DOCSLIB.ORG

Gene Deletion and Functional Analysis of Fetuin-B"

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Gene Deletion and Functional Analysis of Fetuin-B "Gene Deletion and Functional Analysis of Fetuin-B" Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologin Jennifer Wessling geb. Goldstein aus Aachen Berichter: Universitätsprofessor Dr. rer. nat. W. Jahnen-Dechent Universitätsprofessor Dr. rer. nat. L. Elling Tag der mündlichen Prüfung: 21. November 2007 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Contents 1 Introduction 5 1.1 Fetuin Family of Proteins . 5 1.1.1 Protein Structure . 6 1.1.2 Expression . 10 1.1.3 Genetic Organization . 11 1.1.4 Biological Role . 12 1.2 Female Reproduction . 14 1.2.1 Oogenesis and Ovulation . 14 1.2.2 Fertilization . 16 1.2.3 Parthenogenesis . 17 1.3 The Mouse as a Model System . 17 1.3.1 Targeting the Mouse Genome . 18 1.4 Aim of the Study . 20 2 Materials and Methods 21 2.1 Oligonucleotides . 22 2.2 Generating a fetuin-B-de¯cient Mouse Strain . 23 2.2.1 Cloning of the Gene Targeting Vector . 23 2.2.2 Gene Targeting in Embryonic Stem Cells . 25 2.2.3 DNA Extraction from Cells in 96-Well Plates . 28 2.2.4 PCR Screening . 28 2.2.5 Southern Blot . 30 2.2.6 Blastocyst Injection . 30 2.2.7 Embryo Transfer . 30 2.2.8 Animal Husbandry and Breeding . 31 2.2.9 Genotyping . 31 2.3 Analysis of fetuin-B-de¯cient Mice . 32 i 2.3.1 Western Blot . 32 2.3.2 Immunohistochemistry . 33 2.3.3 Collecting Mouse Oocytes . 34 2.3.4 Primary Morphological Analysis . 35 2.3.5 Necropsy . 35 2.3.6 Histology . 36 2.3.7 Weighing . 37 2.3.8 Blood Pressure Measurement . 37 2.3.9 Glucose Tolerance Test . 37 2.3.10 Clinical Chemical Screening . 38 2.4 Recombinant Expression and Puri¯cation . 38 2.4.1 Cloning Murine Fetuin-B . 38 2.4.2 Generating a Fetuin-B Transfecting Adenovirus . 41 2.4.3 Expression and Puri¯cation of Fetuin-B . 41 2.5 In Vitro Analysis of Fetuin-B . 42 2.5.1 Precipitation Inhibition Assay . 42 2.5.2 Mink Lung Cell Reporter Assay . 43 2.6 Promoter Analysis . 44 2.6.1 Cloning of Reporter Constructs . 44 2.6.2 Transfection of Reporter Constructs . 44 3 Results 47 3.1 Generating a Fetuin-B-de¯cient Mouse Strain . 47 3.1.1 Gene Deletion and the Targeting Vector . 47 3.1.2 Establishing the Screening Methods . 50 3.1.3 Gene Targeting in Embryonic Stem Cells . 52 3.1.4 Screening for Recombinant ES Cell Clones . 54 3.1.5 Blastocyst Injection and Chimera . 55 3.1.6 Embryo-Transfer and Breeding . 57 3.1.7 Con¯rmation of Fetuin-B-de¯cient Mice . 58 3.2 Phenotyping Fetuin-B-de¯cient Mice . 59 3.2.1 Fertility . 60 3.2.2 Anatomical and Morphological Analysis . 67 3.2.3 Physiological Parameters . 74 3.2.4 Glucose Tolerance Test . 74 3.2.5 Clinical Chemical Screening . 78 ii 3.2.6 Summarizing the Phenotyping Results . 80 3.3 Recombinant Murine Fetuin-B . 81 3.3.1 Cloning Recombinant Murine Fetuin-B . 81 3.3.2 Generating a Fetuin-B Transfecting Adenovirus . 86 3.3.3 Expressing and Purifying Recombinant Fetuin-B . 86 3.3.4 Precipitation Inhibition Assay . 90 3.3.5 TGF-¯ Reporter Activity in Mink Lung Cells . 90 3.3.6 Serum Concentration of Fetuin-B . 93 3.3.7 Summarizing the Results of the in Vitro Data . 94 3.4 Promoter Analysis . 95 3.4.1 Cloning and Analysis of the Fetub Promoter . 95 4 Discussion 99 4.1 Generation of Fetuin-B-de¯cient Mice . 99 4.1.1 Gene Deletion . 99 4.1.2 Embryonic Stem Cells and Genetic Background . 99 4.1.3 Screening Procedure . 100 4.1.4 Expression of Fetuin Family Proteins . 101 4.1.5 Fetuin Serum Concentrations . 101 4.2 Phenotyping . 102 4.2.1 Vitality . 102 4.2.2 Female Infertility . 102 4.2.3 Anatomy and Morphology . 