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.

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.

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.

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 Missense Mutation in ZP3 Causes Empty Follicle Syndrome and Female Infertility.

Empty follicle syndrome (EFS) is defined as the failure to aspirate oocytes from mature ovarian follicles during in vitro fertilization. Except for some cases caused by pharmacological or iatrogenic problems, the etiology of EFS remains enigmatic. In the present study, we describe a large family with a dominant inheritance pattern of female infertility characterized by recurrent EFS. Genome-wide linkage analyses and whole-exome sequencing revealed a paternally transmitted heterozygous missense mutation of c.400 G>A (p.Ala134Thr) in zona pellucida glycoprotein 3 (ZP3). The same mutation was identified in an unrelated EFS pedigree. Haplotype analysis revealed that the disease allele of these two families came from different origins. Furthermore, in a cohort of 21 cases of EFS, two were also found to have the ZP3 c.400 G>A mutation. Immunofluorescence and histological analysis indicated that the oocytes of the EFS female had degenerated and lacked the zona pellucida (ZP). ZP3 is a major component of the ZP filament. When mutant ZP3 was co-expressed with wild-type ZP3, the interaction between wild-type ZP3 and ZP2 was markedly decreased as a result of the binding of wild-type ZP3 and mutant ZP3, via dominant negative inhibition. As a result, the assembly of ZP was impeded and the communication between cumulus cells and the oocyte was prevented, resulting in oocyte degeneration. These results identified a genetic basis for EFS and oocyte degeneration and, moreover, might pave the way for genetic diagnosis of infertile females with this phenotype.

The application of naked DNA plasmid (DrZP3) and recombinant adenovirus (Ad-rZP3) in rat animal model to determine comparative efficacy of ZP3-Immunocontraceptive vaccines.

The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host.

A Unique Seminal Plasma Protein, Zona Pellucida 3-Like Protein, has Ca2+ -Dependent Sperm Agglutination Activity.

Identification of seminal proteins provides a means of investigating their roles. Despite their importance in the study of protein function, such as regulation of sperm motility, it is difficult to select candidates from the large number of proteins. Analyzing the rate of molecular evolution is a useful strategy for selecting candidates, and expressing the protein allows the examination of its function. In the present study, we investigated seminal plasma proteins of the cichlid Oreochromis mossambicus, which exhibits a unique mode of fertilization and a rapidly evolving gene that encodes a seminal plasma protein, zona-pellucida 3-like (ZP3-like), which does not belong to the same molecular family as other ZPs. Seminal plasma proteins of O. mossambicus were separated by two-dimensional electrophoresis, and 19 major proteins were identified by mass spectrometry (MALDI-Tof Mass). Because proteins that are under positive selection often impact sperm function, the rates of molecular evolution of these proteins were analyzed in terms of non-synonymous/synonymous substitutions (ω). Among the 19 proteins, positive selection was supported for five genes; functional assays were carried out on four of the proteins encoded by these genes. Of the four positively selected proteins, only ZP3-like protein agglutinated sperm in a dose- and Ca2+ -dependent manner. The other three proteins did not affect sperm motility. Because of the unique fertilization type, in which fertilization occurs in the buccal cavity, the need to retain sperm within the cavity during spawning, and the agglutination of sperm, which may be partly assisted by ZP3-like protein, may contribute to fertilization success. Fertilization in the buccal cavity may be related to its rapid molecular evolution.

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

STUDY QUESTION: Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? SUMMARY ANSWER: The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. WHAT IS KNOWN ALREADY: ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. STUDY DESIGN, SIZE, DURATION: This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in mature oocytes than in immature oocytes (all P < 0.05). In mature oocytes, IL-ZP retardance was significantly correlated with the expression of all four ZP mRNAs (all P < 0.05). The oocyte ZP3 expression was the main predictor of the fertilization capacity, next to IL-retardance and IL-thickness. Using stepwise regression analysis, IL-thickness combined with EFNB2 expression in CC and the patient's ovarian response resulted in a noninvasive oocyte fertilization prediction model. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study and the relation of oocyte mRNA levels to fertilization capacity is indirect as oocyte gene expression analysis required lysis of the oocyte. WIDER IMPLICATIONS OF THE FINDINGS: Overall relations between PLM observations, mRNA expression changes and intrinsic oocyte competence were successfully documented. As such PLM and CC gene expression are confirmed as valuable noninvasive techniques to evaluate oocyte competence. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by University of Torino, Italy, WFWG UZ-Brussel and Agentschap voor Innovatie door Wetenschap en Technologie IWT 110680, Belgium. All authors declare that their participation in the study did not involve actual or potential conflicts of interests.

