RESUMEN
EXOC5 is a crucial component of a large multi-subunit tethering complex, the exocyst complex, that is required for fusion of secretory vesicles with the plasma membrane. Exoc5 deleted mice die as early embryos. Therefore, to determine the role of EXOC5 in follicular and oocyte development, it was necessary to produce a conditional knockout (cKO), Zp3-Exoc5-cKO, in which Exoc5 was deleted only in oocytes. The first wave of folliculogenesis appeared histologically normal and progressed to the antral stage. However, after IVF with normal sperm, oocytes collected from the first wave (superovulated 21-day-old cKO mice) were shown to be developmentally incompetent. Adult follicular waves did not progress beyond the secondary follicle stage where they underwent apoptosis. Female cKO mice were infertile. Overall, these data suggest that the first wave of folliculogenesis is less sensitive to oocyte-specific loss of Exoc5, but the resulting gametes have reduced developmental competence. In contrast, subsequent waves of folliculogenesis require oocyte-specific Exoc5 for development past the preantral follicle stage. The Zp3-Exoc5-cKO mouse provides a model for disrupting folliculogenesis that also enables the separation between the first and subsequent waves of folliculogenesis.
Asunto(s)
Ratones Noqueados , Oocitos , Oogénesis , Folículo Ovárico , Animales , Femenino , Oogénesis/genética , Oogénesis/fisiología , Oocitos/metabolismo , Ratones , Folículo Ovárico/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Masculino , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismoRESUMEN
Autoimmune factors play an important role in premature ovarian insufficiency (POI). Human amniotic epithelial stem cells (hAESCs) have recently shown promising treatment effects on chemotherapy-induced POI. However, the therapeutic efficacy and underlying mechanisms of hAESCs in autoimmune POI remain to be investigated. In this study, we showed for the first time that intravenous transplantation of hAESCs could reside in the ovary of zona pellucida 3 peptide (pZP3) induced autoimmune POI mice model for at least 4 weeks. hAESCs could improve ovarian function and fertility, alleviate inflammation and reduce apoptosis of granulosa cells (GCs) in autoimmune POI mice. The transcriptome analysis of mice ovaries and in vitro co-cultivation experiments suggest that activation of the AKT and ERK pathways may be the key mechanism in the therapeutic effect of hAESCs. Our work provides the theoretical and experimental foundation for optimizing the administration of hAESCs, as well as the clinical application of hAESCs in autoimmune POI patients.
Asunto(s)
Amnios , Células Epiteliales , Células de la Granulosa , Sistema de Señalización de MAP Quinasas , Insuficiencia Ovárica Primaria , Proteínas Proto-Oncogénicas c-akt , Células Madre , Femenino , Animales , Insuficiencia Ovárica Primaria/terapia , Insuficiencia Ovárica Primaria/patología , Insuficiencia Ovárica Primaria/metabolismo , Humanos , Ratones , Amnios/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células de la Granulosa/metabolismo , Células Epiteliales/metabolismo , Células Madre/metabolismo , Células Madre/citología , Modelos Animales de Enfermedad , Trasplante de Células Madre , Apoptosis , Enfermedades Autoinmunes/terapia , Enfermedades Autoinmunes/patología , Glicoproteínas de la Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/genéticaAsunto(s)
Fertilización , Fertilización/fisiología , Animales , Humanos , Femenino , Masculino , Zona Pelúcida/metabolismo , Zona Pelúcida/fisiología , Óvulo/metabolismo , Glicoproteínas de la Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/genética , Oocitos/metabolismo , Oocitos/fisiología , Interacciones Espermatozoide-Óvulo/fisiologíaRESUMEN
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.
Asunto(s)
Glicoproteínas de la Zona Pelúcida , Humanos , Masculino , Semen , Espermatozoides/química , Espermatozoides/metabolismo , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/química , Glicoproteínas de la Zona Pelúcida/metabolismo , Óvulo/química , Óvulo/metabolismo , FemeninoRESUMEN
The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2 (ZP2), a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding, and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing ovastacin-substrate interactions as well as the individual characteristics that distinguish ovastacin from similar proteases, such as meprins and tolloid. Eventually, we identified several substrates whose cleavage could affect mammalian fertilization. Consequently, these substrates indicate newly identified functions of ovastacin in mammalian fertilization beyond zona pellucida hardening.