106 4.2.4 Physiology and Learning . 107 4.2.5 Clinical Chemistry Screening . 108 4.3 Recombinant Murine Fetuin-B . 109 4.3.1 Eukaryotic Expression of Recombinant Fetuin-B . 109 4.3.2 Fetuin-B Transfecting Adenovirus . 110 4.3.3 Inhibition of Ectopic Calci¯cation . 110 4.3.4 TGF-¯ Biology . 111 4.4 Outlook . 112 Summary 113 Appendix 115 Pedigrees . 115 Fetub Genomic Sequence . 117 iii Abbreviations . 125 List of Figures . 128 List of Tables . 129 References 142 Acknowledgements 143 Curriculum Vitae 145 iv Chapter 1 Introduction 1.1 Fetuin Family of Proteins For a long time the fetuin family of proteins consisted of a set of orthologous serum proteins in humans, sheep, pigs, cows and rodents but also in birds and reptiles. They were variably designated fetuin in sheep, pig and cow, ®2-HS-glycoprotein (AHSG) in human, phosphoprotein of 63 kDa (pp63) in rat or countertrypin in mouse and gerbil. However, their close sequence ho- mology, especially a set of 12 cysteine residues at ¯xed distances and a fetuin motif (LETXCHXLDPTP), indicated that fetuin, AHSG, pp63 and coun- tertrypin emerged from a single ancestral gene and resembled counterparts (orthologous) among these species [1, 2, 3, 4, 5]. The fetuins were classi¯ed as cystatin superfamily members because they contain cystatin-like domains characterized by the distinct cysteine residues. Further members of the cystatin superfamily, all sharing cystatin-like do- mains, are the cystatins themselves, histidine-rich glycoproteins (HRG) and kininogens (KNG). The cystatins are low-molecular-weight cysteine protease inhibitors and prototypic for the cystatin superfamily [6, 7]. A study that aimed at systematic cloning of hepatic mRNAs corresponding to inflammation-related genes in rat [8] revealed a second member of the fetuin family based on sequence similarity. Sequence analysis showed that the novel protein contained both fetuin signatures, including the set of 12 cysteine residues at ¯xed distances, perfectly conserved, and the fetuin motif that is present in a truncated version (LETGCHVL) [9]. The novel fetuin, fetuin- B was found in rat, mouse and humans, revealing both fetuin signatures 5 6 CHAPTER 1. INTRODUCTION with a 61% overall sequence homology (strictly conserved residues) in these species. When compared to the original fetuin, fetuin-A, fetuin-B shared 17% of strictly conserved amino acids and 24% of similar residues. The similarity was signi¯cant but much lower than the similarity between the rat, mouse and human fetuin-A (58% strictly conserved residues). The sequence analysis indicated that fetuin-B was homologous, yet, clearly di®erent from fetuin-A. Therefore, the novel fetuins were considered a novel entity within the fetuin family. The two paralogous were designated fetuin-A (comprising fetuin, AHSG, pp63 and countertrypin) and fetuin-B (alias fetuin beta) [9]. 1.1.1 Protein Structure Three major domains (D1-D3) characterize fetuin proteins. Domain D1 and D2 are tandemly arranged cystatin-like domains containing 12 cysteine residues at ¯xed distances, and are followed by a unique domain, D3, that is only loosely conserved between species [10]. In fetuin-A, domains D1 and D2 both contain dibasic motifs, db1 and db2 respectively. They are thought to account for protease-inhibition domains at least in human and bovine fetuin-A. Additionally, domain D1 contains a calcium, TGF-¯ and lectin binding sites [1, 11, 12]. Domain D3 can further be subdivided into D3a, a hydrophobic, proline-rich N-terminal half, and D3b, a C-terminal half including a connecting peptide, at least in humans [1, 7, 13, 14]. The