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.

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.

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.

Role of diagnostic intracytoplasmic sperm injection (ICSI) in the management of genetically determined zona pellucida-free oocytes during in vitro fertilization: a case report.

PURPOSE: To report the utilization of diagnostic intracytoplasmic sperm injection (D-ICSI), an ICSI cycle performed in the natural cycle, to obtain information about embryo development potential after sperm injection into zona pellucida (ZP)-free oocytes. MATERIALS AND METHODS: We report the case of a couple with primary unexplained infertility with a history of previous failed, in vitro fertilization intracytoplasmic sperm injection (IVF-ICSI) cycles characterized by the presence of ZP-free oocytes. Whole exome sequencing (WES) was carried out to analyse the possible genetic basis of oocyte abnormality. RESULTS: Diagnostic ICSI provided information about the embryo development potential from ZP-free oocytes and allowed better planning of the subsequent ICSI cycle. WES revealed that the absence of ZP was likely to be due to a new (ZP1) mutation. The subsequent ICSI cycle resulted in the delivery of a healthy baby. DISCUSSION: To the best of our knowledge, our report is the first to describe the use of D-ICSI to determine the feasibility of embryo development and implantation in a patient with ZP1 mutation, resulting in the subsequent delivery of a healthy baby. We used 'diagnostic' ICSI in the normal menstrual cycle to explore the feasibility of embryo development after sperm injection into ZP-free oocytes. Our results may expand the spectrum of diagnostic procedures associated with unexplained infertility.

Novel mutations in ZP1 and ZP2 cause primary infertility due to empty follicle syndrome and abnormal zona pellucida.

PURPOSE: Mutations in the zona pellucida glycoprotein genes have been reported to be associated with empty follicle syndrome (EFS) and abnormal zona pellucida (ZP). In this study, we performed genetic analysis in the patients with female infertility due to abnormal zona pellucida and empty follicle syndrome to identify the disease-causing gene mutations in these patients. METHODS: We characterized three patients from two independent families who had suffered from empty follicle syndrome or abnormal zona pellucida. Whole exome sequencing and Sanger sequencing were used to identify the mutations in the families. Western blot was used to check the expression of wild type and mutant disease genes. RESULTS: We identified two novel mutations in these patients, including a novel compound heterozygous mutation (c.507delC, p. His170fs; c.239 G>A, p. Cys80Tyr and c.241 T>C, p. Tyr81His) in ZP1 gene and a compound mutation in ZP2 gene (c.860_861delTG, p.Val287fs and c.1924 C>T, p.Arg642Ter). Expression of the mutant ZP1 protein (p. Cys80Tyr and p. Tyr81His) is significantly decreased compared with the wild-type ZP1. Other three mutations produce truncated proteins. CONCLUSIONS: Our findings expand the mutational spectrum of ZP1 and ZP2 genes associated with EFS and abnormal oocytes and provide new support for the genetic diagnosis of female infertility.

Novel biallelic loss-of-function variants in ZP1 identified in an infertile female with empty follicle syndrome.

PURPOSE: Empty follicle syndrome (EFS) refers to the inability to obtain mature oocytes after appropriate ovarian stimulation during the process of in vitro fertilization (IVF). However, the specific cause and mechanism of action underlying EFS remain to be further explored. Herein we aimed to investigate the clinical and genetic characteristics of EFS. METHODS: After data were collected in an infertile family, we performed whole-exome sequencing (WES) on the patient and confirmed the pathogenic mutations through Sanger sequencing. Western immunoblotting, immunofluorescence staining, and minigene assay were further used to investigate the negative effects of these mutations. RESULTS: Absence of oocytes was observed over 2 cycles of IVF in the patient, and we evaluated the novel compound heterozygous mutations c.2T>A (p. M1K) and c.1112+1G>T of the zona pellucida glycoprotein 1 gene (ZP1, MIM# 195000) by WES. For the family under study, EFS was classified as an autosomal recessive inheritance pattern. The results of western blotting and immunofluorescence staining analyses confirmed that the missense mutation of c.2T>A [p. M1K] resulted in almost missing protein production. Additionally, using a minigene assay, we demonstrated the deleterious effect on the ZP1 gene of the splice site mutation c.1112+1G>T, which caused truncation of ZP1 protein. CONCLUSIONS: The compound heterozygous mutations of ZP1 gene identified in this study by genetic and functional experiments constituted a novel genetic cause of EFS, and further study will expand its use in the clinical and molecular diagnoses of EFS.

