. On this basis, two consecutive generations of H, cruciferae would be possible on autumn-sown oilseed rape in southern England, but the second would mature fully only after the crop was harvested. In Scotland, two consecutive generations could also occur but the second would be much less mature by harvest: only about 850 day degrees are available compared to almost 1100 in southern England. In practice, however, overlapping generations probably occur due to flushes of hatching of juveniles (i) at sowing, (ii) when soils warm up after winter and (iii) when the first generation completes its development. The proportion of eggs found in egg sacs was never more than 37% and some field cysts contained about 220 eggs; their egg sacs may, therefore, have contained as many as 150 eggs. Any study of population dynamics or damage assessment will require a quantification of the contribution of eggs in egg sacs to population density. Oilseed rape is direct drilled and may, therefore, be more sensitive to a given population density of the nematode than host crops which are transplanted.     

Population growth rate of the parasitic rotifer Proales gigantea,and susceptibility to parasitization in the snail Lymnaea acuminata at different stages of embryonic development

Developing eggs of the host snail Lymnaea acuminata were experimentally parasitized with the parasitic rotifer Proales gigantea to study the population growth rate of the parasite within the snail egg capsule and the susceptibility of the host eggs at different stages of embryonic development. The population growth rate of P. gigantea was 0.46 ± 0.07 individual^–1 day^–1 at the ambient temperature of 18–22 °C. Snail eggs were most susceptible to rotifer attack during the initial stages of development, becoming progressively more resistant after the hippo stage. Yet, regardless of the stage of development, the host embryo was doomed to die without hatching even if one individual rotifer gained entry inside the egg capsule. The presence of P. gigantea within the parasitized egg capsules or in the mucilage had no effect on the developmental rates and hatching success of non-parasitized eggs within the same egg mass.     

Reproductive trade-off of the copepod Acartia tonsa in a hypersaline estuary of the Southwestern Atlantic. Temporal variations in the morphology of eggs

The purpose of the present work was to study the seasonal variations in egg production, morphology and hatching success in the cryptic species Acartia tonsa, taking into account variations in female size, population abundance and environmental factors in a turbid and hypersaline estuary. Sampling was performed during the austral warm (18–23°C and 32–36 salinity) and cold seasons (5–7°C; 32–38) in Bahía Blanca Estuary (BBE), Argentina, during 2007 and 2009. Field-collected females were incubated in the laboratory simulating in situ environmental conditions, and specimens from fixed samples were measured using optical and scanning electronic microscopy. Acartia tonsa’s marked seasonality in its reproductive traits was found to ensure its permanence in the water column all over the year. During the warm season, small-sized females were observed to invest their energy in the production of subitaneous eggs with high hatching success and smooth appearance (12.95?±?2.38 eggs f^?1 day^?1 and specific egg production rate (SEP) of 16.57%C f^?1 day^?1). During the cold season, females invested C in body mass as well as in the production of resting eggs of three different morphotypes (6.56?±?3.2 eggs f^?1 day^?1 and SEP of 7.37%C f^?1 day^?1). Although these morphotypes were found to show differences in surface ornamentation, they exhibited the same delayed hatching behaviour. The eggs with shorter spines were found to integrate the resting egg bank in BBE. Our findings confirming a delayed egg hatching behaviour and a great tolerance to low temperatures and high salinities in the A. tonsa population in BBE suggest that this possible strain is a valuable phenotype for aquaculture.     

Understanding control of calcitic biomineralization—Proteomics to the rescue

The avian eggshell is one of the fastest calcifying processes known and represents a unique model for studying biomineralization. Eggshell strength is a crucial economic trait for table egg production, and ensures that a safe egg reaches the consumer kitchen. However, a common toolkit for eggshell mineralization has not yet been defined. In this issue, label‐free MS‐based protein quantification technology has been used by Sun et al. (Proteomics 2013, 13, 3523–3536) to detect differences in protein abundance between eggshell matrix from strong and weak eggs and between the corresponding uterine fluids bathing strong and weak eggs. Proteins associated with the formation of strong eggshells are identified, which are now candidates for further investigations to define the regulatory relationship between specific eggshell matrix proteins and calcite crystal texture.