general organization of domains is maintained in fetuin-B, as shown in Figure 1.1. However, the dibasic motifs db1 and db2 are only loosely retained and the calcium-binding site in domain D1 is absent. Furthermore, domain D3 does contain a proline-rich N-terminal but no connecting peptide in the C-terminal half. However, fetuin-B contains a short homology to the TGF-¯ receptor type II (depicted in Figure 1.2) and two archetypal Kunitz- type motifs, associated with protease-inhibitory activity, within domain D1 and D2 that is only present in fetuin-A domain D2 [9]. However, none of these domains were functionally veri¯ed. 1.1. FETUIN FAMILY OF PROTEINS 7 8 CHAPTER 1. INTRODUCTION Figure 1.1: Alignment of rat, mouse and human fetuin-B (f-B) with fetuin- A (f-A) from various mammals (Olivier et al. [9]). White residues on black background indicate identical or similar residues conserved in at least 10 out of 11 sequences. Hyphens indicate gaps; boxes represent the proposed signal peptides. The LETXCHXLDPTP fetuin signature is underlined. The 12 crit- ical cysteine residues are marked with a dot. A CPG tripeptide conserved in the cystatin superfamily is double-underlined. Disulphide bonds are indicated by a horizontal connecting line between cysteine residues; the bond connecting the ¯rst and ultimate cysteine is noted with a dashed line: The domains D1, D2 and D3 (D3a and D3b) are marked with broken arrows. The connecting peptide is over-lined and marked with CP. The potential calcium-binding site is over-lined and marked with CBS. The dibasic peptides db1 and db2 are over-lined. 1.1. FETUIN FAMILY OF PROTEINS 9 Figure 1.2: Modeled binding of human TGF-beta to its receptor type II (structure software Pymol 0.9V). The TGF-¯ binding site is depicted in green. The homology between the receptor and fetuin-B is depicted in yellow. Note that it is not part of the TGF-¯ binding site.
Load more

Recommended publications
  • Xenopus Laevis Sperm Receptor Gp69/64 Glycoprotein Is a Homolog
    Proc. Natl. Acad. Sci. USA Vol. 96, pp. 829–834, February 1999 Biochemistry Xenopus laevis sperm receptor gp69y64 glycoprotein is a homolog of the mammalian sperm receptor ZP2 JINGDONG TIAN*, HUI GONG, AND WILLIAM J. LENNARZ† Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 11794-5215 Contributed by William J. Lennarz, November 4, 1998 ABSTRACT Little is known about sperm-binding pro- Much less information is available on the identity of sperm teins in the egg envelope of nonmammalian vertebrate species. receptors in other, nonmammalian vertebrate species. Re- We report here the molecular cloning and characterization of cently, a Xenopus laevis sperm receptor (gp69y64) in the egg a recently identified sperm receptor (gp69y64) in the Xenopus vitelline envelope (VE) was identified (7, 8). It was shown that laevis egg vitelline envelope. Our data indicate that the gp69 the purified gp69y64 proteins, as well as their antibodies, and gp64 glycoproteins are two glycoforms of the receptor and blocked sperm binding to unfertilized eggs or to beads coupled have the same number of N-linked oligosaccharide chains but with gp69y64 proteins. It has been know for some time (9) that differ in the extent of O-glycosylation. The amino acid se- during fertilization, gp69y64 undergoes limited proteolysis. quence of the receptor is closely related to that of the mouse However, the molecular details of this cleavage have been zona pellucida protein ZP2. Most of the sequence conserva- unclear because the primary structure of gp69y64 was un- tion, including a ZP domain, a potential furin cleavage site, known.
