ZP1-Y262C mutation causes abnormal zona pellucida formation and female infertility in humans

Defective oocyte maturation is a common cause of female infertility. The loss of the zona pellucida (ZP) represents a specific condition of impaired oocyte maturation. The extracellular matrix known as the ZP envelops mammalian oocytes and preimplantation embryos, exerting significant influence on oogenesis, fertilization, and embryo implantation. However, the genetic factors leading to the loss of the ZP in oocytes are not well understood. This study focused on patients who underwent oocyte retrieval surgery after ovarian stimulation and were found to have abnormal oocyte maturation without the presence of the ZP. Ultrasonography was performed during the surgical procedure to evaluate follicle development. Peripheral blood samples from the patient were subjected to exome sequencing. Here, a novel, previously unreported heterozygous mutation in the ZP1 gene was identified. Within the ZP1 gene, we discovered a novel heterozygous mutation (ZP1 NM_207341.4:c.785A>G (p.Y262C)), specifically located in the trefoil domain. Bioinformatics comparisons further revealed conservation of the ZP1-Y262C mutation across different species. Model predictions of amino acid mutations on protein structure and cell immunofluorescence/western blot experiments collectively confirmed the detrimental effects of the ZP1-Y262C mutation on the function and expression of the ZP1 protein. The ZP1-Y262C mutation represents the novel mutation in the trefoil domain of the ZP1 protein, which is associated with defective oocyte maturation in humans. Our report enhances comprehension regarding the involvement of ZP-associated genes in female infertility and offers enriched understanding for the genetic diagnosis of this condition.


Introduction
Infertility in humans is characterized by the inability to achieve conception following 1 year of unprotected sexual intercourse, with comparable contributions from both males and females to its etiology.Common genetic causes of female infertility include polycystic ovary syndrome (PCOS), oocyte maturation defects (OMD), or premature ovarian failure (POF) (Yatsenko and Rajkovic, 2019).Women with oocyte maturation defects have varying degrees of defects in oocyte maturation, including oocyte absence, oocyte immaturity, follicular atresia, or failure of oocytes to be fertilized.The diplotene stage of prophase I, commonly referred to as the germinal vesicle (GV) stage, represents the period of arrest for primary oocytes within the ovary.Following a surge in luteinizing hormone (LH), GV-stage oocytes initiate meiosis, leading to the extrusion of the first polar body(Pb1), and subsequently halt at the metaphase II (MII) stage of the second meiotic division (Eppig et al., 1996).
Zona pellucida (ZP) deficiency is a specific phenomenon associated with oocyte maturation defects.The ZP is a glycoprotein matrix that forms during oocyte growth and remains present around the oocyte and early embryos until embryo hatching (Avella et al., 2014).Within the follicle, the ZP forms a physical barrier separating the oocyte from the surrounding granulosa cells.Concurrently, the ZP presents in the follicular fluid in anticipation of the arrival of sperm (Carino et al., 2001;Hasegawa and Koyama, 2007).The main constituents of the ZP in humans are four proteins (hZP1-hZP4), each featuring zona pellucida domain (ZPD), transmembrane domain (TMD), conserved furin cleavage site (CFCS), and a signal sequence.Notably, the trefoil domain is exclusive to ZP1 and ZP4 (Jovine et al., 2002;Litscher and Wassarman, 2020a).The composition of the ZP differs between mice and humans.In mice, the ZP is composed of three proteins (mZP1-mZP3).Female mice with homozygous mutations in Zp2 or Zp3 produce oocytes lacking ZP or degenerate oocytes, resulting in complete infertility (Liu et al., 1996;Rankin et al., 1996;Rankin et al., 2001).Female mice with homozygous Zp1 mutations have a reduced number of implanted embryos and decreased litter size (Rankin et al., 1999).
On the other hand, in the context of assisted reproductive treatments, the color, thickness, and refractive index of the zona pellucida (ZP) are often used as indicators of oocyte quality.However, the genetic factors underlying ZP defects remain unclear, and recent advancements in high-throughput sequencing, particularly whole-exome sequencing (WES), have made it possible to identify pathogenic gene mutation sites (Dai et al., 2019;Yuan et al., 2019;Cao et al., 2020;Luo et al., 2020;Okutman et al., 2020;Loeuillet et al., 2022;Pujalte et al., 2023).
Here, through whole-exome sequencing, a novel heterozygous mutation (ZP1 NM_207341.4:c.785A>G(p.Y262C)) situated at the trefoil domain of the ZP1 protein was identified in this investigation.This novel mutation, which we report here, affects the trefoil domain of ZP1.Despite the presence of several large follicles, the patient in this case exhibited oocytes with missing ZP and was unable to undergo fertilization.Confirmation of the functional consequences of this newly identified mutation on the ZP1 protein was obtained through subsequent bioinformatic analysis and protein expression experiments.

