Vertebrate Eggs Research Articles

Putative branchiopod and vertebrate eggs from the Remigiusberg Lagerstätte (Pennsylvanian-Permian boundary) of the Saar-Nahe Basin, SW Germany

Putative branchiopod and vertebrate eggs from the Remigiusberg Lagerstätte (Pennsylvanian-Permian boundary) of the Saar-Nahe Basin, SW Germany

ZP2 cleavage blocks polyspermy by modulating the architecture of the egg coat

Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.

Zona pellucida family genes in Chinese pond turtle: identification, expression profiles, and role in the spermatozoa acrosome reaction†.

The zona pellucida (ZP) is an extracellular matrix that surrounds all vertebrate eggs, and it is involved in fertilization and species-specific recognition. Numerous in-depth studies of the ZP proteins of mammals, birds, amphibians, and fishes have been conducted, but systematic investigation of the ZP family genes and their role during fertilization in reptiles has not been reported to date. In this study, we identified six turtle ZP (Tu-ZP) gene subfamilies (Tu-ZP1, Tu-ZP2, Tu-ZP3, Tu-ZP4, Tu-ZPD, and Tu-ZPAX) based on whole genome sequence data from Mauremys reevesii. We found that Tu-ZP4 had large segmental duplication and was distributed on three chromosomes, and we also detected gene duplication in the other Tu-ZP genes. To evaluate the role of Tu-ZP proteins in sperm-egg binding, we assessed the expression pattern of these Tu-ZP proteins and their ability to induce the spermatozoa acrosome reaction in M. reevesii. In conclusion, this is the first report of the existence of gene duplication of Tu-ZP genes and that Tu-ZP2, Tu-ZP3, and Tu-ZPD can induce acrosome exocytosis of spermatogenesis in the reptile.

Visualizing Lipid Droplets in Drosophila Oogenesis.

Lipid droplets (LDs) are fat storage organelles highly abundant in oocytes and eggs of many vertebrates and invertebrates. They have roles both during oogenesis and in provisioning the developing embryo. In Drosophila, large numbers of LDs are generated in nurse cells during mid-oogenesis and then transferred to oocytes. Their number and spatial distribution changes developmentally and in response to various experimental manipulations. This chapter demonstrates how to visualize LDs in Drosophila follicles, both in fixed tissues and living samples. For fixed samples, the protocol explains how to prepare female flies, dissect ovaries, isolate follicles, fix, apply stains, mount the tissue, and perform imaging. For live samples, the protocol shows how to dissect ovaries, apply a fluorescent LD dye, and culture follicles such that they remain alive and healthy during imaging. Finally, a method is provided that employs in vivo centrifugation to assess colocalization of markers with LDs.

Domain Expansion and Functional Diversification in Vertebrate Reproductive Proteins.

The rapid evolution of fertilization proteins has generated remarkable diversity in molecular structure and function. Glycoproteins of vertebrate egg coats contain multiple zona pellucida (ZP)-N domains (1–6 copies) that facilitate multiple reproductive functions, including species-specific sperm recognition. In this report, we integrate phylogenetics and machine learning to investigate how ZP-N domains diversify in structure and function. The most C-terminal ZP-N domain of each paralog is associated with another domain type (ZP-C), which together form a “ZP module.” All modular ZP-N domains are phylogenetically distinct from nonmodular or free ZP-N domains. Machine learning–based classification identifies eight residues that form a stabilizing network in modular ZP-N domains that is absent in free domains. Positive selection is identified in some free ZP-N domains. Our findings support that strong purifying selection has conserved an essential structural core in modular ZP-N domains, with the relaxation of this structural constraint allowing free N-terminal domains to functionally diversify.

