Formation mechanism of agar-like zona pellucida and its adverse effects on human oocytes
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE239936 | GEO | 2026/04/25
REPOSITORIES: GEO
Similar Datasets
Project description:Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads 0.02 mg/mL, forming a group named 'Trim-away ZP3' group, in triplicate. As a reference, pronuclear-stage oocytes were microinjected with the same mixture as above, except that the antibody buffer was used in lieu of the antibody itself, in triplicate, forming a group named ‚no Trim'. To identify differently expressed genes we compared group 'Trim-away ZP3' with group ‘no Trim’. Ten hours after microinjection, embryos were collected and lysed for transcriptome analysis. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and group ‘no Trim' differed in gene expression and were resolved in principal component analysis. The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development.
2023-10-12 | GSE232142 | GEO
Project description:The zona pellucida (Zp) genes are quintessential oocyte-specific genes that are transcribed only during oogenesis and whose protein products are secreted in the extracellular space. In mice the Zp genes are three: Zp1, Zp2, Zp3 (Zp4 is a pseudogene). Contrary to longtime knowledge, data from 2023 have begun to challenge the notion that the functions of the Zp proteins operate solely in the extracellular space. Doubts began to arise after the results of proteasomal depletion of ZP3 with the Trim-away method (PMID 29153837): mouse zygotes microinjected with a specific antibody anti-ZP3 together with the ubiquitin-protein ligase TRIM21 died within a few hours (PMID 37930049). Given this background, we designed the present study to test whether the knockdown of another protein of the zona pellucida, namely ZP2, would impinge on preimplantation development and transcriptome composition, as previously observed for ZP3. Unlike our previous analysis (GSE278859) this time we have taken into account the additional information that the mRNAs of Zp genes are associated with the ribosomes of pre-implantation mouse embryos (PMID 35697785), which suggests that ZP proteins may be produced de novo after oogenesis. If that were the case, then the depletion of ZP2 by Trim-away alone would not suffice, as the protein amount depleted could be regenerated by new protein synthesis of ZP2. Therefore, this time we combined Trim-away with antisense oligo-mediated inhibition of Zp2 mRNA translation (morpholino). Pronuclear-stage mouse oocytes were microinjected with a cocktail of Trim21-mRNA, antibody anti-ZP2, and morpholino anti-ZP2, or with a cocktail in which antibody and morpholino were both directed against a protein that is not present in wildtype embryos (green fluorescent protein, GFP). We examined three experimental groups the blastocyst stage, in triplicate (R1, R2, R3), as follows. GROUP 1: blastocysts from pronuclear-stage oocytes that were microinjected a cocktail composed of Trim21 mRNA, fluorescent dextran beads, antibody anti-ZP2 and morpholino anti-Zp2, forming a group named “ZP2 knockdown\" (ZP2 KD R1, R2, R3). GROUP 2: blastocysts from pronuclear-stage oocytes were microinjected with a cocktail composed of Trim21 mRNA and fluorescent dextran beads, and cultured further, forming a group named “micromanipulation control\" (MC R1, R2, R3). GROUP 3: blastocysts from pronuclear-stage oocytes were microinjected with a cocktail composed of Trim21 mRNA, fluorescent dextran beads, antibody anti-GFP and morpholino anti-GFP, and cultured further, forming a group named “mock knockdown" (mock KD R1, R2, R3). Approximately eighty hours after microinjection, at the early blastocyst stage, embryos were collected and lysed for transcriptome analysis. GROUPS 1 and 3 were compared with GROUP 2 for mRNAs that are differently expressed (p<0.05; t test); then, the two sets of differently expressed genes were intersected by Venn diagram to identify the genes that are uniquely altered by knockdown of ZP2. These genes that were uniquely altered after ZP2 depletion were subjected to overrepresentation analysis using Webgestalt. Collectively, these data support a conclusion that ZP2 found inside the embryo was not merely a passive remnant from oogenesis, but also served an active functional role during mouse preimplantation development.