    [Show full text]
  • Rat Zona Pellucida Sperm-Binding Protein 2 (ZP2) ELISA Kit
    Product Datasheet Rat Zona pellucida sperm-binding protein 2 (ZP2) ELISA Kit Catalog No: #EK11690 Orders: [email protected] Package Size: #EK11690-1 48T #EK11690-2 96T Support: [email protected] Description Product Name Rat Zona pellucida sperm-binding protein 2 (ZP2) ELISA Kit Brief Description ELISA Kit Applications ELISA Species Reactivity Rat (Rattus norvegicus) Other Names ZPA; zona pellucida glycoprotein 2|zona pellucida protein A|zona pellucida sperm-binding protein 2 Accession No. P48829 Storage The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37C for 4 and 7 days, and compare O.D.values of the kit kept at 37C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37C can be considered as 6 months at 2 - 8C, which means 7 days at 37C equaling 12 months at 2 - 8C). Application Details Detect Range:0.312-20 ng/mL Sensitivity:0.125 ng/mL Sample Type:Serum, Plasma, Other biological fluids Sample Volume: 1-200 µL Assay Time:1-4.5h Detection wavelength:450 nm Product Description Detection Method:SandwichTest principle:This assay employs a two-site sandwich ELISA to quantitate ZP2 in samples. An antibody specific for ZP2 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and anyZP2 present is bound by the immobilized antibody.
    [Show full text]
  • Positive Darwinian Selection Drives the Evolution of Several Female Reproductive Proteins in Mammals
    Positive Darwinian selection drives the evolution of several female reproductive proteins in mammals Willie J. Swanson*†, Ziheng Yang‡, Mariana F. Wolfner*, and Charles F. Aquadro* *Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703; and ‡Department of Biology, University College London, 4 Stephenson Way, London NW1 2HE, United Kingdom Communicated by M. T. Clegg, University of California, Riverside, CA, December 20, 2000 (received for review May 15, 2000) Rapid evolution driven by positive Darwinian selection is a recur- neutral evolution, purifying selection, and positive diversifying rent theme in male reproductive protein evolution. In contrast, selection, respectively. This criterion has been used to demon- positive selection has never been demonstrated for female repro- strate rapid evolution of male reproductive proteins in a variety ductive proteins. Here, we perform phylogeny-based tests on three of invertebrate and vertebrate species (1–17). Recently, Wyckoff female mammalian fertilization proteins and demonstrate positive et al. (1) used the ␻ ratio and other criteria to demonstrate the selection promoting their divergence. Two of these female fertil- rapid evolution of male reproductive proteins in primates. In ization proteins, the zona pellucida glycoproteins ZP2 and ZP3, are their study, female reproductive proteins, including OGP and part of the mammalian egg coat. Several sites identified in ZP3 as ZP3, were placed within a control group of nonrapidly evolving likely to be under positive selection are located in a region genes expressed in a variety of tissues (1). To elucidate the previously demonstrated to be involved in species-specific sperm- selective forces underlying the rapid divergence of reproductive egg interaction, suggesting the selective pressure is related to proteins, it is important to analyze female, as well as male, male-female interaction.
