Egg Coat Research Articles

Recurrent Independent Pseudogenization Events of the Sperm Fertilization Gene ZP3r in Apes and Monkeys.

Many reproductive proteins show signatures of rapid evolution through sequence divergence and duplication. These features of reproductive genes may complicate the detection of orthologs across taxa, making it difficult to connect studies in model systems to human biology. In mice, ZP3r/sp56 is a binding partner to the egg coat protein ZP3 and may mediate induction of the acrosome reaction, a crucial step in fertilization. In rodents, ZP3r, as a member of the Regulators of Complement Activation cluster, is surrounded by paralogs, some of which have been shown to be evolving under positive selection. Although primate egg coats also contain ZP3, sequence divergence paired with paralogous relationships with neighboring genes has complicated the accurate identification of the human ZP3r ortholog. Here, we phylogenetically and syntenically resolve that the human ortholog of ZP3r is the pseudogene C4BPAP1. We investigate the evolution of this gene within primates. We observe independent pseudogenization events of ZP3r in all Apes with the exception of Orangutans, and independent pseudogenization events in many monkey species. ZP3r in both primates that retain ZP3r and in rodents contains positively selected sites. We hypothesize that redundant mechanisms mediate ZP3 recognition in mammals and ZP3r's relative importance to ZP recognition varies across species.

O-120 The role of CatSper in male infertility - from bench to bedsite and back

Abstract Infertility is on the rise, but the underlying causes still remain mostly unknown. In fact, for about one third of infertile men, semen parameters are within reference limits. This so called ‘unexplained male infertility’ seems to rest on elusive functional defects of the sperm undetectable by semen analysis, precluding early diagnosis of the infertility and evidence-based treatment by medically assisted reproduction (MAR). As a result, affected couples often experience failed MAR attempts, subjecting the ‘healthy’ female to recurring medical risks. We set out to gain insight into the pathomechanisms underlying unexplained male infertility. To this end, we systematically assessed the function of the sperm-specific multisubunit CatSper-channel complex in sperm of 2,300 men undergoing a fertility workup at our center, using a simple motility-based ‘CatSper-Test’ combined with [Ca2+]i fluorimetric and electrophysiological recordings. Thereby, we identified a group of men with normal semen parameters, but defective CatSper function. These men or couples failed to conceive naturally and upon MAR via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, we provide the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

ZP2: the precision regulator of egg coat architecture during fertilization.

ZP2: the precision regulator of egg coat architecture during fertilization.

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.

Human fertilization in vivo and in vitro requires the CatSper channel to initiate sperm hyperactivation.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

Proteomic Analysis Identifies Distinct Protein Patterns for High Ovulation in FecB Mutant Small Tail Han Sheep Granulosa Cells.

The Booroola fecundity (FecB) mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene increases ovulation in sheep. However, its effect on follicular maturation is not fully understood. Therefore, we collected granulosa cells (GCs) at a critical stage of follicle maturation from nine wild-type (WW), nine heterozygous FecB mutant (WB), and nine homozygous FecB mutant (BB) Small Tail Han sheep. The GCs of three ewes were selected at random from each genotype and consolidated into a single group, yielding a total of nine groups (three groups per genotype) for proteomic analysis. The tandem mass tag technique was utilized to ascertain the specific proteins linked to multiple ovulation in the various FecB genotypes. Using a general linear model, we identified 199 proteins significantly affected by the FecB mutation with the LIMMA package (p < 0.05). The differential abundance of proteins was enriched in pathways related to cholesterol metabolism, carbohydrate metabolism, amino acid biosynthesis, and glutathione metabolism. These pathways are involved in important processes for GC-regulated 'conservation' of oocyte maturation. Further, the sparse partial least-squares discriminant analysis and the Fuzzy-C-mean clustering method were combined to estimate weights and cluster differential abundance proteins according to ovulation to screen important ovulation-related proteins. Among them, ZP2 and ZP3 were found to be enriched in the cellular component catalog term "egg coat", as well as some apolipoproteins, such as APOA1, APOA2, and APOA4, enriched in several Gene Ontology terms related to cholesterol metabolism and lipoprotein transport. A higher abundance of these essential proteins for oocyte maturation was observed in BB and WB genotypes compared with WW ewes. These proteins had a high weight in the model for discriminating sheep with different FecB genotypes. These findings provide new insight that the FecB mutant in GCs improves nutrient metabolism, leading to better oocyte maturation by altering the abundance of important proteins (ZP2, ZP3, and APOA1) in favor of increased ovulation or better oocyte quality.

