PMC8170154_01
Genotypic features of the EFS patient. (A) Pedigrees of the family with EFS and female infertility. The filled circle indicates the EFS patient. The arrow indicates the proband. The ZP3 genotype for each subject detected is shown, with WT indicating a normal allele and MT indicating the mutant allele. (B) Sanger genotype of ZP3 for each individual. (C) Location of the mutation (red line) is indicated in the genomic structure (top) and domain organization (bottom) of ZP3. SS, signals sequence; CFCS, consensus furin cleavage site; TMD, transmembrane domain. (D) Thr189_Gly193 is highlighted in red and conserved in the selected species. (E) Expression of wild-type (WT) and mutant ZP3 proteins in Chinese hamster ovary (CHO) cells examined by immunoblot analysis using anti-ZP3 antibodies.
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F1
A patient affected by EFS (Figures 1A,B) was recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. This mutation is highly conserved among species (Figure 1D) and is located within the ZP domain (ZPD) of the ZP3 protein, a structural module essential for the polymerization and assembly of ZP proteins (Figure 1C) (Jovine et al.,). As a result, the proband and her father harbored the same heterozygous deletion c.565-579del, while both her mother and brother had the wild-type, suggesting that the deletion had been paternally transmitted (Figures 1A,B) and had not affected male fertility. The expression level of the mutant ZP3 protein was significantly lower than that of the normal protein (Figure 1E), suggesting that the mutant protein was unstable and degraded. Moreover, in the experiment of co-transfection of wild-type and mutant plasmids, simulating heterozygous mutation in the patient, the total level of ZP3 protein was as low as in the cells transfected with the mutant plasmid (Figure 1E), indicating a dominant negative effect of the mutant ZP3.
Ovarian histology and immunohistochemistry staining of the EFS patient with a heterozygous ZP3 deletion. Ovaries isolated from the EFS patient carrying a ZP3 mutation (A-O) and a normal control (P-Z, AA) were sectioned and stained with antibodies against VASA (A-E, P-S), ZP1 (F-J, T-W), or ZP3 (K-O, X-Z, AA). The scale bars represent 50 mum.
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F2
Using VASA staining, intact follicles containing an oocyte at the primordial to secondary stages were observed in the patient's ovary (Figures 2A-D). It is worth noting that, compared with homogeneous cytoplasm in normal oocytes (Figures 2P-AA), the patient's oocytes showed meshy cytoplasm (Figures 2D,I,N), which suggested that the oocytes might be undergoing degeneration. Using ZP staining, a continuous ZP structure increasing in width along with growing oocyte was observed in the normal control (Figures 2T-AA). In contrast, no clear and intact ZP structure was detected in the patient's secondary follicles (Figure 2N). Moreover, no follicles beyond secondary stage were found, but many empty follicle-like structures were detected (Figures 2E,J,O, 3). However, dispersive ZP3 signals were detected surrounding the patient's oocyte within a secondary follicle (Figure 2N), which might result from secretion and accumulation of the relict ZP3 protein during follicle growth.
Ovarian histology and immunohistochemistry staining of serial sections showing an intact empty follicle-like structure of the EFS patient with a heterozygous ZP3 deletion. There are a total of 30 sections with 5 mum per section and 223 mum in its widest part.
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F3
Moreover, no follicles beyond secondary stage were found, but many empty follicle-like structures were detected (Figures 2E,J,O, 3).