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.

Importin α Partitioning to the Plasma Membrane Regulates Intracellular Scaling.

Early embryogenesis is accompanied by reductive cell divisions requiring that subcellular structures adapt to a range of cell sizes. The interphase nucleus and mitotic spindle scale with cell size through both physical and biochemical mechanisms, but control systems that coordinately scale intracellular structures are unknown. We show that the nuclear transport receptor importin α is modified by palmitoylation, which targets it to the plasma membrane and modulates its binding to nuclear localization signal (NLS)-containing proteins that regulate nuclear and spindle size in Xenopus egg extracts. Reconstitution of importin α targeting to the outer boundary of extract droplets mimicking cell-like compartments recapitulated scaling relationships observed during embryogenesis, which were altered by inhibitors that shift levels of importin α palmitoylation. Modulation of importin α palmitoylation in human cells similarly affected nuclear and spindle size. These experiments identify importin α as a conserved surface area-to-volume sensor that scales intracellular structures to cell size.

Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

Repair of a DNA-protein crosslink by replication-coupled proteolysis.

DNA-protein crosslinks (DPCs) are caused by environmental, endogenous, and chemotherapeutic agents and pose a severe threat to genome stability. We use Xenopus egg extracts to recapitulate DPC repair in vitro and show that this process is coupled to DNA replication. A DPC on the leading strand template arrests the replisome by stalling the CMG helicase. The DPC is then degraded on DNA, yielding a peptide-DNA adduct that is bypassed by CMG. The leading strand subsequently resumes synthesis, stalls again at the adduct, and then progresses past the adduct using DNA polymerase ζ. A DPC on the lagging strand template only transiently stalls the replisome, but it too is degraded, allowing Okazaki fragment bypass. Our experiments describe a versatile, proteolysis-based mechanism of S phase DPC repair that avoids replication fork collapse.

A molecular network of conserved factors keeps ribosomes dormant in the egg.

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2.

The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.

Genetic adaptations for the oceanic success of fish eggs.

Genetic adaptations of organisms living in extreme environments are fundamental to our understanding of where life can evolve. Water is the single limiting parameter in this regard, yet when released in the oceans, the single-celled eggs of marine bony fishes (teleosts) have no means of acquiring it. They are strongly hyposmotic to seawater and lack osmoregulatory systems. Paradoxically, modern teleosts successfully release vast quantities of eggs in the extreme saline environment and recorded the most explosive radiation in vertebrate history. Here, we highlight key genetic adaptations that evolved to solve this paradox by filling the pre-ovulated eggs with water. The degree of water acquisition is uniquely prevalent to marine teleosts, permitting the survival and oceanic dispersal of their eggs.

Genetic Screens to Analyze Pattern Formation of Egg and Embryo in Drosophila: A Personal History.

In Drosophila development, the axes of the egg and future embryo are established during oogenesis. To learn about the underlying genetic and molecular pathways that lead to axis formation, I conducted a large-scale genetic screen at the beginning of my independent career. This led to the eventual understanding that both anterior-posterior and dorsal-ventral pattern information is transmitted from the oocyte to the surrounding follicle cells and in turn from the follicle cells back to the oocyte. How I came to conduct this screen and what further insights were gained by studying the mutants isolated in the screen are the topics of this autobiographical article.

Nucleation and transport organize microtubules in metaphase spindles.

Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of microtubules in these structures. Here we present a method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle in Xenopus laevis egg extracts and found that microtubules are shortest near poles and become progressively longer toward the center of the spindle. These data, in combination with mathematical modeling, imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, and the proper organization of microtubules in the spindle is determined by nonuniform microtubule nucleation and the local sorting of microtubules by transport.

Human Germline Cell Development: from the Perspective of Single-Cell Sequencing.

Germline cells are the beginning of new individuals in multicellular animals, including humans. Our understanding of these cell types is limited by the difficulty of analyzing the precious and heterogeneous germline tissue samples. The rapid development of single-cell sequencing technologies provides a chance for comprehensive profiling of the omics dynamics of human germline development. In this review, we discuss progress in analyzing the development of human germline cells, including preimplantation and implantation embryos, fetal germ cells (FGCs), and adult spermatogenesis by single-cell transcriptome and epigenome sequencing technologies.

Flexible oviposition behavior enabled the evolution of terrestrial reproduction.