Mechanism-based understanding of the potential cellular targets of triclosan in zebrafish larvae

Triclosan (TCS) has become widely distributed due to its widespread use. In this study, we investigated the mechanisms of TCS’s potential effects on cellular targets in zebrafish (Danio rerio) larvae using transcriptome sequencing. The expressions of 772, 368, and 1039 genes were significantly altered in zebrafish after embryos were exposed to 2, 10, and 50 μg/L TCS for consecutive 50 d, respectively, and 33 differentially expressed genes (DEGs) were found. DEGs were significantly enriched in the biological processes, including inflammatory response and purine ribonucleoside bisphosphate biosynthetic process by Go analysis, and in processes such as egg coat formation, binding of sperm to zona pellucida, positive regulation of acrosome reaction, and immune response by Gene set enrichment analysis (GSEA). Both KEGG pathway analysis and GSEA showed that NOD-like receptor signaling pathway and Steroid biosynthesis were significantly affected. Results showed that TCS potentially affected reproduction, immune, and metabolism of zebrafish larvae.

Involvement in Fertilization and Expression of Gamete Ubiquitin-Activating Enzymes UBA1 and UBA6 in the Ascidian Halocynthia roretzi

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the “sperm reaction”. However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.

New insights into the role of microheterogeneity of ZP3 during structural maturation of the avian equivalent of mammalian zona pellucida.

The egg coat including mammalian zona pellucida (ZP) and the avian equivalent, i.e., inner-perivitelline layer (IPVL), is a specialized extracellular matrix being composed of the ZP glycoproteins and surrounds both pre-ovulatory oocytes and ovulated egg cells in vertebrates. The egg coat is well known for its potential importance in both the reproduction and early development, although the underlying molecular mechanisms remain to be fully elucidated. Interestingly, ZP3, one of the ZP-glycoprotein family members forming scaffolds of the egg-coat matrices with other ZP glycoproteins, exhibits extreme but distinctive microheterogeneity to form a large number of isoelectric-point isoforms at least in the chicken IPVL. In the present study, we performed three-dimensional confocal imaging and two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE) of chicken IPVLs that were isolated from the ovarian follicles at different growth stages before ovulation. The results suggest that the relative proportions of the ZP3 isoforms are differentially altered during the structural maturation of the egg-coat matrices. Furthermore, tandem mass spectrometry (MS/MS) analyses and ZP1 binding assays against separated ZP3 isoforms demonstrated that each ZP3 isoform contains characteristic modifications, and there are large differences among ZP3 isoforms in the ZP1 binding affinities. These results suggest that the microheterogeneity of chicken ZP3 might be regulated to be associated with the formation of egg-coat matrices during the structural maturation of chicken IPVL. Our findings may provide new insights into molecular mechanisms of egg-coat assembly processes.