    [Show full text]
  • Zona Pellucida Protein ZP2 Is Expressed in the Oocyte of Japanese Quail (Coturnix Japonica)
    REPRODUCTIONRESEARCH Zona pellucida protein ZP2 is expressed in the oocyte of Japanese quail (Coturnix japonica) Mihoko Kinoshita, Daniela Rodler1, Kenichi Sugiura, Kayoko Matsushima, Norio Kansaku2, Kenichi Tahara3, Akira Tsukada3, Hiroko Ono3, Takashi Yoshimura3, Norio Yoshizaki4, Ryota Tanaka5, Tetsuya Kohsaka and Tomohiro Sasanami Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan, 1Institute of Veterinary Anatomy II, University of Munich, Veterinaerstrasse 13, 80539 Munich, Germany, 2Laboratory of Animal Genetics and Breeding, Azabu University, Fuchinobe, Sagamihara 229-8501, Japan, 3Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, 4Department of Agricultural Science, Gifu University, Gifu 501-1193, Japan and 5Biosafety Research Center, Foods, Drugs, and Pesticides (An-Pyo Center), Iwata 437-1213, Japan Correspondence should be addressed to T Sasanami; Email: [email protected] Abstract The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian oocytes, is composed of at least three glycoproteins. Our previous studies have demonstrated that the matrix’s components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver and is transported to the ovary by blood circulation. In this study, we report the isolation of cDNA encoding quail ZP2 and its expression in the female bird. By RNase protection assay and in situ hybridization, we demonstrate that ZP2 transcripts are restricted to the oocytes of small white follicles (SWF). The expression level of ZP2 decreased dramatically during follicular development, and the highest expression was observed in the SWF. Western blot and immunohistochemical analyses using the specific antibody against ZP2 indicate that the 80 kDa protein is the authentic ZP2, and the immunoreactive ZP2 protein is also present in the oocytes.
    [Show full text]
  • Binding of Mouse ZP2 to Sperm Proacrosin 4129 from the Specific Activity of Labelling and Avogadro’S Constant (Bleil Et Al., 1988)
    RESEARCH ARTICLE 4127 Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization Elizabeth Howes1, John C. Pascall1, Wolfgang Engel2 and Roy Jones1,* 1Signalling Programme, The Babraham Institute, Cambridge CB2 4AT, UK 2Institut fur Humangenetik, Universitat Gottingen, D-37073, Germany *Author for correspondence (e-mail: [email protected]) Accepted 7 August 2001 Journal of Cell Science 114, 4127-4136 (2001) © The Company of Biologists Ltd SUMMARY The mouse zona pellucida glycoprotein, mZP2, is thought complementary binding sites on proacrosin/acrosin in to be the secondary receptor on eggs for retention of solid-phase binding assays. The same competitors also acrosome-reacted sperm during fertilization. Here, we displace attached sperm from the zona pellucida of eggs in present evidence that one of its complementary binding an in vitro fertilization system. This combination of genetic, proteins on sperm is proacrosin/acrosin. mZP2 binds to biochemical and functional data supports the hypothesis proacrosin null sperm considerably less effectively than to that mZP2-proacrosin interactions are important for wild-type sperm. Binding is mediated by a strong ionic retention of acrosome-reacted sperm on the egg surface interaction between polysulphate groups on mZP2 and during fertilization. Safe mimetics of suramin have basic residues on an internal proacrosin peptide. The potential as non-steroidal antifertility agents. stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a Key words: Fertilization, Proacrosin, Zona Pellucida Glycoprotein polysulphonated anticancer drug, compete with mZP2 for mZP2, Secondary binding, Mouse INTRODUCTION consensus of opinion on the nature of the primary mZP3- binding molecules on the mouse sperm surface.
    [Show full text]
  • ZP2 Pathogenic Variants Cause in Vitro Fertilization Failure and Female Infertility
    © American College of Medical Genetics and Genomics ARTICLE ZP2 pathogenic variants cause in vitro fertilization failure and female infertility Can Dai, PhD1, Liang Hu, MD,PhD1,2,3,4, Fei Gong, MD, PhD1,2,3, Yueqiu Tan, PhD1,2,3, Sufen Cai, MD1,2, Shuoping Zhang, MD1,2, Jing Dai, MS1,2, Changfu Lu, PhD1,2,3, Jing Chen, MS2, Yongzhe Chen, MS2, Guangxiu Lu, MD1,3,4, Juan Du, PhD1,2,3 and Ge Lin, MD, PhD1,2,3,4 Purpose: The oocyte-borne genetic causes leading to fertilization families. All oocytes carrying PV were surrounded by a thin ZP that failure are largely unknown. We aimed to identify novel human was defective for sperm-binding. Immunostaining indicated a lack pathogenic variants (PV) and genes causing fertilization failure. of ZP2 protein in the thin ZP. Studies in CHO cells showed that Methods: We performed exome sequencing for a consanguineous both PV resulted in a truncated ZP2 protein, which might be family with a recessive inheritance pattern of female infertility intracellularly sequestered and prematurely interacted with other characterized by oocytes with a thin zona pellucida (ZP) and ZP proteins. fertilization failure in routine in vitro fertilization. Subsequent Conclusion: We identified loss-of-function PV of ZP2 causing a PV screening of ZP2 was performed in additional eight unrelated structurally abnormal and dysfunctional ZP, resulting in fertiliza- infertile women whose oocytes exhibited abnormal ZP and similar tion failure and female infertility. fertilization failure. Expression of ZP proteins was assessed in mutant oocytes by immunostaining, and functional studies of the wild-type Genetics in Medicine (2019) 21:431–440; https://doi.org/10.1038/s41436- and mutant proteins were carried out in CHO-K1 cells.