Among vertebrates, nearly all oviparous animals are considered to have either obligate aquatic or terrestrial oviposition, with eggs that are specialized for developing in those environments. The terrestrial environment has considerably more oxygen but is dry and thus presents both opportunities and challenges for developing embryos, particularly those adapted for aquatic development. Here, we present evidence from field experiments examining egg-laying behavior, egg size, and egg jelly function of 13 species of Central and South American treefrogs in the genus Dendropsophus, which demonstrates that flexible oviposition (individuals laying eggs both in and out of water) and eggs capable of both aquatic and terrestrial development are the likely factors which enable the transition from aquatic to terrestrial reproduction. Nearly half of the species we studied had previously undescribed degrees of flexible oviposition. Species with obligate terrestrial reproduction have larger eggs than species with aquatic reproduction, and species with flexible reproduction have eggs of intermediate sizes. Obligate terrestrial breeding frogs also have egg masses that absorb water more quickly than those with flexible oviposition. We also examined eight populations of a single species, Dendropsophus ebraccatus, and document substantial intraspecific variation in terrestrial oviposition; populations in rainy, stable climates lay fewer eggs in water than those in drier areas. However, no differences in egg size were found, supporting the idea that the behavioral component of oviposition evolves before other adaptations associated with obligate terrestrial reproduction. Collectively, these data demonstrate the key role that behavior can have in facilitating major evolutionary transitions.

Winners vs. losers: Schistosoma mansoni intestinal and liver eggs exhibit striking differences in gene expression and immunogenicity.

The eggs of the blood fluke Schistosoma mansoni are the main cause of the clinical manifestations of chronic schistosomiasis. After laying, the egg "winners" attach to the endothelium of the mesenteric vein and, after a period of development, induce the growth of a small granuloma, which facilitates their passage to the intestinal lumen. Egg "losers" carried by the bloodstream to non-specific tissues also undergo full development and induce large granuloma formation, but their life ends there. Although these trapped eggs represent a dead end in the parasite life cycle, the vast majority of studies attempting to describe the biology of the S. mansoni eggs have studied these liver-trapped "losers" instead of migrating intestinal "winners". This raises the fundamental question of how these eggs differ. With robust comparative transcriptomic analysis performed on S. mansoni eggs isolated 7 weeks post infection, we show that gene expression is critically dependent on tissue localization, both in the early and late stages of development. While mitochondrial genes and venom allergen-like proteins are significantly upregulated in mature intestinal eggs, well-described egg immunomodulators IPSE/alpha-1 and omega-1, together with micro-exon genes, are predominantly expressed in liver eggs. In addition, several proteases and protease inhibitors previously implicated in egg-host interactions display clear tissue-specific gene expression patterns. These major differences in gene expression could be then reflected in the observed different ability of liver and intestinal soluble egg antigens to elicit host immune responses and in the shorter viability of miracidia hatched from liver eggs. Our comparative analysis provides a new perspective on the biology of parasite's eggs in the context of their development and tissue localization. These findings could contribute to a broader and more accurate understanding of parasite eggs interactions with the host, which have historically been often restricted to liver eggs and sometimes inaccurately generalized.

Insights into the micromechanical properties of the metaphase spindle.

The microtubule-based metaphase spindle is subjected to forces that act in diverse orientations and over a wide range of timescales. Currently, we cannot explain how this dynamic structure generates and responds to forces while maintaining overall stability, as we have a poor understanding of its micromechanical properties. Here, we combine the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations to analyze the vertebrate metaphase spindle's timescale- and orientation-dependent viscoelastic properties. We find that spindle viscosity depends on microtubule crosslinking and density. Spindle elasticity can be linked to kinetochore and nonkinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. These data suggest a quantitative model for the micromechanics of this cytoskeletal architecture and provide insight into how structural and functional stability is maintained in the face of forces, such as those that control spindle size and position, and can result from deformations associated with chromosome movement.

1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm-egg interaction.

Sperm acrosomal membrane proteins, such as Izumo sperm-egg fusion 1 (IZUMO1) and sperm acrosome-associated 6 (SPACA6), play essential roles in mammalian gamete binding or fusion. How their biosynthesis is regulated during spermiogenesis has largely remained elusive. Here, we show that 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik is a type-II transmembrane protein expressed in early round spermatids but not in mature spermatozoa. It interacts with proteins involved in N-linked glycosylation, disulfide isomerization, and endoplasmic reticulum (ER)-Golgi trafficking, suggesting a potential role in nascent protein processing. The ablation of 1700029I15Rik destabilizes non-catalytic subunits of the oligosaccharyltransferase (OST) complex that are pivotal for N-glycosylation. The knockout testes exhibit normal expression of sperm plasma membrane proteins, but decreased abundance of multiple acrosomal membrane proteins involved in fertilization. The knockout sperm show upregulated chaperones related to ER-associated degradation (ERAD) and elevated protein ubiquitination; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway underpinning the biosynthesis of acrosomal membrane proteins during spermiogenesis.