Transcriptomic profile of symbiotic accessory nidamental gland during female maturation in bigfin reef squid

The bigfin reef squid, Sepioteuthis lessoniana, are a valuable commercial species in East Asian regions such as Taiwan and Japan. A lack of genomic information limits the application of potential aquaculture techniques, especially in breeding when considering the hatching rate of offspring. In some squids and cuttlefishes, symbiotic bacteria are transmitted from the accessory nidamental gland (ANG) to the jelly coat of eggs. In Hawaiian bobtail squid, these parent-delivered mutualistic bacteria play an important role in preventing lethal biofouling of the eggs and accelerating the hatch rate of offspring. The bacterial consortium, which is housed in the female squids ANG, are governed by host selection during female maturation. Immune functions are typically used to explain the regulatory mechanism of symbioses by host selection. In this study, we evaluated the transcripts featured in bacterial selection and maintenance during ANG development using RNA-seq. Different developmental stages of ANGs (stages 1–4) were sequenced. The de novo transcriptome assembly resulted in 524,918 unigenes. Two groups, non-pigmentation group (stage 1 and stage 3) and pigmentation group (stage 4), were clustered by transcriptome-wide expression profile analysis. The gene expression analyses indicated that 9,475 differential expression genes (DEGs) in three different phases and 1,363 (14.3%) DEGs were matched in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Furthermore, KEGG-enriched analysis results suggested that immune responses are a dominant pathway in the non-pigmentation group (stage 1 and stage 3) whereas lipid metabolism and metabolism of flora fermentation are dominant in the pigmentation group (stage 4). Although the host immunity plays an important role during bacterial colonization of the ANG in bigfin reef squid, our results showed that most immune-related genes had a reduced transcriptomic level in the pigmentation group compared with the non-pigmentation group. Therefore, our results provide new insight to understand the regulatory mechanisms of initial bacterial colonization and later bacterial pigmentation in the bigfin reef squid.

Mechanisms of Sperm-Egg Interactions: What Ascidian Fertilization Research Has Taught Us.

Fertilization is an essential process in terrestrial organisms for creating a new organism with genetic diversity. Before gamete fusion, several steps are required to achieve successful fertilization. Animal spermatozoa are first activated and attracted to the eggs by egg-derived chemoattractants. During the sperm passage of the egg’s extracellular matrix or upon the sperm binding to the proteinaceous egg coat, the sperm undergoes an acrosome reaction, an exocytosis of acrosome. In hermaphrodites such as ascidians, the self/nonself recognition process occurs when the sperm binds to the egg coat. The activated or acrosome-reacted spermatozoa penetrate through the proteinaceous egg coat. The extracellular ubiquitin–proteasome system, the astacin-like metalloproteases, and the trypsin-like proteases play key roles in this process in ascidians. In the present review, we summarize our current understanding and perspectives on gamete recognition and egg coat lysins in ascidians and consider the general mechanisms of fertilization in animals and plants.

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.

Recurrent Duplication and Diversification of Acrosomal Fertilization Proteins in Abalone.

Reproductive proteins mediating fertilization commonly exhibit rapid sequence diversification driven by positive selection. This pattern has been observed among nearly all taxonomic groups, including mammals, invertebrates, and plants, and is remarkable given the essential nature of the molecular interactions mediating fertilization. Gene duplication is another important mechanism that facilitates the generation of molecular novelty through functional divergence. Following duplication, paralogs may partition ancestral gene function (subfunctionalization) or acquire new roles (neofunctionalization). However, the contributions of duplication followed by sequence diversification to the molecular diversity of gamete recognition genes has been understudied in many models of fertilization. The marine gastropod mollusk abalone is a classic model for fertilization. Its two acrosomal proteins (lysin and sp18) are ancient gene duplicates with unique gamete recognition functions. Through detailed genomic and bioinformatic analyses we show how duplication events followed by sequence diversification has played an ongoing role in the evolution of abalone acrosomal proteins. The common ancestor of abalone had four members of its acrosomal protein family in a tandem gene array that repeatedly experienced positive selection. We find that both sp18 paralogs contain positively selected sites located in different regions of the paralogs, suggestive of functional divergence where selection acted upon distinct binding interfaces in each paralog. Further, a more recent species-specific duplication of both lysin and sp18 in the European abalone H. tuberculata is described. Despite clade-specific acrosomal protein paralogs, there are no concomitant duplications of egg coat proteins in H. tuberculata, indicating that duplication of egg proteins per se is not responsible for retention of duplicated acrosomal proteins. We hypothesize that, in a manner analogous to host/pathogen evolution, sperm proteins are selected for increased diversity through extensive sequence divergence and recurrent duplication driven by conflict mechanisms.

