RESUMEN
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.
Asunto(s)
Proteínas del Huevo/fisiología , Oocitos/fisiología , Smegmamorpha/metabolismo , Animales , Citoprotección/fisiología , Proteínas del Huevo/metabolismo , Femenino , Oocitos/citología , Oocitos/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Proteómica , Zona Pelúcida/metabolismo , Zona Pelúcida/fisiología , Glicoproteínas de la Zona Pelúcida/análisis , Glicoproteínas de la Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/fisiologíaRESUMEN
OBJECTIVE: To identify the major causative gene(s) of genuine empty follicle syndrome (GEFS) characterized by oocyte degeneration. DESIGN: Genetic and functional studies. SETTING: University-based reproductive medicine center. PATIENT(S): Thirty-five unrelated women with GEFS and oocyte degeneration. INTERVENTION(S): Whole-exome sequencing (WES) and targeted Sanger sequencing. MAIN OUTCOME MEASURE(S): Variants predicted by software and the functional effects of variants assessed via Western blot and immunofluorescence in Chinese hamster ovary (CHO) cells. RESULT(S): We identified zona pellucida (ZP) gene variants in 18 individuals, which included 20 variants in the ZP1 gene, two variants in the ZP2 gene, and one previously reported recurrent variant in the ZP3 gene. The women carrying ZP variants constituted 51.43% of the GEFS cohort. The ZP1 variants were inherited in an autosomal recessive pattern; the ZP2 and ZP3 variants were inherited in an autosomal dominant pattern. All variants were predicted to be deleterious. Studies in CHO cells suggested that most ZP1 variants led to increased intracytoplasmic protein and some variants influenced the intracellular transportation of other ZP proteins. Variant p.R642Q of ZP2 caused the secretion of ZP2 protein with an increased molecular weight, suggesting altered protein modification. Variant p.I619N of ZP2 resulted in increased ZP2 protein in cell lysate and decreased ZP2 protein in culture medium. These results showed that ZP variants might block the intracellular transportation and secretion of ZP proteins and disrupt the zona pellucida. CONCLUSION(S): We identified novel variants of ZP genes in more than half the cohort with GEFS and oocyte degeneration. Variants of ZP genes caused protein intracellular sequestration and failure to assemble the ZP filaments, resulting in EFS and female infertility. Our findings not only reveal the critical roles of ZP genes but also pave the way for the efficient genetic diagnosis of females with GEFS and oocyte degeneration.
Asunto(s)
Muerte Celular/genética , Infertilidad Femenina/genética , Oocitos/fisiología , Enfermedades del Ovario/genética , Glicoproteínas de la Zona Pelúcida/genética , Adulto , Animales , Células CHO , Cricetinae , Cricetulus , Análisis Mutacional de ADN , Familia , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Infertilidad Femenina/patología , Mutación , Oocitos/patología , Folículo Ovárico/patología , Linaje , Zona Pelúcida/metabolismo , Zona Pelúcida/patología , Glicoproteínas de la Zona Pelúcida/fisiologíaRESUMEN
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.
Asunto(s)
Pollos , Proteínas del Huevo/fisiología , Glicoproteínas de la Zona Pelúcida/fisiología , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Pollos/metabolismo , Pollos/fisiología , Proteínas del Huevo/química , Femenino , Masculino , Modelos Biológicos , Óvulo/química , Conformación Proteica , Interacciones Espermatozoide-Óvulo/fisiología , Zona Pelúcida/química , Zona Pelúcida/fisiología , Glicoproteínas de la Zona Pelúcida/químicaRESUMEN
Mammals evolved from oviparous reptiles that laid eggs in a dry, terrestrial environment, thus requiring large amounts of yolk to support development and tough, outer coats to protect them. Eutherian mammals such as humans and mice exhibit an "extreme" form of viviparity in which yolk and conceptus coats have become largely redundant. However, the "other" mammals-monotremes and marsupials-have retained and modified some features of reptilian development that provide valuable insights into the evolution of viviparity in mammals. Most striking of these are the conceptus coats, which include the zona pellucida, the mucoid coat, and the shell coat. We discuss current knowledge of these coats in monotremes and marsupials, their possible roles, and recently identified components such as the zona pellucida protein ZPAX, conceptus coat mucin (CCM), and nephronectin (NPNT).
Asunto(s)
Embrión de Mamíferos/química , Marsupiales/embriología , Monotremata/embriología , Glicoproteínas de la Zona Pelúcida/fisiología , Cigoto/metabolismo , Animales , Proteínas del Huevo/química , Proteínas del Huevo/fisiología , Embrión de Mamíferos/metabolismo , Óvulo/química , Óvulo/metabolismo , Zona Pelúcida/química , Zona Pelúcida/fisiología , Glicoproteínas de la Zona Pelúcida/química , Cigoto/químicaRESUMEN
Human zona pellucida (ZP) matrix, a delicate network of thin interconnected filaments, is primarily composed of four glycoproteins, namely, ZP1, ZP2, ZP3, and ZP4. All four zona proteins share common structural elements such as signal peptide, "ZP domain," consensus furin cleavage site, transmembrane-like domain, and short cytoplasmic tail. In addition, ZP1 and ZP4 also have "Trefoil domain." Recombinant/native human zona proteins have been used to investigate their binding characteristics to the capacitated and/or acrosome-reacted spermatozoa. These investigations revealed that ZP1, ZP3, and ZP4 primarily bind to the head region of the capacitated human spermatozoa, whereas ZP2 binds to the acrosome-reacted sperm. However, using transgenic mice, N-terminal region of human ZP2 has also been shown to play an important role in binding of sperm to the egg. ZP1, ZP3, and ZP4 lead to dose-dependent increase in acrosome reaction, suggesting that in humans more than one ZP glycoprotein is responsible for induction of acrosome reaction. Glycosylation of these proteins, in particular, N-linked glycosylation as well as sialyl-Lewisx, is essential for inducing acrosome reaction. Studies delineating downstream signaling events associated with induction of acrosome reaction reveal subtle differences between ZP3 and ZP1/ZP4 with respect to activation of Gi protein-coupled receptor and protein kinase A. The role of mutations in the zona proteins and ZP autoantibodies leading to infertility in women is suggestive and needs more rigorous experimentations for confirming their role in female infertility. The above-mentioned aspects of the human ZP glycoproteins have been discussed in this review.