Oocyte glycoproteins regulate the form and function of the follicle basal lamina and theca cells.

Maintaining follicle integrity during development, whereby each follicle is a functional unit containing a single oocyte, is essential for the generation of healthy oocytes. However, the mechanisms that regulate this critical function have not been determined. In this paper we investigate the role of the oocyte in maintaining follicle development. To investigate this role, we use a mouse model with oocyte-specific deletion of C1galt1 which is required for the generation of core 1-derived O-glycans. The loss of oocyte-generated O-glycans results in the joining of follicles and the generation of Multiple-Oocyte Follicles (MOFs). The aim was to determine how Mutant follicle development is modified thus enabling follicles to join. Extracellular matrix and follicle permeability were studied using histology, immunohistochemistry and electron microscopy (EM). In ovaries containing Mutant Oocytes, the Follicle basal lamina (FBL) is altered both functionally and structurally from the primary stage onwards with Mutant follicles possessing unexpectedly thicker FBL. In Mutant ovaries, the theca cell layer is also modified with intermingling of theca between adjacent follicles. MOF function was analysed but despite increased numbers of preantral MOFs in Mutants, these do not reach the preovulatory stage after gonadotrophin stimulation. We propose a model describing how oocyte initiated changes in FBL and theca cells result in follicles joining. These data reveal new and important roles for the oocyte in follicle development and follicle integrity.

Case report: A novel homozygous variant in ZP3 is associated with human empty follicle syndrome.

Empty follicle syndrome (EFS) is a rare condition in female infertility. It is characterized by the inability to retrieve oocytes from visibly large, normally developing follicles in the ovaries, despite ovarian stimulation. The genetic factors contributing to this syndrome remain unclear. This study focused on patients who underwent three consecutive ovarian stimulation procedures for oocyte retrieval but experienced unsuccessful outcomes, despite the presence of observable large follicles. Ultrasound examinations were conducted to assess follicular development during each procedure. In order to investigate potential genetic causes, we performed whole exome sequencing on peripheral blood samples from the patient. Interestingly, we identified that this patient carries a homozygous mutation in the ZP3 genes. Within the ZP3 gene, we identified a homozygous variant [NM_001110354.2, c.176T>A (p.L59H)] specifically located in the zona pellucida (ZP) domain. Further analysis, including bioinformatics methods and protein structure modeling, was carried out to investigate the conservation of the ZP3L59H variant across different species. This homozygous variant exhibited a high degree of conservation across various species. Importantly, the homozygous ZP3L59H variant was associated with the occurrence of empty follicle syndrome in affected female patients. The homozygous ZP3L59H variant represents a newly discovered genetic locus implicated in the development of human empty follicle syndrome. Our findings contribute to a deeper understanding of the role of zona pellucida-related genes in infertility and provide valuable insights for the genetic diagnosis of female infertility.

A tumor cell specific Zona Pellucida glycoprotein 3 RNA transcript encodes an intracellular cancer antigen.

Background: Expression of Zona Pellucida glycoprotein 3 (ZP3) in healthy tissue is restricted to the extracellular Zona Pellucida layer surrounding oocytes of ovarian follicles and to specific cells of the spermatogenic lineage. Ectopic expression of ZP3 has been observed in various types of cancer, rendering it a possible therapeutic target. Methods: To support its validity as therapeutic target, we extended the cancer related data by investigating ZP3 expression using immunohistochemistry (IHC) of tumor biopsies. We performed a ZP3 transcript specific analysis of publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, and validated expression data by independent computational analysis and real-time quantitative PCR (qPCR). A correlation between the ZP3 expression level and pathological and clinical parameters was also investigated. Results: IHC data for several cancer types showed abundant ZP3 protein staining, which was confined to the cytoplasm, contradicting the extracellular protein localization in oocytes. We noticed that an alternative ZP3 RNA transcript, which we term 'ZP3-Cancer', was annotated in gene databases that lacks the genetic information encoding the N-terminal signal peptide that governs entry into the secretory pathway. This explains the intracellular localization of ZP3 in tumor cells. Analysis of publicly available RNA-seq data of 1339 cancer cell lines (CCLs), 10386 tumor tissues (The Cancer Genome Atlas) and 7481 healthy tissues (Genotype-Tissue Expression) indicated that ZP3-Cancer is the dominant ZP3 RNA transcript in tumor cells and is highly enriched in many cancer types, particularly in rectal, ovarian, colorectal, prostate, lung and breast cancer. Expression of ZP3-Cancer in tumor cells was confirmed by qPCR. Higher levels of the ZP3-Cancer transcript were associated with more aggressive tumors and worse survival of patients with various types of cancer. Conclusion: The cancer-restricted expression of ZP3-Cancer renders it an attractive tumor antigen for the development of a therapeutic cancer vaccine, particularly using mRNA expression technologies.

