ZP2 cleavage blocks polyspermy by modulating the architecture of the egg coat.

Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.

LEAFY and APETALA1 down-regulate ZINC FINGER PROTEIN 1 and 8 to release their repression on class B and C floral homeotic genes.

Organ initiation from the shoot apical meristem first gives rise to leaves during vegetative development and then flowers during reproductive development. LEAFY (LFY) is activated after floral induction and together with other factors promotes the floral program. LFY functions redundantly with APETALA1 (AP1) to activate the class B genes APETALA3 (AP3) and PISTILLATA (PI), the class C gene AGAMOUS (AG), and the class E gene SEPALLATA3, which leads to the specification of stamens and carpels, the reproductive organs of flowers. Molecular and genetic networks that control the activation of AP3, PI, and AG in flowers have been well studied; however, much less is known about how these genes are repressed in leaves and how their repression is lifted in flowers. Here, we showed that two genes encoding Arabidopsis C2H2 ZINC FINGER PROTEIN (ZFP) transcription factors, ZP1 and ZFP8, act redundantly to directly repress AP3, PI, and AG in leaves. After LFY and AP1 are activated in floral meristems, they down-regulate ZP1 and ZFP8 directly to lift the repression on AP3, PI, and AG. Our results reveal a mechanism for how floral homeotic genes are repressed and derepressed before and after floral induction.

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.

Cryo-EM structure of native human uromodulin, a zona pellucida module polymer.

Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) "domain". Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo-electron microscopy study of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of heteromeric vertebrate egg coat filaments identify a common sperm-binding region at the interface between subunits.

Recurrent Independent Pseudogenization Events of the Sperm Fertilization Gene ZP3r in Apes and Monkeys.

Many reproductive proteins show signatures of rapid evolution through sequence divergence and duplication. These features of reproductive genes may complicate the detection of orthologs across taxa, making it difficult to connect studies in model systems to human biology. In mice, ZP3r/sp56 is a binding partner to the egg coat protein ZP3 and may mediate induction of the acrosome reaction, a crucial step in fertilization. In rodents, ZP3r, as a member of the Regulators of Complement Activation cluster, is surrounded by paralogs, some of which have been shown to be evolving under positive selection. Although primate egg coats also contain ZP3, sequence divergence paired with paralogous relationships with neighboring genes has complicated the accurate identification of the human ZP3r ortholog. Here, we phylogenetically and syntenically resolve that the human ortholog of ZP3r is the pseudogene C4BPAP1. We investigate the evolution of this gene within primates. We observe independent pseudogenization events of ZP3r in all Apes with the exception of Orangutans, and independent pseudogenization events in many monkey species. ZP3r in both primates that retain ZP3r and in rodents contains positively selected sites. We hypothesize that redundant mechanisms mediate ZP3 recognition in mammals and ZP3r's relative importance to ZP recognition varies across species.

Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

A Novel Homozygous Missense ZP1 Variant Result in Human Female Empty Follicle Syndrome.

Empty follicle syndrome (EFS) is a disorder characterised by the unsuccessful retrieval of oocytes from matured follicles following ovarian stimulation for in vitro fertilisation (IVF). Genetic factors significantly contribute to this pathology. To date, an increasing number of genetic mutations associated with GEFS have been documented, however, some cases still remain unexplained by these previously reported mutations. Here, we identified a novel homozygous missense ZP1 variant (c.1096 C > T, p.Arg366Trp) in a female patient with GEFS from a consanguineous family who failed to retrieve any oocytes during two cycles of IVF treatment. We conducted a molecular dynamics simulation analysis on the mutant ZP1 model, revealing that the mutant ZP1 protein has an altered 3D structure, lower fluctuation, higher compactness and higher instability than wild-type ZP1. Immunostaining, immunoblotting and co-immunoprecipitation results showed that the homozygous missense mutation in ZP1 impaired protein secretion and weakened interactions between ZP1 and other ZP proteins, which may affect the ZP assembly. This study contributes to a more comprehensive understanding of the genetic aetiopathogenesis of GEFS.

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.

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.

Effects of indented zona pellucida on oocyte growth and development explored from changes of gene expression in cumulus cells.