2025-12-19 | E-MTAB-16339 | biostudies-arrayexpress
Project description:Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + buffer of ZP3 antibody + dextran beads 0.02 mg/mL as tracer, forming a group named 'Trim21 overexpression', in quadruplicate. As a reference, pronuclear-stage oocytes were microinjected with mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads, forming a group named 'Trim-away ZP3' group, in triplicate. To identify differently expressed genes we compared group 'Trim-away ZP3' with group 'Trim21 overexpression'. In addition, a single 'non-manipulated' sample was also incuded merely to confirm that Trim21 was detectable in the two experimental groups, but not in the non-microinjected embryos. On the day after microinjection, embryos were collected and lysed for transcriptome analysis. Those of group 'Trim21 overexpression' were at the 2-cell stage so as the non-manipulated embryos, whereas those of group 'Trim-away ZP3' were arrested at the 1-cell stage. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and 'group 'Trim21 overexpression' differed in the expression of 197 of 11137 genes (t test, FDR<0.05). The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development.
2023-10-12 | GSE203626 | GEO
Project description:Zona pellucida (ZP) proteins, which play an important role in oocyte development, undergo post-translational regulation through furin-mediated cleavage. However, our understanding of the functional significance of furin-mediated cleavage of ZP proteins in female reproduction remains limited. Here, using mouse models with disrupted furin cleavage sites in ZP1, ZP2, and ZP3, we found that loss of the furin site in ZP2 caused female infertility associated with empty follicle syndrome (EFS), characterized by the absence of oocyte retrieval after ovarian hyperstimulation. In contrast, female mice carrying cleavage-resistant variants at the furin sites of ZP1 and ZP3 exhibited defective zona pellucidae in a subset of oocytes, leading to reduced fecundity. Mechanistically, disruption of the furin cleavage site in ZP2 impaired the transmembrane transport of the non-cleaved ZP2 protein and subsequently reduced the levels of SNARE proteins, ultimately triggering oocyte apoptosis through activation of the p53 and PI3K signaling pathways. Collectively, we uncovered the essential role of furin-mediated cleavage of ZP proteins in female fertility and provided new mechanistic insights into the pathogenesis of EFS. These findings open new avenues for investigating the contribution of post-translational modifications to female reproduction and for developing potential therapeutic strategies to treat female infertility.
2025-10-29 | PXD070013 |
Project description:After the end of the last ice age, 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. Stickleback populations are reproductively isolated to varying degrees, despite the fact that they can be crossed in the lab to produce viable offspring. Ecological and behavioral factors have been suggested to underlie incipient stickleback speciation. However, reproductive proteins represent a previously unexplored driver of speciation. As mediators of gamete recognition during fertilization, reproductive proteins both create and maintain species boundaries. Gamete recognition proteins are also frequently found to be rapidly evolving, and their divergence may culminate in reproductive isolation and ultimately speciation. As an initial investigation into the contribution of reproductive proteins to stickleback reproductive isolation, we characterized the egg coat proteome of threespine stickleback eggs. In agreement with other teleosts, we find that stickleback egg coats are comprised of homologs to the zona pellucida (ZP) proteins ZP1 and ZP3. We explore aspects of stickleback ZP protein biology, including glycosylation, disulfide bonding, and sites of synthesis, and find many substantial differences compared to their mammalian homologs. Furthermore, molecular evolutionary analyses indicate that ZP3, but not ZP1, has experienced positive Darwinian selection across teleost fish. Taken together, these changes to 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.
2021-05-26 | PXD017488 | Pride
Project description:After the end of the last ice age, 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. Stickleback populations are reproductively isolated to varying degrees, despite the fact that they can be crossed in the lab to produce viable offspring. Ecological and behavioral factors have been suggested to underlie incipient stickleback speciation. However, reproductive proteins represent a previously unexplored driver of speciation. As mediators of gamete recognition during fertilization, reproductive proteins both create and maintain species boundaries. Gamete recognition proteins are also frequently found to be rapidly evolving, and their divergence may culminate in reproductive isolation and ultimately speciation. As an initial investigation into the contribution of reproductive proteins to stickleback reproductive isolation, we characterized the egg coat proteome of threespine stickleback eggs. In agreement with other teleosts, we find that stickleback egg coats are comprised of homologs to the zona pellucida (ZP) proteins ZP1 and ZP3. We explore aspects of stickleback ZP protein biology, including glycosylation, disulfide bonding, and sites of synthesis, and find many substantial differences compared to their mammalian homologs. Furthermore, molecular evolutionary analyses indicate that ZP3, but not ZP1, has experienced positive Darwinian selection across teleost fish. Taken together, these changes to 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.