Glacial-interglacial evolution of seasonal cooling events documented by land-snail eggs from Chinese loess

The alternations of glacial and interglacial cycles are a classical feature of Quaternary climatic evolution and have been demonstrated to be closely related to seasonal insolation changes at high northern latitudes. Therefore, seasonal features may provide insights into glacial-interglacial cycles. However, mainly due to the lack of long time series of seasonally sensitive proxies, little is known about seasonal changes on the glacial-interglacial scale. The unhatched eggs preserved in sediments can serve as a proxy of seasonal cooling events (e.g., cold spells) since biological principles indicate that egg hatching is sensitive to temperature changes, and cooling-event-induced low temperatures during the reproductive season are unfavorable for eggs to hatch. Vertebrate eggs are well documented in the geological records, but they rarely provide continuous records through time. Here we present a high-resolution time series of land-snail eggs from the Chinese Loess Plateau, spanning the last three glacial-interglacial cycles. The results show that seasonal cooling events, indicated by peaks in egg abundance, are strong during glacial inceptions and climate cooling shifts of the marine isotope stages (MIS) 7e/7d, MIS 5e/5d, MIS 5c/5b and MIS 3/2. They tend not to occur during deglacials. They may result in low temperatures unfavorable for egg hatching during the reproductive season. Although several factors may be involved, seasonal cooling events in the Chinese Loess Plateau seem to be positively and more closely related to high-northern-latitude ice sheet growth. This finding may provide a new perspective for understanding glacial-interglacial evolution.

Oophagy in spiders: consumption of invertebrate and vertebrate eggs

In this paper, we present an update on our knowledge on egg predation (oophagy) by spiders. Based on a survey of 233 reports, ghost spiders (Anyphaenidae), lynx spiders (Oxyopidae), jumping spiders (Salticidae), and yellow sac spiders (Cheiracanthiidae) were the most prominent groups of spiders engaged in oophagy. Around 75% of the reports referred to the consumption of lepidopteran and spider eggs worldwide. Another 10% referred to the consumption of eggs/embryos of anurans – especially predation upon embryos of glass frogs (Centrolenidae) by spiders from the families Anyphaenidae and Trechaleidae in the Neotropics. The remaining 17% included rare instances of feeding on eggs of coleopterans, dermapterans, dipterans, heteropterans, homopterans, hymenopterans, acarids, neuropterans, opilionids, and squamates. Our study demonstrates that oophagy in spiders is much more widespread than previously thought, both geographically and taxonomically. The finding that spiders feed on eggs/embryos from so many different invertebrate and vertebrate taxa is novel.

Phylogeny and evolutionary history of the amniote egg.

We review morphological features of the amniote egg and embryos in a comparative phylogenetic framework, including all major clades of extant vertebrates. We discuss 40 characters that are relevant for an analysis of the evolutionary history of the vertebrate egg. Special attention is given to the morphology of the cellular yolk sac, the eggshell, and extraembryonic membranes. Many features that are typically assigned to amniotes, such as a large yolk sac, delayed egg deposition, and terrestrial reproduction have evolved independently and convergently in numerous clades of vertebrates. We use phylogenetic character mapping and ancestral character state reconstruction as tools to recognize sequence, order, and patterns of morphological evolution and deduce a hypothesis of the evolutionary history of the amniote egg. Besides amnion and chorioallantois, amniotes ancestrally possess copulatory organs (secondarily reduced in most birds), internal fertilization, and delayed deposition of eggs that contain an embryo in the primitive streak or early somite stage. Except for the amnion, chorioallantois, and amniote type of eggshell, these features evolved convergently in almost all major clades of aquatic vertebrates possibly in response to selective factors such as egg predation, hostile environmental conditions for egg development, or to adjust hatching of young to favorable season. A functionally important feature of the amnion membrane is its myogenic contractility that moves the (early) embryo and prevents adhering of the growing embryo to extraembryonic materials. This function of the amnion membrane and the liquid-filled amnion cavity may have evolved under the requirements of delayed deposition of eggs that contain developing embryos. The chorioallantois is a temporary embryonic exchange organ that supports embryonic development. A possible evolutionary scenario is that the amniote egg presents an exaptation that paved the evolutionary pathway for reproduction on land. As shown by numerous examples from anamniotes, reproduction on land has occurred multiple times among vertebrates-the amniote egg presenting one "solution" that enabled the conquest of land for reproduction.