Asunto(s)
Fibroblastos , Semen , Masculino , Animales , Ratones , Glicoproteínas de la Zona Pelúcida/metabolismo , Fibroblastos/metabolismo , Semen/metabolismo , Metaloproteasas/metabolismo , Mamíferos/metabolismo , Endopeptidasas , Fertilización/fisiologíaRESUMEN
Mammalian fertilization is a species-selective event that involves a series of interactions between sperm proteins and the oocyte's zona pellucida (ZP) glycoproteins. Bovine ZP consists of three glycoproteins: bZP2, bZP3, and bZP4. In our previous study, we demonstrated that bovine sperm binds to plastic wells coated with recombinant bZP4 and identified that the N-terminal domain and the middle region of bZP4 are critical for sperm-binding activity. Here, we investigated the sperm-binding site in the middle region (residues 290 to 340) of bZP4, which includes the hinge region. We showed that bovine sperm binds to bZP4's middle region in a species-selective manner. We mapped the function of bZP4's middle region to its N-glycosylation site at Asn-314 using several recombinant mutated proteins. Moreover, we showed that mutations of the N-glycosylation sites at Asn-314 close to the hinge region and Asn-146 of the hinge region of bZP4 and bZP3, respectively, reduced the sperm-binding activity of the complex of the bZP3 (from 32 to 178) and bZP4 (from 136 to 464) fragments. Together, these results suggest that ZP's middle regions of bZP3 and bZP4 form one of the sperm-binding sites of bovine ZP.
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Glicoproteínas de Membrana , Receptores de Superficie Celular , Masculino , Bovinos , Animales , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Glicosilación , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas del Huevo/genética , Proteínas del Huevo/química , Proteínas del Huevo/metabolismo , Zona Pelúcida/metabolismo , Semen/metabolismo , Espermatozoides/metabolismo , Glicoproteínas/metabolismo , Mamíferos/metabolismoRESUMEN
In oocyte biology, the zona pellucida has long been known to operate three extracellular functions downstream of the secretory pathway, namely, encasing the oocytes in ovarian follicles, mediating sperm-oocyte interaction, and preventing premature embryo contact with oviductal epithelium. The present study uncovers a fourth function that is fundamentally distinct from the other three, being critical for embryonic cell survival in mice. Intriguingly, the three proteins of the mouse zona pellucida (ZP1, ZP2, ZP3) were found abundantly present also inside the embryo 4 days after fertilization, as shown by mass spectrometry, immunoblotting, and immunofluorescence. Contrary to current understanding of the roles of ZP proteins, ZP3 was associated more with the cytoskeleton than with secretory vesicles in the subcortical region of metaphase II oocytes and zygotes, and was excluded from regions of cell-cell contact in cleavage-stage embryos. Trim-away-mediated knockdown of ZP3 in fertilized oocytes hampered the first zygotic cleavage, while ZP3 overexpression supported blastocyst formation. Transcriptome analysis of ZP3-knockdown embryos pointed at defects of cytoplasmic translation in the context of embryonic genome activation. This conclusion was supported by reduced protein synthesis in the ZP3-knockdown and by the lack of cleavage arrest when Trim-away was postponed from the one-cell to the late two-cell stage. These data place constraints on the notion that zona proteins only operate in the extracellular space, revealing also a role during the oocyte-to-embryo transition. Ultimately, these data recruit ZP3 into the family of maternal factors that contribute to developmental competence of mouse oocytes.
