case_images: PMC10613883_01
case_images: Morphological characteristics of mutant ZP3 follicles and dynamic expression of ZP3 genes in different species. (A) The illustration presents an ultrasound image as a representative sample of a ZP3 mutant follicle. The luminal follicles are denoted by "*". The scale bar is 20 mm. (B) The table shows the number and retrieval period of oocytes obtained from three oocyte retrieval procedures in this particular patient. (C) Single-cell RNA sequencing (scRNA-seq) transcriptome analysis exhibits the dynamics of ZP3 mRNA from the zygote stage to the inner cell mass (ICM) stage in mice, marmosets, and humans. (D) The dynamic changes in ribosome-bound RNA expression (RPF) of ZP3 mRNA from the oocyte stage to the ICM stage in mice are represented in the graph. The RPF folding line chart illustrates the alterations in RNA molecules bound to ribosomes using low-input Ribo-seq (Ribo-lite). The RNA folding line chart represents regular mRNA sequencing (mRNA-seq). RPF refers to ribosome-protected fragments. Other abbreviations include FGOs (fully grown oocytes), LPI (late prometaphase I), MII (metaphase II), PN3 (early one-cell stage), PN5 (late one-cell stage), E2C (early two-cell stage), L2C (late two-cell stage), 4C (four-cell stage), 8C (eight-cell stage), ICM (inner cell mass of blastocyst), and mESC (mouse embryonic stem cells).
case_images: fgene-14-1256549-g001.jpg
case_images: PMC10613883_01_fgene-14-1256549-g001.jpg
case_images: F1
case_images: No significant abnormalities were observed in her reproductive organs, including the ovaries and uterus, and multiple large follicles were visible (Figure 1A). Interestingly, despite undergoing three oocyte retrieval procedures, no oocytes were obtained (Figure 1B). Interestingly, the expression levels of ZP3 in 2PN embryos were most abundant at various stages of preimplantation embryo development in mice and marmosets (Figure 1C). To further investigate the dynamic changes in ZP3 during oocyte meiosis, ribosome profiling (low-input Ribo-seq) was used to capture ZP3 mRNA expression, which was found to be 20-fold higher than that in 2PN zygotes (Figure 1D).
case_images: Genealogy and bioinformatics analysis of the proband. (A) The pedigree of the patient's family is depicted on the left, with arrows indicating the proband. The right panel illustrates the mutant ZP3 loci identified through whole-exome sequencing and comparison with the hg19 reference genome. A homozygous variant in ZP3 (NM_001110354.2, c.176T>A (p. L59H)) is highlighted. (B) The DNA electropherogram demonstrates the heterozygous state of the 176th nucleotide (T > A) in the ZP3 gene (NM_001110354.2) for both the patient's father and mother. (C) An illustrative diagram showcases the locations of mutated sites within the structural domain of the ZP3 protein. Previously reported sites are indicated in yellow, while the newly identified L59H mutation from this study is highlighted in red. The ZP domain represents the zona pellucida domain. (D) The residue L59 within the ZP3 protein is highly conserved across six species. Human-specific sites are marked in red, with a specific emphasis on the L59 position denoted by boxes. (E) Utilizing SWISS-MODEL software, spatial structure pattern maps were simulated for both the wild-type and mutant ZP3 proteins. These simulations revealed that the L59H variant alters the protein's overall shape. PolyPhen-2 was employed to predict the potential impact of the L59H variant on the structure and function of the human ZP3 protein. This mutation was predicted to be potentially damaging, with a score of 1.000 (sensitivity: 0.00; specificity: 1.00).
case_images: fgene-14-1256549-g002.jpg
case_images: PMC10613883_01_fgene-14-1256549-g002.jpg
case_images: F2
case_images: Further validation in the patient's parents revealed that both of them were carriers, with DNA sequencing showing heterozygous ZP3L59H mutations (Figures 2A, B). These four sites are located within the zona pellucida (ZP) domain of ZP3 (Figure 2C). The ZP3L59H mutation is conserved across six representative species (mouse, rat, sheep, bovine, macaque, and human) (Figure 2D). Moreover, both PolyPhen-2 and Mutation Assessor predicted high levels of structural damage caused by this mutation, with a score close to 100% (PolyPhen-2: PROBABLY DAMAGING; score 1.000; specificity: 1.00) (Figure 2E).