Knock out of specific maternal vitellogenins in zebrafish (Danio rerio) evokes vital changes in egg proteomic profiles that resemble the phenotype of poor quality eggs

BackgroundWe previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts.ResultsThe vtg-KO caused severe changes in the proteome of 1-cell stage zebrafish eggs. These changes were disclosed by molecular signatures that highly resembled the proteomic phenotype of poor quality zebrafish eggs reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs to protein and in energy homeostasis. The lack of this contribution appears to have a significant impact on endoplasmic reticulum and mitochondrial functions, and thus embryonic development, even after zygotic genome activation. Increased endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and in human neurodegenerative disease related activities in both vtg1- and vtg3-KO eggs were found to be indicators of the aforementioned conditions. Distinctive increase in apoptosis and Parkinson disease pathways, as well as the decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms have been identified as strong candidate markers for studying the details of these mechanisms during early embryonic development in zebrafish and possibly other vertebrates.ConclusionsThese findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

Bacterial avidins are a widely distributed protein family in Actinobacteria, Proteobacteria and Bacteroidetes

BackgroundAvidins are biotin-binding proteins commonly found in the vertebrate eggs. In addition to streptavidin from Streptomyces avidinii, a growing number of avidins have been characterized from divergent bacterial species. However, a systematic research concerning their taxonomy and ecological role has never been done. We performed a search for avidin encoding genes among bacteria using available databases and classified potential avidins according to taxonomy and the ecological niches utilized by host bacteria.ResultsNumerous avidin-encoding genes were found in the phyla Actinobacteria and Proteobacteria. The diversity of protein sequences was high and several new variants of genes encoding biotin-binding avidins were found. The living strategies of bacteria hosting avidin encoding genes fall mainly into two categories. Human and animal pathogens were overrepresented among the found bacteria carrying avidin genes. The other widespread category were bacteria that either fix nitrogen or live in root nodules/rhizospheres of plants hosting nitrogen-fixing bacteria.ConclusionsBacterial avidins are a taxonomically and ecologically diverse group mainly found in Actinobacteria, Proteobacteria and Bacteroidetes, associated often with plant invasiveness. Avidin encoding genes in plasmids hint that avidins may be horizontally transferred. The current survey may be used as a basis in attempts to understand the ecological significance of biotin-binding capacity.

Cryo‐EM structure of native human uromodulin, a zona pellucida module polymer

Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo‐electron microscopy study of uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant protein in human urine and an archetypal ZP module‐containing molecule, in its mature homopolymeric state. UMOD forms a one‐start helix with an unprecedented 180‐degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD‐based models of heteromeric vertebrate egg coat filaments identify a common sperm‐binding region at the interface between subunits.

Thermal variability during ectotherm egg incubation: A synthesis and framework.

Natural populations of ectothermic oviparous vertebrates typically experience thermal variability in their incubation environment. Yet an overwhelming number of laboratory studies incubate animals under constant thermal conditions that cannot capture natural thermal variability. Here, we systematically searched for studies that incubated eggs of ectothermic vertebrates, including both fishes and herpetofauna, under thermally variable regimes. We ultimately developed a compendium of 66 studies that used thermally variable conditions for egg incubation. In this review, we qualitatively discuss key findings from literature in the compendium, including the phenotypic effects resulting from different patterns of thermally variable incubation, as well as the ontogenetic persistence of these effects. We also describe a physiological framework for contextualizing some of these effects, based on thermal performance theory. Lastly, we highlight key gaps in our understanding of thermally variable incubation and offer suggestions for future studies.

Egg predation and vertebrates associated with wild crocodilian nests in Mexico determined using camera-traps

ABSTRACT Interactions between species and individuals can determine their survival in the wild. Most of the time these relationships are difficult to study in situ by direct observation. However, technology can help collect these data with minimal impact on animals’ behaviour. Egg stage is certainly the most vulnerable life stage in crocodilians, but few studies have focused on animal species visiting crocodilian nests and associated egg predation. Herein, we use camera-traps in four Mexican states (Chiapas, Oaxaca, Jalisco, Tabasco) to determine vertebrate species and egg predators associated with wild nests of Caiman crocodilus chiapasius, Crocodylus acutus and Crocodylus moreletii. We recorded 72 species of vertebrates at nesting sites and obtained the first photographic evidence of crocodilian egg predation by Caracara cheriway, Cuniculus paca, Didelphis virginiana and Procyon lotor. We also identified commensalism, cooperation, and predation as types of interactions within observed nesting areas, which indicates the importance of crocodilian nesting areas for other wild vertebrates. Finally, we found that crocodilian egg predation depends on species richness present in the area of study, as well as crocodilian size.