    [Show full text]
  • Biology of Mammalian Fertilization: Role of the Zona Pellucida
    Perspectives Biology of Mammalian Fertilization: Role of the Zona Pellucida Jurrien Dean Laboratory of Cellular and Developmental Biology, National Institute ofDiabetes and Digestive and Kidney Diseases, National Institutes ofHealth, Bethesda, Maryland 20892 Introduction before approaching the ovulated egg in the oviduct (Fig. 1 A). In mammals, a series of carefully orchestrated events culmi- Motile sperm pass through the enveloping cumulus oophorus, nate in the fusion of a sperm and egg to form a one-cell zygote, which is composed of a glycosylaminoglycan matrix and cu- the obligatory precursor of all cells in the embryo. The overall mulus cells. They then bind to the zona pellucida that rate of fertilization in humans has contributed to sustained surrounds the mammalian egg (1, 2). The mouse and human increases in the world population and added urgency to the zonae pellucidae are composed of three major glycoproteins, need to develop new, effective contraceptive agents. For some, ZP1, ZP2, and ZP3. Solubilized zonae pellucidae from unfertil- however, the success rate is considerably lower, and there are ized mouse eggs (but not from two-cell embryos) can inhibit millions of infertile couples in the United States. Dramatic ad- sperm binding to ovulated eggs (3). This sperm-receptor activ- vances in reproductive biology have provided some relief for ity of the zona has been ascribed to a class of 3.9-kD 0-linked these individuals through the development of in vitro fertiliza- oligosaccharides on ZP3 (4). ZP2 has been implicated as a sec- tion techniques. Further progress requires additional under- ondary sperm receptor that binds sperm only after the induc- standing ofthe molecular basis for normal fertilization and our tion of the sperm acrosome reaction (5).
    [Show full text]
  • ZP2 Antibody Cat
    ZP2 Antibody Cat. No.: 30-070 ZP2 Antibody Specifications HOST SPECIES: Rabbit SPECIES REACTIVITY: Human Antibody produced in rabbits immunized with a synthetic peptide corresponding a region IMMUNOGEN: of human ZP2. TESTED APPLICATIONS: ELISA, WB ZP2 antibody can be used for detection of ZP2 by ELISA at 1:62500. ZP2 antibody can be APPLICATIONS: used for detection of ZP2 by western blot at 2.5 μg/mL, and HRP conjugated secondary antibody should be diluted 1:50,000 - 100,000. POSITIVE CONTROL: 1) Cat. No. 1211 - HepG2 Cell Lysate PREDICTED MOLECULAR 68 kDa WEIGHT: Properties PURIFICATION: Antibody is purified by protein A chromatography method. CLONALITY: Polyclonal CONJUGATE: Unconjugated PHYSICAL STATE: Liquid September 25, 2021 1 https://www.prosci-inc.com/zp2-antibody-30-070.html Purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% BUFFER: sucrose. CONCENTRATION: batch dependent For short periods of storage (days) store at 4˚C. For longer periods of storage, store ZP2 STORAGE CONDITIONS: antibody at -20˚C. As with any antibody avoid repeat freeze-thaw cycles. Additional Info OFFICIAL SYMBOL: ZP2 ALTERNATE NAMES: ZP2, ZPA, Zp-2 ACCESSION NO.: NP_003451 PROTEIN GI NO.: 4508045 GENE ID: 7783 USER NOTE: Optimal dilutions for each application to be determined by the researcher. Background and References The zona pellucida is an extracellular matrix that surrounds the oocyte and early embryo. It is composed primarily of three or four glycoproteins with various functions during fertilization and preimplantation development. ZP2 is a structural component of the zona pellucida and functions in secondary binding and penetration of acrosome-reacted spermatozoa.