Ethical approval
Approval for this study was granted by the Ethics Committee of Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University (2021-384-01).The embryos analyzed were obtained from the Reproductive Medicine Center of Nanjing Drum Tower Hospital, Nanjing University Medical School.All participants providing clinical samples for this study provided informed consent.

Searching for genetic mutations
Peripheral blood samples were collected from the patients for the extraction of genomic DNA.Fragmentation and library preparation were performed on the extracted DNA.The resulting DNA sequences were aligned with the human genome (hg19) to evaluate the coverage and quality of the specific regions of interest.Identified variants underwent bioinformatics analysis to determine their pathogenicity.The classification of variants adhered to the guidelines established by the Human Genome Variation Society (HGVS) (website: http://varnomen.hgvs.org/).Criteria for assessing variant pathogenicity were established based on the variant interpretation standards and guidelines developed by the American College of Medical Genetics and Genomics (Richards et al., 2015;Kalia et al., 2017).It should be acknowledged that our approach may have limitations in identifying potentially pathogenic variants within deep intronic or gene regulatory regions with microvariants (deletions or insertions) spanning less than 10 base pairs.Moreover, our method is not suitable for detecting specific genomic structural variations such as complex rearrangements, large-scale deletions or duplications, inversions, dynamic mutations, or translocations.

Conservation analysis and protein modeling
The IBS 2.0 software was employed to visualize the mutation sites in the ZP1 gene.Conservation analysis of ZP1 amino acids across multiple species, including mouse, macaque, rat, horse, rabbit, and human, was conducted using the Align feature on the UniProt website (https://www.uniprot.org/).Using the SWISS-MODEL website (https://swissmodel.expasy.org),schematic diagrams of the wild-type (WT) and mutant (ZP1, NM_207341.4, c.785A>G (p.Y262C)) ZP1 proteins were generated.The model was based on the reference template (3nk3.1.A.pdb).

Functional impact prediction of mutant proteins
The impaired function of ZP1-Y262C mutations in proteins, including disease-associated variants, was predicted using the PolyPhen-2 tool.PolyPhen-2 (website: http://genetics.bwh.harvard.edu/pph2/), integrates sequence, structure, and conservation information to evaluate the effect of mutations on protein function.It provides predictive scores categorizing the mutations as benign, possibly deleterious, or deleterious.Once loading the amino acid sequences of the ZP1-WT and ZP1-Y262C mutant into these softs and interpreting the results, insights into the impact of the mutation on protein function can be derived.Higher scores, closer to 1, indicate a greater degree of functional impairment.

Changes in ZP1 expression levels
To underscore the significance of ZP1 in the progression of oocyte maturation and embryonic development, we aimed to elucidate the temporal expression patterns of ZP1 across diverse species at distinct developmental stages.Leveraging the singlecell transcriptome repository, we reexamined the spatiotemporal dynamics of ZP1 mRNA expression throughout different embryonic milestones, including 2-PN-zygote, 2-Cell-embryo, 4-Cell-embryo, 8-Cell-embryo, blastocyst, early inner cell mass, and late inner cell mass stages (E-MTAB-7078) (Boroviak et al., 2018).Additionally, the mRNA translatome data was used to reanalyzed dynamic expression of Zp1 mRNA associated with ribosomes in mouse oocytes and embryos (GSE165782) (Xiong et al., 2022).In summary, RNA sequencing library preparation and sequencing steps were performed using the Smart-seq2 protocol (Picelli et al., 2014) for samples at different time points.Initially, M2 medium was used to perform two washes on oocytes or embryos.Subsequently, cell lysis was conducted using Rnase inhibitorcontaining lysis buffer.Following this, cDNA libraries were prepared and subjected to high-throughput sequencing.The alterations in ribosome-associated RNA molecules were depicted using RPF (ribosome-associated RNA expression) profile plots obtained through the utilization of ultra-lowinput Ribo-seq (Ribo-lite) methodology (Xiong et al., 2022).