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.

Novel expression of zona pellucida 3 protein in normal testis; potential functional implications.

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

Contraceptive efficacy of recombinant porcine zona proteins and fusion protein encompassing canine ZP3 fragment and GnRH in female beagle dogs.

PROBLEM: To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs. METHOD OF STUDY: Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs. RESULTS: Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control. CONCLUSION: These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.

Zona Pellucida sperm-binding protein 3 receptor distribution during Gopc-/- globozoospermic spermatogenesis.

Globozoospermia is a type of teratozoospermia characterized by round morphology of the sperm head. Gopc-/- infertile globozoospermic murine model has failures during spermiogenesis, such as the incorrect biogenesis of the acrosome, disorganized acroplaxome and manchette, round nuclei and spiral flagella. In this study, Western blot, RT-PCR, immunohistochemistry and immunogold were done for the localization of the acrosome protein Zona Pellucida sperm-binding protein 3 receptor (ZP3R), also called sp56, in wild type and Gopc-/- mice testis. The ZP3R protein was located in the acrosome and pseudo-acrosome vesicles of wild type and Gopc-/- mice, respectively. Also, it is distributed through the cytoplasm of the haploid spermatids only. The incorrect spermiogenesis of Gopc-/- mice causes a deregulation in the expression of ZP3R in the globozoospermic spermatids. Our results suggest that although the lack of GOPC causes a failure during the transport of the pre-acrosomal vesicles, the acrosome protein ZP3R is localized in the acrosome and is distributed through the cytoplasm only during spermiogenesis. Furthermore, the failure in spermiogenesis does not impair the synthesis of ZP3R and its localization in the pre-acrosomal vesicles.

Immunocontraceptive potential of a GnRH receptor-based fusion recombinant protein.

BACKGROUND: The management of stray dog population has been of utmost importance due to their overpopulation, increase in dog bites incidence, and rabies. Contraceptive vaccines, a non-surgical alternative to spaying and neutering are viewed as a valuable option for the management of dog population. In this study, the contraceptive potential of a recombinant fusion protein containing the three genes GnRH, GnRH receptor, and ZP3 was explored. RESULTS: The gene fragment encoding GnRH, GnRHR, and ZP3 along with the antigenic epitopes of canine distemper virus and tetanus toxoid was assembled, synthesized, and cloned into pET28a expression vector. The resulting construct GVAC08 was successfully transformed into BL21DE3 strain of E. coli and confirmed by colony PCR. The recombinant GVAC08 protein was expressed and purified using Ni-NTA and was confirmed to be a 50-KDa protein by SDS PAGE and Western blot. Mice were immunized with the GVAC08 protein using Freund's complete adjuvant followed by a booster using Freund's incomplete adjuvant. This induced a high antibody titer against GnRH, GnRH receptor, and ZP3 which was determined by ELISA. CONCLUSION: Mating studies showed that the GVAC08 recombinant protein was able to reduce the litter size in immunized mice showing improved efficacy. However, the vaccine candidate with further improvements will be a viable contraceptive vaccine.

Empty Zona Pellucida Only Case: A Critical Review of the Literature.

The presence of empty zona pellucida (EZP) in oocytes following oocyte retrieval (OR) during an in vitro fertilization (IVF) cycle presents a major clinical and laboratory challenge in assisted reproduction. It has been attributed to several factors such as the ovarian stimulation protocol employed, the damaging of the follicles during oocyte retrieval (OR) mainly through the high aspiration pressure, during the denudation technique, and the degeneration of oolemma within the zona pellucida (ZP) through apoptosis. The role of ZP is pivotal from the early stages of follicular development up to the preimplantation embryo development and embryo hatching. Polymorphisms or alterations on the genes that encode ZP proteins may contribute to EZP. We present a critical review of the published literature hitherto on EZP and available options when encountered with the phenomenon of EZP. Concerning the former, we found that there is rare data on this phenomenon that merits documentation. The latter includes technical, genetic, and pathophysiological perspectives, along with specific treatment options. In conclusion, we identify the lack of a definitive management proposal for couples presenting with this phenomenon, we underline the need for an algorithm, and indicate the questions raised that point towards our goal for a strategy when addressing a previous finding of EZP.