Evolutionary ecology of insect egg coloration: a review

Body coloration in animals is often adaptive and used for defense against biotic (e.g., predators, competitors) and abiotic (e.g., solar radiation, desiccation) threats. The study of adaptive coloration in insects usually favors obvious model species such as yellowjacket wasps (Vespula spp.) and longwing butterflies (Heliconius spp.), partly because they actively interact with their environment. Yet, one life stage has received less attention because of its immobility: the egg. So far, vertebrate eggs, especially avian eggs, have held the ‘‘big end of the stick’’ when it comes to research effort on adaptive egg coloration. In species where eggs are not provided with parental care and left to survive on their own until hatching, studying the defensive roles played by their colors is imperative to understand their evolutionary ecology. Adaptive functions provided by egg coloration such as crypsis, aposematism and photoprotection against ultraviolet radiation potentially have huge fitness impacts and deserve more attention. Here, the current literature on insect egg coloration is reviewed, reporting its known adaptive significance. Clear distinctions are made between functions tested empirically and functions that remain hypothetical despite often being treated as facts. Avenues for future work in the field are also provided.

Variation in avian egg shape and nest structure is explained by climatic conditions

Why are avian eggs ovoid, while the eggs of most other vertebrates are symmetrical? The interaction between an egg and its environment likely drives selection that will shape eggs across evolutionary time. For example, eggs incubated in hot, arid regions face acute exposure to harsh climatic conditions relative to those in temperate zones, and this exposure will differ across nest types, with eggs in open nests being more exposed to direct solar radiation than those in enclosed nests. We examined the idea that the geographical distribution of both egg shapes and nest types should reflect selective pressures of key environmental parameters, such as ambient temperature and the drying capacity of air. We took a comparative approach, using 310 passerine species from Australia, many of which are found in some of the most extreme climates on earth. We found that, across the continent, egg elongation decreases and the proportion of species with domed nests with roofs increases in hotter and drier areas with sparse plant canopies. Eggs are most spherical in open nests in the hottest environments, and most elongate in domed nests in wetter, shadier environments. Our findings suggest that climatic conditions played a key role in the evolution of passerine egg shape.

Microbiome assembly of avian eggshells and their potential as transgenerational carriers of maternal microbiota.

The microbiome is essential for development, health and homeostasis throughout an animal's life. Yet, the origins and transmission processes governing animal microbiomes remain elusive for non-human vertebrates, oviparous vertebrates in particular. Eggs may function as transgenerational carriers of the maternal microbiome, warranting characterisation of egg microbiome assembly. Here, we investigated maternal and environmental contributions to avian eggshell microbiota in wild passerine birds: woodlark Lullula arborea and skylark Alauda arvensis. Using 16S rRNA gene sequencing, we demonstrated in both lark species, at the population and within-nest levels, that bacterial communities of freshly laid eggs were distinct from the female cloacal microbiome. Instead, soil-borne bacteria appeared to thrive on freshly laid eggs, and eggshell microbiota composition strongly resembled maternal skin, body feather and nest material communities, sources in direct contact with laid eggs. Finally, phylogenetic structure analysis and microbial source tracking underscored species sorting from directly contacting sources rather than in vivo-transferred symbionts. The female-egg-nest system allowed an integrative assessment of avian egg microbiome assembly, revealing mixed modes of symbiont acquisition not previously documented for vertebrate eggs. Our findings illuminated egg microbiome origins, which suggested a limited potential of eggshells for transgenerational transmission, encouraging further investigation of eggshell microbiome functions in vertebrates.

Egg-Coat and Zona Pellucida Proteins of Chicken as a Typical Species of Aves.