Plasmid construction, cell culture, and transfection
Human ZP1 and its mutant form (p.Thy262Cys) were generated and integrated into the eukaryotic expression vector pcDNA3.1,with His and GFP tags attached to the C-terminus and N-terminus of ZP1.These constructs were synthesized by Genescript (Nanjing, China).HEK293T cells were maintained in DMEM (Gibco, 12,491,015) supplemented with 100 mg/mL streptomycin/penicillin (Beyotime Biotechnology, C0222) and 10% fetal bovine serum (FBS, Clark, FB25015) under 37 °C and 5% CO2.Upon reaching 80% confluence, cells were transiently transfected with Lipofectamine 2000 reagent (Invitrogen,11,668,019).Following transfection, cells were rinsed with PBS and incubated in serum-free medium for 2 days prior to harvesting.

Protein immunoblotting
Preparation of cell lysates and supernatants involved the utilization of RIPA cell lysis buffer (Thermo Scientific, 89,900).
Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific,23,227).Subsequently, proteins underwent separation on a 10% SDS-PAGE gel and subsequent transfer onto a PVDF membrane.To mitigate non-specific binding, a 2-h room temperature incubation in 5% non-fat milk diluted in Tris-buffered saline containing 0.05% Tween-20 was performed to block the membrane.The PVDF membrane was subsequently incubated overnight at 4 °C with diluted antibodies for GAPDH (Abclonal, AC002) and ZP1 (Santa Cruz, sc-365435).After three washes with TBST, at room temperature, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (diluted 1:10,000) for 1 h in TBST.The proteins were visualized using chemiluminescence (Bio-Rad) after another three rounds of washing the membrane with TBST.

Statistical analysis
Between-group mean comparisons were conducted using one-way analysis of variance (ANOVA).All experiments were carried out three times, and the results are expressed as SEM (mean ± standard error).Statistical significance was determined at a p-value less than 0.05.SPSS 16.0 was utilized for all statistical analyses.

Phenotype of a patient with zona pellucidadeficient oocytes
Upon presentation, a 28-year-old female patient with a 2-year history of primary infertility was admitted to our center.There were no obvious abnormalities in her reproductive organs, including the ovaries and uterus, and we can observe multiple large follicles (Figure 1A).The patient exhibited abnormal oocyte development, with over 10 follicles observed after hormonal stimulation, but only three oocytes could be retrieved.Zona pellucida-deficient oocytes, lacking the transparent layer, were observed in all specimens (Figure 1B).All her hormone levels were within the normal limits, including FSH and AMH.It has been shown by routine semen analysis that her husband has normal fertility potential.Furthermore, the absence of reproductive disorders in the patient's family lineage over two consecutive generations, coupled with the lack of siblings, suggests that her condition appears to be isolated.

Expression of the human ZP1 and mouse Zp1 gene
The expression of the human ZP1 gene is highly pronounced in one-cell embryos with two pronuclei (2PN), and the level of ZP1 expression gradually diminishes.Notably, in mice and human, Zp1/ ZP1 expression is most prominent in 2PN-embryos during various stages of pre-implantation embryo development (Figure 1C) (E-MTAB-7078) (Boroviak et al., 2018).Given the substantial storage of maternal mRNA in oocytes, the ribosome-bound mRNA provides a more comprehensive representation of protein expression alterations.To investigate the dynamic changes of Zp1 during mouse oocyte meiosis, we reanalyzed the recently published ribosome profiling sequencing (Ribo-seq) data (GSE165782) (Xiong et al., 2022).This publicly available Ribo-seq data provides expression levels of mRNA and ribosome-bound RNA at different stages, ranging from oocytes to blastocysts in mice.The analysis reveals that the expression of Zp1 mRNA decreases by approximately 11-fold from the oocyte (FGO) to the 2-pronuclear zygotes (PN3) (Figure 1D).