    [Show full text]
  • Three-Dimensional Structure of the Zona Pellucida
    Reviews of Reproduction (1997) 2, 147–156 Three-dimensional structure of the zona pellucida David P. L. Green Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago Medical School, PO Box 913, Dunedin, New Zealand The zona pellucida is the extracellular coat that surrounds the mammalian oocyte. It forms a spherical shell of remarkably uniform thickness (5–10 µm in eutherian mammals). The mouse is currently the largest source of data on the zona pellucida and this review is built largely on these data. The zona pellucida is composed of three proteins in both mice and humans: ZP1, ZP2 and ZP3. These proteins are glycosylated and, in mice, have mature relative molecular masses of 200 000, 120 000 and 83 000, respectively. ZP1 is a dimer of two apparently identical subunits. All three mouse proteins have been sequenced and possess transmembrane domains at their C-terminal ends coupled with furin cleavage sites immediately upstream. Sequence data have been used to provide an accurate assessment of the mole ratios of the three proteins. The ratio of ZP2:ZP3 is close to 1:1, whereas ZP1 is approximately 9% of the combined mole amounts of ZP2 and ZP3. Ultrastructural evidence suggests that the mouse zona pellucida is composed of filaments constructed by head-to-tail association of globular proteins. The co- ordinate synthesis of the three zona pellucida proteins coupled with the near 1:1 stoichiometry of ZP2 and ZP3 is consistent with a model in which ZP2–ZP3 heterodimers are the basic re- peating units of the filament, with cross-linking of filaments by dimeric ZP1.
    [Show full text]
  • Coordinate Expression of the Three Zona Pellucida Genes During Mouse Oogenesis
    Development 121, 1947-1956 (1995) 1947 Printed in Great Britain © The Company of Biologists Limited 1995 Coordinate expression of the three zona pellucida genes during mouse oogenesis Olga Epifano1,*, Li-fang Liang1, Mary Familari1, Malcolm C. Moos, Jr2 and Jurrien Dean1 1Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA 2Laboratory of Developmental Biology, Center for Biologics Evaluation and Research, Bethesda, MD 20892, USA *Author for correspondence SUMMARY The mammalian zona pellucida is an extracellular matrix restricted to the oocyte. ZP2 transcripts, but not ZP1 or that surrounds growing oocytes, ovulated eggs and early ZP3, are detected in resting (15 µm diameter) oocytes, and embryos. The mouse zona is composed of three sulfated all three zona transcripts coordinately accumulate as glycoproteins: ZP1, ZP2 and ZP3. Each is critically oocytes begin to grow. Together they represent approxi- involved in fertilization, the postfertilization block to mately 1.5% of the total poly(A)+ RNA in 50-60 µm oocytes. polyspermy and protection of the preimplantation embryo. In the latter stages of oogenesis, their abundance declines We have previously isolated cDNAs encoding mouse ZP2 and each zona transcript is present in ovulated eggs at less and ZP3 and now report the isolation of a full-length cDNA than 5% of its maximal level. No zona transcripts were encoding ZP1. Mouse ZP1 is composed of a 623 amino acid detected above background signal in granulosa cells. We polypeptide chain with a signal peptide and a carboxyl conclude that, in mice, the three zona pellucida genes are terminal transmembrane domain, typical of all zona expressed in a coordinate, oocyte-specific manner during proteins.