Compound heterozygous ZP1 mutations cause empty follicle syndrome in infertile sisters.

Empty follicle syndrome (EFS) is a condition in which no oocyte is retrieved from mature follicles after proper ovarian stimulation in an in vitro fertilization procedure. Genetic evidence accumulates for the etiology of recurrent EFS without pharmacological or iatrogenic problems. In this study, we present two infertile sisters in a family with EFS after three cycles of standard ovarian stimulation with human chorionic gonadotrophin and/or gonadotropin-releasing hormone agonist therapy. Via whole-exome sequencing and cosegregation test, we identified compound heterozygous mutations in the gene of ZP1 in both of the infertile sisters. Coimmunoprecipitation tests and homology modeling analysis confirmed that both mutated ZP1 disrupt the formation of oocyte zona pellucida by interrupting the interaction among ZP1, ZP2, and ZP3. We thus propose that the specific mutations in ZP1 gene render a causality for the intractable EFS.

ZP1 mutations are associated with empty follicle syndrome: evidence for the existence of an intact oocyte and a zona pellucida in follicles up to the early antral stage. A case report.

Empty follicle syndrome (EFS) is the complete failure to retrieve oocytes after ovarian stimulation. Although LHCGR and ZP3 were identified as causative genes, it is still unclear what happens to these patients' oocytes, and the pathogenesis of EFS remains obscure. Here, we identified six novel ZP1 mutations associated with EFS and female infertility that was inherited recessively in five unrelated families. Studies in CHO-K1 cells showed that these mutations resulted in either degradation or truncation of ZP1 protein. Immunohistochemistry using ovarian serial sections demonstrated that all preantral follicles had normal architecture, but with a thin ZP, lacking ZP1, surrounding the growing oocytes. The antral follicles were also defective in normal cumulus-oocyte complex organisation, leading us to speculate that the lack of ZP1 might lead to oocyte degeneration or increased fragility of the oocyte during follicular puncture, ultimately resulting in EFS. To our knowledge, this is the first study that presents morphological evidence showing normal preantral folliculogenesis with abnormal ZP assembly in EFS patients. Our data provides a better understanding of the biological functions of ZP1 in human ZP assembly and folliculogenesis and gives new insights into the pathogenesis of EFS and possible therapeutic developments.

Novel mutation in the ZP1 gene and clinical implications.

PURPOSE: Empty follicle syndrome (EFS) is a complex reproductive disorder characterized by the repeated failure to aspirate oocytes from mature ovarian follicles during in vitro fertilization (IVF). In addition to some cases caused by iatrogenic problems and known genetic factors, there are still many unexplained aspects of EFS. Here, we aimed to assess the clinical and genetic characteristics of two EFS patients. METHODS: We have characterized two primary infertility patients with EFS in a nonconsanguineous family from China. Both the patients presented similar clinical phenotypes, that is a few granulosa cells but no oocytes could be retrieved during repeated cycles with normal follicular development, E2 levels, and bioavailable hCG plasma levels. Abnormal oocytes were obtained once or twice between multiple IVF cycles. We performed Sanger sequencing of the LHCGR and ZP1~ZP4 genes in the patients, and further bioinformatics analysis was performed to identify pathogenic elements in the genes. RESULTS: A novel mutation, c.181C>T (p.Arg61Cys), and a known mutation, c.1169_1176delTTTTCCCA (p.Ile390Thrfs*16), in the ZP1 gene were both identified in patient 2, but no mutations were identified in patient 1. The novel mutation inherited from her mother was absent in the control cohort and the ExAc database. The arginine residue is conserved at this position, and its replacement by cysteine was predicted to be deleterious. In another allele, a paternal frameshift mutation was predicted to introduce premature stop codons, resulting in the deletion of 234 amino acids from the C-terminus of the ZP1 protein. CONCLUSIONS: Our findings presented compound heterozygous mutations in ZP1 associated with EFS and abnormal oocytes and provided further new evidence for the genetic basis of EFS and support for the genetic diagnosis of infertile individuals.