Asunto(s)
Zona Pelúcida/fisiología , Autoanticuerpos/sangre , Femenino , Fertilización/fisiología , Glicoproteínas/química , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Glicosilación , Humanos , Infertilidad Femenina/sangre , Infertilidad Femenina/inmunología , Masculino , Dominios Proteicos , Procesamiento Proteico-Postraduccional/fisiología , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/química , Glicoproteínas de la Zona Pelúcida/inmunología , Glicoproteínas de la Zona Pelúcida/fisiologíaRESUMEN
The egg coat, an extracellular matrix made up of glycoprotein filaments, plays a key role in animal fertilization by acting as a gatekeeper for sperm. Egg coat components polymerize using a common zona pellucida (ZP) "domain" module that consists of two related immunoglobulin-like domains, called ZP-N and ZP-C. The ZP module has also been recognized in a large number of other secreted proteins with different biological functions, whose mutations are linked to severe human diseases. During the last decade, tremendous progress has been made toward understanding the atomic architecture of the ZP module and the structural basis of its polymerization. Moreover, sperm-binding regions at the N-terminus of mollusk and mammalian egg coat subunits were found to consist of domain repeats that also adopt a ZP-N fold. This discovery revealed an unexpected link between invertebrate and vertebrate fertilization and led to the first structure of an egg coat-sperm protein recognition complex. In this review we summarize these exciting findings, discuss their functional implications, and outline future challenges that must be addressed in order to develop a comprehensive view of this family of biomedically important extracellular molecules.
Asunto(s)
Glicoproteínas de la Zona Pelúcida/química , Secuencia de Aminoácidos , Animales , Femenino , Fertilización/fisiología , Humanos , Masculino , Dominios Proteicos , Multimerización de Proteína/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/fisiologíaRESUMEN
All animal oocytes are surrounded by a glycoproteinaceous egg coat, a specialized extracellular matrix that serves both structural and species-specific roles during fertilization. Egg coat glycoproteins polymerize into the extracellular matrix of the egg coat using a conserved protein-protein interaction module-the zona pellucida (ZP) domain-common to both vertebrates and invertebrates, suggesting that the basic structural features of egg coats have been conserved across hundreds of millions of years of evolution. Egg coat proteins, as with other proteins involved in reproduction, are frequently found to be rapidly evolving. Given that gamete compatibility must be maintained for the fitness of sexually reproducing organisms, this finding is somewhat paradoxical and suggests a role for adaptive diversification in reproductive protein evolution. Here we review the structure and function of metazoan egg coat proteins, with an emphasis on the potential role their evolution has played in the creation and maintenance of species boundaries.
Asunto(s)
Evolución Biológica , Proteínas del Huevo/química , Proteínas del Huevo/metabolismo , Glicoproteínas de la Zona Pelúcida/química , Glicoproteínas de la Zona Pelúcida/metabolismo , Animales , Proteínas del Huevo/fisiología , Femenino , Humanos , Invertebrados/química , Invertebrados/embriología , Invertebrados/metabolismo , Dominios Proteicos , Multimerización de Proteína/fisiología , Vertebrados/embriología , Vertebrados/metabolismo , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/fisiologíaRESUMEN
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.
Asunto(s)
Pollos/anatomía & histología , Ovario/anatomía & histología , Óvulo/fisiología , Glicoproteínas de la Zona Pelúcida/fisiología , Animales , Pollos/fisiología , Femenino , Ovario/fisiología , Oviposición , Glicoproteínas de la Zona Pelúcida/clasificaciónRESUMEN
Epithelia are bound by both basal and apical extracellular matrices (ECM). Although the composition and function of the former have been intensively investigated, less is known about the latter. The embryonic sheath, the ECM apical to the Caenorhabditis elegans embryonic epidermis, has been suggested to promote elongation of the embryo. In an RNAi screen for the components of the sheath, we identified the zona pellucida domain proteins NOAH-1 and NOAH-2. We found that these proteins act in the same pathway, and in parallel to three other putative sheath proteins, the leucine-rich repeat proteins SYM-1, LET-4 and FBN-1/Fibrillin, to ensure embryonic integrity and promote elongation. Laser nano-ablation experiments to map the stress field show that NOAH-1 and NOAH-2, together with PAK-1/p21-activated kinase, maintain and relay the actomyosin-dependent stress generated within the lateral epidermis before muscles become active. Subsequently, loss-of-function experiments show that apical ECM proteins are essential for muscle anchoring and for relaying the mechanical input from muscle contractions, which are essential for elongation. Hence, the apical ECM contributes to morphogenesis by maintaining embryonic integrity and relaying mechanical stress.