Oocyte-specific EXOC5 expression is required for mouse oogenesis and folliculogenesis.

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.

Human Amniotic Epithelial Stem Cells Alleviate Autoimmune Premature Ovarian Insufficiency in Mice by Targeting Granulosa Cells via AKT/ERK Pathways.

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.

N-Glycosylation Site in the Middle Region Is Involved in the Sperm-Binding Activity of Bovine Zona Pellucida Glycoproteins ZP3 and ZP4.

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.

Intracellular fraction of zona pellucida protein 3 is required for the oocyte-to-embryo transition in mice.

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.

Identification of zona pellucida defects revealed a novel loss-of-function mutation in ZP2 in humans and rats.

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.

Zona pellucida family genes in Chinese pond turtle: identification, expression profiles, and role in the spermatozoa acrosome reaction†.

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.

Bi-allelic variants in ASTL cause abnormal fertilization or oocyte maturation defects.

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.

Egg envelope formation of medaka Oryzias latipes requires ZP proteins originating from both the liver and ovary.

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.

Novel variants in ZP1, ZP2 and ZP3 associated with empty follicle syndrome and abnormal zona pellucida.

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.

Transfer of the zp3a gene results in changes in egg adhesiveness and buoyancy in transgenic zebrafish.

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.

A ZP1 gene mutation in a patient with empty follicle syndrome: A case report and literature review.

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.

ZP3 and AIPL1 participate in GVBD of mouse oocytes by affecting the nuclear membrane localization and maturation of farnesylated prelamin A.

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.

Effect of In Vitro Maturation of Human Oocytes Obtained After Controlled Ovarian Hormonal Stimulation on the Expression of Development- and Zona Pellucida-Related Genes and Their Interactions.

In an in vitro fertilization program, approximately 10-15% of oocytes obtained after controlled ovarian stimulation are immature, with germinal vesicles (GVs). These oocytes are usually discarded in clinical practice; however, an in vitro maturation (IVM) procedure can be applied to mature them. There are scarce data in the literature on the effect of IVM on the expression of important development- and zona pellucida (ZP)-related genes in human oocytes; therefore, we wanted to determine this. One hundred nine human oocytes were collected from patients enrolled in an intracytoplasmic sperm injection program. The expression of the BMP4, GDF9, ZP1, ZP2, ZP3, and ZP4 genes was analyzed using RT-qPCR in oocytes matured in vitro with different reproductive hormones in the IVM medium (AMH, FSH + hCG, FSH + hCG + AMH), in in vivo matured oocytes and in immature oocytes with GVs. No statistically significant differences in the expression of selected genes in oocytes were observed among groups with different reproductive hormones in IVM medium. However, several interesting significant correlations were found between BMP4 and GDF9, and ZP1 and ZP4; between GDF9 and ZP1, and ZP2 and ZP4; and between ZP1 and ZP3 and ZP4 in the in vitro matured oocytes, while no such correlations were present in other groups of oocytes. The type of reproductive hormone in the maturation medium does not affect the expression of the analyzed genes in oocytes during the maturation process. However, the in vitro maturation procedure itself generated correlations among analyzed genes that were otherwise not present in in vivo matured and immature oocytes.

Novel mutations in TUBB8 and ZP3 cause human oocyte maturation arrest and female infertility.

PURPOSE: Variations in many genes may lead to the occurrence of oocyte maturation defectsand female infertility. The objective was to describe newly discovered mutations in TUBB8 and ZP3, and to characterise the accompanying spectrum of phenotypes and modes of inheritance. METHODS: TUBB8 and ZP3 were sequenced from genomic DNA samples extracted from peripheral blood of patients and their family members by the whole-exome sequencing. The TUBB8 and ZP3 sequences are then aligned with cryptographic software to identify rare variations. Sanger sequencing and mass spectrometry were used to validate mutations. ExAC database was used to retrieve the frequency of corresponding mutations. PolyPhen-2 and PROVEAN were analyzed for mutations using silicon. RESULTS: We identified Three novel mutations and two known variant in TUBB8 and ZP3 associated with maturation in five families, and fertilization and developmental arrest are in these patients. These mutations include four heterozygous mutations in TUBB8 (c.730G > A, p.Gly244Ser, c.124C > G, p.Leu42Val, c.1172G > T, p.Arg391Leu and c.178G > A, p.Val60Met), and a heterozygous mutation in ZP3 (c.400G > A, p.Ala134Thr). Among them, these variants of TUBB8 were highly conserved among primates. CONCLUSION: As far as we know, the TUBB8 mutations detected in our study at four sites have not been reported before, and the variant of ZP3 has been published as pathogenic. Our findings extend the known mutant spectrum of TUBB8 and ZP3, and provide insights into the etiology of infertility in human women. The exact molecular mechanism has not been analyzed and should be further investigated in the future.

Expression Analysis of ZPB2a and Its Regulatory Role in Sperm-Binding in Viviparous Teleost Black Rockfish.

Black rockfish is a viviparous teleost whose sperm could be stored in the female ovary for five months. We previously proposed that zona pellucida (ZP) proteins of black rockfish play a similar sperm-binding role as in mammals. In this study, SsZPB2a and SsZPB2c were identified as the most similar genes with human ZPA, ZPB1 and ZPB2 by Blastp method. Immunohistochemistry showed that ovary-specific SsZPB2a was initially expressed in the cytoplasm of oocytes at stage III. Then it gradually transferred to the region close to the cell membrane and zona pellucida of oocytes at stage IV. The most obvious protein signal was observed at the zona pellucida region of oocytes at stage V. Furthermore, we found that the recombinant prokaryotic proteins rSsZPB2a and rSsZPB2c could bind with the posterior end of sperm head and rSsZPB2a was able to facilitate the sperm survival in vitro. After knocking down Sszpb2a in ovarian tissues cultivated in vitro, the expressions of sperm-specific genes were down-regulated (p < 0.05). These results illustrated the regulatory role of ZP protein to the sperm in viviparous teleost for the first time, which could advance our understanding about the biological function of ZP proteins in the teleost.