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Semen , Zona Pelúcida , Femenino , Ratones , Masculino , Animales , Zona Pelúcida/metabolismo , Semen/metabolismo , Oocitos/metabolismo , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Folículo Ovárico/metabolismoRESUMEN
Membrane expansion integrates multiple forces to mediate precise tube growth and network formation. Defects lead to deformations, as found in diseases such as polycystic kidney diseases, aortic aneurysms, stenosis, and tortuosity. We identified a mechanism of sensing and responding to the membrane-driven expansion of tracheal tubes. The apical membrane is anchored to the apical extracellular matrix (aECM) and causes expansion forces that elongate the tracheal tubes. The aECM provides a mechanical tension that balances the resulting expansion forces, with Dumpy being an elastic molecule that modulates the mechanical stress on the matrix during tracheal tube expansion. We show in Drosophila that the zona pellucida (ZP) domain protein Piopio interacts and cooperates with the ZP protein Dumpy at tracheal cells. To resist shear stresses which arise during tube expansion, Piopio undergoes ectodomain shedding by the Matriptase homolog Notopleural, which releases Piopio-Dumpy-mediated linkages between membranes and extracellular matrix. Failure of this process leads to deformations of the apical membrane, tears the apical matrix, and impairs tubular network function. We also show conserved ectodomain shedding of the human TGFß type III receptor by Notopleural and the human Matriptase, providing novel findings for in-depth analysis of diseases caused by cell and tube shape changes.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Humanos , Drosophila/metabolismo , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/metabolismo , Proteínas de Drosophila/metabolismo , Proteolisis , Matriz Extracelular/metabolismo , Estructuras de la Membrana Celular/metabolismo , Tráquea/metabolismoRESUMEN
Independent cell volume regulation is first acquired by the oocyte in two steps that occur during meiotic maturation: (1) activation of the glycine transporter GLYT1 (Slc6a9) that mediates the intracellular accumulation of glycine to provide osmotic support in the mature egg and early preimplantation embryo, and (2) release of the oocyte from the strong attachment to its rigid extracellular matrix shell, the zona pellucida (ZP). It was recently shown that oocyte-ZP detachment requires metallopeptidase activity that is proposed to cleave transmembrane ZP proteins connecting the oocyte to the ZP. It is unknown, however, how GLYT1 is activated. We hypothesized that oocyte-ZP detachment precedes and may be required for GLYT1 activation. In identically treated pools of oocytes, oocyte-ZP detachment occurred ~20 min before GLYT1 activation. In individual oocytes, GLYT1 activity was detected only in those that were mostly or fully detached. Blocking detachment using previously validated small molecule metallopeptidase inhibitors partly suppressed GLYT1 activation. However, removal of the ZP did not accelerate GLYT1 activation. This indicates that oocyte-ZP detachment or cleavage of transmembrane ZP proteins may be required for GLYT1 to become fully activated, or alternatively that metallopeptidase activity independently affects both detachment and GLYT1 activation.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática , Zona Pelúcida , Zona Pelúcida/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Glicoproteínas de la Zona Pelúcida/metabolismo , Oocitos/metabolismo , Metaloproteasas/metabolismo , Tamaño de la CélulaRESUMEN
The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.
