ZP3 Gene Research Articles

A Compound Heterozygous Pathogenic Variant in ZP2 Gene Causes Female Infertility.

The oocyte maturation defect 6 is an autosomal recessive hereditary disease caused by a homozygous variant in ZP2 gene. It is characterized by female primary infertility due to an abnormally thin zona pellucida (ZP) and defective sperm binding. Here we identified a compound heterozygous variant (c.1924C > T and c.1695-2A > G) in ZP2 gene in a Chinese Han family. Quantitative real-time PCR showed that the variant c.1924C > T significantly decreased the expression of truncated ZP2message RNA by the nonsense-mediated decay pathway. Minigene assays showed the c.1695-2A > G variant led to an extra-61-nt preservation of intron 15 at the junction between exons 15 and 16 during transcription. Both variants (c.1924C > T and c.1695-2A > G) resulted in truncated ZP2 proteins (p.R642X and p.C566Hfs*2) that lost the transmembrane domain, which prevented the secretion of the mutant ZP2 proteins and produced a structurally abnormal ZP, thus resulting in female infertility. This study further elucidated the pathogenic mechanism of these two variants and provided new support for the genetic diagnosis of female infertility.

Effects of Different Photoperiods on the Transcriptome of the Ovary and Small White Follicles in Zhedong White Geese.

Photoperiod can regulate the broodiness of geese and thus increase their egg-laying rate. The laying performance of geese is mainly determined by ovary and follicle development. To understand the effect of photoperiod on the ovary and small white follicles, sixteen 220-day-old healthy female Zhedong white geese were randomly divided into two groups for long photoperiods (15L:9D) and short photoperiods (9L:15D). The geese were euthanized after two months of feeding, and their ovaries and follicles were collected for transcriptome sequencing. RNA-seq analysis identified 187 and 448 differentially expressed genes in ovaries and small white follicles of different photoperiod groups, respectively. A long photoperiod promotes high expression of SPP1, C6, MZB1, GP1BA, and FCGBP genes in the ovaries, and increases the expression of SPP1, ANGPTL5, ALPL, ZP1, and CHRNA4 genes in small white follicles. Functional enrichment analysis showed that photoperiod could affect respiratory system development, smooth muscle cell proliferation in ovaries, and extracellular matrix-related function in small white follicles. WGCNA revealed 31 gene modules, of which 2 were significantly associated with ovarian weight and 17 with the number of small white follicles. Our results provide a better understanding of the molecular regulation in the photoperiod affecting goose reproduction.

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.

Transcriptomics and metabolomics reveal the regulation of reproductive performance and egg quality of blotched snakehead Channa maculata broodstock induced by a high protein diet

To evaluate the effects of dietary protein on reproductive performance, egg quality and larval health of the blotched snakehead Channa maculata, three groups were designed: a high protein group (HP) with 48% protein content, a low protein group (LP) with 44% protein content, and a control group (C) fed with commercial feed containing 40% protein. The results demonstrated that the HP diet significantly enhanced maternal absolute fecundity, egg size and offspring health; however, it had no impact on fertilization rate and hatching rate. The maternal plasma levels of 11-ketotestosterone, 17β-estradiol, and vitellogenin in the HP group were found to be significantly higher compared to those in the C group. Plasma SOD activity and T-AOC capacity in the HP group were significantly higher than those in the C group, while MDA content showed an opposite trend. The trend of T-AOC in 7 days post-hatching larvae exhibited a consistent pattern with that observed in the maternal. Metabolomics and transcriptomics results indirectly indicated that the differences in reproductive and egg quality of the broodstock were associated with the regulation of arachidonic acid metabolism, steroid hormone biosynthesis, cholesterol metabolism, glycerophospholipid metabolism, and glycerolipid metabolism. The expression levels of star, zp3, foxo3, tgf-β, ghr, igf, cyp17a1, and cyp19a1 genes associated with reproduction and were significantly upregulated in the HP group. By depleting the abundance of metabolites in key pathway such as the pentose phosphate pathway, glycolysis/gluconeogenesis, nicotinate and nicotinamide metabolism, vitamin B6 metabolism within the ovary, there is an elevation in arachidonic acid metabolite abundance, thereby facilitating ovarian maturation and regulating egg quality. In conclusion, the high protein diet may affect the reproductive process and egg quality by inducing an upregulation of TGF-beta signaling pathway and ovarian steroidogenesis and arachidonic acid metabolism, leading to increased plasma cholesterol levels and enhanced steroid hormone synthesis.