A Novel Homozygous Nonsense Mutation in ZP1 Causes Female Infertility due to Empty Follicle Syndrome.

ZP1 is a critical glycoprotein in the formation of the zona pellucida. It plays an indispensable role in the maturation of oocytes. To identify the causative gene of empty follicle syndrome (EFS) in a patient from a consanguineous family, whole-exome sequencing was performed in the proband. We identified a novel homozygous nonsense mutation c.1260C > G (p. Tyr420X) in the ZP1 gene from two primary infertile patients. Western blot showed that Y420X mutation in ZP1 gene produced a truncated protein. However, the mutation had no significant effect on subcellular localization of the mutant protein. Our findings confirmed the important role of the ZP1 gene in human female reproduction, enriched the mutation spectrums of ZP1 gene, and expanded its applications in the clinical and molecular diagnoses of EFS.

Novel Heterozygous Mutations in ZP2 Cause Abnormal Zona Pellucida and Female Infertility.

Zona pellucida (ZP) which is an extracellular matrix consisting of ZP1, ZP2, ZP3, and ZP4 plays a vital role in oocyte maturity, early embryonic development, and fertilization process. Any alterations of structure or function may lead to the abnormal formation of ZP and female infertility. Two novel heterozygous mutations c.1859G > A (p.Cys620Tyr) and c.1421 T > C (p.Leu474Pro) in ZP2 gene were recognized in three patients from two unrelated families with abnormal ZP and female infertility in this study. The expression constructs carrying wild-type ZP2 gene, c.1859G > A (p.Cys620Tyr) mutant ZP2 gene, and c.1421 T > C (p.Leu474Pro) mutant ZP2 gene were transfected into CHO cells respectively. There was a remarkable decrease in the expression of p.Cys620Tyr mutant protein with western blot. In addition, secretion of p.Leu474Pro mutant protein in the culture medium reduced markedly compared with that of wild-type ZP2 protein. Furthermore, co-immunoprecipitation showed that the p.Leu474Pro mutation affected the interaction between ZP2 and ZP3. Prediction of three-dimensional (3D) structure of the proteins showed that p.Cys620Tyr mutation altered the disulfide bond of ZP2 protein and may affect its function. These findings extend the ranges of mutations of ZP2 gene. Meanwhile, it will be helpful to the precise diagnosis of abnormal ZP.

Novel mutations in ZP2 and ZP3 cause female infertility in three patients.

PURPOSE: The aim of this study was to identify the disease-causing mutations found in three infertile female patients who were diagnosed with abnormal zona pellucida (ZP) and empty follicle syndrome (EFS). METHODS: We performed whole-exome sequencing and Sanger sequencing to identify and verify the disease-causing mutations. Additionally, we performed Western blotting and mini-gene splicing assay to assess the effects of the mutations. RESULTS: We identified two novel compound heterozygous mutations in the ZP2 gene, a patient with an abnormal ZP carrying a novel compound heterozygous mutation (c.1695-2A>G and c.1831G>T, p.V611F) and a patient with EFS carrying a novel compound heterozygous mutation (c.1695-2A>G and c.1924 C>T, p.R642*). Furthermore, we identified a patient with typical abnormal ZP carrying a novel heterozygous mutation (c.400G>T, p.A134S) in the ZP3 gene. The splice site mutation (c.1695-2A>G) can cause abnormal pre-mRNA splicing that inserts an extra sequence of 61 bp in the mRNA of ZP2, and the missense mutation (c.1831G>T) can cause a decrease of ZP2 protein in HEK293 cells. CONCLUSION: We identified three novel mutations in the ZP2 gene and the ZP3 gene in three Chinese female patients with infertility. Our study expands the spectrum of ZP gene mutations and phenotypes and thus is beneficial in the genetic diagnosis of infertility in females.

A recurrent ZP1 variant is responsible for oocyte maturation defect with degenerated oocytes in infertile females.

A female factor is present in approximately 70% of couple infertility, often due to ovulatory disorders. In oocyte maturation defect (OMD), affected patients have a primary infertility with normal menstrual cycles but produce no oocyte, degenerated (atretic) or abnormal oocytes blocked at different stages of maturation. Four genes have so far been associated with OMD: PATL2, TUBB8, WEE2, and ZP1. In our initial study, 6 out of 23 OMD subjects were shown to carry the same PATL2 homozygous loss of function variant and one patient had a TUBB8 truncating variant. Here, we included four additional OMD patients and reanalyzed all 27 subjects. In addition to the seven patients with a previously identified defect, five carried the same deleterious homozygous ZP1 variant (c.1097G>A; p.Arg366Gln). All the oocytes from ZP1-associated patients appeared shriveled and dark indicating that the abnormal ZP1 protein induced oocyte death and degeneration. Overall ZP1-associated patients had degenerated or absent oocytes contrary to PATL2-associated subjects who had immature oocytes blocked mainly at the germinal vesicle stage. In this cohort of North African OMD patients, whole exome sequencing permitted to diagnose 44% of the patients studied and to identify a new frequent ZP1 variant.