Recurrent Co-Option and Recombination of Cytokine and Three Finger Proteins in Multiple Reproductive Tissues Throughout Salamander Evolution.

Reproductive proteins evolve at unparalleled rates, resulting in tremendous diversity of both molecular composition and biochemical function between gametes of different taxonomic clades. To date, the proteomic composition of amphibian gametes is largely a molecular mystery, particularly for Urodeles (salamanders and newts) for which few genomic-scale resources exist. In this study, we provide the first detailed molecular characterization of gametes from two salamander species (Plethodon shermani and Desmognathus ocoee) that are models of reproductive behavior. Long-read PacBio transcriptome sequencing of testis and ovary of both species revealed sex-specific expression of many genes common to vertebrate gametes, including a similar expression profile to the egg coat genes of Xenopus oocytes. In contrast to broad conservation of oocyte genes, major testis transcripts included paralogs of salamander-specific courtship pheromones (PRF, PMF, and SPF) that were confirmed as major sperm proteins by mass spectrometry proteomics. Sperm-specific paralogs of PMF and SPF are likely the most abundant secreted proteins in P. shermani and D. ocoee, respectively. In contrast, sperm PRF lacks a signal peptide and may be expressed in cytoplasm. PRF pheromone genes evolved independently multiple times by repeated gene duplication of sperm PRF genes with signal peptides recovered through recombination with PMF genes. Phylogenetic analysis of courtship pheromones and their sperm paralogs support that each protein family evolved for these two reproductive contexts at distinct evolutionary time points between 17 and 360 million years ago. Our combined phylogenetic, transcriptomic and proteomic analyses of plethodontid reproductive tissues support that the recurrent co-option and recombination of TFPs and cytokine-like proteins have been a novel driving force throughout salamander evolution and reproduction.

Fertilization of Ascidians: Gamete Interaction, Self/Nonself Recognition and Sperm Penetration of Egg Coat.

Fertilization is one of the most important events in living organisms to generate a new life with a mixed genetic background. To achieve successful fertilization, sperm and eggs must undergo complex processes in a sequential order. Fertilization of marine invertebrate Ciona intestinalis type A (Ciona robusta) has been studied for more than a hundred years. Ascidian sperm are attracted by chemoattractants from eggs and bind to the vitelline coat. Subsequently, sperm penetrate through the vitelline coat proteolytically and finally fuse with the egg plasma membrane. Here, we summarize the fertilization mechanisms of ascidians, particularly from sperm-egg interactions to sperm penetration of the egg coat. Since ascidians are hermaphrodites, inbreeding depression is a serious problem. To avoid self-fertilization, ascidians possess a self-incompatibility system. In this review, we also describe the molecular mechanisms of the self-incompatibility system in C. intestinalis type A governed by three allelic gene pairs of s-Themis and v-Themis.

Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4.

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in mice (ZP1–ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.

Dynamic transcriptome analysis of ovarian follicles in artificial maturing Japanese eel (Anguilla japonica)