Effect of the chicken zp1 gene on osteoblast mineralization

The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.

O-207 zp1 mutation affects oocyte energy metabolism and leads to oocyte apoptosis

Abstract Study question How does the zp1 variation leads to apoptosis oocytes in antral follicular and infertility in females? Summary answer Mutant zp1 induces abnormal pyruvate transport leading to the accumulation of reactive oxygen species (ROS), which activates the mitochondria-mediated apoptosis pathway leading to oocytes apoptosis. What is known already Previous research reported a zp1 mutation in human infertility, and we develop a homologous rat strain to investigate the pathogenic effect. The ovaries of homozygous rat posses growing and fully grown oocytes, the oocytes completely lack a zona pellucida. Only 1-2 eggs were recovered from oviducts of per superovulated homozygous rat, and were not surrounded by a zona pellucida thus lost their fertilization capacity in vitro.Further studies showed that the homozygous female rats not only lacked ZP but also TZPs (an important communication bridge between granulosa cells and oocytes), and the caval follicles showed obvious apoptotic signals. Study design, size, duration In this study, 10 Sprague-Dawley rats were used at 8 weeks of age (5 wild-type and 5 mutant respectively). Participants/materials, setting, methods We used CRISPR/Cas 9-mediated genomic editing technology to develop a rat model carrying the homologous 8bp deleted mutation in Zp1 gene(Zp1mt/mt). The rats were sacrificed by cervical vertebra dislocation and their ovaries were isolated immediately. Then using bulk RNA sequencing of ovaries from Zp1mt/mt and wild-type female rats to identify the differential expression gene between the two groups.The differentially expressed genes enriched from KEGG were verified by qRT-PCR and in vitro functional experiments. Main results and the role of chance Total of 293 DEGs were obtained, of which 144 were up-regulated and 149 were down-regulated. The results of biological process showed that up-regulated genes were mainly involved in redox reaction and negative regulation of cell migration.Down-regulated genes are mainly involved in the follicular growth and development,and related to oocyte energy metabolism. In order to verify the DEG results obtained from RNA sequencing, the differentially expressed gene Gcgr, Plcβ2, Aox3 and Hpgd enriched from KEGG in cell metabolism were verified by qRT-PCR. The results showed that the differentially expressed gene results were consistent with the expression trend of sequencing data.The results showed that there was no significant difference in the expression of Pkm in granulosa cells, while there was a significant difference in the expression of Pdk4 in Zp1mt/mt oocytes. For ROS detection, 52 eggs of 4 Zp1mt/mt rats and 48 eggs of 6 wide-type rats were analyzed for reactive oxygen species. The results showed that the IOD of mutant eggs was significantly higher than that of wild-type,indicating that ROS content in mutant eggs was significantly increased, which was statistically significant.The detection of apoptotic protein expression in eggs shows that the apoptosis probability of Zp1mt/mt eggs is significantly higher than that ofwild-type. Limitations, reasons for caution This study provides more experimental basis and data support for the involvement of ZP in oogenesis, but the effect of ZP deficiency on the dynamic transcriptional regulation of developing oocytes-granulosa cells and the specific molecular mechanism remain to be further explored. Wider implications of the findings The work provides key insights into the crucial features of the transcriptional regulation in Zp1 mutant animal ovaries, and offers important clues for exploring the potential mechanism of abnormal oocyte development due to ZP deficiency. Trial registration number none