Asunto(s)
Pollos , Glicoproteínas de Membrana , Femenino , Animales , Glicoproteínas de la Zona Pelúcida , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Pollos/genética , Proteínas del Huevo/química , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Receptores de Superficie Celular , EstrógenosRESUMEN
Introduction: Human zona pellucida (ZP) plays an important role in reproductive process. Several rare mutations in the encoding genes (ZP1, ZP2, and ZP3) have been demonstrated to cause women infertility. Mutations in ZP2 have been reported to cause ZP defects or empty follicle syndrome. We aimed to identify pathogenic variants in an infertile woman with a thin zona pellucida (ZP) phenotype and investigated the effect of ZP defects on oocyte gene transcription. Methods: We performed whole-exome sequencing and Sanger sequencing of genes were performed for infertilite patients characterized by fertilization failure in routine in vitro fertilization (IVF). Immunofluorescence (IF) and intracytoplasmic sperm injection (ICSI) were used in the mutant oocytes. Single-cell RNA sequencing was used to investigate transcriptomes of the gene-edited (Zp2mut/mut) rat model. Biological function enrichment analysis, quantitative real-time PCR (qRT-PCR), and IF were performed. Results: We identified a novel homozygous nonsense mutation of ZP2 (c.1924C > T, p.Arg642X) in a patient with non-consanguineous married parents. All oocytes showed a thin or no ZP under a light microscope and were fertilized after ICSI. The patient successfully conceived by receiving the only two embryos that developed to the blastocyst stage. The immunofluorescence staining showed an apparently abnormal form of the stopped oocytes. We further demonstrated a total of 374 differentially expressed genes (DEGs) in the transcriptome profiles of Zp2mut/mut rats oocytes and highlighted the signal communication between oocytes and granulosa cells. The pathway enrichment results of DEGs showed that they were enriched in multiple signaling pathways, especially the transforming growth factor-ß (TGF-ß) signaling pathway in oocyte development. qRT-PCR, IF, and phosphorylation analysis showed significantly downregulated expressions of Acvr2b, Smad2, p38MAPK, and Bcl2 and increased cleaved-caspase 3 protein expression. Discussion: Our findings expanded the known mutational spectrum of ZP2 associated with thin ZP and natural fertilization failure. Disruption of the integrity of the ZP impaired the TGF-ß signaling pathway between oocytes and surrounding granulosa cells, leading to increased apoptosis and decreased developmental potential of oocytes.
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Semen , Zona Pelúcida , Humanos , Masculino , Femenino , Ratas , Animales , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Semen/metabolismo , Mutación , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3'-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 µg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3'-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(ß1-4)GlcNAc further supports the abundance of 3'-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3'-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes.
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Búfalos , Zona Pelúcida , Femenino , Masculino , Animales , Zona Pelúcida/metabolismo , Búfalos/metabolismo , Semen/metabolismo , Espermatozoides/metabolismo , Fertilización/fisiología , Polisacáridos , Glicoproteínas de la Zona Pelúcida , Lectinas/metabolismo , Oviductos/metabolismo , Trisacáridos/metabolismo , Trisacáridos/farmacología , Epitelio/metabolismo , Interacciones Espermatozoide-ÓvuloRESUMEN
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.
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Reacción Acrosómica , Tortugas , Animales , Masculino , Acrosoma/metabolismo , Proteínas del Huevo/genética , Mamíferos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reptiles/metabolismo , Semen/metabolismo , Espermatozoides/metabolismo , Tortugas/genética , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , FemeninoRESUMEN
Fertilization is a fundamental process of development, and the blocking mechanisms act at the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, permeating and fusing after fertilization. In clinical practice, some couples undergoing recurrent IVF failures that mature oocytes had abnormal fertilization for unknown reason. Ovastacin encoded by ASTL cleave the ZP protein ZP2 and play a key role in preventing polyspermy. Here, we identified bi-allelic variants in ASTL that are mainly characterized by fertilization problems in humans. All four independent affected individuals had bi-allelic frameshift variants or predicted damaging missense variants, which follow a Mendelian recessive inheritance pattern. The frameshift variants significantly decreased the quantity of ASTL protein in vitro. And all missense variants affected the enzymatic activity that cleaves ZP2 in mouse egg in vitro. Three knock-in female mice (corresponding to three missense variants in patients) all show subfertility due to low embryo developmental potential. This work presents strong evidence that pathogenic variants in ASTL cause female infertility and provides a new genetic marker for the diagnosis of fertilization problems.