Effect of ZP1-Y262C mutation on predicted structure
In a patient (II-1) displaying abnormal zona pellucida formation, we successfully identified the heterozygous ZP1 mutation (ZP1-Y262C) via whole-exome sequencing.Subsequent exome sequencing of the patient's father (I-2) confirmed his carrier status for the ZP1 mutation, while the mother (I-1) exhibited a normal genotype (Figures 2A,B).The ZP1-Y262C mutation specifically affects the trefoil domain, a compact structure composed of three intramolecular disulfide bonds formed by six cysteine residues.Infertility-associated mutations in hZP1 have been reported at a minimum of 13 distinct sites, each located within different structural domains of the protein (Figure 2C).Notably, the ZP1-Y262C mutation exhibits conservation across six representative species, including human, mouse, macaque, rat, rabbit and horse (Figure 2D).Differing from previously reported mutation sites, our newly identified mutation occurs at the 262nd amino acid residue, specifically situated within trefoil domain of the ZP1 protein.This novel mutation results in the substitution of tyrosine (Tyr), a phenolic group, with cysteine (Cys), which contains a thiol group (-SH).Consequently, the mutation at the 262nd residue impacts the hydrophobic interactions and polarity of the amino acid, leading to alterations in the protein structure of ZP1 (Figure 2E).Importantly, PolyPhen-2 tool predicts the severe structural damage caused by this mutation, with scores approaching 1.000 (PolyPhen-2 predicted a damaging effect with a score of 1.000 and a specificity of 1.00) (Figure 2F).

Effects of ZP1-Y262C mutation on ZP1 protein expression
The impact of the specific mutation on ZP1 protein expression was evaluated by transfecting HEK293T cells with wild-type and mutant expression plasmids.Immunofluorescence analysis indicated a significant reduction in the number of ZP1-Y262C mutant protein-positive cells compared to equimolar transfection with wild-type plasmids (Figures 3A,B).To further validate the effect of the ZP1-Y262C mutation on ZP1 protein expression, Western blot analysis was conducted (Figures 3C,D).These findings provide evidence that the ZP1-Y262C protein mutation leads to a reduction in ZP1 protein expression.Human-specific sites are highlighted in red, with Y262 position indicated in a box.(E) Simulated spatial structure models of wild-type and mutant ZP1 protein using SWISS-MODEL software.(F) The potential impact of the Y262C variant on the structure and function of human ZP1 protein was predicted using PolyPhen-2.The mutation was predicted to be potentially damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00).

Discussion
Surrounding the oocyte and early embryo, the zona pellucida (ZP) constitutes a multi-layered glycoprotein matrix, ensuring their protection.It plays a crucial role in ensuring recognition between human oocytes and sperm, preventing the entry of sperm from other species.Upon binding to the ZP, sperm can undergo reactions that facilitate penetration into the oocyte.Once a sperm enters the oocyte, the ZP functions to block the entry of other sperm (Litscher and Wassarman, 2020a).However, various issues related to the ZP are commonly observed in clinical settings, including thinning, excessive thickness, dark coloration, appearance of separations, serrations, gel-like consistency, or complete absence of the ZP.Genetic mutations in the ZP proteins (ZP1-4) can lead to ZP thinning or loss, resulting in infertility in patients (Mannikko et al., 2005;Dai et al., 2019;Yuan et al., 2019;Cao et al., 2020;Luo et al., 2020;Okutman et al., 2020;Wassarman and Litscher, 2021;Loeuillet et al., 2022;Shen et al., 2022;Pujalte et al., 2023).
In this paper, whole-exome sequencing was conducted on a patient with characteristic oocyte ZP deficiency following hormonal stimulation.Subsequent analysis led to the identification of a novel heterozygous mutation (ZP1 NM_207341.4:c.785A>G(p.Y262C)) situated within the trefoil domain of the ZP1 protein.Further bioinformatic analysis and functional experiments confirmed that this heterozygous ZP1 mutation led to the dysfunction of ZP1 proteins.
ZP1 plays a crucial role in cross-linking the long fibers that form the matrix of the zona pellucida (ZP).The presence of ZP1 protein is essential for maintaining the structural integrity of the ZP in mice.The N-terminal region of ZP1 in both mice and humans is rich in proline (Pro), providing flexibility to the elasticity of the ZP (Litscher and Wassarman, 2020b).Female mice with Zp1 gene deletion exhibit a reduced number of two-cell embryos compared to the normal control group, and the number of offspring born from Zp1deficient mice is significantly decreased, leading to decreased fertility (Rankin et al., 1999).Some points mutations in the ZP1 protein have been identified to cause functional impairments of ZP1 (Jovine et al., 2005;Litscher and Wassarman, 2020a;Luo et al., 2020;Wassarman and Litscher, 2021;Wu et al., 2021;Loeuillet et al., 2022) (Table 1).Mutations in the ZP1 gene are commonly observed as homozygous or compound heterozygous mutations, with some cases showing heterozygous mutations.Mutations in the ZP1 gene can lead to two abnormal oocyte phenotypes: zona pellucida deficiency and empty follicle syndrome (EFS).The novel mutation we present in this study has been identified in the trefoil domain of ZP1, similar to what were previously reported (Metwalley et al., 2020;Xu et al., 2020).Interestingly, the trefoil domain is unique to the hZP1 protein and is not found in hZP2 and hZP3.This trefoil domain is a compact structure composed of three intramolecular disulfide bonds formed by six cysteine residues (Thim, 1997;Tomasetto et al., 1990).The trefoil domain has also been found in two intracellular enzymes, lysosomal α-glucosidase and sucrase-isomaltase (Thim, 1997;Tomasetto et al., 1990), suggesting its potential role in carbohydrate binding.In addition, mounting evidence suggests that the trefoil domain primarily serves a structural function rather than binding to sperm (Braun et al., 2009).