PURPOSE: Abnormal Zona Pellucida (ZP) of human oocytes is an extracellular oocyte abnormality leading to subfertility or infertility, among which indented ZP (iZP) is a common clinical case, and there is currently no effective clinical solution. The study aimed to find out the influence of this abnormal ZP on the growth and development of GC and further explore its influence on the growth and development of oocytes, hoping to provide new ideas for the etiology and treatment of such patients. METHODS: In this study, we collected granulosa cells GC from oocytes with iZP(four cases) and GC from oocytes with a normal appearance of the ZP(eight cases) during ICSI treatment cycles, and submitted them to transcriptomic analysis using next-generation RNA sequencing (RNAseq). RESULTS: 177 Differentially Expressed Genes (DEG) were identified by RNAseq analysis of Granulosa Cells (GC) from oocytes with a normal ZP morphological appearance and those with iZP. Correlation analysis of these DEGs showed that the expression levels of the immune factor CD274 and the inflammatory factors IL4R and IL-7R, which are positively associated with ovulation, were significantly down-regulated in the GC of oocytes with iZP. Hippo, PI3K-AKT, Ras and calcium signaling pathways related to oocyte growth and development, NTRK2 and its ligands (BDNF and NT5E) from the neurotrophin family that are trophic to the oocyte were also significantly down-regulated in the GC of oocytes with iZP. In addition, the expression of cadherin family members CDH6, CDH12 and CDH19 were significantly down-regulated in DEGs, and the down-regulation of these proteins may affect the gap junction between Granulosa cells and oocytes. CONCLUSION: IZP might cause obstacles to dialogue and material exchange between GC and oocytes and further affect the growth and development of oocytes.

A novel gene mutation in ZP3 loop region identified in patients with empty follicle syndrome.

The zona pellucida (ZP) is an extracellular matrix surrounding mammalian oocytes. It is composed of three to four glycoproteins, ZP1-ZP4. ZP3 is essential for sperm binding and zona matrix formation. Here, we identified a novel heterozygous mutation (NM_001110354.2:c.502_504delGAG) of ZP3, occurring in a pair of sisters with empty follicle syndrome (EFS). A mouse model with the same mutation was established using the CRISPR/Cas9 gene-editing system. As in the above family, F0 -, F1 -, and F2 -generation female mice with the mutation were all infertile. Further analysis using the Chinese hamster ovary cells (CHO-K1) also showed that this mutation weakens the strength of binding between ZP3 and ZP2, which hinders the assembly of ZP and results in unstable ZP formation. Immunohistochemical analysis using ovarian serial sections in both humans and mice demonstrated that the ZP of preantral follicles was thinner than normal control, or even absent. Our study presents a new gene mutation that leads to EFS, providing new evidence and support for the genetic diagnosis of infertile individuals with similar phenotypes. Our results also show that the loop of ZP3 is not only a linker between two amphiphilic helices but may play a critical role in specifying the correct heterodimerization partner.

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

STUDY QUESTION: Is a recurrent heterozygous mutation in ZP2, c.1925G>A (p.R642Q), associated with the Empty follicle syndrome (EFS)? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: This was a basic medical research project to assess the pathogenicity of a heterozygous mutation in the ZP2 gene in EFS. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: These results are expected to open new avenues for researchers in the exploration of potential therapeutic strategies in treating EFS. STUDY FUNDING/COMPETING INTEREST(S): This project is funded by the National Key Research and Development Program of China (2018YFC1002804, 2017YFC1001500 and 2016YFC1000200). All authors declared no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Can successful pregnancy be achieved and predicted from patients with identified ZP mutations? A literature review.

BACKGROUND: In mammals, normal fertilization depends on the structural and functional integrity of the zona pellucida (ZP), which is an extracellular matrix surrounding oocytes. Mutations in ZP may affect oogenesis, fertilization and early embryonic development, which may cause female infertility. METHODS: A PubMed literature search using the keywords 'zona pellucida', 'mutation' and 'variant' limited to humans was performed, with the last research on June 30, 2022. The mutation types, clinical phenotypes and pregnancy outcomes were summarized and analyzed. The naive Bayes classifier was used to predict clinical pregnancy outcomes for patients with ZP mutations. RESULTS: A total of 29 publications were included in the final analysis. Sixty-nine mutations of the ZP genes were reported in 87 patients with different clinical phenotypes, including empty follicle syndrome (EFS), ZP-free oocytes (ZFO), ZP-thin oocytes (ZTO), degenerated and immature oocytes. The phenotypes of patients were influenced by the types and location of the mutations. The most common effects of ZP mutations are protein truncation and dysfunction. Three patients with ZP1 mutations, two with ZP2 mutations, and three with ZP4 mutations had successful pregnancies through Intracytoplasmic sperm injection (ICSI) from ZFO or ZTO. A prediction model of pregnancy outcome in patients with ZP mutation was constructed to assess the chance of pregnancy with the area under the curve (AUC) of 0.898. The normalized confusion matrix showed the true positive rate was 1.00 and the true negative rate was 0.38. CONCLUSION: Phenotypes in patients with ZP mutations might be associated with mutation sites or the degree of protein dysfunction. Successful pregnancy outcomes could be achieved in some patients with identified ZP mutations. Clinical pregnancy prediction model based on ZP mutations and clinical characteristics will be helpful to precisely evaluate pregnancy chance and provide references and guidance for the clinical treatment of relevant patients.