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Infertilidad Femenina , Semen , Humanos , Masculino , Femenino , Ratones , Animales , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Semen/metabolismo , Oocitos/metabolismo , Infertilidad Femenina/genética , Fertilización/genética , Metaloproteasas/genéticaRESUMEN
Teleost oocytes are surrounded by a structure called chorion or egg envelopes, which is composed of zona pellucida (ZP) proteins. As a result of the gene duplication in teleost, the expression site of the zp genes, coding the major component protein of egg envelopes, changed from the ovary to the maternal liver. In Euteleostei, there are three liver-expressed zp genes, named choriogenin (chg) h, chg hm, and chg l, and the composition of the egg envelope is mostly made up of these Chgs. In addition, ovary-expressed zp genes are also conserved in the medaka genomes, and their proteins have also been found to be minor components of the egg envelopes. However, the specific role of liver-expressed versus ovary-expressed zp genes was unclear. In the present study, we showed that ovary-synthesized ZP proteins first form the base layer of the egg envelope and then Chgs polymerize inwardly to thicken the egg envelope. To analyze the effects of dysfunction of the chg gene, we generated some chg knockout medaka. All knockout females failed to produce normally fertilized eggs by the natural spawning. The egg envelopes lacking Chgs were significantly thinner, but layers formed by ZP proteins synthesized in the ovary were found in the thin egg envelope of knockout as well as wildtype eggs. These results suggest that the ovary-expressed zp gene is well conserved in all teleosts, including those species in which liver-derived ZP proteins are the major component, because it is essential for the initiation of egg envelope formation.
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Proteínas de Peces , Hígado , Oryzias , Ovario , Glicoproteínas de la Zona Pelúcida , Animales , Femenino , Secuencia de Aminoácidos , Hígado/metabolismo , Oryzias/genética , Oryzias/metabolismo , Ovario/anatomía & histología , Ovario/metabolismo , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Técnicas de Inactivación de Genes , Expresión Génica , Óvulo/citología , Óvulo/metabolismoRESUMEN
RESEARCH QUESTION: Which genetic variants might explain the causes of empty follicle syndrome (EFS) and abnormal zona pellucida (ZP) and affect the success of treatment with assisted reproductive technologies (ART)? DESIGN: Whole-exome sequencing was performed in probands with EFS and abnormal ZP. Sanger sequencing was used for variant validation. Using HEK-293T cells, the effects of ZP1 and ZP2 variants on protein expression were explored by western blotting, and the effect of the ZP1 variant on protein location was investigated via immunofluorescence. The protein structure was also analysed to investigate the pathogenicity of variants. RESULTS: A homozygous nonsense variant in ZP1 (c.874C>T, p.Gln292*) was detected in a patient with EFS. A novel homozygous frameshift variant in ZP2 (c.836_837delAG, p.Glu279Valfs*6) and a novel heterozygous missense variant in ZP3 (c.1159G>A, p.Val387Met) were identified in two patients with ZP morphological abnormalities, respectively. Western blotting and immunofluorescence analysis showed that the ZP1 variant results in a premature stop codon, leading to the truncated ZP1 protein. The ZP2 variant, which is situated in the N-terminus, triggers the degradation of a premature termination protein. Additionally, the patient with the ZP3 variant achieved clinical pregnancy following intracytoplasmic sperm injection treatment. CONCLUSIONS: These findings expand the mutational spectrum of ZP1, ZP2 and ZP3, and provide new evidence for genetic diagnosis of female infertility. The targeted genetic diagnosis of ZP genes is recommended to choose appropriate fertilization methods and improve success rates of treatment with ART.
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Enfermedades del Ovario , Zona Pelúcida , Embarazo , Humanos , Masculino , Femenino , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Semen , Heterocigoto , Mutación , Enfermedades del Ovario/genética , Enfermedades del Ovario/metabolismoRESUMEN
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.