Sex Determination in the Mammalian Germline.

Sexual reproduction crucially depends on the production of sperm in males and oocytes in females. Both types of gamete arise from the same precursor, the germ cells. We review the events that characterize the development of germ cells during fetal life as they commit to, and prepare for, oogenesis or spermatogenesis. In females, fetal germ cells enter meiosis, whereas in males they delay meiosis and instead lose pluripotency, activate an irreversible program of prospermatogonial differentiation, and temporarily cease dividing. Both pathways involve sex-specific molecular signals from the somatic cells of the developing gonads and a suite of intrinsic receptors, signal transducers, transcription factors, RNA stability factors, and epigenetic modulators that act in complex, interconnected positive and negative regulatory networks. Understanding these networks is important in the contexts of the etiology, diagnosis, and treatment of infertility and gonadal cancers, and in efforts to augment human and animal fertility using stem cell approaches.

Bacterial contact induces polar plug disintegration to mediate whipworm egg hatching.

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.

An insight into the gut microbiota after Schistosoma japonicum eggs immunization in an experimental ulcerative colitis model.

Schistosome infection and schistosome-derived products have been implicated in the prevention and alleviation of inflammatory bowel disease by manipulating the host immune response, whereas the role of gut microbiota in this protective effect remains poorly understood. In this study, we found that the intraperitoneal immunization with Schistosoma japonicum eggs prior to dextran sulfate sodium (DSS) application significantly ameliorated the symptoms of DSS-induced acute colitis, which was characterized by higher body weight, lower disease activity index score and macroscopic inflammatory scores. We demonstrated that the immunomodulatory effects of S. japonicum eggs were accompanied by an influence on gut microbiota composition, abundance, and diversity, which increased the abundance of genus Turicibacter, family Erysipelotrichaceae, phylum Firmicutes, and decreased the abundance of genus Odoribacter, family Marinifilaceae, order Bacteroidales, class Bacteroidia, phylum Bacteroidota. In addition, Lactobacillus was identified as a biomarker that distinguishes healthy control mice from DSS-induced colitis mice. The present study revealed the importance of the gut microbiota in S. japonicum eggs exerting protective effects in an experimental ulcerative colitis (UC) model, providing an alternative strategy for the discovery of UC prevention and treatment drugs.

Terrestrial reproduction and parental care drive rapid evolution in the trade-off between offspring size and number across amphibians.

The trade-off between offspring size and number is central to life history strategies. Both the evolutionary gain of parental care or more favorable habitats for offspring development are predicted to result in fewer, larger offspring. However, despite much research, it remains unclear whether and how different forms of care and habitats drive the evolution of the trade-off. Using data for over 800 amphibian species, we demonstrate that, after controlling for allometry, amphibians with direct development and those that lay eggs in terrestrial environments have larger eggs and smaller clutches, while different care behaviors and adaptations vary in their effects on the trade-off. Specifically, among the 11 care forms we considered at the egg, tadpole and juvenile stage, egg brooding, male egg attendance, and female egg attendance increase egg size; female tadpole attendance and tadpole feeding decrease egg size, while egg brooding, tadpole feeding, male tadpole attendance, and male tadpole transport decrease clutch size. Unlike egg size that shows exceptionally high rates of phenotypic change in just 19 branches of the amphibian phylogeny, clutch size has evolved at exceptionally high rates in 135 branches, indicating episodes of strong selection; egg and tadpole environment, direct development, egg brooding, tadpole feeding, male tadpole attendance, and tadpole transport explain 80% of these events. By explicitly considering diversity in parental care and offspring habitat by stage of offspring development, this study demonstrates that more favorable conditions for offspring development promote the evolution of larger offspring in smaller broods and reveals that the diversity of parental care forms influences the trade-off in more nuanced ways than previously appreciated.

Restarting life: fertilization and the transition from meiosis to mitosis.

Fertilization triggers a complex cellular programme that transforms two highly specialized meiotic germ cells, the oocyte and the sperm, into a totipotent mitotic embryo. Linkages between sister chromatids are remodelled to support the switch from reductional meiotic to equational mitotic divisions; the centrosome, which is absent from the egg, is reintroduced; cell division shifts from being extremely asymmetric to symmetric; genomic imprinting is selectively erased and re-established; and protein expression shifts from translational control to transcriptional control. Recent work has started to reveal how this remarkable transition from meiosis to mitosis is achieved.