FIGURE 1
FIGURE 1 Morphological features of ZP1 mutant follicles and dynamic expression of ZP1 gene in different species.(A) Ultrasound images of ZP1 mutant follicles as representative samples.The follicles within the cavity are indicated by "*".Scale bar is 20 μm.(B) The image presented depicts representative pictures of control group oocytes and patient oocytes with zona pellucida defects.Scale bar is 100 µm.(C) Single-cell RNA sequencing (scRNA-seq) transcriptome analysis depicting the dynamic expression of Zp1/ZP1 mRNA from fertilization to inner cell mass (ICM) stage in mouse and human embryos.(D) Dynamic changes in ribosome-associated RNA expression (RPF) of Zp1 mRNA from oocyte stage to ICM stage in mouse.The RPF line graph represents changes in RNA molecules bound to ribosomes using low-input Ribo-seq (Ribo-lite).The RNA line graph represents conventional mRNA sequencing (mRNA-seq).RPF refers to ribosome-protected fragments.Other abbreviations include FGOs (fully-grown oocytes), LPI (late prophase I), MII (metaphase II), PN3 (early one-cell stage), PN5 (late one-cell stage), E2C (early 2-cell stage), L2C (late 2-cell stage), 4C (4-cell stage), 8C (8-cell stage), ICM (inner cell mass), and mESC (mouse embryonic stem cells).

FIGURE 2
FIGURE 2 Pedigree and bioinformatics analysis of the pathogenic mutation.(A) Left panel shows the pedigree of the patient's family with an arrow indicating the patient.(B) DNA sequencing results demonstrates that the father(I-2) and the patient (II-1) are heterozygous at nucleotide 785 (A>G) of the ZP1 gene (NM_207341.4).Heterozygous variation of ZP1 (NM_207341.4,(C)785A>G (p.Y262C)) is highlighted.(C) Schematic representation of the location of the mutation site within the ZP1 structural domains.Previously reported sites are highlighted in yellow, while the newly identified Y262C mutation in this study is marked in red.The ZP domain represents the zona pellucida domain.(D) Residue Y262 in ZP1 protein is highly conserved across six species.Human-specific sites are highlighted in red, with Y262 position indicated in a box.(E) Simulated spatial structure models of wild-type and mutant ZP1 protein using SWISS-MODEL software.(F) The potential impact of the Y262C variant on the structure and function of human ZP1 protein was predicted using PolyPhen-2.The mutation was predicted to be potentially damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00).

FIGURE 3 ZP1-
FIGURE 3 ZP1-Y262C mutation decreases the expression of ZP1 protein.(A) Localization of ZP1 wild-type and ZP1-Y262C mutant proteins in the cell line.Scale bar is 20 µm.(B) Proportion of ZP1 wild-type and ZP1-Y262C mutant positive cells.(C) Western blot experiment validating the expression of ZP1 wild-type and ZP1-Y262C mutant proteins.Significant reduction in the expression of ZP1-Y262C mutant protein is observed.(D) Quantitative grayscale analysis of the western blot experiment in Fig3C.

TABLE 1
Reported mutated sites of the ZP1 gene.Note: NA, represents protein alterations that have not been reported; EFS, refers to Empty Follicle Syndrome; ZP, indicates zona pellucida; * indicates a premature STOP, codon.