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Espectrometría de Masas en Tándem , Zona Pelúcida , Animales , Femenino , Zona Pelúcida/metabolismo , Proteínas del Huevo/metabolismo , Glicoproteínas de la Zona Pelúcida/metabolismo , Pollos/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Mamíferos/metabolismoRESUMEN
Reproductive strategies and spawning habits play key roles in the evolution of endemic East Asian cyprinids. However, the molecular mechanisms underlying the regulation of spawning habits are not well understood. We recently identified zona pellucida (Zp) as the top differentially expressed protein between East Asian cyprinids that produce adhesive and semi-buoyant eggs, suggesting that Zp protein may play important roles in the regulation of egg type. In this work, we generated transgenic zebrafish in which oocyte-specific expression of zp genes from rare minnow ( Gobiocypris rarus), an East Asian cyprinid laying adhesive eggs, was driven by a zebrafish zp3.2 gene promoter. We found that the transgenic eggs obtained partial adhesiveness and exhibited alteration in hydration and buoyancy. Abnormal metabolism of vitellogenin (VTG) may contribute to enhanced hydration and/or buoyancy. Our work shows that expression of the exogenous zp3a gene from an adhesive-egg producing fish is sufficient to induce changes in both egg adhesiveness and buoyancy in zebrafish, emphasizing the important role of zp genes in the regulation of spawning habits. Our results thus provide new insights into how endemic East Asian cyprinids may have adapted to the Yangtze river-lake system via changes in spawning habits.
Asunto(s)
Cyprinidae , Pez Cebra , Animales , Pez Cebra/genética , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Adhesividad , Receptores de Superficie Celular/genética , Animales Modificados Genéticamente/genéticaRESUMEN
Genuine empty follicle syndrome (gEFS) is a rare cause of female infertility; it is defined as the presence of cumulus-oocyte complexes (COCs) in follicular fluid but the absence of oocytes after denudation in an in vitro fertilization (IVF) programme. Mutations in one of the four genes encoding zona pellucida (ZP) proteins have been implicated in gEFS. The objectives of the present study were to explore the molecular basis of idiopathic infertility in a 35-year-old woman with gEFS (observed after four ovarian retrievals), compare her phenotype and genotype with those of other patients described in the literature, and discuss therapeutic approaches that could be adopted by reproductive health centres in this situation. Sequencing of the ZP genes revealed a new homozygous missense variant in ZP1: c.1097G > A;p.(Arg366Gln). The variant is located in the ZP-N domain, which is essential for ZP protein polymerization. An immunohistochemical assessment of an ovarian biopsy confirmed the absence of ZP1 protein. The novel variant appears to prevent ZP assembly, which would explain the absence of normal oocytes after denudation in our patient (and despite the retrieval of COCs). ZP gene sequencing should be considered for patients with a phenotype suggestive of gEFS. An etiological genetic diagnosis enables appropriate genetic counselling and a switch to an IVF programme (with a suitable denudation technique) or an oocyte donation programme.
Asunto(s)
Oocitos , Zona Pelúcida , Humanos , Femenino , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Oocitos/metabolismo , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Zona Pelúcida/patología , Mutación , GenotipoRESUMEN
The low maturation rate of oocytes is an important reason for female infertility and failure of assisted pregnancy. The germinal vesicle breakdown (GVBD) is a landmark event of oocyte maturation. In our previous studies, we found that zona pellucida 3 (ZP3) was strongly concentrated in the nuclear region of germinal vesicle (GV) oocytes and interacted with aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) and lamin A to promote GVBD. In the current study, we found that lamin A is mainly concentrated in the nuclear membrane. When ZP3 is knocked down, lamin A will be partially transferred to the nucleus of oocytes. The prelamin A is increased in both the nuclear membrane and nucleus, while phosphorylated lamin A (p-lamin A) is significantly reduced. AIPL1 was also proved to accumulate in the GV region of oocytes, and ZP3 deletion can significantly inhibit the aggregation of AIPL1 in the nuclear region. Similar to ZP3 knockdown, the absence of AIPL1 resulted in a decrease in the occurrence of GVBD, an increase in the amount of prelamin A, and a significant decrease in p-lamin A in oocytes developed in vitro. Finally, we propose the hypothesis that ZP3 can stabilize farnesylated prelamin A on the nuclear membrane of AIPL1, and promote its further processing into mature lamin A, therefore promoting the occurrence of GVBD. This study may be an important supplement for the mechanism of oocyte meiotic resumption and provide new diagnostic targets and treatment clues for infertility patients with oocyte maturation disorder.