In-vivo oogenesis of oogonial and mesenchymal stem cells seeded in transplanted ovarian extracellular matrix

Objective (s)One way to overcome the recurrence of cancer cells following ovarian tissue transplantation is to use decellularized tissues as a scaffold that does not have any cellular components. These cell-free scaffolds can be seeded with different type of stem cells for ovarian restoration.Materials and methodsOSCs, PMSCs and BMSCs (oogonial, peritoneal and bone marrow mesenchymal stem cells, respectively) were seeded into human decellularized ovarian tissue as 4 groups: Scaffold + OSCs (SO), Scaffold + OSC + PMSCs (SOP), Scaffold + OSC + BMSCs (SOB) and Scaffold + OSC + PMSCs + BMSCs (SOPB). The produced grafts were transplanted into the sub-peritoneal space of ovariectomized NMRI mice as artificial ovary (AO). The expression of Vegf, CD34, Gdf9, Zp3, Ddx4, Amh and Lhr genes in AOs were measured by qRT-PCR. Also, histotechniques were considered to detect the anti GFP, PCNA, VEGF, GDF9, ZP3 and AMH proteins.ResultsH & E staining showed follicle-like structures in all groups; the number of these structures, in the SOP and SOB groups, were the highest. In SO group, differentiation ability to oocyte and granulosa cells was observed. Endothelial, oocyte, germ, and granulosa cell-like cells were specially seen in SOP and angiogenesis capability was more in SOB group. However, angiogenesis ability and differentiation to theca cell-like cells were more often in SOPB group. While none of the groups showed a significant difference in AMH level, estradiol levels were significantly higher in SOPB group.ConclusionIntegration of OSCs + PMSCs and those OSCs + BMSCs were more conducive to oogenesis.

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.

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.

Detection of Putative Novel Mutation in Zp1 Gene Causing Empty Follicle Syndrome

Abstract Introduction/Objective Mutations in the zona pellucida glycoprotein genes have been reported to be associated with empty follicle syndrome (EFS) and abnormal zona pellucida (ZP). At our clinic, in a cohort of infertile patients, we found three cases with empty follicle syndrome as a root cause of infertility. Further, we designed a study to identify the disease-causing gene mutations in these patients. At present, there are no citations or functional evidence in ClinVar for NM_207341.3:c.1168del variant indicating the involvement of this variant in empty follicle syndrome. But it has been interpreted to be Likely pathogenic causing female infertility due to zona pellucida defect. Methods/Case Report In this prospective study conducted at Al Ain Fertility Center (AAFC), U.A.E, we performed genetic analysis on three patients (first-degree sisters) having a mean age of 31.7 (2.52) years. Patient indications were primary female infertility and IVF failures due to no eggs present in the follicles when egg retrieval was attempted. We characterized these three patients as suffering from empty follicle syndrome. Along with the patient's sample, we also included their common parent's sample for Whole Exome Sequencing and Sanger sequencing, to rule out the inheritance pattern in the family. The effects of this mutation were further characterized through mRNA and protein studies. Results (if a Case Study enter NA) N/A. Conclusion To our knowledge, this is the first study in the U.A.E. showing the familial novel variant in the ZP1 gene associated with empty follicle syndrome. We identified a novel mutation in these patients (NM_207341.3:c.1168del) in the ZP1 gene in the homozygous state and the same variant in the heterozygous state in the parents. Our findings add to the existing mutational spectrum of the ZP1 gene associated with empty follicle syndrome and abnormal oocytes and provide new support for the genetic diagnosis of female infertility.