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.

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.

[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.

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

Genuine empty follicle syndrome (gEFS) is a rare cause of female infertility; it is defined as the presence of cumulus-oocyte complexes (COCs) in follicular fluid but the absence of oocytes after denudation in an in vitro fertilization (IVF) programme. Mutations in one of the four genes encoding zona pellucida (ZP) proteins have been implicated in gEFS. The objectives of the present study were to explore the molecular basis of idiopathic infertility in a 35-year-old woman with gEFS (observed after four ovarian retrievals), compare her phenotype and genotype with those of other patients described in the literature, and discuss therapeutic approaches that could be adopted by reproductive health centres in this situation. Sequencing of the ZP genes revealed a new homozygous missense variant in ZP1: c.1097G > A;p.(Arg366Gln). The variant is located in the ZP-N domain, which is essential for ZP protein polymerization. An immunohistochemical assessment of an ovarian biopsy confirmed the absence of ZP1 protein. The novel variant appears to prevent ZP assembly, which would explain the absence of normal oocytes after denudation in our patient (and despite the retrieval of COCs). ZP gene sequencing should be considered for patients with a phenotype suggestive of gEFS. An etiological genetic diagnosis enables appropriate genetic counselling and a switch to an IVF programme (with a suitable denudation technique) or an oocyte donation programme.

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.

A novel homozygous nonsense ZP1 variant causes human female infertility associated with empty follicle syndrome (EFS).

BACKGROUND: Empty follicle syndrome (EFS) is a rare but severe condition in which no oocyte is recovered in female patients undergoing in vitro fertilization (IVF) after sufficient ovarian response to hormonal trigger. Accumulating evidence highlights the genetic basis of EFS occurrence. METHODS: In this study, we report a patient with primary infertility showing the characteristics of EFS from a consanguineous family. Under the treatment of assisted reproductive technique (ART), no oocyte was retrieved following the aspiration of mature follicles. Through whole-exome sequencing (WES), we discovered a novel recessively transmitted mutation in ZP1 (c.769 C>T, p. Q257*). RESULTS: In vitro Co-immunoprecipitation assays showed that mutant ZP1 protein failed to interact with either ZP2 or ZP3, which explains the degenerated oocytes in the patient with EFS. CONCLUSION: Together, our data further expand the spectrum of ZP1 mutations that are associated with human EFS and thus provide novel insight into the diagnosis of EFS patients.

Effect of the chicken zp1 gene on osteoblast mineralization / 生物工程学报

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.

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 Mouse Egg's Zona Pellucida.

All mammalian eggs are surrounded by a highly specialized extracellular matrix (ECM), called the zona pellucida (ZP), that functions before, during, and after fertilization. Unlike somatic cell ECM the mouse ZP is composed of three different proteins, ZP1-3, that are synthesized and secreted by growing oocytes and assembled into long interconnected fibrils. ECM or vitelline envelope (VE) that surrounds fish, reptilian, amphibian, and avian eggs also consists of a limited number of proteins all closely related to ZP1-3. Messenger RNAs encoding ZP1-3 are expressed only by growing oocytes at very high levels from single-copy genes present on different chromosomes. Processing at the amino- and carboxy-termini of nascent ZP1-3 permits secretion of mature proteins into the extracellular space and assembly into fibrils and matrix. Structural features of nascent ZP proteins prevent assembly within secretory vesicles of growing oocytes. Homozygous knockout female mice that fail to synthesize either ZP2 or ZP3 are unable to construct a ZP, ovulate few if any eggs, and are infertile. ZP1-3 have a common structural feature, the ZP domain (ZPD), that has been conserved through 600 million years of evolution and is essential for ZP protein assembly into fibrils. The ZPD consists of two subdomains, each with four conserved cysteine residues present as two intramolecular disulfides, and resembles an immunoglobulin (Ig) domain found in a wide variety of proteins that have diverse functions, from receptors to mechanical transducers. ZP2 and ZP3 function as receptors for acrosome-reacted and acrosome-intact sperm, respectively, during fertilization of ovulated eggs, but are inactivated as sperm receptors as a result of fertilization.