Nisin ZP, an Antimicrobial Peptide, Induces Cell Death and Inhibits Non-Small Cell Lung Cancer (NSCLC) Progression in vitro in 2D and 3D Cell Culture.

Lung cancer is the leading cause of cancer deaths globally with most of the reported cases (> 85%) associated with non-small cell lung cancer (NSCLC). Current therapies have enhanced the overall survival rate of patients but treatment-related adverse effects and increase in drug-resistance limit the success of these treatment options. Antimicrobial peptides (AMPs) have gained interest as anticancer agents as they selectively target cancer cells and decrease the possibility of resistance. Nisin ZP is a polycyclic antimicrobial peptide produced by the Gram-positive bacterium, Lactococcus lactis and is commonly used as a food preservative. Nisin ZP has recently demonstrated anticancer activity in melanoma, head and neck squamous cell carcinoma, hepatic, colon, and blood cancer. In this study, we evaluated the anticancer potential of nisin ZP and assessed the underlying mechanisms in NSCLC cells. The results revealed that nisin ZP induced selective toxicity in cancer (A549 and H1299) cells compared to healthy (HEK293) cells after 48 h of treatment. Nisin ZP exposure induced apoptosis and cell cycle arrest (G0/G1 phase) in NSCLC cells irrespective of tumor protein p53 expression. The cancer cell proliferation was inhibited via non-membranolytic pathways by mitochondrial membrane depolarization and elevation in reactive oxygen species (ROS) generation. Furthermore, nisin ZP decreased cancer cells' clonal expansion and migration, demonstrating potential use against highly metastatic NSCLC. The 3D spheroid growth and cell viability of the A549 cells were significantly inhibited by nisin ZP compared to control. Overall, the results suggest an excellent antitumor potential in vitro and, thus, can further be developed as a novel therapeutic for NSCLC.

The effect of Escherichia coli ZP strain with a conjugation-based colicin E7 delivery on growth performance, hematological, biochemical, and histological parameters, gut microbiota, and nonspecific immunity of broilers.

The Escherichia coli ZP strain (ZP) was constructed based on the known probiotic E. coli strain Nissle 1917. It was genetically modified to carry the colicin E7 synthesis gene encoding DNase on a conjugative plasmid and the colicin E7 immunity gene in the chromosome. The aim of this study was to evaluate the effects of the daily ZP per oral administration (5 × 108 or 5 × 1010 CFU per bird) on the growth performance, hematological, biochemical, histological parameters, gut microbiota, and nonspecific immunity of the 4-24 days old broilers. The ZP administration increased the abundance of genera Bacillus, Butyrivibrio, and Clostridium and did not influence the weight gain of 4-16 days old broilers. The biochemical parameters were within normal ranges for poultry in experimental and control groups. The ZP administration had no effect on the erythrocyte numbers, hemoglobin and immunoglobulin Y concentrations, but significantly increased the serum lysozyme concentration, leukocyte numbers, and reactive oxygen species production by phagocytes compared with the control group. It did not cause inflammatory changes in intestinal mucosa, Peyer's patches, and spleen. Thus, the ZP had no detrimental effects on broiler health and could be an efficient probiotic for the broiler colibacillosis prophylaxis.

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.

Zona Pellucida like Domain Protein 1 (ZPLD1) Polymerization Is Regulated by Two Distinguished Hydrophobic Motifs.

Zona Pellucida Like Domain 1 Protein (ZPLD1) is a main component of the cupula, a gelatinous structure located in the labyrinth organ of the inner ear and involved in vestibular function. The N-glycosylated protein is likely able to organize high-molecular-weight polymers via its zona pellucida (ZP) module, which is common for many extracellular proteins that self-assemble into matrices. In this work, we confirmed that ZPLD1 can form multimers while setting up a cellular model leveraging Madin-Darby canine kidney (MDCK) cells to study protein polymerization. We identified two motifs within ZPLD1 which regulate its polymerization and follow previously published conserved regions, identified across ZP proteins. Mutational depletion of either one of these modules led to diminished or abnormal polymer formation outside of the cells, likely due to altered processing at the plasma membrane. Further, intracellular polymer formation was observed. Proteolytic cleavage during secretion, separating the regulatory motif located distinct of the ZP module from the mature monomer, seems to be necessary to enable polymerization. While the molecular interactions of the identified motifs remain to be proven, our findings suggest that ZPLD1 is a polymer forming ZP protein following an orchestrated mechanism of protein polymerization to finally build up a gelatinous hydrogel.