The chromosome-scale genome of the raccoon dog: Insights into its evolutionary characteristics

SummaryThe raccoon dog (Nyctereutes procyonoides) is an invasive canid species native to East Asia with several distinct characteristics. Here, we report a chromosome-scale genome of the raccoon dog with high contiguity, completeness, and accuracy. The intact taste receptor genes, expanded gene families, and positively selected genes related to digestion, absorption, foraging, and detoxification likely support the omnivory of raccoon dogs. Several positively selected genes and raccoon dog-specific mutations in TDRD6 and ZP3 genes may explain their high reproductivity. Enriched GO terms in energy metabolism and positively selected immune genes were speculated to be closely related to the diverse immune system of raccoon dogs. In addition, we found that several expanded gene families and positively selected genes related to lipid metabolism and insulin resistance may contribute to winter sleep of the raccoon dog. This high-quality genome provides a valuable resource for understanding the evolutionary characteristics of this species.

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.

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.1421T > 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.1421T > 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.

Identification of a heterozygous variant of ZP2 as a novel cause of empty follicle syndrome in humans and mice.

Is a recurrent heterozygous mutation in ZP2, c.1925G>A (p.R642Q), associated with the Empty follicle syndrome (EFS)? ZP2, c.1925G>A (p.R642Q), led to female infertility related to EFS in humans and mice and resulted in ZP2 accumulation in the cytoplasm of oocytes. EFS is a complex disease defined as a complete failure of oocyte retrieval after ovarian stimulation and after repeated aspirations and flushing of mature ovarian follicles. Furin-mediated cleavage is a post-translational modification (PTM) involved in various physiological processes, but the clear role of PTM mediated by furin cleavage of ZP2 protein on female fertility needs to be further explored. PTM is required for proteins to function in physiological conditions, and its perturbation has been linked to a growing number of human pathologies. Zona pellucida (ZP) proteins, which are important for oocyte development, are regulated post-translationally by well-characterized glycosylation events, as well as by furin-mediated cleavage. However, knowledge of the relevance of the consensus furin cleavage site of ZP proteins in female reproduction remains lacking. This was a basic medical research project to assess the pathogenicity of a heterozygous mutation in the ZP2 gene in EFS. We studied 3 families with EFS and a control group 2213 women with proven fertility. Whole-exome sequencing detected a heterozygous mutation in the ZP2 gene in all EFS patients. The mouse strain Zp2Arg635Gln/+ (ZP2R642Q) was generated by CRISPR-Cas9-mediated genome editing. RNA-sequencing was applied to investigate transcriptional changes in the ovaries of heterozygous ZP2R642Q knock-in (KI) mice compared to WT mice. We found a heterozygous mutation of ZP2, c.1925G>A (p.R642Q), in unrelated females with EFS, which was inherited in an autosomal-dominant manner. We used CRISPR-Cas9 to generate a mouse model encoding the orthologous variant of ZP2R642Q detected in humans, and the female ZP2R642Q KI mice recapitulated the human EFS phenotype. We further found the decreased expression of key genes involved in oocyte maturation in ZP2R642Q KI mice compared to WT mice by RNA-sequencing analysis. N/A. Only three families affected by EFS with the mutation were available because of its rare incidence. Although we have found different expressions of the several indispensable genes related to oocyte development between WT mice and ZP2R642Q KI mice through RNA-sequencing analysis, the specific regulatory mechanisms of the oocyte apoptosis in ZP2R642Q KI mice need to be studied further. These results are expected to open new avenues for researchers in the exploration of potential therapeutic strategies in treating EFS. This project is funded by the National Key Research and Development Program of China (2018YFC1002804, 2017YFC1001500 and 2016YFC1000200). All authors declared no competing interests. N/A.

Identification and in silico Characterization of Deleterious Single Nucleotide Variations in Human ZP2 Gene.