    [Show full text]
  • Human Homolog of the Mouse Sperm Receptor (Human Zona Pellucida/ZP3/Oocyte-Specific Gene Expression/Cis-Acting Elements/Human Sperm Receptor) MARGARET E
    Proc. Nati. Acad. Sci. USA Vol. 87, pp. 6014-6018, August 1990 Developmental Biology Human homolog of the mouse sperm receptor (human zona pellucida/ZP3/oocyte-specific gene expression/cis-acting elements/human sperm receptor) MARGARET E. CHAMBERLIN AND JURRIEN DEAN Laboratory of Cellular and Developmental Biology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Christian B. Anfinsen, May 31, 1990 ABSTRACT The human zona pellucida, composed of see ref. 7). The recent cloning of mouse ZP2 and ZP3 cDNAs three glycoproteins (ZP1, ZP2, and ZP3), forms an extracel- (8-10) and the characterization of their genomic loci (8, 11, lular matrix that surrounds ovulated eggs and mediates species- 12) has provided a wealth of molecular detail on the primary specific fertilization. The genes that code for at least two of the structure of the zona proteins and their developmentally zona proteins (ZP2 and ZP3) cross-hybridize with other mam- regulated expression during oogenesis (8, 13). malian DNA. The recently characterized mouse sperm receptor Less is known about the primary structure and the func- gene (Zp-3) was used to isolate its human homolog. The human tions of the zona pellucida proteins from other species, homolog spans =18.3 kilobase pairs (kbp) (compared to 8.6 including that of human. The human zona pellucida is com- kbp for the mouse gene) and contains eight exons, the sizes of posed of three glycoproteins, ZP1 (90-110 kDa), ZP2 (64-76 which are strictly conserved between the two species. Four kDa), and ZP3 (57-73 kDa) (14, 15), at least one of which short (8-15 bp) sequences within the first 250 bp of the 5' appears to be modified following fertilization (15).
    [Show full text]
  • Sperm-Borne Phospholipase C Zeta-1 Ensures Monospermic Fertilization
    www.nature.com/scientificreports OPEN Sperm-borne phospholipase C zeta-1 ensures monospermic fertilization in mice Received: 1 September 2017 Kaori Nozawa1,2, Yuhkoh Satouh 1, Takao Fujimoto1,3, Asami Oji1,3 & Masahito Ikawa 1,2,3,4 Accepted: 3 January 2018 Sperm entry in mammalian oocytes triggers intracellular Ca2+ oscillations that initiate resumption of Published: xx xx xxxx the meiotic cell cycle and subsequent activations. Here, we show that phospholipase C zeta 1 (PLCζ1) is the long-sought sperm-borne oocyte activation factor (SOAF). Plcz1 gene knockout (KO) mouse spermatozoa fail to induce Ca2+ changes in intracytoplasmic sperm injection (ICSI). In contrast to ICSI, Plcz1 KO spermatozoa induced atypical patterns of Ca2+ changes in normal fertilizations, and most of the fertilized oocytes ceased development at the 1–2-cell stage because of oocyte activation failure or polyspermy. We further discovered that both zona pellucida block to polyspermy (ZPBP) and plasma membrane block to polyspermy (PMBP) were delayed in oocytes fertilized with Plcz1 KO spermatozoa. With the observation that polyspermy is rare in astacin-like metalloendopeptidase (Astl) KO female oocytes that lack ZPBP, we conclude that PMPB plays more critical role than ZPBP in vivo. Finally, we obtained healthy pups from male mice carrying human infertile PLCZ1 mutation by single sperm ICSI supplemented with Plcz1 mRNA injection. These results suggest that mammalian spermatozoa have a primitive oocyte activation mechanism and that PLCζ1 is a SOAF that ensures oocyte activation steps for monospermic fertilization in mammals. At mammalian fertilization, sperm entry causes Ca2+ oscillations, repetitive acute increases and decreases in cytosolic Ca2+ levels lasting for several hours in human and mouse eggs, which trigger not only resumption of the meiotic cell cycle but also block polyspermy, the entry of multiple sperm heads into the ooplasm1.
    [Show full text]