Birds are oviparous vertebrates in terrestrial animals. Birds' eggs accumulate mass of egg yolk during the egg development and are accordingly much larger than the eggs of viviparous vertebrates. Despite such difference in size and contents, the birds' eggs are surrounded with the egg-coat morphologically and compositionally resembling the mammalian egg-coat, zona pellucida. On the other hand, there are some differences in part between the two egg-coats, though relationships of such structural differences to any biological roles specific for the extracellular matrix of birds' eggs are not fully understood. In birds, unlike mammals, ZP proteins constituting the egg-coat are highly conserved and therefore those of chicken are described as a representative of birds. The egg-coat ZP proteins, ZP1, ZP3, and ZPD as the majors, accumulate and form the matrix by self-assembly around the egg rapidly growing in the ovarian follicle, in which ZP1 is from liver and both ZP3 and ZPD are from follicular granulosa cells. Although details of the egg-coat-sperm interaction on fertilization remain to be investigated, the lytic degradation process of egg-coat matrix for the sperm penetration has become to be clarified gradually. ZP1 is the primary target of sperm acrosin, and the limited cleavage in the specific region leading to the loss of intermolecular cross-linkages is crucial for the lysis of egg-coat matrix. Possible roles of the ZP1 with the additional sequence characteristic to birds are discussed from a viewpoint of giving both robustness and elastomeric nature to the egg-coat matrix for the birds' eggs.

Microtubule-actin crosslinking factor 1 (Macf1) domain function in Balbiani body dissociation and nuclear positioning.

Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead our results suggest that Macf1a functions by linking one cytoskeletal system, cortical actin, to another structure, the Bb, where Macf1a is localized. Through this novel linking process, it dissociates the Bb at the oocyte cortex, thus specifying the AV axis of the oocyte and future egg. To our knowledge, this is also the first study to use genome editing to unravel the module-dependent function of a cytoskeletal linker.

A Novel Pattern of Yolk Processing in Developing Snake Eggs (Colubridae: Lampropeltini) and its Functional and Evolutionary Implications.

Early amniotic vertebrates evolved large-yolked eggs that permitted production of well-developed, terrestrial hatchlings. This reproductive pattern required new mechanisms for cellularizing the yolk and mobilizing it for embryonic use. In birds, cells that line the yolk sac cavity phagocytose and digest the yolk material, a pattern that is commonly assumed to be universal among oviparous amniotes. However, recent evidence challenges the assumption that all squamate reptiles conform to the avian developmental pattern. In this paper, scanning electron microscopy and histology were used to study mechanisms of yolk processing in two colubrid snakes, the kingsnake Lampropeltis getula and the milksnake L. triangulum. Endodermal cells from the yolk sac splanchnopleure proliferate massively as they invade the yolk sac cavity, forming elaborate chains of interlinked cells. These cells grow in size as they phagocytose yolk material. Subsequently, vitelline capillaries invade the masses of yolk-laden cells and become coated with the endodermal cells, forming an elaborate meshwork of cell-coated strands. The close association of cells, yolk, and blood vessels allows yolk material to be cellularized, digested, and transported for embryonic use. The overall pattern is like that of the corn snake Pantherophis guttatus, but contrasts markedly with that of birds. Given recent evidence that this developmental pattern may also occur in certain lizards, we postulate that it is ancestral for squamates. Studies of lizards, crocodilians, and turtles are needed to clarify the evolutionary history of this pattern and its implications for the evolution of the amniotic (terrestrial) vertebrate egg.

Avian Egg and Egg Coat.

An ovulated egg of vertebrates is surrounded by unique extracellular matrix, the egg coat or zona pellucida, playing important roles in fertilization and early development. The vertebrate egg coat is composed of two to six zona pellucida (ZP) glycoproteins that are characterized by the evolutionarily conserved ZP-domain module and classified into six subfamilies based on phylogenetic analyses. Interestingly, investigations of biochemical and functional features of the ZP glycoproteins show that the roles of each ZP-glycoprotein family member in the egg-coat formation and the egg-sperm interactions seemingly vary across vertebrates. This might be one reason why comprehensive understandings of the molecular basis of either architecture or physiological functions of egg coat still remain elusive despite more than 3 decades of intensive investigations. In this chapter, an overview of avian egg focusing on the oogenesis are provided in the first section, and unique features of avian egg coat, i.e., perivitelline layer, including the morphology, biogenesis pathway, and physiological functions are discussed mainly on chicken and quail in terms of the characteristics of ZP glycoproteins in the following sections. In addition, these features of avian egg coat are compared to mammalian zona pellucida, from the viewpoint that the structural and functional varieties of ZP glycoproteins might be associated with the evolutionary adaptation to their reproductive strategies. By comparing the egg coat of birds and mammals whose reproductive strategies are largely different, new insights into the molecular mechanisms of vertebrate egg-sperm interactions might be provided.