ZP2, an important component of the zona matrix, surrounds mammalian oocytes and facilitates fertilization. Recently, some studies have documented the association of mutations in genes encoding the zona matrix with the infertile status of human females. Single nucleotide polymorphisms are the most common type of genetic variations observed in a population and as per the dbSNP database, around 5,152 SNPs are reported to exist in the human ZP2 (hZP2) gene. Although a wide range of computational tools are publicly available, yet no computational studies have been done to date to identify and analyze structural and functional effects of deleterious SNPs on hZP2. In this study, we conducted a comprehensive in silico analysis of all the SNPs found in hZP2. Six different computational tools including SIFT and PolyPhen-2 predicted 18 common nsSNPs as deleterious of which 12 were predicted to most likely affect the structure/functional properties. These were either present in the N-term region crucial for sperm-zona interaction or in the zona domain. 31 additional SNPs in both coding and non-coding regions were also identified. Interestingly, some of these SNPs have been found to be present in infertile females in some recent studies.

Does combination of estradiol and sesame oil improve the oocyte quality, embryo development and expressions of Zp3, E-cad, and Ctnnb1 genes in mice? An experimental study.

BackgroundAging may reduce oocyte maturation, embryo quality, and fertility potential.ObjectiveTo compare the effect of estradiol (E2) and sesame oil on oocyte and embryo quality between young and old mice.Materials and MethodsSixty old and young female mice were divided in to two groups (30 mice/group, grouped by age). Each group was divided into three subgroups of mice treated with sesame oil, E2 + sesame oil, and normal saline as control group. After ovulation induction, some oocytes were considered for in vitro fertilization and the rest were assessed for morphological status. After obtaining the two-cell embryos, the embryos were collected to determine the expression of zona pellucida (ZP) glycoprotein 3, E-cadherin, and β-catenin genes and some of them followed until the blastocysts stage to evaluate the viability.Results The findings showed that the mean ZP and perivitelline space thickness increased in the old mice that received the E2 + sesame oil treatment. The number of 2-cell embryos, blastocysts, and live cells were significantly higher in the old group treated with sesame oil respectively (p = 0.018, 0.002, and 0.0001, respectively). The normal ZP shape and refractile body numbers increased in the old mice that were treated with sesame oil, respectively. The E-cadherin gene was downregulated in the treatment groups compared to the controls.ConclusionSesame oil showed a better response in the old mice, because aging is associated with an increased rate of reactive oxygen species, causing deficiencies in both oocyte and embryo qualities.

Validation of the existence of genuine Empty follicle syndrome, versus false empty follicle syndrome to make definitive decisions in cases where recurrent IVF failure encountered secondary to absence of oocytes on ovum pick up-a short communication

Aim: Worldwide a big altercation exists with regards to the actual existence of the term “ Empty follicle syndrome’’(EFS), so much so that certain big authorities in the field have been believing that true EFS does not exist. Basically EFS is a syndrome when no functionally intact oocyte get retrieved when attempting an oocyte pick up (OPU) for a successful in vitro fertilization (IVF), however such patients encounter recurrent IVF failures. Since it is has become a big problem for the treating reproductive endocrinologist, besides the patient encountering recurrent IVF failures, it has become essential to differentiate the true EFS alias genuine Empty follicle syndrome (gEFS) from what is labeled today as the false empty follicle syndrome (fEFS). In view of the recently documented presence of mutations, gEFS got verified and appears to silence this biggest conflict that arose secondary to the existence of a false empty follicle syndrome(fEFS), where one could manage to get successful IVF outcomes subsequent to repeated hCG injections/ gonadotropin releasing hormone (GnRH) agonist in addition to pregnancy, with lot of clinicians believing there is no true term like EFS. Methods: Recently Yang et al., performed a study In tertiary a university based reproductive center in China that was comprised of a big cohort of patients that presented with gEFS. Genetic evaluation was conducted on 35 non correlated infertile patients who went through 16 failed IVF cycles in addition to oocyte degeneration, besides the subjects got a diagnosis of possessing a particular kind of EFS- cumulus oocytes complexes (COC’s) but possessed oocytes that were undergoing degeneration, with the utilization of whole –exome sequencing along with targeted Sanger sequencing. Results: Yang et al., found 22 innovative genetic variant of zona pellucida (ZP), genes in 18 subjects, that were inclusive of 20 variants in ZP 1 gene, 2 in ZP 2 gene in addition to 1 recurring variant in ZP3 gene that had been earlier documented. The homogenous /compound heterogenous ZP 1 mutations were inherited in an autosomal recessive manner, while the heterogenous variants of ZP 2 as well as ZP3 genes possessed an autosomal dominant manner of inheritance. Conclusions: These mutations were anticipated to be harmful in silico along with got further experimentally corroborated to be functionally null dependent on their ectopic expression in vitro. Thus with this further evidence that has been recently provided with regards to the existence of genuine Empty follicle syndrome (gEFS), it is significant for the youngsters to realize if they encounter similar cases after trial of rescue hCG injections / GnRH) agonist, not to further keep waiting, but evaluate further with regards to the existence of mutations for Zona Pellucida (ZP), ZP 1, ZP2 as well as ZP3 genes mutations, or LH/ chorionic gonadotropins receptor (LHCGR) gene mutation without subjecting the patient to repeated IVF, with her psychological as well as financial health in mind