Garlic and ginger essential oil-based neomycin nano-emulsions as effective and accelerated treatment for skin wounds' healing and inflammation: In-vivo and in-vitro studies.

Skin, largest organ of human, is directly exposed to environment and hence is prone to high rates of injuries and microbial infections. Over the passage of time these microbes have developed resistance to antibiotics making them ineffective especially in lower doses and hence, higher dosages or new drugs are required. The current study deals with designing of nano-emulsion (NE) formulations composed of garlic and ginger oils (0.1 %) with neomycin sulphate used in different ratios (0.001, 0.01 and 0.1 %) and combinations. The resulting NEs were characterized for droplet size (145-304 nm), zetapotential (-3.0-0.9 mV), refractive index (1.331-1.344), viscosity (1.10-1.23cP), transmittance (96-99 %), FT-IR and HPLC and found stable over a period of three months. All NEs were also found effective against both gram positive and negative bacterial strains i.e., B. spizizenii, S. aureus, E. coli and S. enterica as compared to pure neomycin sulphate (NS) used as control with highest activity recorded for NE-2 and NE-4 against all strains showing zone of inhibition in range of 22-30 mm and 21-19 mm, respectively. NEs were also tested using rabbit skin excision wound model which potentiates that all the NEs resulted in early recovery with 86-100 % wound healing achieved in 9 days as compared to NS ointment (71 %). The studies confirmed that essential oils when used in combination with traditional drug can lead to much higher efficacies as compared to pure drugs.

Mutant Zp1 impedes incorporation of ZP3 and ZP4 in the zona pellucida, resulting in zona absence and female infertility in rats†.

The zona pellucida (ZP) plays vital roles in reproductive processes including oogenesis, fertilization, and preimplantation development. Both human and rat ZP consist of four glycoproteins, called ZP1, ZP2, ZP3, and ZP4. Our previous research reported a novel Zp1 mutation in cases of human infertility, associated with an abnormal phenotype involving the absence of the ZP. Here, we developed a homologous rat strain to investigate the pathogenic effect. The ovaries of homozygous (Zp1MT/MT) females possessed both growing and fully grown oocytes; the oocytes completely lacked a ZP, but ZP1 was detectable inside the cytoplasm. Only 1-2 eggs were recovered from oviducts of superovulated Zp1MT/MT females, while an average of 21 eggs were recovered from superovulated Zp1WT/WT per female. The eggs of Zp1MT/MT females were not surrounded by a ZP and lost their fertilization capacity in vitro. Zp1MT/MT females mated with wild-type males failed to become pregnant. Studies in 293T cells showed that mutant Zp1 resulted in a truncated ZP1 protein, which might be intracellularly sequestered and interacted with wild-type ZP3 or ZP4. Our results suggest that the Zp1 point mutation led to infertility and loss of the ZP in oocytes in rats.

Novel mutations in ZP1: Expanding the mutational spectrum associated with empty follicle syndrome in infertile women.

Empty follicle syndrome (EFS) is a serious and complex reproductive complication for infertile women suffering from the recurrent failure of oocyte retrieval in an in vitro fertilization procedure, and its pathogenesis remains obscure. Increasing evidence highlights the genetic basis of EFS occurrence. In this study, we identified two novel missense mutations (c.1127G > A, p.C376Y and c.325C > T, p.R109C), two novel frameshift mutations (c.800_801delAG, p.E267Gfs*80 and c.1815_1825delGGTCCTTTTGC, p.V606Afs*42), one novel nonsense mutation (c.199G > T, p.E67Ter), and three reported mutations (c.769C > T, p.Q257Ter; c.1430 + 1G > T, p.C478Ter and c.1169_1176delTTTTCCCA, p.I390Tfs*16) in five unrelated probands, showing similar EFS manifestations, which expands the mutational spectrum of individuals with autosomal recessive ZP1. Current research will provide a better understanding of the biological functions of ZP1, and some insight into the determination of ZP1 variation as an additional rule for assessing the EFS disease.