Case Report: A Novel Heterozygous ZP3 Deletion Associated With Empty Follicle Syndrome and Abnormal Follicular Development.

Background: Empty follicle syndrome (EFS) is defined as the complete failure to retrieve oocytes after ovarian stimulation. Although several mutations in ZP1, ZP2, ZP3, and LHCGR have been identified as genetic causes of EFS, its pathogenesis is still not well-understood. Methods: Whole-exome sequencing (WES) was employed to identify the candidate pathogenic mutations, which were then verified by Sanger sequencing. A study in CHO-K1 cells was performed to analyze the effect of the mutation on protein expression. Additionally, immunohistochemistry (IHC) staining was used to examine follicular development and zona pellucida (ZP) assembly in the ovary of an EFS patient. Results: A novel heterozygous deletion in ZP3 (c.565_579del[p.Thr189_Gly193del]) was identified in the EFS patient. It was inherited dominantly and resulted in significant degradation of the ZP3 protein. Oocytes with degenerated cytoplasm and abnormal ZP assembly were observed in follicles up to the secondary stage, and many empty follicle-like structures were present. Conclusion: We identified a novel ZP3 mutation that expands the mutational spectrum associated with human EFS. We also showed the abnormal follicular development and ZP assembly of the EFS patient with the heterozygous ZP3 mutation, which provides new insights into the pathogenesis of EFS.

The Fish Egg's Zona Pellucida.

All fish eggs are surrounded by an envelope, called the zona pellucida (ZP), that plays various roles during oogenesis, egg deposition, fertilization, and embryogenesis. The fish egg ZP consists of only a few proteins that are homologs of mammalian ZP proteins ZP1, ZP3, and ZP4. Unlike the situation in mammals, in fishes there are often multiple copies of ZP genes, perhaps a consequence of ancient polyploidization, gene amplification, and mutation. Like mammalian ZP proteins, fish egg ZP1-like proteins exhibit conserved organization with distinct domains and motifs, but unlike mammalian ZP1 and ZP4 have a glutamine (Q)- and/or proline (P)-rich stretch as an N-terminal extension. Such extensions may play a role in assembly of ZP fibrils and/or account for certain properties of the fish egg ZP, such as elasticity. Recent proposals suggest that fish egg ZP proteins can adopt amyloid-like structures, serve as antifreeze proteins in Antarctic icefishes, and protect eggs subjected to desiccating conditions in small shallow pools. In this chapter, these and other aspects of fish egg ZP proteins are presented.

The Human Egg's Zona Pellucida.

Human zona pellucida (ZP) matrix, a delicate network of thin interconnected filaments, is primarily composed of four glycoproteins, namely, ZP1, ZP2, ZP3, and ZP4. All four zona proteins share common structural elements such as signal peptide, "ZP domain," consensus furin cleavage site, transmembrane-like domain, and short cytoplasmic tail. In addition, ZP1 and ZP4 also have "Trefoil domain." Recombinant/native human zona proteins have been used to investigate their binding characteristics to the capacitated and/or acrosome-reacted spermatozoa. These investigations revealed that ZP1, ZP3, and ZP4 primarily bind to the head region of the capacitated human spermatozoa, whereas ZP2 binds to the acrosome-reacted sperm. However, using transgenic mice, N-terminal region of human ZP2 has also been shown to play an important role in binding of sperm to the egg. ZP1, ZP3, and ZP4 lead to dose-dependent increase in acrosome reaction, suggesting that in humans more than one ZP glycoprotein is responsible for induction of acrosome reaction. Glycosylation of these proteins, in particular, N-linked glycosylation as well as sialyl-Lewisx, is essential for inducing acrosome reaction. Studies delineating downstream signaling events associated with induction of acrosome reaction reveal subtle differences between ZP3 and ZP1/ZP4 with respect to activation of Gi protein-coupled receptor and protein kinase A. The role of mutations in the zona proteins and ZP autoantibodies leading to infertility in women is suggestive and needs more rigorous experimentations for confirming their role in female infertility. The above-mentioned aspects of the human ZP glycoproteins have been discussed in this review.