Inducing maturation of the ovaries to enable the production of good-quality eggs is critical for the successful artificial breeding of Anguilla japonica. During the spawning season, however, the ovaries of A. japonica have been found to develop into asynchronous clutches, impeding the success of artificial breeding on a commercial scale. The dynamic molecular regulation of follicular development in the same individual was assessed by transcriptome analysis of the five stages of follicles, the pre-vitellogenic, early vitellogenic, midvitellogenic, late vitellogenic, and migratory nucleus stages in artificial maturing A. japonica. Comparisons across these developmental stages identified a total of 19,298 differentially expressed transcripts (DETs). Short time-series expression miner analysis across these DETs revealed four significant expression profiles. Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses found that some of the significantly enriched biological processes and metabolic pathways included those related to steroid hormone biosynthesis (cyp11a1, cyp17a1, cyp17a2, hsd17b1, and hsd17b12), cargo receptor activity (vtgr and vldlr), meiosis and ovulation (pgrs and mPRγ), hydration (cts and aqp1), and egg coat formation (zp). These genes and pathways were associated with serum 17β-estradiol concentrations and morphological changes. The levels of hsd17b12 and mPRγ mRNAs were much higher during the migratory nucleus stage, suggesting their respective involvement in the biosynthesis and functional pathway of the maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one. The gene subtypes aqp1b and ctsd may regulate water influx into oocytes and yolk protein proteolysis, respectively. To our knowledge, the present study is the first to describe combined transcriptome profiling of asynchronously developing follicles in the same individual. The findings suggest that steroid hormone synthesis and nutrient absorption in follicular somatic cells play important roles during follicular development and maturation, despite the same external physiological surroundings.

Using machine learning to study protein-protein interactions: From the uromodulin polymer to egg zona pellucida filaments.

Neural network-based models for protein structure prediction have recently reached near-experimental accuracy and are fast becoming a powerful tool in the arsenal of biologists. As suggested by initial studies using RoseTTAFold or the ColabFold implementation of AlphaFold2, a particularly interesting future development will be the optimization of these computational methods to also routinely yield high-confidence predictions of protein-protein interactions. Here I use AlphaFold2 and ColabFold to investigate the activation and polymerization of uromodulin (UMOD)/Tamm-Horsfall protein, a zona pellucida (ZP) module-containing protein whose precursor and filamentous structures have been previously determined experimentally by X-ray crystallography and cryo-EM, respectively. Despite having no knowledge of the UMOD polymer structure (coordinates for which were neither used for model training nor as template), AlphaFold2/ColabFold are able to recapitulate a crucial conformational change underlying UMOD polymerization, as well as the general organization of protein subunits within the resulting filament. This surprising result is achieved by simply deleting from the input sequence a stretch of residues that correspond to a polymerization-inhibiting C-terminal propeptide. By mimicking in silico the activating effect of propeptide dissociation triggered by site-specific proteolysis of the protein precursor, this example has implications for the assembly of egg coat proteins and the many other molecules that also contain a ZP module. Most importantly, it shows the potential of exploiting machine learning not only to accurately predict the structures of individual proteins or complexes, but also to carry out computational experiments replicating specific molecular events.

Proteomics support the threespine stickleback egg coat as a protective oocyte envelope.

Ancestrally marine threespine stickleback fish (Gasterosteus aculeatus) have undergone an adaptive radiation into freshwater environments throughout the Northern Hemisphere, creating an excellent model system for studying molecular adaptation and speciation. Ecological and behavioral factors have been suggested to underlie stickleback reproductive isolation and incipient speciation, but reproductive proteins mediating gamete recognition during fertilization have so far remained unexplored. To begin to investigate the contribution of reproductive proteins to stickleback reproductive isolation, we have characterized the stickleback egg coat proteome. We find that stickleback egg coats are comprised of homologs to the zona pellucida (ZP) proteins ZP1 and ZP3, as in other teleost fish. Our molecular evolutionary analyses indicate that across teleosts, ZP3 but not ZP1 has experienced positive Darwinian selection. Mammalian ZP3 is also rapidly evolving, and surprisingly some residues under selection in stickleback and mammalian ZP3 directly align. Despite broad homology, however, we find differences between mammalian and stickleback ZP proteins with respect to glycosylation, disulfide bonding, and sites of synthesis. Taken together, the changes we observe in stickleback ZP protein architecture suggest that the egg coats of stickleback fish, and perhaps fish more generally, have evolved to fulfill a more protective functional role than their mammalian counterparts.

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.