2021-05-26 | PXD017489 | Pride
Project description:Background Fertilization involves interactions between sperm and metaphase II (MII) oocyte, initiating a cascade of events including oocyte activation, calcium release, resumption of meiosis, and zygote formation. Total fertilization failure (TFF), characterized by disruption of any of these processes, occurs in 1-3% of intracytoplasmic sperm injection (ICSI) cycles. The genetic basis of TFF remains largely unexplored. Methods We recruited a familial case of TFF. We assessed sperm parameters, binding ability, and fertilization outcomes using molecular assays and confocal microscopy. The whole genome sequencing and comprehensive genetic analyses were performed on the patients and family members to identify potential loss-of-function variants in genes involved in oogenesis and fertilization. Additionally, single-cell RNA sequencing of the patient's MII oocytes was conducted to assess transcriptomic profile. Two ICSI cycles were performed, one of which included assisted oocyte activation (AOA) using a calcium ionophore. Integrated bioinformatic analysis using RNA-seq and ATAC-seq was used to map genome-wide regulatory landscape in normal oocytes and early embryos. An enhancer-mediated gene regulatory network (eGRN) was constructed to assess the impact of mutations on transcription factor binding and gene regulation across the different GO BP and Reactome pathways. Results Despite normal sperm parameters in the husband and the absence of genetic variants related to oocyte activation in both partners, RNA-seq revealed aberrant expression of genes involved in meiosis, zona pellucida biogenesis, and oocyte activation. RNA-seq and ATAC-seq data identified 22 key transcription factors whose binding is significantly affected by the mutational landscape of the patient’s oocytes and that may disrupt downstream pathways involved in oogenesis, fertilization, and early embryonic development. ZNF121, TFAP2D, VEZF1 and YY1 were the top 4 hub TFs discovered in GO terms network. Additionally, TFAP2D, YY1, ZFP28 and ZNF121 were the most connected TFs to different Reactome pathways. Conclusions Our findings suggest that transcriptional dysregulations play a central role in total fertilization failure (TFF), by impacting downstream gene expression. Although, assisted oocyte activation with calcium ionophore helped successfully achieve fertilization, leading to the development of an 8-cell embryo. The insights from our study provide valuable opportunities for targeted interventions and potential therapeutic strategies in reproductive medicine.
2025-12-03 | GSE289190 | GEO
Project description:Vertebrate oocytes are coated with a special extracellular matrix termed egg-coat or Zona pellucida (ZP). Egg-coat is constituted by several ZP glycoproteins. In chicken fertilization, ejaculated sperm releases enzyme(s) to solubilize the ZP glycoprotein(s) for penetrating the egg-coat. In this study, we analyzed the solubilized ZP glycoprotein fragment. We also analyzed the glycosylation property of its fragment.
2019-08-01 | PXD013994 | JPOST Repository
Project description:A long-standing question in mammalian embryology is whether regional differences of oocyte composition matter for the properties of blastomeres receiving those regions after fertilization. A hitherto untested hypothesis is that allocation depends on the orientation of 1st cleavage. However, the orientation is influenced by the site of sperm entry in the oocyte, which can be almost anywhere on the membrane of oocytes when these are inseminated. Therefore, we harnessed the intracytoplasmic sperm injection to impose the site of fertilization in three specific ooplasmic regions (animal pole, vegetal pole, equator) of mouse oocytes. Notwithstanding this categorical distinction, the sister blastomeres of resultant 2-cell embryos differed from each other in the same way, as measured by gene expression and twin blastocysts formation following bisection. We reasoned that either the oocyte territories did not matter, or their effect was obscured by other factors. To shed light on these possibilities, we immobilized the oocytes on the micromanipulation stage during sperm injection and for 24 h thereafter. Imaging revealed that the orientation of 1st cleavage, instead of depending on the fertilization site, depended on the shorter diameter of the unfertilized oocyte. This even led to the segregation of animal and vegetal hemispheres in the blastomeres of 2-cell embryos. We conclude that 1stcleavage is a manifestation of the intrinsic organization of the oocyte prior to fertilization, and that oocyte geometry factors in to the patterning of mouse embryos more so than fertilization topology.
2024-03-01 | GSE241089 | GEO
Project description:Formation mechanism of agar-like zona pellucida and its adverse effects on human oocytes
| PRJNA1001502 | ENA