Oocyte-specific gene knockdown by intronic artificial microRNAs driven by Zp3 transcription in mice.

Conditional knockout technology is a powerful tool for investigating the spatiotemporal functions of target genes. However, generation of conditional knockout mice involves complicated breeding programs and considerable time. A recent study has shown that artificially designed microRNAs (amiRNAs), inserted into an intron of the constitutively expressed gene, induce knockdown of the targeted gene in mice, thus creating a simpler method to analyze the functions of target genes in oocytes. Here, to establish an oocyte-specific knockdown system, amiRNA sequences against enhanced green fluorescent protein (EGFP) were knocked into the intronic sites of the Zp3 gene. Knock-in mice were then bred with EGFP transgenic mice. Our results showed that Zp3-derived amiRNA successfully reduced EGFP fluorescence in the oocytes in a size-dependent manner. Importantly, knockdown of EGFP did not occur in somatic cells. Thus, we present our knockdown system as a tool for screening gene functions in mouse oocytes.

The critical role of ZP genes in female infertility characterized by empty follicle syndrome and oocyte degeneration

To identify the major causative gene(s) of genuine empty follicle syndrome (GEFS) characterized by oocyte degeneration. Genetic and functional studies. University-based reproductive medicine center. Thirty-five unrelated women with GEFS and oocyte degeneration. Whole-exome sequencing (WES) and targeted Sanger sequencing. Variants predicted by software and the functional effects of variants assessed via Western blot and immunofluorescence in Chinese hamster ovary (CHO) cells. We identified zona pellucida (ZP) gene variants in 18 individuals, which included 20 variants in the ZP1 gene, two variants in the ZP2 gene, and one previously reported recurrent variant in the ZP3 gene. The women carrying ZP variants constituted 51.43% of the GEFS cohort. The ZP1 variants were inherited in an autosomal recessive pattern; the ZP2 and ZP3 variants were inherited in an autosomal dominant pattern. All variants were predicted to be deleterious. Studies in CHO cells suggested that most ZP1 variants led to increased intracytoplasmic protein and some variants influenced the intracellular transportation of other ZP proteins. Variant p.R642Q of ZP2 caused the secretion of ZP2 protein with an increased molecular weight, suggesting altered protein modification. Variant p.I619N of ZP2 resulted in increased ZP2 protein in cell lysate and decreased ZP2 protein in culture medium. These results showed that ZP variants might block the intracellular transportation and secretion of ZP proteins and disrupt the zona pellucida. We identified novel variants of ZP genes in more than half the cohort with GEFS and oocyte degeneration. Variants of ZP genes caused protein intracellular sequestration and failure to assemble the ZP filaments, resulting in EFS and female infertility. Our findings not only reveal the critical roles of ZP genes but also pave the way for the efficient genetic diagnosis of females with GEFS and oocyte degeneration.